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Chapter-3

Somatic Embryogenesis in C. borivilianum and Scale-up of

Fast Growing Somatic Embryo Cultures in Liquid Medium

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3.1 INTRODUCTION

Mass propagation via tissue culture (micropropagation, clonal propagation) of selected,

improved plant species of economic importance is advantageous over conventional

methods of vegetative propagation (cutting, grafting). During the last thirty years,

micropropagation and other in vitro techniques have been widely used for the mass

propagation of horticultural, crop, ornamental and medicinal plants and for conservation of

genetic resources, particularly in those crops which are vegetatively propagated or have

recalcitrant seeds which cannot be stored under conventional seed bank conditions

(George and Sherrington, 1984; George, 1993).

Plants can be multiplied by tissue culture technique employing either of the two

different pathways: organogenesis or somatic embryogenesis. Generally in the first case,

shoots and roots are formed sequentially and in response to appropriate culture conditions

(mainly to the type and concentration of plant growth regulators present in the culture

medium). This type of development is also characterized by the presence of vascular

connections between the mother tissue and the regenerating organs (Terzi and Lo Schiavo,

1990). On the other hand, somatic embryogenesis can be described as the process through

which haploid or diploid somatic cells develop into structures that resemble to zygotic

embryos being bipolar structures and without any vascular connection with the parental

tissue through an orderly series of characteristic embryological stages (globular, heart-

shaped, torpedo-shaped and finally cotyledonary stage) without fusion of gametes

(Williams and Maheswaran, 1986; Emons, 1994; Raemakers et al., 1995). Somatic

embryogenesis is recently much advocated technology for cloning plants using tissue

culture. One striking characteristic of the somatic embryo is its continuous growth

resulting from the absence of developmental arrest (Faure et al., 1998). Both processes,

organogenesis as well as somatic embryogenesis have been reported to occur in the same

explant of a particular plant species (He et al., 1990), but also to originate from particular

tissue layers or cells within explants (Osternack et al., 1999).

The first demonstration of in vitro somatic embryo production was carried out

independently by Steward et al. (1958) and Reinert (1959) working with carrot, a member

of family Umbelliferae. Since then, the number of higher plants species from which

somatic embryos could be obtained and regenerated into plants has continuously

increased. This phenomenon has been documented in at least 200 gymnosperm and

angiosperm species (Raemakers et al., 1995). Some species, however, are more

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recalcitrant than others regarding both initiation of embryogenic cultures and regeneration

of plants (Rao, 1996). This aspect of somatic embryogenesis has been the subject of

several reviews (Tomes, 1985; Durzan and Gupta, 1988; Bannikova and Barabanova,

1990; Gray and Purohit, 1991; Tautorus et al., 1991).

Somatic embryogenesis has several advantages compared to other propagation methods:

• The mass propagation of plants in a shorter time through multiplication of

embryogenic propagules is one of the most commercially attractive application of

somatic embryogenesis (Merkle et al., 1990). The somatic embryogenesis is

preffered over over axillary or adventitious shoot propagation. Embryos are bipolar

structures, bearing both root and shoot apices which are necessary for complete

plant development, therefore in most of the cases the procedure of cutting plantlets

into segments and transferring segments onto new media in the proliferation phase

are not necessary. Due to the simultaneous development of both the shoot and root

meristems and its origin from single cell, propagation via somatic embryogenesis

results into the production of uniform plants in a process involving fewer culture

transfers resulting in economies of time, cost efficiency and increased yields of

somatic seedlings and regeneration in bioreactors.

• The high volume multiplication of embryogenic propagules can be utilised directly

in various studies as in regeneratation of genetically transformed plants (Litz and

Gray, 1995; Vicient and Martinez, 1998) and somatic hybridization.

• Somatic embryos can be cryopreserved for germplasm storage, which makes it

possible to maintain a large number of genotypes. The success in inducing

dormancy and the accomplishment of long-term storage, together with the

achievement of encapsulation of somatic embryos, has also opened up the

possibility for their use in the synthetic seed technology (Gray and Purohit, 1991;

Gray et al., 1995; Litz and Gray, 1995).

• In vitro embryogenesis in addition to its usefulness for cloning and vegetative

propagation of a given plant, can serve as a model system to study the molecular,

cytological, physiological and developmental events underlying embryogenesis

(Kiyosue et al., 1993; Zimmerman, 1993; Dodeman et al., 1997).

• The use of embryogenic callus and cell suspension cultures as well as somatic

embryos themselves as a source of protoplasts has been exploited for a range of

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species taking advantage of the totipotency of these embryogenic cultures

(McCown and Russell, 1987; Merkle et al., 1990; Finch et al., 1991; Chang and

Wong, 1994; Funatsuki et al., 1996, Jimenez, 1996).

• Secondary or recurrent embryogenesis which is reported in a large number of plant

species (Raemakers et al., 1995), offers a great potential for in vitro production of

embryo metabolites such as lipids and seed storage proteins. Embryogenic property

can be maintained for prolonged period of time by repeated cycles of secondary

embryogenesis (Raemakers et al., 1995).

• Finally, the embryogenic development of somatic cells appears to be more

sensitive to the application of exogenous chemical compounds than the growth of

whole plants or even callus cultures. This offers the possibility of using in vitro

screening and selection procedures to identify plant genotypes resistant to certain

characters such as aluminum toxicity or toxins produced by pathogens (Merkle

et al., 1990).

The use of liquid cultures for the cultivation of somatic cells in recapitulating

embryogeny was reported by Steward et al. (1958) and Reinert (1959) in carrot (Daucus

carota L.). Bioereactor technology employing liquid medium for plant micropropagation

is advancing much faster for somatic embryo based culture systems (Vasil, 1994; Ziv,

1995; Takayama and Akita, 1998). Somatic embryo driven routes are best suited for the

technology. Somatic embryos are small and can be adequately handled in scaled-up

procedures with low labour inputs since embryos can be grown individually and freely

floating in liquid medium. Induction of somatic embryogenesis in liquid cultures using

bioreactor systems offers opportunities for bioprocess automation and environmental

control, and consequently is expected to reduce manual labour and costs. They are

amenable to sorting and separation by image anlysis, dispensing by automated systems

and can be encapsulated as synthetic seeds and either stored or planted directly with the

aid of mechanized systems (Ammirato and Styer 1985; Cazzulino et al. 1991; Cervelli and

Senaratna 1995; Sakamoto et al. 1995).

However, the potential of somatic embryogenesis in many plant species is yet to be

utilized due to certain problems faced during the process including low germination rate of

somatic embryos, genotypic influences and induction of somatic embryogenesis in a limited

number of explants. A sharp focus is needed to solve these problems. Further research is

required at both the biochemical and molecular levels to understand the mechanism of

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somatic embryogenesis, enabling the induction of somatic embryogenesis in other

recalcitrant plant species. The combination of this technique with other modern technologies

including computer-aided image analysis, robotics, bioreactors (including temporary

immersion systems), somatic embryo encapsulation, development of appropriate somatic

embryo coating material etc., require further investigations for producing millions of

somatic seeds in a short time and to cut down the cost of seed production.

C. borivilianum commonly known as safed musli is widely used for its tonic and

aphrodisiac properties due to the prescence of steroidal saponins viz. neotigogenin,

neohecogenin, stigmasterol and tokorogenin (Tandon and Shukla, 1995b). Tuberous roots

of this plant are of commercial importance containing steroidal saponins as one of the

important phytochemical constituents. They are used as an important ingredient for

various therapeutic applications in the Ayurvedic and Unani (Oudhia and Tripathi, 1999)

systems of medicine. Generally C. borivilianum has been used along with other plants

such as Asparagus ascendense, A. racemosum, Curculigo orchioides and Withania

somnifera in several formulations in Indian system of medicine (Kirtikar and Basu, 1975;

Ramawat et al., 1998). Dried roots are exported in bulk quantity and there is considerable

demand for dried roots of the safed musli in Indian as well as international drug market.

In the present study attempts have been made to standardize various aspects related

to somatic embryogenesis in C. borivilianum with a view to use the protocol for large

scale multiplication of C. borivilianum employing liquid culture medium. For this various

aspects related to induction, development, maturation and germination of somatic embryos

were studied in the present investigation. In this context present part of the investigation

deals with following objectives:

(a) Selection of suitable explant and to work out the nutrient and other cultural

requirements for induction and growth of callus in C. borivilianum.

(b) Studies on induction of somatic embryos from embryogenic callus and maturation

of somatic embryos in semi-solid medium.

(c) Germination and plantlet regeneration from somatic embryos in semi-solid

medium.

(d) Assessment of growth and development of somatic embryos in liquid medium.

(e) Molecular characterization of in vitro grown plants derived from somatic embryos

through molecular techniques.

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(f) 3.2 REVIEW OF LITERATURE

(g) Somatic embryogenesis is the process of a single cell or group of cells initiating

the developmental pathway that leads to reproducible regeneration of non-zygotic

embryos capable of germinating to form complete plants. Under natural conditions

this pathway is not normally followed, but employing tissue culture methods,

somatic embryogenesis occurs more frequently and as an alternative to

organogenesis for regeneration of whole plants. Adherence to this pattern of

morphogenesis depends on co-ordinated behaviour of a cell or group of cells to

establish polarity as a unit and thereby initiating gene action sequentially specific

to emerging tissue regions. Thus, a complete sexual apparatus is not an essential

prerequisite for embryogeny in tissue cultures. Developed structures commonly

referred to as ‘somatic embryos’ were first discovered in cultures of carrot cultures

and related members of family Umbelliferae in the late 1950s (Waris, 1957;

Steward et al., 1958; Reinert, 1959). Waris (1957) working with Oenanthe

aquatica (Umbelliferae) described development of embryo-like structures from the

cells of aseptically cultured Oenanthe aquatica root tips. These early studies were

significant, because they confirmed Haberlandts prediction that embryos can arise

from single cells in culture (i.e., cellular totipotency). Soon after the discovery of

somatic embryogenesis it was asked how and when somatic cells become

embryogenic. F.C. Steward (1958) believed that some special chemical stimuli,

such as those found in embryosac fluids like coconut milk (more accurately,

coconut water), cause ‘free’ cultured cells to embark upon an embryogenic

pathway. Kohlenbach (1978, 1982) seems to have been the first to raise the point

in print that established embryogenic cultures are already determined. Many have

since investigated the nature of somatic embryogenesis and aimed to develop its

potential for applied uses like clonal micropropagation (Krikorian, 1982; Halperin,

1995, Krikorian and Simola, 1999).

(h) The early history of somatic embryogenesis has been reviewed earlier (Halperin,

1995; Krikorian and Simola, 1999). Many species that were once considered

recalcitrant have been shown to respond in vitro and a significant body of

information has been gathered to establish the embryogenic potential of somatic

plant cells in many plant species including medicinal and aromatic plants (Table-

22).

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(i) Acquisition of embryogenic competency largely relies on dedifferentiation, a

process whereby existing transcriptional and translational profiles are erased or

altered in order to allow cells to set a new developmental programme. The

activation of cell division is required to maintain the dedifferentiated cell fate, as

well as for embryo differentiation. This is not only true for those embryogenic

systems where embryogenic callus formation precedes somatic embryo

development (indirect somatic embryogenesis) but also for those systems where

somatic embryos develop on primary explants without an intervening callus phase

(direct embryogenesis).

(j) Table-22: Some important plant species in which somatic embryogenesis has

been reported

Group Family Plant species Active constituent(s)

Explant(s) used for micropropagation

Reference(s)

Dicotyledons Amaryllidaceae Narcissus confusus

Alkaloid (Galanthamine)

Mature zygotic embryos

Selles et al., 1999

Apocynaceae Catharanthus roseus

Alkaloids (Vinblastine and vincristine)

Hypocotyls of in vitro germinated seeds

Junaid et al., 2006

Thevetia peruviana

Glycoside (Peruvoside)

Leaf discs Sharma and Kumar, 1994

Araliaceae Eleutherococcus sessiliflorus

Saponins (Ginsenosides)

Zygotic embryos Choi et al., 2002

Panax spp. Cotyledons Choi et al., 2003

Asclepiadaceae Gymnema sylvestre

Gymnemic acid

Cotyledons, hypocotyls and young leaves from germinated seedlings

Ashok et al., 2002

Hemidesmus indicus

Phytosterols (Hemidesmol, hemidesterol), saponins

Leaf stem explants Sarasan et al., 1994

Tylophora indica Alkaloids (Tylophorine, tylophorenine and tylophorinidine)

Leaves Jayanthi and Mandal, 2001

Berberidaceae Dysosma pleintha Lignan (Podophyllotoxin)

Immature seeds and mature zygotic embryos

Chuang and Chang, 1987

Dioscoreaceae Dioscorea zinziberensis

Steroids (Diosgenin) Stem explants Shu et al., 2005

Echinaceae Echinacea purpurea

High molecular weight polysaccharides and isobutylamides

Petiole explants Choffe et al., 2000

Fagaceae Quercus spp. Tannins (Tannic acid)

Immature zygotic embryos

Chalupa, 1990

Gentianaceae Gentiana pneumonanthe

Secoiridoid glycoside (Gentiopicroside)

Leaves and apical meristem

Bach and Pawlowska, 2003

Lamiaceae Salvia fruticosa Caffeic acid ester (Rosamarinic acid)

Leaf explants Kintzios et al., 1999

Meliaceae Azadirachta indica

Gedunin Cotyledons,hypocotyl Su et al., 1997

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Papaveraceae Eschcholzia californica

Benzophenanthridine alkaloids (Sanguinarine)

Seeds Park and Facchini, 2000; Park and Facchini, 2001

Papaver somniferum

Morphinan alkaloids (Morphine, codeine and thebaine)

Seedling hypocotyls Nessler, 1982 Septa of capsules

Septa of capsules Ovecka et al., 2000

Piperaceae Piper nigrum Alkaloids (Piperine), amides (Futoamide), alkamide

Zygotic embryos Joseph et al., 1996

Polygonaceae Fagopyrum esculentum

Flavonoid glycoside (Rutin)

Immature embryos Nescovic et al., 1986

Ranunculaceae Aconitum heterophyllum

Aconites (Atisin, heteratisin and hetasin)

Leaf and petiole explants

Giri et al., 1993

Scrophulariaceae Bacopa monniera Bacosides Nodal explants Tiwari et al., 1998

Digitalis obscura Cardiac glycosides Hypocotyl Arrillaga et al., 1987

Solanaceae Hyoscyamus niger

Tropane alkaloids (Scopolamine, hyoscyamine)

Seedling, cotyledon, petiole

Cheng and Raghavan,1985

Solanum surattense

Steroidal alkaloids (Solasodine)

Cotyledon, leaf explants

Ramaswamy et al., 2004

Ammi majus Linear furanocoumarins (Psoralens)

Hypocotyl explants Grewal et al., 1976

Angelica sinensis Ligustilide Immature embryos Tsay and Huang, 1998

Carum carvi Caraway oil ketone (Carvone) and terpene (d-limonene)

Hypocotyl Furmanowa et al., 1991

Centella asiatica Glycosides (Asiaticoside, indocentelloside, brahmoside, theankuniside)

Leaf segments Paramageetham et al., 2004

Coriandrum sativum

Fatty acid (Petroselenic acid)

Zygotic embryos, hypocotyls segments

Kim et al., 1996

Valerianaceae Nardostachys jatamansi

Valepotriates Petiole explants Mathur, 1993

Zingiberaceae Zingiberofficinale Sesquiterpenes Leaf segments Kackar et al., 1993

Monocotyledons Liliaceae

Allium sativum Allicin Leaf and root sections

Fereol et al., 2002

Asparagus officinalis

Phytoestrogens (Asparagosides)

Shoot segments Reuther, 1977

Orchidaceae Phalaenopsis amabilis

Anthocyanins Leaf explants Chen and Chang, 2006

Gymnosperms Pinaceae Pinus brutia Terpenes (α-pinene) Immature zygotic embryos

Yildirim et al., 2006

Taxaceae Taxus brevifolia Taxol Immature zygotic embryos

Chee, 1996

(k)

(l) The process of somatic embryogenesis can be divided into different steps: (1)

initiation of embryogenic tissue from the primary explant, (2) multiplication and

proliferation of embryogenic tissue: during this proliferation phase, embryogenic

tissue can be frozen and stored in liquid nitrogen. The tissue can be used for

preservation of elite germplasm and later to resume the somatic embryogenesis

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process, (3) maturation of somatic embryos: once a sufficient quantity of

embryogenic tissue is obtained, the next phase is to stop proliferation and allow the

tissue to form mature somatic embryos. Usually clumps of embryogenic tissue are

transferred to a maturation medium which promotes maturation of the somatic

embryos, (4) germination of somatic embryos; mature somatic embryos are picked

from the clumps and placed onto germination medium for early plant development.

(m) 3.2.1 Development of somatic embryogenesis

(n) Somatic embryogenesis development has two main phases, (a) differentiated

somatic cells acquire embryogenic competence and proliferate as embryogenic

cells; (b) the embryogenic cells display their embryogenic competence and

differentiate into somatic embryos. Both processes appear to be independent from

each other. The former phase, called phase 0 by Komamine et al. (1992),

determination phase by Rao (1996) and induction phase by Dodeman et al. (1997)

has no direct counterpart in zygotic embryogenesis (Emons, 1994).

(o) 3.2.1.1 Induction of somatic embryogenesis

(p) Induction of embryogenic growth in carrot and many other species appears to

occur in one of the two ways. Somatic embryos can be formed directly on the

surface of an organized tissue such as leaf or stem segments, cotyledons,

hypocotyls portion of seedlings, zygotic embryos, from protoplasts or from

microspores. They can also be formed indirectly via an intermediate step of callus

or suspension culture (in these cases additional factors are needed to induce

dedifferentiation and reinitiation of cell division of already differentiated cells

before they can express embryogenic competence. (Williams and Maheswaran,

1986; Emons, 1994). Direct and indirect somatic embryogenesis have been

considered as two extremes of a continum (Williams and Maheswaran, 1986;

Carman, 1990). Once induction of embryogenic determined cells has been

achieved, there appears to be no fundamental differences between indirect and

direct somatic embryogenesis (Williams and Maheswaran, 1986).

(q) Embryogenic cells are unique: superficially they resemble meristematic cells

though generally they are smaller, more isodiametric in shape, have larger, more

densely staining nuclei and nucleoli and have a denser cytoplasm (Williams and

Maheswaran, 1986; Carman, 1990).

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(r) 3.2.1.2 Expression of somatic embryogenesis

(s) Once the induction of an embryogenic state is complete, the mechanism of pattern

formation that leads to the zygotic embryos is common to all other forms of

embryogenesis (Mordhorst et al., 1997). Thus, somatic and zygotic embryos share

similar gross ontogenies with both typically passing through globular, heart-

shaped, torpedo-shaped and cotyledonary stages (Gray et al., 1995; Toonen and de

Vries, 1996). Schiavone and Cooke (1985) described an intermediate growth stage

between globular and heart-shaped embryos and termed it oblong embryo.

Although heart and torpedo-shaped embryos have traditionally been defined as

separate stages of the embryo development, the distinction between them is

apparently based on the difference in size (Schiavone and Cooke, 1985). Yet

another type of embryo development takes place in conifers (Tautorus et al., 1991)

which includes three stages: globular, early cotyledonary and late cotyledonary

embryos (Dong and Dunstan, 2000).

(t) In the model proposed by Komamine et al. (1992) explaining the early process of

embryogenesis, the first phase describes the expression of somatic embryogenesis.

During this phase cell clusters (already induced to express embryogenic

development) proliferate slowly and apparently without differentiation. In the

second phase rapid cell division occurs in certain parts of cell clusters leading to

the formation of globular embryos. In the third phase plantlets develop from

globular embryos after passing through heart and torpedo-shaped embryo stages.

Induction and expression of somatic embryogenesis might be triggered by different

factors depending on explant types, genotypes (species, cultivars), structural

factors, plant growth regulators, physiological conditions and other chemical and

physical factors.

(u) 3.2.2 Factors

associated with embryogenic competence

(v) 3.2.2.1 Explant

(w) The success in obtaining regenerating cultures of several plant species

which were once regarded recalcitrant has been possible largely due to shift in

emphasis from media manipulation to explant selection. Totipotent embryogenic

cells have been most commonly obtained from explants of embryonic or young

seedling tissues. Excised small tissues from young inflorescences (before

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maturation of floral primordia) are equally effective for induction of somatic

embryogenesis in cultures. Other explants used are the scutellum, young roots,

petioles, immature leaves and hypocotyls. For the establishment of embryogenic

cultures of alfalfa petiole sections from 2-3 youngest fully expanded leaves were

suitable. (McKersie et al., 1989).

(x) Unlike dicotyledonous plants, the vegetative parts of monocotyledonous plant do

not readily proliferate in cultures. Embryogenic or meristematic tissues i.e. young

inflorescences, seedlings and leaves are the explants generally used. Immature

zygotic embryos have proved to be good explants to raise embryogenic cultures.

Success was obtained in regeneration of plants from embryogenic cultures of

cereals and grass species (Vasil and Vasil, 1991). In cereals, zygotic embryos

exhibit the potential to form somatic embryos shortly after histogenesis and prior

to embryo maturation (Williams and Maheswaran, 1986) which corresponds to a

period from 11-14 days post anthesis (DPA) in Triticum aestivum. For initiation of

embryogenic cultures in coniferous species, immature zygotic embryos have been

used as the most preffered explants (Bornman, 1993). In these cases embryogenic

tissue arises from cells in the suspensor region of the zygotic embryo.

(y) Age, physiological state, genotype and orientation of the explants on the medium

influence the induction of somatic embryogenesis. These aspects govern the

disruption of explant tissue integrity, callus friability, isolation of cells and other

requirements in order to enhance somatic embryogenesis in various species

(Merkle et al., 1995). In general only a very limited number of cells in any given

explant respond by becoming embryogenic (Toonen and de Vries, 1996).

(z) 3.2.2.2 Genotype

(aa) Genotypic effects on somatic embryogenesis has also been reported to play

a vital role. Of the 500 varieties of rice screened, 19 showed 65-100%

embryogenesis, 41 showed 35-64% embryogenesis and the remaining 440 cultivars

were less efficient (Kamiya et al., 1988). Genotypic variations could also be due to

endogenous levels of hormones (Carman, 1990). Somatic embryogenesis in

orchard grass is also shown to be a heritable dominant trait (Gavin et al., 1989).

Somatic embryogenesis in alfalfa is a genetically controlled process (Hernandez-

Fernandez and Christie, 1989; Kielly and Bowley, 1992). Cytoplasmic factors have

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also been implicated in the control of somatic embryogenesis (Rode et al., 1988;

Peng and Hodges, 1989).

(bb) 3

.2.2.3

Structural factors

(cc) T

he initiation of polarity in embryos is often regarded as the first step in

embryogenesis (Warren and Warren, 1993). In carrot and Medicago it clearly

appears that in order to confer embryogenic competence to the individual cells

capable of producing embryos, the first division has to be asymmetric, producing

two cells of different sizes (Dudits et al., 1991). In most species showing

embryogenic capacity, the asymmetric division does not form an embryo directly

but forms a proembryogenic mass (PEM), in which only one or a few cells

subsequently develop into an embryo (Komamine et al., 1992; Nuti and Giorgetti,

1995). The rest of the PEM cells are probably eliminated through a cycle of

programmed cell death as observed in Norway spruce (Filonova et al., 2000).

(dd) McCabe et al. (1997) recently observed that a cell-wall antigen on cells

destined to form embryos segregates asymmetrically during a formative division,

producing one daughter cell with a cell wall antigen recognized by the antibody

JIM8 and the other without it, and the epitope-free cells ultimately form somatic

embryos.

(ee) Another important point is the necessity of the cells subjected to

embryogenic induction for physical isolation from the surroundings. This is the

case with somatic embryos formed in suspension cultures. This physical isolation

leads to a more or less varying degree of physiological isolation caused by loss of

plasmodesmata between surrounding cells, interrupting symplastic continuity and

reducing electrical coupling (Warren and Warren, 1993).

(ff) 3.2.2.4 Plant growth regulators

(gg) (a) Effect of exogenously applied plant growth regulators on

somatic embryogenesis

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(hh) Hormones are one of the most important factor in the regulation of somatic

embryogenesis. Although auxins which are known to mediate the transition from

somatic to embryogenic cells, are the agents generally used to induce

embryogenesis, the effect of other plant growth regulators on this phenomenon can

not be overlooked. While in monocots, primary embryogenesis in most of the cases

was induced by auxin-supplemented media, many other growth regulators are used

to induce somatic embryogenesis in dicot species. Amongst 65 dicot species,

somatic embryogenesis was induced in 17 species on hormone-free media, in 29

species on auxin-containing media and in 25 species on cytokinin-supplemented

media (Raemakers et al., 1995). Among auxins, 2,4-dichlorophenoxyacetic acid

(50%) was the most frequently used auxin followed by naphthalene acetic acid

(28%), indole-3-acetic acid (6%), indole-3-butyric acid (6%), picloram (5%) and

dicamba (5%), (Fig.-12). In the case of cytokinins, N6-benzylaminopurine was

used most often (57%), followed by kinetin (37%), zeatin (3%) and thidiazuron

(3%).

(ii)

(jj)

(kk)

(ll)

(mm)

(nn)

(oo)

(pp)

(qq) Fig.- 12:

Percentagewise use of different auxins in somatic embryogenesis

(rr) The influence of exogenously applied auxins preferentially 2,4-D on the induction

of somatic embryogenesis is well documented (Dudits et al., 1991; Yeung, 1995).

Among different auxin analogues used to induce somatic embryogenesis, 2,4-D is

by far the most efficient and therefore this synthetic growth regulator is used in the

majority of embryogenic cell and tissue culture systems. It can be suggested that

2,4-D above a certain specified concentration has a dual effect in these cultures, as

50%

28%

6%6%

5% 5%

2,4-D NAA IAA IBA P Dicamba

127

an auxin (directly or through endogenous IAA metabolism) and as a stressor

(Feher et al., 2001, 2002). It has also been suggested that 2,4-D (or other auxin-

type herbicides) affect electrical patterns (Goldsworthy and Mina, 1991),

membrane permeability (Schauf et al., 1987), IAA binding to the auxin binding

protein ABP1 (Deshpande and Hall, 2000). Recently, auxininic herbicides have

been shown to interact with ethylene and ABA synthesis, increasing the cellular

levels of these so called stress hormones (Grossmann, 2000; Wei et al., 2000).

(ss) In some of the cases in which the exogenous application of auxins has proved to be

beneficial for somatic embryogenesis induction, further development of the

existing somatic embryos can be achieved by reducing its level or removing auxin

from the culture media.

(tt)

The effect of the addition of other plant growth regulators is not so well

documented. It has been observed that the addition of abscissic acid (ABA) inhibits

the precocious germination of the somatic embryos and allows them to mature into

normal-shaped plantlets as observed in grapevine (Rajasekaran et al., 1982;

Goebel-Tourand et al., 1993). Nishiwaki et al. (2000) observed that seedlings of

carrot formed somatic embryos when cultured on medium containing ABA and the

number of embryos originating per explant depended on ABA concentration

employed. Endogenous levels of ABA also appear to be significant in some

monocots for initiation of embryogenic cultures (Bhaskaran and Smith, 1990). The

role of ABA in somatic embryogenesis may be exerted through regulation of

certain genes (e.g., DC8) that are thought to be involved in desiccation and

maturation phases of embryogenesis (Hatzopoulos et al., 1990). Rajasekaran et al.

(1987a, b) proposed that ABA could exert its role on somatic embryogenesis by

regulating carbohydrate metabolism via inhibition of α-amylase activity.

(uu) Despite the wide range of physiological effects of gibberellins, their effect

primarily as gibberellic acid (GA3), has only been minimal when added to culture

media (Krikorian, 1995). Exogenous application of GA3 has been reported to

inhibit somatic embryogenesis and somatic embryo development in several species

but it has been reported that this plant growth regulator is required for germination

of the mature somatic embryos if chilling treatment is not applied (Takeno et al.,

1983).

128

(vv) Embryo development in somatic tissues has also been reported also in the

prescence of other growth regulators such as cytokinin (Sagare et al., 2000).

Although the addition of cytokinins as the sole plant regulator has proved to be

effective in inducing somatic embryogenesis, in certain cases, the stimulatory

effects of these plant growth regulators are not universal and many times their

addition should often be coupled with auxins to obtain the desired effect (Merkle

et al., 1995).

(ww) Embryo development in somatic tissues has been reported even in the

absence of growth regulators (Choi et al., 1998). Non hormonal inducers can also

be used to promote the transition from somatic to embryogenic phase. Such

inducers include high sucrose concentration or osmotic stress (Kamada et al.,

1993), heavy metal ions (Kiyosue et al., 1990; Pasternak et al., 2002) and high

temperature (Kamada et al., 1989). Explant (non-embryogenic) cells can be

induced to an embryogenic state by a variety of procedures that usually include pH

shock or treatment with various chemical substances. In carrot, pH changes in the

culture medium can direct the transition from somatic cells to cells which are able

to form embryo-like structures (Smith and Krikorian, 1990a, b) while in Brassica

microspores a temperature shock has been shown to have this capability (Pechan

and Keller, 1988). Wounding and high salt concentrations positively influnced

somatic embryo induction in diverse plant species (Dudits et al., 1995). These

procedures were accompanied by increased expression of diverse stress related

genes evoking the hypothesis that somatic embryogenesis is an adaptation process

of in vitro cultured plant cells (Dudits et al., 1995). In alfalfa leaf protoplasts,

embryogenic cells could be formed in response to different oxidative stress

inducing compounds in the prescence of auxins and cytokinins (Feher et al, 2001,

2002; Pasternak et al., 2002). Mitogen activated protein kinase (MAPK)

phosphorylation cascades may link oxidative stress response to auxin signaling and

cell cycle regulation (Hirt, 2000).

(xx) However, it is still not clear what changes a somatic cell to undergo

transition into an embryogenic cell capable of forming an embryo. There appears

to be the absence of a single, universally applicable signal that renders cells

embryogenic (Mordhorst et al., 1997).

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(yy) (b) Effect of endogenous hormones on somatic

embryogenesis

(zz) Anatomical and physiological differences between embryogenic and non-

embryogenic cultures are thought to be significant factors in the competency for

somatic embryogenesis. Amongst these differences, the endogenous hormone

levels play an important role since they regulate the process of explant

differentiation in culture (Grieb et al., 1997) and are postulated to be the main

cause of difference between genotypes showing different grades of competence

(Bhaskaran and Smith, 1990). The observed responses in in vitro tissue and cell

culture systems after a growth regulator supplemention, are related to interactions

of an endogenous phytohormone system with the exogenous growth regulators

supplied to the nutrient medium (Neumann, 1988; Carman, 1990). Evidence for

this sense has been reported by Liu et al. (1998) who observed an accumulation of

endogenous IAA in soybean hypocotyl explants after their treatment with two

exogenously applied auxins, NAA and IBA.

(aaa) During the last few years, a large body of experimental observations has

accumulated on the central role of endogenous IAA and ABA levels during the

early phases of embryogenesis. Higher endogenous IAA concentration has been

shown to be associated with increased embryogenic response in various

species/explants (Ivanova et al., 1994; Michalczuk and Druart, 1999; Jimenez and

Bangerth, 2001a, b). In carrot cells, exogenous 2,4-D stimulated the accumulation

of large amounts of endogenous IAA (Michalczuk et al., 1992 a, b). These authors

hypothesized that embryogenic competence of carrot cells is closely associated

with the several fold increase in endogenous IAA levels due to the prescence of

2,4-D. It was also suggested that this 2,4-D acts by disturbing endogenous auxin

metabolism and the direct auxin effect of 2,4-D is less significant. The polar

transport of endogenous auxin was found to be an important factor in somatic

embryo formation on cotyledon explants of ginseng, which did not require

exogenous growth regulator application (Choi et al., 1997). These observations

suggest that temporal and spatial changes in endogenous auxin levels are important

factors controlling the embryogenic cell fate. It has also been observed that the

polar transport of auxin is essential for the establishment of bilateral symmetry

during embryogenesis in dicotyledonous somatic (Schiavone and Cooke, 1987)

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and zygotic (Liu et al., 1993) embryos and this was also demonstrated for

monocotyledonous zygotic embryos (Fischer and Neuhaus, 1996).

(bbb) (c) Hormone concentration vs. sensitivity

(ccc) T

rewavas (1981) postulated that the sensitivity of the tissues to a change in the

hormone concentration is more important than the change in the concentration

itself. There are some evidences, that sensitivity to hormones may be important in

conferring embryogenic competence to tissue culture explants. The phytohormone

content of the culture medium is a major factor regulating growth and

differentiation of plant tissue in in vitro conditions only if a responsive tissue is

used as experimental system (Bell et al., 1993; Somleva et al., 1995). Although

plant growth regulators play a key role in inducing somatic embryogenesis, many

other factors also affect the disposition of a particular tissue to undergo somatic

embryogenesis.

(ddd) 3

.2.2.5

Other chemical and physical factors affecting somatic embryogenesis

(eee) (1) Nitrogen source

(fff) The form of nitrogen in the medium significantly affects in vitro

embryogenesis. Halperin and Wetherell (1965) reported that in the wild carrot

cultures raised from petiolar segments, embryo development occurred only if the

medium contained some amount of reduced nitrogen. Meijer and Brown (1987)

found an absolute requirement for ammonium during induction and differentiation

of somatic embryos in alfalfa. In carrot the change in NH4+ concentration could

induce somatic cells to form embryo like structures (Smith and Krikorian, 1989).

(ggg) The suspension cultures of orchardgrass maintained on a modified medium

containing Schek and Haberlandt’s (SH) salts, sucrose, inositol, thiamine

hydrochloride and dicamba exhibited sustained proliferation of cell masses that

differentiated only root primordia. The addition of casein hydrolysate (CH) to the

established cultures stimulated rapid development of somatic embryos which

matured directly in the liquid medium. Subsitution of CH with amino acids did not

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support the production and maturation of embryos. However, a combination of

proline and serine or threonine, each at concentration of 12.5 mM proved even

superior to CH for embryo production particularly with respect to the quality of

somatic embryos (Trigiano and Conger, 1987; Trigiano et al., 1992). The yield of

alfalfa somatic embryos was also considerably improved when amino acids such as

proline, alanine, arginine and glutamine were added to the callus maintainence

medium resulting in upto 100 times more embryo production (Redenbaugh et al.,

1991).

(hhh) (2) Polyamines

(iii)There is some evidence to suggest that polyamines are required for embryo

development in vivo and in vitro (Altman et al., 1990; Mengoli and Bagni, 1992).

Increase in the endogenous level of polyamines (Montague et al., 1978) and the

enzymes for their biosynthesis (Fienberg et al., 1984) concomitant with the

induction of somatic embryogenesis in carrot and the suppression of somatic

embryogenesis by the inhibitors of polyamine biosynthesis (Minocha et al., 1990)

suggest the involvement of polyamines in somatic embryogenesis. The role of the

observed changes in polyamine content and biosynthesis and their causal

relationship to somatic embryogenesis remains to be established.

(jjj)Besides this there are other factors which affect somatic embryogenesis in one or

other way such as antibiotics (Mathias and Boyd, 1986) ethylene action inhibitors,

including AgNO3 (Songstad et al., 1988), oxygen concentration (Carman, 1990;

Nishimura et al., 1993), electrical stimulation (Dijak et al., 1986) and selective

subculture (Vasil and Vasil, 1991; Bornman, 1993), the light quality (Torne et al.,

2001), pre-treatment of donor plants and subculture duration (Morocz et al., 1990).

A more detailed description of some of these factors was given by Tulecke (1987)

and Harada (1999).

(kkk) In case of embryogenic suspension cultures, culture density of cell

suspensions could be another important factor that affects somatic embryogenesis.

While a high cell density is required for the formation of embryogenic cell clusters

from single cells (Nomura and Komamine, 1985), a relatively lower cell density

favours the development of embryos from embryogenic cells (Fujimura and

Komamine, 1979). This may be related to the secretion of proteins and/or other

cellular factors into the culture medium. Secreted (extracellular) and constitutive

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(intracellular) proteins are considered to play a very important part in induction of

somatic embryogenesis (de Vries et al., 1988; Gavish et al., 1991, 1992). The

addition of arabinogalactan proteins, isolated from the culture medium of

embryogenic carrot lines and from dry carrot seeds was capable of promoting the

formation of proembryogenic masses even in previously non-embryogenic carrot

cell lines (de Jong et al., 1993).

(lll)3.2.3 Gene expression in somatic embryogenesis

(mmm) Plant development and differentiation are regulated directly or indirectly by

changes of gene expression especially during embryogenesis (Dong and Dunstan,

2000). Although the process of embryo induction from cells in culture is not fully

understood, it is now generally believed that in the continued presence of auxin, a

differential change in gene expression probably associated with increased

demethylation of DNA in proembryogenic masses (PEMs) occurs (Lo Schiavo

et al., 1989; Litz and Gray, 1995). Under these circumstances, the PEMs within the

culture synthesize all the gene products necessary to complete the globular stage of

embryogenesis. At that point, the PEMs also contain many other mRNAs and

proteins whose continued presence generally inhibits the continuation of the

embryogenic programme. The removal of auxin in some cases results in the

inactivation of a number of genes which supports embryogenic events. The

observation that some carrot cell lines are able to develop only upto the globular

stage in the continued prescence of auxin, suggests that new gene products are

needed for the transition from globular to the heart stage and that these new

products are synthesized only when exogenous auxin is removed from the medium

(Zimmerman, 1993). Gene expression studies during the different stages of this

process of embryogenesis have been carried out (Henry et al., 1994; Kawahara and

Komamine, 1995; Dong and Dunstan, 2000) and suggest that the number of genes

specifically expressed during these events is rather limited (Ermakov and

Matveeva, 1994; Dodeman and Ducreux, 1996; Schrader et al., 1997). Dodeman

and Ducreux (1996) indicated that changes in hormonal levels in tissue cultures

may modify the synthesis of some somatic embryogenesis specific proteins.

(nnn) Finding out the right conditions to induce somatic embryogenesis in

different species and cultivars is yet based on trial and error experiments

(Jacobsen, 1991; Henry et al., 1994), analyzing the effect of different culture

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conditions and media and modifying especially the type and levels of plant growth

regulators. However, the role that the genotype and its physiological condition play

in this process is also important. The induction of somatic embryos and subsequent

recovery of viable plants from somatic embryos is not common for majority of

species (Merkle et al., 1995). The ability to understand the mechanisms involved in

the induction and expression of somatic embryogenesis in different species will

certainly increase the number of species capable of being regenerated by this

process.

(ooo) 3.2.4 Mass propagation of plants through somatic embryogenesis

employing liquid medium in shake flasks/bioreactors

(ppp) Somatic embryogenesis offers potential for scaling-up mass propagation of

plants in bioreactors. Conventional micropropagation requires intensive labour

which often limits its viability and application. Somatic embryos could be easier to

handle since they are relatively small and uniform in size, and do not require

cutting into segments and individual implanting on media during proliferation. In

addition, somatic embryos have the potential for long term storage through

cryopreservation or dessication, which facilitates flexibility in scheduling

production and transportation and therefore fits large-scale production. There have

been several reports on the large scale propagation of several plant species

including horticultural and medicinal plants through somatic embryogensis using

bioreactors (Table-23).

(qqq) Table-23: Some important plants propagated in bioreactors through

somatic embryogenesis pathway

Plant species Response Reference(s) Apium graveolens Somatic embryos Nadel et al., 1990 Camellia sinensis Somatic embryos, plantlets Akula et al., 2000 Citrus deliciosa Somatic embryos Cabasson et al., 1997 Coffea arabica Somatic embryos

Somatic embryos, plantlets Etienne et al., 1997a Etienne- Barry et al., 1999

Cyclamen persicum Callus, somatic embryos Hvoslef-Eide and Munster, 1998 Daucus carota Callus, somatic embryos Jay et al., 1994; Archambault et

al., 1995 Eleutherococcus senticosus Somatic embryos, emblings,

plantlets Paek et al., 2001; Kim and Kim, 2001

Eschcholtzia californica Somatic embryos Archambault et al., 1994 Euphorbia pulcherrima Somatic embryos Preil, 1991; Luttman et al., 1994 Hevea brasiliensis Somatic embryos Etienne et al., 1997b Medicago sativa Callus, somatic embryos Stuart et al., 1985, 1987;

McDonald and Jackman, 1989; Denchev et al., 1992

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Musa spp. Somatic embryos Escalant et al., 1994 Nerine sarniensis Proembryogenic clusters, somatic

embryos, bulblets Lilien-Kipnis et al., 1994

Phoenix dactylifera Embryogenic callus Tisserat and Vandercook, 1985 Picea glauca Somatic embryos Attree et al., 1994 Picea glauca-engelmannii Somatic embryos Tautorus et al., 1994 Picea marianna Soamtic embryos Tautorus et al., 1994

(rrr) The production of synthetic seeds through successful encapsulation of

somatic embryos from shake-liquid or bioreactor cultures has been reported for

some species i.e. carrot (Redenbaugh et al., 1991; Sakamoto et al., 1995), alfalfa

(Fujii et al., 1992; Senaratna, 1992), celery (Kim and Janick, 1989; Onishi et al.,

1994) and white spruce (Attree et al., 1994) and is currently under continuous

investigation for several other species.

(sss) For efficient large scale cultures of both somatic embryos and organogenic

plant tissues, the bioreactor configuration and volume of the medium in the culture

vessel must be determined along with mixing, the intensity of shear stress and

aeration requirements of the plant tissue to be propagated (Doran, 1993; Hvoslef-

Eide and Munster, 1998). The wide array of bioreactor designs have been

developed depending on plant tissue being propagated. For different bioreactor

configurations, the principles of mixing, oxygen transfer and biological oxygen

demand are more or less same. (Curtis, 2005). There are many reports on up-

scaling of somatic embryogenesis employing different bioreactor configurations

(Table-24).

(ttt) Table-24: Bioreactor modifications used for up-scaling somatic

embryogenesis in different plant species

Bioreactor modifications Plant species Reference(s) Stirred tank bioreactor Picea sitchensis Ingram and Marvituna, 2000 Hanging stirred bar bioreactor Picea sitchensis

Piper spp. Ingram and Marvituna, 2000 Marvituna and Buyukalaca, 1996

Aeration-agitation bioreactor Hordeum vulgare Medicago sativa Picea sitchensis Zea mays

Stirn et al., 1994 Stuart et al., 1985 Moorhouse et al., 1996 Stirn et al., 1994

Spin filter bioreactor with a spinning filter for harvesting spent medium

Daucus carota Styer, 1985

Ballon type bubble bioreactor Aralia elata Acanthopanax koreanum

Paek et al., 2001, 2005

Silicone tubing aerated bioreactor for bubble free oxygen supply

Clematis tangutica Euphorbia pulcherrima

Luttman et al., 1994

Silicone tubing aerated bioreactor with a slow speed stirrer that regularly changes direction of rotation

Betula pendula Cyclamen persicum

Hvoslef-Eide, 2000 Hvoslef-Eide and Munster, 1997, 1998

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Temporary immersion sytems Coffea arbica Hevea brasiliensis Phoenix dactylifera

Teisson and Alvard, 1995 Etienna et al., 1997b Tisserat and Vandercook, 1985

(uuu)

(vvv) Kim and Kim (2001) reported the efficient mass production of Siberian

ginseng somatic embryos in bioreactors where the somatic embryos at the torpedo

stage were transferred to 5-10 l airlift bioreactors and cultured for 10-15 days and

somatic embryos developed into emblings. Paek et al. (2005) developed a protocol

for large-scale production of Siberian ginseng somatic embryos in a 500 l ballon

type bubble bioreactor (BTBB). This protocol is being applied on large-scale in

Korea for the commercial production of secondary metabolites (ginsenosides) from

mature somatic embryos of Siberian ginseng (Microplants Co. Ltd., Daejon, South

Korea and CBN Biotech., Chungbuk National University, Cheongju, South Korea).

Using the same protocol, more than 500,000 somatic embryos of thornless Aralia

elata at different developmental stages were harvested from a 10 l BTBB after 6

weeks of culture (Paek et al., 2005).

(www) Hvoslef-Eide et al. (2005) designed and configured six identical bioreactors

to provide optimal conditions for somatic embryogenesis from cultured cells.

There are two features with their bioreactors that make them more gentle to the

cultured plant cells compared to various other commercial designs. Their

bioreactor design provides gentle stirring through a slow speed stirrer that regularly

changes direction of rotation to prevent quiet zones in the suspension in which

cells can settle and grow. Secondly, bubble free oxygen is provided through

hanging silicone tubing loops. All cultures recorded growth in the bioreactors

compared to Erlenmeyer flasks. So far, the bioreactors have been successfully used

for embryogenic cultures of birch (Betula pendula), (Hvoslef-Eide, 2000),

cyclamen (Cyclamen persicum), (Hvoslef-Eide and Munster, 1997, 1998).

(xxx) 3.2.4.1 Physico-chemical parameters affecting somatic embryogenesis

in shake flasks/bioreactors

(yyy) (a) Dissolevd oxygen

(zzz) High aeration rates were found to inhibit growth of cell suspensions

cultured in airlift bioreactors.Somatic embryo development in alfalfa and poinsettia

suspension cultures was enhanced at increased levels (78% and 60% respectively)

of O2 (Stuart et al., 1985; Preil, 1991). This could be explained due to an effect of

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‘stripping’ of the volatiles produced by the plant cells which are apparently

necessary for cell growth (Smart and Fowler, 1984). Increasing O2 levels from

21% to 80% in bioreactor cultures of Boston fern clusters enhanced their growth.

The best production of somatic embryos from embryogenic cultures of Eschcholtza

californica and carrot in a helical ribbon impellar bioreactor was achieved under

20% oxygen concentration. Low O2 concentration (5-10%) inhibited biomass and

somatic embryo production while high O2 concentration (60%) resulted in

undifferentiated biomass production (Archambault et al., 1994, 1995). Additional

studies are required to provide information on optimal dissolved O2 requirements

in large-scale liquid cultures.

(aaaa) (b) Mineral Nutrient consumption

(bbbb) The availability of mineral nutrients during culture period depends on the

physical state of culture medium (whether agar-gelled or liquid), the type and size

of the plant biomass and physical properties of the culture. Factors such as pH,

temperature, light, aeration, the concentration of minerals, the medium volume and

the viscosity of the medium will determine the rate of absorption of various

nutritional constituents (Williams, 1992; Debergh et al., 1994). In several species

the depletion of NH4+ is the first limiting factor in biomass growth and somatic

embryo development. Increasing the concentration of NH4+ resulted in maximum

somatic embryo production in carrot cultures in a helical ribbon impellar bioreactor

(Archambault et al., 1995).

(cccc) A thorough and detailed study of nutrient uptake in E. californica

embryogenic cultures was achieved in a helical ribbon impeller bioreactor

(Archambult et al., 1994). In somatic embryos of spruce cultured in bioreactors,

80% of the ammonium was consumed by the growing biomass (Ilan et al., 1995).

Paek et al. (2005) investigated detailed anlysis of various nutrient compounds

during Lilium bulblet growth in ballon type bubble bioreactor (BTBB). In general,

biomass growth is limited by the availability of phosphate, nitrogen and

carbohydrates and to a lesser extent by the availability of calcium, magnesium, and

other ions. Inspite of these results, there is still need for detailed investigation on

hormonal interactions and dynamics of various nutrient compounds. Offline

analysis of changes in nutrient and hormone concentrations during bioreactor

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culture will present new possibilities for the better manipulation of embryogenesis

and organogenesis. (Paek et al., 2005).

(dddd) (c) Carbohydrate supply and utilization

(eeee) Cultured tissues require a constant supply of carbohydrates as the source of

energy. Sucrose and to a lesser extent glucose, fructose or sorbitol are the most

commonly used carbohydrates in vitro. Carbohydrate sources are well known to

affect the somatic embryogenesis (Stuart et al., 1987; Archambault et al., 1994;

Tautorus et al., 1994). A higher yield of alfafa embryos was obtained when 30g/l

maltose combined with NH4+ was used instead of 30g/l sucrose in combination

with various nitrogen sources (Stuart et al., 1987). In embryogenic suspension

cultures of celery, the addition of mannitol reduced cell lysis and enhanced somatic

embryogenesis (Nadel et al., 1989). In mechanically stirred bioreactors, 60 mM

sucrose resulted in the highest cell biomass and somatic embryos number. The effect

of various carbohydrates on spruce somatic embryos revealed that the response was

species dependent (Tautorus et al., 1994). In embryogenic cultures of E. californica

grown in a helical ribbon impeller bioreactor, sucrose uptake started after 100 h of

cell/tissue inoculation and sucrose was depleted after 600-800 h in culture

(Archambault et al., 1994).

(ffff) (d) Effects of pH of culture medium

(gggg) In alfalfa development of the somatic embryos was affected by the pH ion

concentration of the culture medium. A higher rate of embryo production was

observed at a constant pH of 5.5 than at a non-buffered medium or at lower pH

levels (Stuart et al., 1985).

(hhhh) Carrot cell differentiation into somatic embryos was affected by the pH in a

controlled bioreactor; the highest rate of embryo production was observed at pH

level of 4.3. However, the embryo development was arrested before the embryos

reached the torpedo stage and further development continued only when the pH ion

concentration of the medium was 5.8. The changes in carrot embryo development

were associated with sugar uptake and ammonium depletion and can be attributed

to enzyme and metabolic activity at an optimal pH (Jay et al., 1994). It appears that

pH requirements are species specific and developmental stage dependent. Precise

recording of fluctuations in parameters like pH in computer controlled bioreactors

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will improve repeatability of complex biological processes like somatic

embryogenesis (Paek et al., 2005).

(iiii) (e) Effect of growth regulators

(jjjj) The use of plant growth regulators in liquid cultures can be more effective

in controlling the proliferation and regeneration potential than in agar-gelled

medium due to the direct contact of plant cells and tissues with the medium (Ziv,

1989; Smith and Spoomer, 1994; Sandal et al., 2001).

(kkkk) In embryogenic cultures of Nerine sarniensis, both auxin and cytokinin

were used to induce proembryogenic clusters. Embryogenic expression was

however achieved only after a short exposure to 2-isopentenyladenine (2-iP) and

further subculture in growth regulator free medium (Lilien-Kipnis et al., 1994).

(llll) The information on use of abscissic acid for induction of somatic

embryogenesis is limited and was found effective mainly in the later stages of

somatic embryo development promoting normal embryo growth and maturation in

carrot and alfalfa embryogenic cultures (Ammirato and Styer, 1985; Denchev et

al., 1990).

(mmmm) (f) Cell and aggregate density, foaming and medium rheology in

bioreactors

(nnnn) Growth and proliferation of the biomass in bioreactors depends on the air

flow supply for the aeration and mixing and to avoid the plant biomass

sedimentation. In many plants cultivated in bioreactors continuous aeration, mixing

and circulation causes shearing damage, cell wall breakdown and accumulation of

cell debris which is made up mainly of polysaccharides. Cell debris accumulation

results in foaming, adhesion of cells and aggregates to the culture vessel walls and

the development of a ‘crust’ at the upper part of the bioreactor vessel. This layer

prevents adequate circulation, causing additional cell debris formation and a

demand for higher aeration rates that intensify the clogging problem (Scragg,

1992). As the biomass increases and the cultures become viscous, higher rates of

aeration are required to allow for oxygen supply and circulation. Ziv (1992a)

observed that medium viscosity and foaming were reduced by using half strength

of MS minerals.

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(oooo) Shearing stress and cell wall damage were greatly reduced in a Vibra-mix

bubble free oxygenated bioreactor, in which silicone tubing was used for air supply

(Preil, 1991; Luttman et al., 1994). Embryogenic cells cultured in a helical ribbon

impeller bioreactor produced poor quality embryos due to shearing stress and cell

damage when the mixing speed was increased from 60 to 100 rpm (Archambault et

al., 1994). The introduction of polyethylene glycol 6000 changed the rheology of

the medium in alfalfa liquid cultures and improved somatic embryo development

beyond the globular stage while it was arrested in a less viscous medium (Denchev

et al., 1990).

(pppp) 3.2.4.2 Analysis and monitoring of biomass growth

(qqqq) Carbon dioxide measurements are a good indicator of growth and may

serve as an online indicator for monitoring cell biomass growth inspection. The gas

concentration in headspace or dissolved in the medium may be measured. Optical

density may also be used as indicators of growth. Application of image anlysis

technology is another alternative for measuring cell density (Cazzulino et al., 1991;

Pepin et al., 1999). Besides measuring biomass concentration, these tools will give

information about aggregate size and distribution, pigmentation and morphology of

the culture, which is a great advantage when growing embryos.

(rrrr) The application of liquid cultures for micropropagation in bioreactors using

the embryogenic pathway is becoming a more efficient alternative system for

scale-up and automation in vitro (Aitken-Christie et al., 1995). However,

inconsistencies in optimizing bioreactor types and culture parameters have been

reported. Although the main cause of these inconsistencies may be due to species-

to-species variations, careful consideration is needed in interpreting these results.

Therefore once the culture conditions have been established in a small-scale

bioreactor, cultures can be easily scaled up to large-scale bioreactors (Paek et al.,

2005).

(ssss) The successful exploitation of bioreactors as a commercial

micropropagation system will depend on careful studies of plant morphogenesis in

liquid media and the understanding of the control mechanism of embryo

development from proembryogenic cell masses. For plant regeneration in

bioreactors through somatic embryogenic pathway, the chemical and physical

environment in relation to biomass growth and controlled regeneration should be

140

further investigated in particular plant species. The level of carbohydrates and the

levels and ratios of growth regulators will need to be studied further in more detail.

Improvements are needed for the practical automatic somatic embryo production

systems that can cope with synchronization of the somatic embryo development

and overcoming the difficulty in embling acaclimatization (Paek et al., 2005).

(tttt) Bioreactor cultures for somatic embryogenesis in many plant species are

being established in several commercial laboratories. The technique is estimated to

render a great reduction in propagule unit costs compared to propagation through

the oragnogenic pathway. Using somatic embryogenesis as the propagation

pathway in a semi-automated system (Cervelli and Senaratna, 1995) production

costs were reduced by 24%. Simple bioreactors combined with automated

inoculation, sorting and delivery systems are the solution for efficient and low cost

micropropagation through somatic embryogenic pathway.

(uuuu) 3.2.5 In vitro somatic embryogenesis studies in Chlorophytum species

(vvvv) Attempts have been made to propagate Chlorophytum through somatic

embryogenesis by various workers (Kukda et al., 1994; Purohit et al., 1994b; Jain

et al., 1997; Arora, 1999; Arora et al., 1999; Joshi et al., 2003; Rizvi et al., 2007b)

wherein various factors influencing induction, germination etc. of somatic embryos

were studied.

(wwww) Kukda et al. (1994) described a method for somatic embryogenesis and

plant regeneration in C. borivilianum. They induced callus from immature embryos

inoculated on MS medium containing 1.0 mgl-1 2,4-D. Maturation of these

embryos was observed on MS medium supplemented with 0.1 mgl-1 2,4-D.

Somatic embryos successfully converted into plantlets on plant growth regulator

free basal MS medium.

(xxxx) Purohit et al. (1994b) also reported somatic embryogenesis and plant

regeneration in Chlorophytum. Callus was induced from immature zygotic

embryos inoculated on MS medium containing 1.0 mgl-1 2,4-D. Initially callus was

slow growing, soft, watery and slimy. After six weeks of growth and subsequent

subcultures on MS medium containing 0.5 mgl-1 2,4-D, yellow, compact, hard,

nodular, shiny somatic embryos developed. Embryogenic cultures were maintained

by repeated subculturing after every four weeks on MS medium containing 0.25

mgl-1 2,4-D. Plantlets could be recovered from 20% of these embryos when

141

inoculated on auxin free MS medium and the cultures were transferred to light

conditions. Precocious germination of somatic embryos and profuse rooting was

observed in this study.

(yyyy) Jain et al. (1997) reported induction of somatic embryos in C. borivilianum

from seedling derived callus on B5 medium supplemented with 0.5 mgl-1 2,4-D or

0.1 mgl-1 2,4,5-T. Plantlet formation was observed on B5 medium without growth

regulators.

(zzzz) Arora et al. (1999) reported multiplication of C. borivilianum plants

through somatic embryogenesis in callus cultures obtained from seedling explants.

Somatic embryos were obtained on MS medium containing 2.25 µM 2,4-D and

1.15 µM Kn. In this study ammonium to nitrate nitrogen in the ratio of 1:4 at low

total nitrogen level (250 or 500 mgNl-1) in the medium was favourable for the

growth and somatic embryogenesis. Somatic embryos germinated on modified MS

medium supplemented with BAP.

(aaaaa) Leaf explants obtained from in vitro maintained shoots/plantlets were used

(Arora, 1999). Inspite of use of a large number of combinations and concentrations

of different cytokinins and auxins explant response percentage was erratic, low and

associated with slow callus growth. Little callus produced on certain treatments did

not survive on subculture. Callus produced on high concentration of TDZ from a

few leaf explants grew and produced embryos. However, these attempts using leaf

explants could not form a system for a continuous production of somatic embryos.

(bbbbb) Joshi et al. (2003) established callus cultures of C. borivilianum using

young shoot bases as explants on MS mediunm containing various cytokinins and

auxins either individually or in combination. They obtained fluffy unorganized,

loose and shiny mass of callus at the base of shoots on MS medium containg 5.0

mgl-1 BAP. When callus was subcultred on fresh medium, shoots differentiated

from this callus mass. Subsequently shoots rooted on three-fourth strength MS

medium containing 1.0 mgl-1 IBA. They reported that frequency of shoot

regeneration from callus cultures was little higher than shoot multiplication

obtained during clonal multiplication.

(ccccc) Rizvi et al. (2007b) reported somatic embryogenesis in C. borivilianum and

standardized various aspects related to induction and germination of somatic

embryos obtained from seedling explants. Moderate to good callus induction

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frequency was observed from seedling explants cultured on MS medium

supplemented with 0.25 mgl-1 Kn and 0.25–0.50 mgl-1 2,4-D. Regular subculturing

of callus induced on Kn and 2,4-D supplemented medium induced somatic

embryogenesis. Higher concentrations of 2,4-D were inhibitory for somatic

embryogenesis, therefore embryogenic cultures were further maintained on lower

concentration (0.25 mgl-1) of 2,4-D and 0.25 mgl-1Kn. Modified MS medium

containing 143 mgl-1 NH4NO3 and 1083 mgl-1 KNO3 was found optimal for somatic

embryogenesis. Effect of various plant growth regulators i.e. Kn, 2-iP and TDZ on

somatic embryo growth and development was also studied. Observations revealed

that amongst different growth regulators tested, 2-iP showed better response at

higher levels. Best response was observed at 1.5 mgl-1 level of 2-iP. Amongst

different amino acids tested for their effect on somatic embryogenesis, proline

gave better response than glutamine. Germination of somatic embryos was

achieved on MS medium supplemented with 3.5 mgl-1 BAP resulting in

multiplication of C. borivilianum plants.

(ddddd) 3.2.6 Analysis of genetic stability of plants regenerated via somatic

embryogenesis through RAPD technique

(eeeee) The genetic stability of in vitro regenerated plants is an essential

prerequisite for the large-scale propagation of any plant species especially those

which are of commercial importance. In vitro regenerated plants are usually

susceptible to genetic changes such as ‘somaclonal variations’ due to culture stress

(Dunstan and Thorpe, 1986; Cecchini et al., 1992; Rani and Raina, 1998).

(fffff) Methods for early detection of genetic variations include morphological

observations, cytological methods, isozymes and DNA analysis (Rani and Raina,

2003). In particular, the examination of randomly amplified DNA sequences for

polymorphism i.e. RAPD analysis (Williams et al., 1990) has been used to

ascertain the genetic stability of embryogenic systems for numerous plant species

such as gymnosperms: Picea mariana (Isabel et al., 1993), P. glauca (De Verno et

al., 1999), P. taeda (Tang, 2001) and the angiosperms such as Pennisetum

purpureum (Haydu and Vasil, 1981), peach (Hashmi et al., 1997), mango

(Jayasankar et al., 1998), Phoenix dactylifera (Javouhey et al., 2000) and

Tylophora indica (Jayanthi and Mandal, 2001). In the studies of Quercus species,

RAPD analysis has not detected genetic variation within embryogenic lines of Q.

143

suber (Gallego et al., 1997), Q. serrate (Ishii et al., 1999) or Q. ruber (Sanchez et

al., 2003) or between these lines and the somatic embryo derived plantlets.

(ggggg) In the present part of investigation, a procedure for rapid multiplication of

C. borivilianum through somatic embryogenesis pathway has been standardized in

seedling derived embryogenic callus of C. borivilianum. Subsequently RAPD

analysis was performed to assess the genetic homogeneity of randomly selected

plants regenerated in vitro through somatic embryogenesis.

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3.3 MATERIALS AND METHODS

3.3.1 Induction and maintenance of callus

3.3.1.1 Explant

Experiments were conducted on callus induction and two different explants i.e. leaf

segments and shoot bases regularly obtained from the sterile in vitro grown plants of

Chlorophytum borivilianum maintained on MS medium supplemented with 5.0 mgl-1 BAP

were used as explants. The details of the establishment of aseptic cultures of C.

borivilianum have been described earlier in chapter-2 (2.3). Similarly in vitro germinated

seedlings were also used for induction of callus in the present study.

3.3.1.2 Callus induction in C. borivilianum

Seeds of C. borivilianum obtained from mother plants (control plants) maintained in the

glass house of the institute were used. Callus was induced from the seedlings. Seeds were

treated with a liquid detergent, Teepol 4% (v/v) for 4-5 min. followed by washing under

running tap water for 2 hrs. Seeds were surface sterilized with 0.1% (w/v) aqueous

mercuric chloride solution for 5-6 min. and then rinsed several times with sterile distilled

water before inoculation. Seeds were cultured on MS (Murashige and Skoog) basal

medium supplemented with 20.0 mgl-1 GA3 for germination (Jain et al., 1997). Two

different basal media compositions i.e. MS (Murashige and Skoog, 1962) and B5

(Gamborg et al., 1968) supplemented with different concentrations (0.1-1.0 mgl-1) of 2,4-

D and Kn (0.1-0.5 mgl-1) were employed in the callus induction studies. Details of these

two basal salt media are presented in Table- 25.

For studies involving callus induction from leaves and shoot bases explants, five

different auxins viz. indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-

naphthalene acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D) and picloram (P),

(0.05-2.0 mgl-1) were added to MS basal medium individually while a cytokinin

thidiazuron (TDZ), (0.05-6.0 mgl-1) either individually or in combination with adenine

sulphate (AS), (5.0-40.0 mgl-1) were also tested.

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Table-25: Basic composition of different media used in the present study

Constituent(s) (mgl-1)

MS medium (Murashige and Skoogs, 1962)

B5 medium (Gamborg et al. , 1968)

KNO3 1900 2500 NH4NO3 1650 -

(NH4)2SO4 - 134

CaCl2.2H2O 440 150

MgSO4.7H2O 370 250 MnSO4.4H2O 22.3 10

ZnSO4.7H2O 8.6 2.0 CuSO4.5H2O 0.025 0.025

KH2PO4 170 -

NaH2PO4.2H2O - 150

H3BO3 6.2 3.0

KI 0.83 0.75 Na2MO4.2H2O 0.25 0.25

CoCl2.6H2O 0.025 0.025

FeSO4.7H2O 27.85 27.85

Na2EDTA 37.35 37.35

Thiamine HCL 0.1 10.0 Pyridoxine HCL 0.5 10.0

Nicotinic acid 0.5 1.0

Glycine 2.0 - Myoinositol 100 100

Sucrose 30,000 30,000

3.3.1.3 Establishment and maintenance of embryogenic callus

Embryogenic callus induced from seedlings on the optimal callus induction medium (MS

basal medium supplemented with 0.25 mgl-1 each of 2,4-D and Kn) was maintained by

subculturing after every 3-4 weeks on the fresh medium of the same composition.

3.3.2 Standardization of somatic embryogenesis in C. borivilianum

3.3.2.1 Optimization of culture medium

A substantial amount of nitrogen is required for somatic embryogenesis response. Optimal

levels of nitrogen in the medium have to be worked out to obtain the desired embryogenic

response. Cytokinins also have an important role to play in somatic embryogenesis such as

in induction of embryogenic callus and/or somatic embryos. Therefore certain experiments

were performed to observe the effect of these different factors on induction of somatic

embryogenesis.

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(a) Effect of different inorganic nitrogen sources on somatic embryogenesis

Effect of prescence of different levels of inorganic nitrogen sources i.e. NH4NO3 (143–

856 mgl-1) and KNO3 (360–2166 mgl-1) in the MS basal medium on somatic

embryogenesis were studied. Callus of seedling origin was inoculated on MS medium

containing 0.25 mgl-1 each of 2,4–D and Kn and different concentrations and

combinations of NH4NO3 (143, 285, 428, 571, 714, 856 mgl-1) and KNO3 (360, 722, 1083,

1444, 1805, 2166 mgl-1).

(b) Effect of different cytokinins on somatic embryogenesis

To study the effect of different cytokinins on somatic embryogenesis, seedling derived

callus was inoculated on modified MS medium (143 mgl-1 NH4NO3 and 1083 mgl-1 KNO3)

supplemented with 0.25 mgl-1 2,4–D and different concentrations of cytokinins i.e. Kn, 2-

iP (0.2-1.5 mgl-1) or TDZ (0.1-1.0 mgl-1).

(c) Role of different amino acids in enhancement of somatic embryogenesis

response

An experiment was designed to study the effect of amino acids i.e. glutamine and proline

on somatic embryogenesis in C. borivilianum. In a quest for the further enhancement of

somatic embryogenesis response, embryogenic callus cultures were transferred on

modified MS medium containing 0.25 mgl-1 2,4-D, 1.5 mgl-1 2-iP and different

concentrations (50-400 mgl-1) of proline or glutamine.

3.3.2.2 Somatic embryo development and maturation

Once induction of somatic embryos was standardized, attention was focussed on the

development and maturation of somatic embryos which later on lead to their germination

and may act as a critical factor. Therefore in the present study, experiments were also

performed for observing effect of different carbohydrates, different osmotica and abscissic

acid (ABA) on somatic embryo development and maturation.

(a) Effect of different carbohydrate sources

An appropriate concentration and type of carbohydrate supplemented to the culture

medium can enhance the development and maturation of somatic embryos by inducing

partial dessication. Therefore, experiments involving effect of different carbohydrates on

somatic embryo development and maturation were performed. Different carbohydrate

sources i.e. sucrose, glucose, fructose, lactose, galactose, cellobiose, rhamnose, xylose,

arabinose, maltose, mannose or soluble starch were tested in the present study. Each

147

carbohydrate source was tested at 3% concentration and was supplemented to previously

standardized embryogenic medium (modified MS medium fortified with 0.25 mgl-1 2,4-D,

1.5 mgl-1 2-iP and 100 mgl-1 proline).

(b) Effect of different osmotica

Embryo maturation in most of the cases of somatic embryogenesis is frequently associated

with the state of osmotic potential in tissues or medium surrounding the embryos. With

this background information an experiment was designed to observe the effect of different

plasmolyzing and non-plasmolyzing osmotica on somatic embryo maturation.

Plasmolyzing osmotica (D-mannitol or D-sorbitol, each tested at 3% concentration) were

supplemented to standardized embryogenic medium containing 3% sucrose. Non-

plasmolyzing osmoticum, polyethylene glycol (PEG) was added in the range of 1-11% to

standardized medium containing 3% sucrose as carbohydrate source.

(c) Effect of abscissic acid

The exogenous application of abscissic acid (ABA) to somatic embryo cultures has been

shown to reduce the rate of precocious germination and favouring their synchronized

maturation. Therfore the effect of ABA on somatic embryo maturation was studied in the

present part of investigation. ABA was supplemented to standardized embryogenic

medium at 0.03-1.85 mgl-1 concentrations. ABA was filter sterilized and added to culture

media after it was cooled to about 40°C. Because ABA is sensitive to light, ABA

supplemented medium was stored in dark.

3.3.3 Induction and development of somatic embryos in liquid culture medium

Liquid medium has been shown to increase the induction and growth of somatic embryos.

But for obtaining the desired response various factors such as inoculum density, pH level

should be optimized which may have their bearing on the induction and development of

somatic embryos. Casein acid hydrolysate, in some cases has been shown to improve the

embryogenic response and quality of embrogenic cultures. Therefore experiments were

conducted to study the effect of different physical states (semi-solid or liquid), different

inoculum densities, different pH and casein acid hydrolysate levels in liquid culture

medium on induction and development of somatic embryos.

(a) Physical state of the culture medium

Embryogenic callus of seedling origin was inoculated on standardized embryogenic

medium with agar (semi-solid medium) or without (liquid medium) agar (0.8%, w/v), (Hi-

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Media) for observing effect of different physical states of medium on somatic

embryogenesis.

(b) Determination of optimal inoculum density

To study the effect of different inoculum densities on somatic embryo induction in callus

and their maturation, 0.1-1.0g of embryogenic callus was inoculated in 40 ml of

standardized liquid culture medim.

(c) Somatic embryogenesis in liquid culture medium at varying levels of pH

Effect of different pH levels of standardized culture medium on somatic embryogenesis

was investigated. Embryogenic cell aggregates (0.4g) were inoculated in same

standardized liquid culture medium having different pH levels ranging between 3.86±0.1

to 7.86±0.1.

(d) Effect of casein acid hydrolysate

Effect of casein acid hydrolysate on somatic embryo growth and development was also

studied. Embryogenic cell aggregates were inoculated in standardized liquid medium

supplemented with different levels (1- 4%) of casein acid hydrolysate.

In the experiments involving semi-solid medium, the medium was solidified with

0.8% (w/v) agar (Hi-Media) while in case of liquid medium agar was excluded. The pH of

the medium was adjusted to 5.8±0.1 (except where mentioned otherwise) with 0.1 N HCl

and/or 0.1 N NaOH prior to autoclaving at 121ºC for 20 minutes. Each conical flask (100

ml capacity, Borosil) containing 40 ml semi-solid or liquid medium was plugged with

non-absorbent cotton. The liquid cultures were kept on a rotary shaker (New Brunswick

Scientific, USA) at agitation speed of 100 rpm. Cultures were maintained at 25±2ºC

temperature and 40–50% relative humidity under 16 hrs. photoperiod (cool white

fluorescent tubes, 45 µ mol m-2 s-1). In the present study in all experiments on somatic

embryogenesis about 150 mg (except where mentioned otherwise) callus of seedling

origin was employed as initial inoculum. Two-three explants (callus pieces) were

inoculated per culture flask. Observations on induction and growth of somatic embryos

were recorded after three weeks of growth period. While in other experiments,

observations for development and maturation of somatic embryos (number of globular and

cotyledonary stage embryos) and fresh weight were recorded after four weeks of growth

period. The cotyledonary stage embryos were embryos with an elongated embryonic

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region with cotyledons. Different stages of somatic embryos were photographed using

photomicroscope (Leica, Apo, Germany).

3.3.4 Germination of somatic embryos

Somatic embryos developed from seedling derived embryogenic callus were transferred to

MS medium supplemented with different levels of BAP (1.5-6.5 mgl-1) or MS medium

without phytohormones which served as control medium. The germination percentage of

somatic embryos was recorded after 4 weeks of culture period.

3.3.5 Hardening of somatic embryo derived plants

Plantlets obtained from in vitro germinated somatic embryos after six weeks of culture on

MS medium supplemented with 3.5 mgl-1 BAP, were washed with water to remove agar

without damaging the delicate root system and placed at top of culture tubes filled with

plain double distilled water (only the roots were dipped in water). The culture tubes

containing somatic embryo derived plantlets were kept in the culture room for two weeks

for hardening under above mentioned conditions. Then these plants were transferred to

earthen pots containing soil, sand and farmyard manure in 1:1:1 (v/v) ratio and potted

plants were covered with transparent polythene bags with small holes for air ventilation to

ensure high humidity in initial stages. The polythene bags were removed after two weeks

and the surviving plants were maintained in the green house. The temperature in the green

house was around 25±5ºC.

3.3.6 Analysis of data

Experiments were performed in a randomized block design. In all experiments standard

deviation (SD) was calculated by using following formula:

√ n ∑ χ2 – ( ∑ χ )2

n (n-1)

Where,

n = Number of replicates observed; ∑ = Summation; χ = Observation of the replicate

3.3.7 Histology of somatic embryogenesis in C. borivilianum

To ascertain the embryogenic nature of differentiating structures, randomly selected

subcultured embryogenic tissues of C. borivilianum during 4-6 weeks of culture duration

were subjected to histological study. Embryogenic calli bearing somatic embryos at

different developmental stages were fixed in FAA (formaldehyde:glacial acetic acid:50%

ethyl alcohol of 5:5:90, v/v) solution for 24 hrs. at room temperature. The fixed tissue was

150

washed in double distilled water for 15 min. with 3 changes of 5 min. each. Then samples

were dehydrated through a graded ethanol series (50, 70, 90 and100%). Subsequently they

were infiltrated with xylene overnight at 37ºC and ethyl alcohol was replaced with xylene

by passing the samples through a xylen-ethanol series ending finally in pure xylene.

Thereafter they were infiltrated with and embedded in paraffin wax (congealing point 58-

60ºC, Qualigens, India). Serial sections (8-10 µM thick) were cut using a rotary

microtome (Heidelberg, Germany). After dewaxing in xylene for approximately 10-15

min., sections were passed again through a graded ethanol series in descending order (100,

90, 70, 50 and 30% ethanol), then washed with water and passed through 30 and 50%

ethanol and stained with safranin (dissolved in 70% ethanol) for 40-50 min. After this

sections were passed quickly through 70 and 90% ethanol and stained in light green

(dissolved in 90% ethanol) for 5-6 min. Again quick dips in 90 and 100% ethanol were

made. Sections were passed through fresh xylene mixed with 1-2 drops of clove oil and

mounted in Canada Balsom (Thomas Baker, India). Finally sections were observed and

photographed under a photomicroscope (Nikon FX-35 A, Japan).

3.3.8 Analysis of genetic fidelity of plants of C. borivilianum derived from somatic

embryos through PCR based Random Amplified Polymorphic DNA method

The genetic stability of plants regenerated in vitro through somatic embryogenesis was

evaluated by RAPD analysis comparing leaves taken from wild type (control plant) with

leaves taken from randomly selected in vitro raised plants derived from somatic embryos.

Plants raised through organogenesis i.e from shoot base explants were also included in the

present study to compare the level of genetic similarity with plants raised through somatic

embryogenesis. The in vitro procedure for the initiation, establishment and maintenance of

plants regenerated through organogenesis has been described earlier in section 2.3. The

details of induction and maintenance of embryogenic callus derived from seedling

explants, development of somatic embryos, their germination and regeneration of plants

from somatic embryos has been described earlier in section 3.3.1 and 3.3.2. High

molecular weight DNA from all plants included in the study was isolated following the

modified CTAB method (Khanuja et al., 1999) and later this DNA was amplified through

PCR using twenty different primers MAP 01 to MAP 20 obtained from M/s Bangalore

Genei (India). The protocol for DNA isolation, PCR amplification reaction and the

sequences of these primers used to study DNA polymorphism in the present study has also

been described earlier in section 2.3.9.

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3.4 RESULTS

3. 4.1. Callus induction, establishment and maintenance of embryogenic callus

3.4.1.1. Selection of suitable explant for callus induction

(a) Leaf and shoot base explants

Two different explants i.e. leaf segments and shoot bases obtained from the sterile in vitro grown plants of C. borivilianum maintained on MS medium supplemented with 5.0 mgl-1 BAP were employed in callus induction studies. Similarly seedlings obtained by germinating seeds of C. borivilianum on MS basal medium supplemented with 20 mgl-1 GA3 were also used for induction of callus.

MS basal medium was supplemented with different auxins i.e. IAA, IBA,

NAA, 2,4-D or P individually in the range of 0.05-2.0 mgl-1. No morphogenetic

response was observed in leaf segments cultured on any media combination. The

explants became blackish-brown and necrotic after 3-4 weeks of culture. TDZ either

individually in the range of 0.05-6.0 mgl-1 or in combination with AS (5.0-40.0 mgl-1)

also could not evoke callus induction response from leaf explants (Table-26).

Table-26: Callus induction in shoot bases and leaf explants on MS medium fortified

with different levels of auxins

Explant Morphogenetic response

Auxin (mgl-1) 2,4-D NAA

0.05 0.10 0.25 0.50 1.00 2.00 0.05 0.10 0.25 0.50 1.00 2.00 Leaf

Initiation of callus (days)

- - - - - - - - - - - -

Growth of callus - - - - - - - - - - - - Colour - - - - - - - - - - - -

Shoot base

Initiation of callus (days)

23 - - - - - 10 callus

10 callus with roots

10 callus with roots

10 callus with roots

10 callus with roots

10 callus with roots

Growth of callus + + - - - - - + + + + + + + + + + + + + Colour yello-

wish green

- - - - - green green green light yellowish

light yellow

dull yellowish

+ Poor; + + Average; + + + Moderate; - No response

Similarly, auxins IAA and IBA also could not induce callus formation from

cultured shoot base explants. Amongst all auxins tested NAA in the range of 0.05-2.0

mgl-1 could provoke callus induction response only in shoot base explants. Green to

light yellowish green callus was initiated after 10 days of culture and optimal growth

152

of callus was obtained on 2.0 mgl-1 concentration of NAA. 2-4,D only at 0.05 mgl-1

level could induce callus formation in cultured shoot bases. Callus induction was

observed on all levels of NAA tested for callus induction and rooting was observed in

calli induced on NAA concentrations ranging from 0.1 to 2.0 mgl-1 (Table-26). TDZ

(0.05-6.0 mgl-1) was added to MS basal medium to test its efficacy on callus induction

in both the cultured explants. Light yellow to yellowish-green callus was observed in

cultured shoot bases after 28-35 days of culture incubation (Table-27). Observations

revealed that calli induced on TDZ (0.05-1.0 mgl-1) containing media were compact

and nodular while the calli induced on TDZ (2.0 to 6.0 mgl-1) containing media were

more friable. It was also observed that less time (10-23 days) was required for callus

initiation in NAA supplemented media compared to TDZ supplemented media (28-35

days). Taking into account these observations, MS medium supplemented with TDZ

at 5.0 mgl-1 concentration was used in further studies. NAA supplemented media

were not considered for further studies because rooting response was observed in

calli induced on NAA containing media although time taken for callus initiation was

comparatively less on these media.

Table-27: Callus induction in shoot bases or leaf explants on MS medium fortified with

different levels of TDZ

Explant(s) Morphogenetic response

TDZ (mgl -1) 0.05 0.10 0.25 0.50 1.00 2.00 3.00 4.00 5.00 6.00

Leaf

Initiation of callus (days)

- - - - - - - - - -

Growth of callus - - - - - - - - - - Colour - - - - - - - - - -

Shoot base Initiation of callus (days)

35 35 - - 28 35 - 35 28 35

Growth of callus + + + + + - - + + + + + - + + + + + + + Colour yello-

wish green

yellowish green

- - yellowish green

light yellow

- yellowish green

yellowish green

yellowish green

+ Poor; + + Average; + + + Moderate; - No response

In an effort to further enhance callus induction response, Kn (0.05-2.0 mgl-1)

or AS (5.0-40.0 mgl-1) were supplemented to TDZ (5.0 mgl-1) containing medium.

Addition of AS (10.0-30.0 mgl-1) could increase callus-inducing efficiency of cultured

shoot bases only marginally (Table-28). On other hand, addition of Kn had no

beneficial effect on callus induction response in shoot base explants.

153

Table-28: Callus induction in shoot base or leaf explants on MS medium fortified with

TDZ (5.0 mgl-1) and AS

Explant(s) Morphogenetic response

AS (mgl-1) 5.00 10.00 20.00 30.00 40.00

Leaf

Initiation of callus (days)

- - - - -

Growth of callus - - - - - Colour - - - - -

Shoot base Initiation of callus (days)

- 35 30 35 -

Growth of callus - + + + + + + + + + - Colour - yellowish green yellowish green yellowish green -

+ Poor; + + Average; + + + Moderate; Good + + + +; - No response

(b) Induction of somatic embryogenesis in seedling derived callus of C.

borivilianum

In another set of experiment, efficacy of two different basal media viz. MS and B5

medium,each supplemented with combinations of 0.1-1.0 mgl-1 2,4-D and 0.1-0.5 mgl-1

Kn was tested for induction of somatic embryogenesis in seedling explants of C.

borivilianum (Table-29). MS and B5 basal medium without any hormone served as

control.

It was observed that seeds cultured on MS basal medium exhibited very low

(<10%) germination response. However, addition of GA3 (20 mgl-1) to MS basal medium

enhanced (> 40%) seed germination (Plate-6a).

Among MS and B5 media supplemented with different concentrations and

combinations of 2,4–D and Kn, in general MS medium supplemented with 2,4-D and Kn

exhibited better response than B5 medium and moderate to good callus induction

frequency was observed on MS medium containing 0.25 mgl-1 of Kn and 0.25-0.50 mgl-1

of 2,4-D. The explants turned brownish, swelled and increased in size on these media after

2 weeks ultimately resulting in callus induction after three weeks of incubation (Plate-6b).

Induction of somatic embryogenesis was observed during regular subculturing of calli

maintained on MS basal medium supplemented with different concentrations and

combinations of 2,4-D and Kn and small rounded globular shaped somatic embryos were

induced regularly in yellowish-white callus at various frequencies in different media

combinations (Table-29; Plate-6c, Plate7a-d).

154

Table-29: Effect of different concentrations and combinations of 2,4-D and Kn on

somatic embryogenesis in seedling derived callus of C. borivilianum

MS basal medium Morphogenetic response - No. of somatic embryos

Kn (mgl -1) 2,4-D (mgl-1) 0.00 0.10 0.25 0.50 1.00

Control* 0.00 1.00 ± 0.00† 0.10 - 8.0 ± 1.00 4.00 ± 0.71 - 0.25 6.40 ± 0.54 12.20 ± 1.30 10.20 ± 1.30 3.20 ± 0.83 0.50 - - 5.60 ± 0.55 1.40 ± 0.55

B5 basal medium Morphogenetic response - No. of somatic embryos

Kn (mgl -1) 2,4-D (mgl-1) 0.00 0.10 0.25 0.50 1.00

Control** 0.00 1.20 ± 0.44†

0.10 - 6.00 ± 1.00 2.80 ± 0.71 1.60 ± 0.55 0.25 5.00 ± 1.00 10.00 ± 1.51 8.00 ± 1.22 2.80 ± 0.44 0.50 - - 2.40 ± 0.54 0.80 ± 0.40

* MS basal medium without phytohormones; ** B5 basal medium without phytohormones; † Average value ± SE

(Standard Error); - No response

Observations revealed that amongst different concentrations and combinations of

2,4–D and Kn tested, higher number of somatic embryos were observed on MS basal

medium containing 0.25-0.50 mgl-1 2,4–D and 0.25 mgl-1 Kn. MS or B5 basal media without

any phytohormones exhibited poor somatic embryogenesis response and the number of

somatic embryos induced was lower than that observed on either basal media supplemented

with different concentrations and combinations of 2,4-D and Kn (Table-29). It was also

observed that cultures at higher concentration of 2,4-D (0.5 mgl-1) were slightly more friable

than the culures grown at a lower concentration of 2,4-D but higher concentrations of 2,4-D

were found to be inhibitory for somatic embryogenesis therefore embryogenic cultures were

further maintained on lower concentration (0.25 mgl-1) of 2,4-D.

3.4.1.2 Establishment and maintenance of embryogenic callus

Embryogenic callus induced on callus induction medium (0.25 mgl-1 each of 2,4-D

and Kn) from seedlings was maintained by subculturing regularly after every 3-4

weeks on the fresh medium of the same composition.

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3.4.2 Standardization of somatic embryogenesis in C. borivilianum: Optimization of

culture medium

3.4.2.1 Effect of different inorganic nitrogen sources on somatic embryogenesis

Initially seedling derived callus grew slowly producing both non-embryogenic callus as well as somatic embryos from embryogenic portion of callus. Growth of cultures containing non-embryogenic callus was slow, thereby acting as a limiting factor for getting sufficient embryogenic callus for further experiments. Therefore, to further fasten the callus growth to enhance somatic embryos formation in seedling derived callus, manipulations in MS medium constituents were made and in this context experiments were laid to study effect of different levels of inorganic nitrogen sources i.e. NH4NO3 (143–856 mgl-1) and KNO3 (360–2166 mgl-1) in the MS basal medium containing 0.25 mgl-1 each of 2,4–D and Kn on somatic embryogenesis. Nitrogen source in culture medium has been shown to affect somatic embryogeneis in a significant way and proper levels have to be worked out for a specific species/genotype for obtaining desired response. In the present study, observations revealed that maximum number of somatic embryos (19/inoculum) were obtained on MS medium containing 143 mgl-1 NH4NO3 and 1083 mgl-1 KNO3 followed by 17.6/inoculum on MS medium containing 285 mgl-1 NH4NO3 and 1444 mgl-1 KNO3 (Table-30).

Table-30: Effect of different levels of NH4NO3 and KNO3 on callus growth and somatic embryogenesis

NH4NO3 (mgl-1)

Growth parameter(s)

KNO3 (mgl-1) 360 722 1083 1444 1805 2166

Control* No. of somatic embryos

12.20 ± 1.30∗∗

Callus f.wt. (g) 0.27 ± 0.03 143 No. of somatic

embryos 3.00 ± 0.54 9.00 ± 1.00 19.00 ± 1.58 17.00 ± 1.87 15.00 ± 2.00 6.00 ± 1.00

Callus f.wt. (g) 0.22 ± 0.22 0.29 ± 0.01 0.32 ± 0.02 0.37 ± 0.02 0.30 ± 0.02 0.28 ± 0.01 285 No. of somatic

embryos 12.60 ± 2.07 16.60 ± 1.67 14.40 ± 1.51 17.60 ± 1.81 9.00 ± 1.30 14.00 ± 1.58

Callus f.wt. (g) 0.28 ± 0.02 0.31 ± 0.03 0.33 ± 0.02 0.29 ± 0.02 0.29 ± 0.01 0.25 ± 0.02 428 No. of somatic

embryos 9.60 ± 1.51 13.40 ± 1.67 15.40 ± 2.07 17.00 ± 2.12 13.60 ± 1.67 13.00 ± 2.23

Callus f.wt. (g) 0.27 ± 0.02 0.29 ± 0.01 0.30 ± 0.01 0.30 ± 0.02 0.26 ± 0.01 0.26 ± 0.02 571 No. of somatic

embryos 8.20 ± 1.48 14.40 ± 1.14 16.00 ± 2.16 16.80 ± 1.78 17.20 ± 1.51 14.80 ± 1.64

Callus f.wt. (g) 0.28 ± 0.01 0.26 ± 0.02 0.32 ± 0.02 0.29 ± 0.02 0.27 ± 0.01 0.24 ± 0.02 714 No. of somatic

embryos 7.80 ± 1.30 11.60 ± 2.07 12.80 ± 1.64 14.60 ± 1.81 11.00 ± 1.58 4.90 ± 1.09

Callus f.wt. (g) 0.25 ± 0.02 0.26 ± 0.01 0.28 ± 0.02 0.28 ± 0.03 0.31 ± 0.03 0.23 ± 0.02 856 No. of somatic

embryos 6.00 ± 1.00 8.40 ± 1.58 10.80 ± 1.81 12.60 ± 1.94 12.10 ± 1.58 1.00 ± 0.00

Callus f.wt. (g) 0.22 ± 0.03 0.25 ± 0.02 0.27 ± 0.02 0.24 ± 0.03 0.25 ± 0.01 0.19 ± 0.03

* Standard MS basal medium supplemented with 0.25 mgl-1 each of 2,4-D and Kn (Standard MS basal medium contains

1650 mgl-1 NH4NO3 and 1900 mgl-1 KNO3); ∗∗Average value ± SE (Standard Error)

156

The callus was further subcultured on these two selected media but it was observed that non-embryogenic callus formation increased on the media containing 285 mgl-1 NH4NO3 and 1444 mgl-1 KNO3. High levels of NH4NO3 or KNO 3 were not beneficial for somatic embryogenesis. Growth of the callus tissue was more or less same on most of the media combinations except those at high nitrogen levels on which it was reduced. In the present study levels of NH4NO3 and KNO3 optimal for somatic embryogenesis were much lower than in normal strength MS medium (1650 mgl-1 NH4NO3 and 1900 mgl-1 KNO 3). Therefore, MS medium supplemented with 143 mgl-1 NH4NO3 and 1083 mgl-1 KNO3 found to be optimal for somatic embryogenesis in C. borivilianum in the present study was used for further experiments.

3.4.2.2 Effect of different phytohormones on somatic embryogenesis

Effect of different cytokinins on callus growth and somatic embryogenesis was

studied in another experiment. Observations revealed that lower levels of Kn or TDZ

were more effective for somatic embryogenesis while 2-iP supplemented media

showed optimal response at higher levels (Table-31, Fig.-13).

Table-31: Effect of different cytokinins on callus growth and somatic embryogenesis

S. No. Cytokinins (mgl-1) Callus f. wt. (g) Number of somatic embryos/inoculum 1. Control* 0. 32 ± 0. 02** 19.00 ± 1. 58 2. Kn

0.25 0.32 ± 0. 02 20.40 ± 2.07 0.50 0.26 ± 0. 01 19.20 ± 2.16 1.00 0.24 ± 0. 02 13.40 ± 1.34 1.50 0.25 ± 0. 03 6.50 ± 1.58

3. TDZ 0.10 0.30 ± 0. 01 21.60 ± 2.07 0.25 0.24 ± 0. 03 13.60 ± 1.81 0.50 0.25 ± 0. 03 9.40 ± 1.64 1.00 0.26 ± 0.02 6.80 ± 1.30

4. 2-iP 0.25 0.27 ± 0.02 7.60 ± 1.14 0.50 0.34 ± 0.02 11.40 ± 1.67 1.00 0.29 ± 0.03 16.20 ± 1.92 1.50 0.30 ± 0.02 23.00 ± 2.54

* MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3 and 0.25 mgl-1 2,4-D; ** Average value ± SE (Standard

Error)

The maximum number of somatic embryos (23/inoculum) were obtained on

modified MS medium (containing 143 mgl-1 NH4NO3 and 1083 mgl-1 KNO3)

supplemented with 0.25 mgl-1 2,4–D and 1.5 mgl-1 2-iP followed by an average

number of 21.6 somatic embryos/inoculum on modified MS medium supplemented

with 0.1 mgl-1 TDZ. Thus, addition of 2-iP at 1.5 mgl-1 level to the control medium

157

(MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3 and 0.25 mgl-1 2,4-D)

showed 1.21 fold increase over control in terms of number of somatic embryos.

The other cytokinins TDZ and Kn were comparatively less effective for

somatic embryogenesis. However, no significant difference in biomass production of

callus could be observed between the different levels of all the three cytokinins tested

during the investigation (Table-31). It was also observed that the cultures in which 2-

iP was included were more greener than the cultures maintained on other hormones

tested in the present study. In the present study, 2-iP at 1.5 mgl-1 concentration

showed optimal response for somatic embryogenesis amongst different cytokinins

tested and therfore 2-iP at same concentration was used for subsequent experiments.

Control*Kn (0.25)

TDZ (0.1)2-iP (1.5)

0

5

10

15

20

25

No

. o

f so

ma

tic e

mb

ryo

s /

ino

culu

m

Fig.-13: Effect of different cytokinins (optimal level of each cytokinin in mgl-1) on somatic embryogenesis in

callus raised from seedling explants of C. borivilianum; * MS basal medium containing 143 mgl-1 NH4NO3,

1083 mgl-1 KNO3 (modified MS medium) and 0.25 mgl-1 2,4-D

3.4.2.3 Effect of different amino acids on somatic embryogenesis

Amino acids have been reported to affect morphogenetic response especially somatic

embryogenesis in many plant species, therefore in the present study for the further

enhancement of somatic embryogenesis response two amino acids viz. glutamine and

proline at different levels (10-400 mgl-1) were tested (Table-32). It was observed that

incorporation of these amino acids to the control medium in the present study (modified

MS medium containing 0.25 mgl-1 2,4-D and 1.5 mgl-1 2-iP) had a stimulatory effect on

growth of cultures and somatic embryogenesis (production of average number of somatic

158

embryos/inoculum). Supplementation of glutamine to the medium was less responsive

compared to proline. The medium fortified with 100 mgl-1 proline gave best response

(Fig.-14) both in terms of culture growth (an average callus f. wt. of 0.34 g) and somatic

embryogenesis (an average number of 43.2 somatic embryos/inoculum).

Table-32: Effect of different amino acids on callus growth and somatic

embryogenesis

S. No. Amino acids (mgl-1) Morphogenetic response Callus f. wt. ( g) Number of somatic embryos/inoculum

1. Control* 0.30 ± 0. 02** 23.00 ± 2.54 2. Glutamine

10 0.31 ± 0.03 20.00 ± 2.91 50 0.29 ± 0.01 20.40 ± 2.79 100 0.32 ± 0.02 23.00 ± 2.16 200 0.30 ± 0.02 22.40 ± 2.30 400 0.28 ± 0.03 16.80 ± 3.11

3.

Proline 10 0.29 ± 0.03 25.00 ± 2.64 50 0.33 ± 0.02 37.00 ± 3.53 100 0.34 ± 0.01 43.20 ± 3.36 200 0.31 ± 0.02 32.60 ± 2.38 400 0.30 ± 0.03 29.00 ± 3.74

* Modified MS medium supplemented with 0.25 mgl-1 2, 4 - D and 1.5 mgl-1 2-iP; ** Average value ± SE (Standard Error)

Control*Glutamine (100)

Proline (100)

0

5

10

15

20

25

30

35

40

45

No

. o

f so

ma

tic e

mb

ryo

s /

ino

culu

m

Fig 14: Effect of different amino acids (optimal level of each amino acid in mgl-1) on somatic embryogenesis

in C. borivilianum; * Modified MS medium supplemented with 0.25 mgl-1 2,4- D and 1.5 mgl-1 2iP

Thus, 1.88-fold higher response (Table-32, Fig.-14) in terms of number of somatic

embryos was obtained on medium supplemented with 100 mgl-1 proline compared to the

control followed by 50 mgl-1 proline (with an average callus f. wt. of 0.33 g and an

average number of 37 somatic embryos). Therefore based upon these two parameters

159

(callus f. wt. and number of somatic embryos) modified MS medium (143 mgl-1 NH4NO3

and 1083 mgl-1 KNO3) supplemented with 0.25 mgl-1 2,4-D, 1.5 mgl-1 2-iP and 100 mgl-1

proline was found optimal for somatic embryogenesis and growth of cultures. It was

observed that the cultures maintained on proline containing media were more friable than

the cultures maintained on glutamine containing media.

3.4.3 Germination of somatic embryos

Somatic embryos isolated from 4-5 month old seedling derived callus germinated into

plantlets upon transfer to MS medium supplemented with BAP (1.5–6.5 mgl-1) within

4 weeks with germination frequencies between 3% to 30%. Maximum number of

somatic embryos germinated (30%) on MS medium supplemented with 3.5 mgl-1

BAP while the lowest germination frequency (3%) was observed on control medium

(Table-33, Fig.-15).

Table-33: Percent germination of somatic embryos

BAP (mgl-1) No. of embryos germinated/ 20 embryos Percent Germination Control∗ 0.60 ± 0.55** 3 1.50 1.00 ± 0.00 5 2.50 2.60 ± 0.54 13 3.50 6.00 ± 1.00 30 4.50 3.20 ± 0.45 16 5.50 2.00 ± 0.70 10 6.50 -

∗ MS basal medium without phytohormones; **Average value ± SE (Standard Error); - No response

0

1

2

3

4

5

6

7

*Control 1.5 2.5 3.5 4.5 5.5 6.5

BAP (mg l-1)

No. of embryosgerminated / 20embryos

Fig.-15: Germination of somatic embryos on different BAP levels; ∗ MS basal medium without

phytohormones

160

3.4.5 Hardening of somatic embryo derived plantlets

The survival percentage of somatic embryo derived plantlets was 60-65% in green

house. Plants developed through somatic embryogenesis were morphologically

similar to the parent plant.

3.4.6 Somatic embryo development and maturation

Although in earlier experiments somatic embryogenesis was observed but embryo

maturation rate was low. Therefore experiments were performed for observing effect of

different carbohydrates, osmotica and ABA on somatic embryo development and

maturation in C. borivilianum.

3.4.6.1 Effect of different carbohydrate sources

(a) Maturation and development of somatic embryos

Experiments were performed for testing effect of different carbohydrate sources on

somatic embryo maturation and germination (Plate-8a-f). It was observed that

carbohydrate source and concentration had a marked influence on maturation and

germination rate of somatic embryos. Sucrose employed as carbohydrate source produced

the highest average number of total somatic embryos (44) followed by glucose (41),

maltose (30), mannose (28), fructose (27) or cellobiose (22). The number of total somatic

embryos produced was low in xylose (17) or rhamnose (11), (Table-34).

Table-34: Effect of different carbohydrate sources on callus growth and somatic

embryo development in C. borivilianum

S. No. Carbohydrate source (3%)

No. of total somatic embryos No. of mature (cotyledonary) embryos

Callus f. wt. (g)

1 Control* - - 0.17 ± 0.02 2. Sucrose 44.00 ± 2.21† 17.00 ± 1.24 0.43 ± 0.03 3. Glucose 41.00 ± 2.10 14.00 ± 1.05 0.39 ± 0.03 4. Fructose 27.00 ± 1.24 3.00 ± 0.66 0.30 ± 0.04 5. Lactose - - 0.18 ± 0.03 6. Galactose - - 0.20 ± 0.02 7. Cellobiose 22.00 ± 1.22 2.00 ± 0.66 0.22 ± 0.03 8. Rhamnose 11.00±1.05 - 0.19 ± 0.02 9. Xylose 17.00 ± 1.41 - 0.22 ± 0.03

161

10. Arabinose - - 0.16 ± 0.02 11. Maltose 30.00 ± 1.33 6.00 ± 0.81 0.53 ± 0.04 12. Mannose 28.00 ± 1.33 2.00 ± 0.47 0.31 ± 0.30 13. Soluble starch - - 0.10 ± 0.01 14. Mannitol - - 0.17 ± 0.02 15. Sorbitol - - 0.14 ± 0.02

* Modified MS medium (containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3) supplemented with 0.25 mgl-1 2, 4-D, 1.5

mgl-1 2-iP, 100 mgl-1 proline and without any carbohydrate source; †Average value ± SE (Standard Error); - No

Response

Somatic embryogenesis was not observed in cultures inoculated on media containing other

carbohydrates i.e. arabinose, galactose, lactose soluble starch, mannitol or sorbitol.

Somatic embryo maturation (in terms of average number of cotyledonary stage embryos)

was highest in sucrose (17) followed by glucose (14) and maltose (6); (Fig.-16).

0

5

10

15

20

25

30

35

40

45

Control *Sucrose

GlucoseFructose

Cel lobioseRhamnose

XyloseMaltose

Mannose

No. of total somatic embryos No. of cotyledonary embryos

Fig-16: Effect of different carbohydrates on somatic embryogenesis in C. borivilianum; * Modified MS

medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3 supplemented with 0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-

iP, 100 mgl-1 proline and without any carbohydrate source

On control medium somatic embryogenesis was not observed. The morphogenetic

response in terms of number of cotyledonary stage embryos was low in fructose (3),

cellobiose (2) or mannose (2). Embryo maturation was not observed and cotyledonary

stage embryos were not seen on media supplemented with rhamnose or xylose (Table-34).

The cotyledonary stage embryos observed in the present study were embryos with an

162

elongated embryonic region with cotyledons. It was observed that callus growth was good

on media supplemented with sucrose, glucose, maltose, fructose or mannose. Although

callus growth was higher on medium supplemented with maltose than medium

supplemented with sucrose or glucose but number of total somatic embryos and mature

(cotyledonary stage) embryos were lower than sucrose or glucose containing medium

indicating that the medium supplemented with sucrose or glucose as carbohydrate sources

was more supportive for embryogenic callus growth and subsequently for induction and

development of somatic embryos as compared to medium supplemented with maltose.

Callus growth on media supplemented with lactose, arabinose or soluble starch was poor

(Table-34). On media supplemented with sucrose or glucose comparable number of total

and mature (cotyledonary stage) embryos were observed but sucrose was better than

glucose in terms of average number of total and mature embryos as well as callus growth.

In control medium somatic embryogenesis was not observed although growth of non-

embryogenic callus was observed on this medium. The embryos developed on sucrose

supplemented media were yellowish to yellowish-green in colour whereas those developed

on glucose and maltose containing media were light-yellowish to whitish and dark yellow

to yellowish- brown in colour. On media supplemented with plasmolyzing osmotica viz.

D-mannitol or D-sorbitol, callus growth was poor and somatic embryogenesis response

was not observed (Table-34). The globular stage somatic embryo clusters cultured on

these osmotica failed to grow further and became brownish-blackish and necrotic in due

course of time.

(b) Germination of somatic embryos cultured on different carbohydrate sources

When the effect of different carbohydrates on embryo germination was studied (Plate-9a-

h), it was observed that the cultures which were previously incubated on sucrose or

glucose supplemented media showed higher germination response of somatic embryos as

compared to cultures raised on other carbohydrate sources (Table-35, Fig.-17). As

observed earlier these carbohydrate sources supported better callus growth and induction

163

and development of somatic embryos. The germination frequencies ranged from 5% to

30% on different carbohydrate sources tested in the present study. Germination frequency

of somatic embryos was higher on sucrose (30%) than on glucose (20%) supplemented

medium followed by maltose (15%). The frequency of somatic embryo germination

decreased considerably (5% only) in the prescence of fructose or cellobiose and was

completely inhibited in cultures on media supplemented with lactose, galactose, rhamnose,

xylose, arabinose, mannose or soluble starch as well as on control medium.

Supplementation of the medium with mannitol or sorbitol as osmoticum also completely

inhibited the germination of somatic embryos. It was observed that plantlets developed

from germinated somatic embryos which were previously cultured on sucrose or glucose

supplemented media were taller compared to plantlets developed from somatic embryos

on media containing other carbohydrate sources while in cultures taken from maltose,

lactose or cellobiose supplemented media although plantlet development was observed but

growth was poor especially in the case of lactose and cellobiose supplemented media

where after intial development growth stopped. Therefore, based on these observations

(callus f. wt., number of total and cotyledonary stage embryos and percentage germination

of somatic embryos) sucrose as a carbohydrate source was used for further experiments.

Table-35: Effect of different carbohydrate sources on germination*of somatic

embryos in C. borivilianum

S. No. Carbohydrate source (3%) No.of germinated embryos/20 embryos Percent germination 1 Control** - - 2. Sucrose 6.00 ± 1.00† 30 3. Glucose 4.00 ± 0.67 20 4. Fructose 1.00 ± 0.47 5 5. Lactose - - 6. Galactose - - 7. Cellobiose 1.00 ± 0.47 5 8. Rhamnose - - 9. Xylose - - 10. Arabinose - - 11. Maltose 3.00 ± 0.81 15 12. Mannose - - 13. Soluble starch - - 14. Mannitol - - 15. Sorbitol - -

* For germination somatic embryos cultured on control medium and media supplemented with different carbohydrate

sources were transferred to MS basal medium supplemented with 3.5 mgl-1 BAP; ** Modified MS medium (containing

143 mgl-1 NH4NO3, 1083 mgl-1 KNO3) supplemented with 0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP, 100 mgl-1 proline and

without any carbohydrate source; †Average value ± SE (Standard Error); - No Response

164

0 1 2 3 4 5 6 7

Control*

Sucrose

Glucose

Fructose

Cellobiose

Maltose

No. of embryos germinated / 20 embryos

Fig-17: Effect of carbohydrates on germination of somatic embryos of C. borivilianum; *

Modified MS medium supplemented with 0.25 mgl-1 2,4-D, 1.5 mgl-1 2-iP, 100 mgl-1

proline and without any carbohydrate source

3.4.6.2 Effect of different sucrose concentrations on somatic embryogenesis in C.

borivilianum

(a) Maturation and development of somatic embryos

After optimizing carbohydrate source i.e. sucrose for somatic embryogenesis in C.

borivilianum, another experiment was conducted to optimize the sucrose concentration.

Various sucrose levels (1, 3, 5, 7, 9 or 11%) were tested in the present study and highest

response (average number of cotyledonary stage embryos) was observed at 3% sucrose

level (control medium) followed by 5% sucrose level. On media supplemented with >7%

sucrose level, low frequency of induction of somatic embryos was observed and their

further development was also inhibited (Table-36, Fig.-18).Therefore based on these

experiments, 3% sucrose level was selected for all further experiments. In general effect of

sucrose levels on growth of callus also showed same pattern as observed for somatic

165

embryos. The medium supplemented with 3% sucrose supported highest callus growth

followed by 5% sucrose level.

Table-36: Effect of different sucrose concentrations on callus growth and somatic

embryo maturation in C. borivilianum

S. No. Sucrose concentration (%)

No. of total somatic embryos

No. of mature (cotyledonary) embryos

Callus f. wt. (g)

1. 1 18.00 ± 1.56† 5.00 ± 0.81 0.29 ± 0.03 2. 3 (control) * 44.00 ± 2.21 17.00 ± 1.05 0.43 ± 0.03 3. 5 29.00 ± 1.76 9.00 ± 1.22 0.35 ± 0.04 4. 7 12.00 ± 1.56 2.00 ± 0.47 0.27 ± 0.03 5. 9 9.00 ± 1.3 - 0.20 ± 0.02 6. 11 2.00 ± 0.66 - 0.17 ± 0.02

* Modified MS medium (containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3) supplemented with 0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP and 100 mgl-1 proline; †Average value ± SE (Standard Error); - No Response

Fig 18: Effect of different sucrose concentrations on somatic embryo development in C. borivilianum

(b) Effect of different sucrose concentrations on germination of somatic embryos

Somatic embryos developed on media supplemented with different sucrose concentrations

were tested for their germination ability by transferring them on MS basal medium

supplemented with 3.5 mgl-1 BAP which was optimized for somatic embryo germination

in an earlier experiment of the present investigation. The germination frequencies of

somatic embryos on different levels of sucrose tested in the present study ranged between

10-30%. It was observed that 3% sucrose level besides supporting the highest number of

total and mature embryos also supported their germination at highest frequency (30%)

amongst all other levels of sucrose tested in the present study (Table-37, Fig.-19). The

lowest germination frequency (10%) was observed on medium supplemented with 1%

0

5

10

15

20

25

30

35

40

45

50

1 3 5 7 9 11

Sucrose (%)

No. of total somaticembryosNo. of cotyledonaryembryos

166

sucrose level. Sucrose levels above 5% completely inhibited the germination of embryos.

As observed earlier these levels also did not supported somatic embryo development and

maturation.

Table-37: Effect of different sucrose concentrations on germination* of somatic

embryos in C. borivilianum

S. No. Sucrose concentration (%) No. of germinated embryos/20 embryos

Percent germination

1. 1 2.00 ± 0.66† 10 2. 3 (control) ** 6.00 ± 1.00 30 3. 5 3.00 ± 0.66 15 4. 7 - - 5. 9 - - 6. 11 - -

*For germination somatic embryos cultured on different sucrose concentrations were transferred to MS basal medium

supplemented with 3.5 mgl-1 BAP; ** Modified MS medium (containing 143 mgl-1 NH4NO3 and 1083 mgl-1 KNO3)

supplemented with 0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP and 100 mgl-1 proline; †Average value ± SE (Standard Error); - No

Response

Fig 19: Effect of different sucrose concentrations on somatic embryo germination

3.4.6.3 Effect of non-plasmolyzing osmotica (polyethylene glycol) on somatic

embryogenesis

(a) Maturation and development of somatic embryos

Polyethylene glycol (PEG) at 3% level was found to be best for embryo maturation and

highest number (32) of cotyledonary stage embryos were observed at this level. The

number of total somatic embryos was also highest (56) at this level (Table-38). There was

1.27-fold increase in number of total embryos and 1.88-fold increase was observed in

number of mature (cotyledonary) embryos as compared to control medium. The

0

1

2

3

4

5

6

7

1 3 5 7 9 11

Sucrose (%)

No. of embryosgerminated / 20embryos

167

morphogenetic response in terms of number of cotyledonary stage embryos at 3% PEG

level was followed by 1% and 5% of PEG. At >5% level of PEG there was decrease in

embryogenic response (Table-38, Fig.-20). Lowest average number of cotyledonary stage

Table-38: Effect of different concentrations of polyethylene glycol (PEG) on somatic

embryo maturation in C. borivilianum

S. No.

PEG concentration (%)

No. of total somatic embryos No. of mature (cotyledonary) embryos

Callus f. wt. (g)

1. Control* 44.00 ± 2.21† 17.00 ± 1.24 0.43 ± 0.03 2. 1 51.00 ± 3.62 28.00 ± 2.0 0.53 ± 0.04 3. 3 56.00 ± 3.05 32.00 ± 3.67 0.60 ± 0.05 4. 5 40.00 ± 3.43 18.00 ± 2.16 0.38 ± 0.04 5. 7 30.00 ± 3.19 12.00 ± 1.19 0.28 ± 0.03 6. 9 26.00 ± 2.35 8.00 ± 1.05 0.21 ± 0.02 7. 11 15.00 ± 1.69 4.00 ± 0.81 0.18 ± 0.02

*Modified MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3, 0.25 mgl-1 2,4-D, 1.5 mgl-1 2-iP,100 mgl- 1

proline and 3% sucrose; †Average value ± SE (Standard Error)

Fig-20: Effect of different PEG levels on somatic embryo development; * Modified MS medium containing

0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP, 100 mgl 1 proline and 3% sucrose

somatic embryos was recorded on medium containing 11% PEG. An average number of

28-18 mature (cotyledonary stage embryos were obtained on 1-5% levels of PEG

respectively as compared to 17 mature embryos obtained on control medium. The embryos

cultured on PEG containing media were whitish to yellowish-white in colour and

yellowish to yellowish-green in colour on control medium (without PEG) which probably

indicates their advancement towards physiological maturity on PEG containing media

0

10

20

30

40

50

60

Control* 1 3 5 7 9 11

PEG (%)

No. of total somaticembryos No. of cotyledonarystage embryos

168

(Plate-8e, f). The best callus growth was obtained at medium supplemented with 3% PEG

level (0.6 g) followed by 1% PEG level.

(b) Effect of PEG on somatic embryo germination in C. borivilianum

It was observed that supplementation of the medium previously standardized for somatic

embryo induction and growth (Modified MS medium containing 143 mgl-1 NH4NO3, 1083

mgl-1 KNO3, 0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP, 100 mgl1 proline and 3% sucrose) with PEG

(1-3%) improved germination frequencies without limiting embryo development and

maturation. Supplementation with 3% PEG resulted in 1.67-fold increase in germination

frequencies over the control medium while 1% PEG level resulted in 1.33-fold increase in

germination frequencies over the control. As observed previously these two levels were also

most supportive for somatic embryo maturation amongst other levels tested in the present

study. About 50% somatic embryo germination was recorded at 3% PEG level followed by

1% PEG level (40%). The levels beyond 5% resulted in reduction in germination

frequencies (Table-39, Fig.-21). As observed previously these levels were also not

supportive for somatic embryo maturation. The germination percentage of somatic embryos

ranged from 12% to 50% at different PEG levels tested in the present study. PEG at 3%

level supported 50% germination of somatic embryos (Plate-9f) while lowest (12%)

germination response was observed on culture medium containing 9% PEG (Table-39).

Table-39: Effect of different concentrations of polyethylene glycol (PEG) on germination*

of somatic embryos in C. borivilianum

S. No. PEG concentration (%) No.of germinated embryos/20 embryos Percent germination 1. Control** 6.00 ± 1.00† 30 2. 1 8.00 ± 1.05 40 3. 3 10.00 ± 1.24 50 4. 5 6.00 ± 0.94 30 5. 7 4.00 ± 0.66 20 6. 9 2.00 ± 0.63 12 7. 11 - -

*For germination somatic embryos cultured on media with different PEG concentrations were transferred to MS basal

medium supplemented with 3.5 mgl-1 BAP; ** Modified MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3,

0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP, 100 mgl 1 proline and 3% sucrose; †Average value ± SE (Standard Error); - No

Response

169

Fig-21: Effect of different PEG levels on somatic embryo germination; * Modified MS medium containing

0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP, 100 mgl 1 proline and 3% sucrose

3.4.6.4 Effect of abscissic acid

(a) Maturation and development of somatic embryos

In the experiments conducted to analyze the effect of ABA on somatic embryo maturation,

it was observed that ABA was not much effective in maturation of embryos. Amongst

different ABA concentrations (0.03, 0.13, 0.26, 0.80, 1.32 and 1.85 mgl-1) tested, ABA at

0.26 mgl-1 level gave better response follwed by 0.13 mgl-1. But the number of

cotyledonary stage embryos on these levels of ABA was lower than obtained on control

medium. On other ABA concentrations (0.03, 0.80, 1.32 or 1.85 mgl-1) embryo maturation

was completely inhibited (Table-40). Embryos became brown and necrotic on these

Table-40: Effect of different concentrations of ABA on somatic embryo maturation

in C. borivilianum

S. No.

ABA concentration (mgl-1)

No. of total somatic embryos No. of mature (cotyledonary) embryos

Callus f. wt. (g)

1. Control* 44.00 ± 2.21† 17.00 ± 1.24 0.43 ± 0.03 2. 0.03 5.00 ± 0.63 - 0.18 ± 0.02 3. 0.13 14.00 ± 1.22 2.00 ± 0.47 0.20 ± 0.03 4. 0.26 20.00 ± 1.41 4.00 ± 0.66 0.24 ± 0.03 5. 0.80 - - 0.16 ± 0.02 6. 1.32 - - 0.15 ± 0.02 7. 1.85 - - 0.13 ± 0.02

* Modified MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3, 0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP, 100 mgl- 1

proline and 3% sucrose; † Average value ± SE (Standard Error); - No Response

0

2

4

6

8

10

12

Control* 1 3 5 7 9 11

PEG (%)

No. of embryosgerminated / 20embryos

170

concentrations and did not develop further. Callus growth was reduced at all ABA

concentrations tested in the present study compared to control medium (Table-40). Thus,

different ABA levels were not supportive to both callus growth and embryo maturation.

(b) Germination of somatic embryos previously cultured on ABA supplemented

medium

Supplementation of the medium with ABA (0.03-1.85 mgl-1) inhibited germination of

somatic embryos. Somtic embryo germination was observed only at 0.26 mgl-1 level of

ABA recording only 5% germination frequency which was significantly lower than even

control medium (30%). At all other levels of ABA tested in the present study, embryo

germination was completely inhibited (Table-41).

Table-41: Effect of different concentrations of ABA*on germination of somatic

embryos in C. borivilianum

S. No. ABA concentration (mgl-1) No. of germinated embryos/20 embryos

Percent germination

1. Control** 6.00 ±1.00† 30 2. 0.03 - - 3. 0.13 - 4. 0.26 1.00 ± 0.47 5 5. 0.80 - - 6. 1.32 - - 7. 1.85 - -

*For germination somatic embryos cultured on different ABA concentrations were transferred to MS basal medium

supplemented with 3.5 mgl-1 BAP; ** Modified MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3, 0.25 mgl-1

2, 4-D, 1.5 mgl-1 2-iP, 100 mgl- 1 proline and 3% sucrose; †Average value ± SE (Standard Error); - No Response

3.4.7 Induction and development of somatic embryos in liquid culture medium

3.4.7.1 Physical state of medium

(a) Maturation and development of somatic embryos

Effect of two different physical states (semi-solid and liquid) of culture medium was

studied on somatic embryo growth and maturation in the present study. Better

growth and maturation of somatic embryos was observed on liquid culture medium

as compared to semi-solid medium (Table-42). On liquid culture medium an average

number of 66 somatic embryos were induced as compared to 54 on semi-solid

medium. The number of globular and cotyledonary stage somatic embryos was also

higher in liquid medium (20 and 32 respectively) as compared to semi-solid medium

(16 and 26 respectively). The total fresh weight of somatic embryo clusters was

higher (0.28g) on liquid culture medium as compared to semi-solid medium (0.19g)

171

which indicated better growth of somatic embryos in liquid medium compared to

semi-solid medium. Therefore liquid medium exhibited better response for induction

and proliferation of globular somatic embryos and their maturation. The cell

suspension in the present study consisted of small cell aggregates which grew faster

than the bigger embryogenic callus pieces (Plate-10a, b). The small embryogenic cell

aggregates in liquid culture were subcultured every 3-4 weeks to enable proliferation

of somatic embryos. It was observed that somatic embryos induced and developed in

liquid medium were transluscent, more whitish and slightly longer than that

developed on semi-solid medium (Plate-10c-f).

Table-42: Effect of different physical states of medium* on somatic embryogenesis in

C. borivilianum

S. No. Physical state of medium

No. of total somatic embryos

No. of globular somatic embryos

No. of mature (cotyledonary)

embryos

Fresh wt. (g) of total somatic

embryos 1. Semi-solid

(control) 54.00 ± 3.16** 16.00 ± 1.58 26.00 ± 2.12 0.19 ± 0.02

2. Liquid 66.00 ± 3.87 20.00 ± 2.23 32.00 ± 2.34 0.28 ± 0.03

* Modified MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3, 0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP and 100 mgl-1

proline, 3% polyethylene glycol and 3% sucrose; ** Average value ± SE (Standard Error)

0

10

20

30

40

50

60

70

Semi-solid Liquid

No. of total somaticembryosNo. of globularsomatic embryosNo. of cotyledonarysomatic embryos

Fig.-22: Effect of semi-solid or liquid medium on induction and development of somatic embryos in C.

borivilianum

172

(b) Germination of somatic embryos on semi-solid or liquid medium

Germination response of somatic embryos cultured on semi-solid (control medium) or

liquid medium was almost similar. Liquid medium exhibited marginally better response

(germination percentage of 53% in liquid medium as against 50% in case of semi-solid

medium), (Table 43, Plate-10g).

Table-43: Effect of different physical states of medium*on germination of somatic

embryos in C. borivilianum

S. No. Physical state of medium** No. of germinated embryos/20 embryos

Germination percentage

1. Semi-solid (control) 10.00 ± 1.24† 50.00

2. Liquid 10.60 ± 1.34 53.00

*For germination somatic embryos cultured on semi-solid or liquid culture medium were transferred to MS basal

medium supplemented with 3.5 mgl-1 BAP; ** Modified MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3,

0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP, 100 mg- 1proline and 3% sucrose; †Average value ± SE (Standard Error)

9.7

9.8

9.9

10

10.1

10.2

10.3

10.4

10.5

10.6

No

. o

f e

mb

ryo

s g

erm

ina

ted

/ 2

0

em

bry

os

Semi-solid Liquid

Fig 23: Somatic embryo germination on semi-solid or liquid medium

3.4.7.2 Detrmination of optimal inoculum density

The effect of different inoculum densities on somatic embryo growth and maturation was

studied. Optimal results were obtained with an initial inoculum of 0.4 g/40 ml of culture

medium and an average number of 79 somatic embryos were produced. The number of

globular (22) and cotyledonary (38) embryos was also higher at this inoculum density

compared to other inoculum densities (0.01-1.0 g/40 ml). As compared to control medium

(0.15 g initial inoculum density) about 1.2-fold increase in number of total somatic

173

embryos was observed. At 0.6 g/40 ml inoculum density and beyond, inhibition in somatic

embryo development and maturation was observed (Table-44). The number of

cotyledonary stage embryos was lowest (4) in cultures having 1.0 g initial inoculum

density. The average number of globular embryos was more compared to cotyledonary

embryos at higher inoculum densities (0.6, 0.8 or 1.0 g/40 ml of culture medium inoculum

densities) while the reverse was true in case of lower inoculum densities (<0.60 g/40 ml).

At 0.4 g/40 ml culture medium initial inoculum density, 1.14-fold increase in fresh weight

of total somatic embryos was observed compared to control medium. Thus, it can be

concluded that best growth of somatic embryos was observed at inoculum density of 0.4

g/40 ml of culture medium. At higher inoculum densities (>0.6 g/40 ml of culture

medium) lower fresh weight and growth of somatic embryos was observed. At 1.0 g/40 ml

inoculum density, cultures became brown in colour and their further development and

maturation was inhibited. Therefore in this study based upon these three parameters i.e.

number of total somatic embryos, number of mature (cotyledonary stage) embryos and

fresh weight of total somatic embryo clusters, 0.4 g/40 ml of culture medium inoculum

density was observed to be best for somatic embryo growth and maturation in C.

borivilianum.

Table-44: Effect of different initial inoculum densities in liquid culture medium* on

somatic embryogenesis in C. borivilianum

S. No.

Initial inoculum (g/40 ml of culture medium) of cell aggregates

No. of total somatic embryos

No. of globular somatic embryos

No. of mature (cotytledonary)

somatic embryos

Fresh wt. (g) of total somatic embryos

1. 0.10 40.00 ± 3.39** 14.00 ± 1.58 16.00 ± 1.22 0.16 ± 0.02 2. 0.15 (control) 66.00 ± 3.87 20.00 ± 2.23 32.00 ± 2.34 0.28 ± 0.03 3. 0.20 61.00 ± 3.80 17.00 ± 1.87 28.00 ± 2.00 0.24 ± 0.02 4. 0.40 79.00 ± 5.09 22.00 ± 1.87 38.00 ± 2.34 0.32 ± 0.02 5. 0.60 55.00 ± 3.80 23.00 ± 1.87 18.00 ± 1.58 0.21 ± 0.02 6. 0.80 38.00 ± 3.16 19.00 ± 1.58 8.00 ± 1.00 0.15 ± 0.01

7. 1.00 30.00 ± 2.24 21.00 ± 2.12 4.00 ± 1.00 0.13 ± 0.01

* Modified MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3, 0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP, 100 mgl-1

proline, 3% polyethylene glycol and 3% sucrose; **Average value ± SE (Standard Error)

174

Fig.-24: Induction and development of somatic embryos in C. borivilianum as affected by different inoculum

density levels

3.4.7.3 Somatic embryogenesis in liquid culture medium at varying levels of pH

(a) Maturation and development of somatic embryos

Effect of different pH levels on somatic embryogenesis in liquid culture medium was

studied and it was observed that 5.86 pH level (control) was optimal compared to

other pH levels tested in the present study. The number of total somatic embryos was

79 and number of globular and cotyledonary embryos was 22 and 38 respectively.

The number of cotyledonary stage embryos obtained at different pH levels tested in

the present study ranged between 7 to 38 with highest number obtained at 5.86 pH

level (control) and lowest number at pH level 3.86 and 7.86. It was also observed that

maturation of somatic embryos was promoted between 4.86 to 6.86 pH levels. The

number of mature (cotyledonary) stage somatic embryos was more than globular

somatic embryos at these pH levels (Table-45) while reverse was true in case of pH

levels lower/higher than this range. At pH level 7.86, the number of globular and

mature (cotyledonary) embryos was significantly lower (13 and 7) than the control

medium. The total fresh weight of somatic embryos was also highest at pH level 5.86

(0.32g) while at pH level 6.86 and beyond significant decrease in fresh weight of total

somatic embryos was observed. Thus, in the present study pH levels 4.86 to 6.86 were

favourable for development and maturation of somatic embryos while pH levels 3.86

or 7.86 favoured differentiation of globular stage embryos only and suppressed their

further development.

0

10

20

30

40

50

60

70

80

90

0.1 0.15 0.2 0.4 0.6 0.8 1

Inoculum density (g / 40 ml culture medium)

No. of total somaticembryosNo. of globularsomatic embryosNo. of cotyledonarysomatic embryos

175

Table-45: Effect of different pH levels in liquid culture medium* on somatic

embryogenesis of C. borivilianum

S. No. pH levels No. of total somatic embryos

No. of globular somatic embryos

No. of mature (cotytledonary)

somatic embryos

Fresh wt. (g) of total somatic

embryos 1. 3.86 39.00 ± 2.55** 19.00 ± 1.58 7.00 ± 1.22 0.16 ± 0.02 2. 4.86 68.00 ± 4.47 21.00 ± 1.41 29.00 ± 2.12 0.27 ± 0.03 3. 5.86 (control) 79.00 ± 5.09 22.00 ± 1.87 38.00 ± 2.34 0.32 ± 0.02 4. 6.86 52.00 ± 4.06 15.00 ± 1.00 23.00 ± 2.12 0.21 ± 0.02 5. 7.86 27.00 ± 2.73 13.00 ± 1.22 7.00 ± 1.0 0.11 ± 0.01

* Modified MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3, 0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP, 100 mgl-1

proline, 3% polyethylene glycol and 3% sucrose. About 0.4 g/40 ml of culture medium of initial inoculum (cell

aggregates) was inoculated in case of each treatment; **Average value ± SE (Standard Error)

Fig 25: Effect of pH of the culture medium on induction and development of somatic embryos

(b) Effect of different pH levels on germination of somatic embryos in C. borivilianum

Somatic embryos cultured in liquid media having different pH levels exhibited

germination frequencies ranging between 35.0 to 57.5% (Table-46, Fig.-26). The best

response (57.5%) in terms of embryo germination was obtained at 5.86 pH level followed

by 6.86 (38%) and 4.86 (35%) pH levels. As observed earlier (Table-45) these levels were

also supportive for somatic embryo maturation. At pH levels lower than 4.86 or higher

than 6.86, germination was completely inhibited. As noted earlier these levels were also

least supportive for maturation of somatic embryos.

0

10

20

30

40

50

60

70

80

90

3.86 4.86 5.86 6.86 7.86

Culture medium pH

No. of total somaticembryos No. of globular somatic embryos No. of cotyledonarysomatic embryos

176

Table-46: Effect of different pH levels* on germination of somatic embryos in C.

borivilianum

S. No. pH levels of the culture medium

No. of embryos germinated/20 embryos

Germination percentage

1. 3.86 - - 2. 4.86 7.00 ± 0.71 35

3. 5.86 (control) ** 11.50 ± 1.22 57.5

4. 6.86 7.60 ± 0.89 38

5. 7.86 -

*For germination somatic embryos cultured on liquid medium at different pH levels were transferred to MS basal

medium supplemented with 3.5 mgl-1 BAP; ** Modified MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3,

0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP, 100 mgl- 1proline and 3% sucrose; †Average value ± SE (Standard Error)

Fig 26: Germination of somatic embryos at different pH levels

3.4.7.4 Effect of casein acid hydrolysate

Effect of different levels of casein acid hydrolysate on somatic embryogenesis was

also investigated in the present study. It was observed that CH did not have any

stimulatory effect on somatic embryogenesis especially on embryo maturation in C.

borivilianum. Tested at different levels (1-4%), CH only at 2% level showed better

response followed by 1% level (Table-47). At 2% level, an average number of 44 total

somatic embryos were obtained. Amongst them 26 were at globular stage while only

7 were in mature (cotyledonary) stage Thus, the number of mature (cotyledonary

stage) embryos was significantly lower compared to control medium. Embryo

maturation was inhibited at

0

2

4

6

8

10

12

14

3.86 4.86 5.86 6.86 7.86

pH levels of culture medium

No. of embryosgerminated / 20embryos

177

Table-47: Effect of different levels of casein acid hydrolysate in liquid culture

medium on somatic embryogenesis of C. borivilianum

S. No. Casein acid hydrolysate (%)

No. of total somatic embryos

No. of globular somatic embryos

No. of mature (cotytledonary)

somatic embryos

Fresh wt. (g) of total somatic embryos

1. Control* 79.00 ± 5.09** 22.00 ± 1.87 38.00 ± 2.34 0.32 ± 0.02 2. 1 35.00 ± 2.83 21.00 ± 2.12 4.00 ± 0.70 0.14 ± 0.02 3. 2 44.00 ± 3.93 26.00 ± 2.34 7.00 ± 0.80 0.18 ± 0.03 4. 3 20.00 ± 2.0 15.00 ± 1.58 - 0.08 ± 0.01 5. 4 15.00 ± 2.0 12.00 ± 1.22 - 0.06 ± 0.02

* Modified MS medium containing 143 mgl-1 NH4NO3, 1083 mgl-1 KNO3, 0.25 mgl-1 2, 4-D, 1.5 mgl-1 2-iP, 100 mgl-1

proline, 3% polyethylene glycol and 3% sucrose; **Average value ± SE (Standard Error); - No response

other levels tested (3% and 4%) and most of the embryos were in the globular stage.

In liquid culture having different CH levels some root like structures developed from

cell aggregates and these cultures turned non-embryogenic. Germination of somatic

embryos was completely inhibited at different levels of the casein acid hydrolysate

and root like structures protruded from cell masses.

3.4.8 Histological analysis of somatic embryogenesis in C. borivilianum

Histological examinations revealed the prescence of embryos at different stages of growth

among nondifferentiated callus cells. In light microscopic observations of the serial

sections of embryogenic cultures, cell masses of darker stained colour were observed

which were embryogenic cell masses or meristematic cell clusters. Globular embryos

appeared from embryogenic cell masses at the surface of cells as evidenced from the

histological studies (Plate-11a, b), which later on differentiated into cotyledonary stage

embryos having bipolar structures (Plate-11c, d). Thus in the present histological studies

somatic embryos of C. borivilianum are similar to zygotic embryos in their developmental

process.

3.4.9 PCR-based Random Amplified Polymorphic DNA analysis of plants regenerated

through organogenesis and embryogenesis

A set of 20 primers (MAP01 to MAP20) with the sequences as described previously in

section 2.7 were used to study DNA polymorphism for determining the similarity index

(degree of similarity) among the 17 randomly selected plants of C. borivilianum

regenerated through organogenesis or embryogenesis along with parent plant. Plants were

grown in glass house conditions along with control plant (parent plant).

178

PCR amplification by using MAP01 to MAP20 primers was carried out for the

isolated genomic DNA of all the 18 sample plants. Among 20 primers, 5 primers i.e.,

MAP 03, MAP 05, MAP 06, MAP 07 and MAP 11 accounting for 25% of the total

primers used did not exhibited any amplification. The remaining 15 primers i.e. MAP 01,

MAP 02, MAP 04, MAP 08, MAP 09, MAP 10, MAP 12, MAP 13, MAP 14, MAP 15,

MAP 16, MAP 17, MAP 18, MAP 19 and MAP 20, which accounted for 75% of the total

primers used, exhibited amplification. Five primers i.e. MAP 01, MAP 04, MAP13, MAP

15 and MAP 19 did not gave clear reproducible bands. MAP 02, MAP 08, MAP 09,

MAP10, MAP12, MAP 14, MAP 16 MAP 17, MAP 18 and MAP 20 were selected on the

basis of their amplification products which revealed clear fragmentation of DNA isolated

from selected plants (Plate-12a-d). The pattern of amplification was reproducible.

Although total 51 RAPD bands were produced through amplification with these primers

but only 47 bands were taken into consideration, out of which 38 bands were found to be

monomorphic and 9 were polymorphic in nature. All the responding primers tested in the

present study produced monomorphic bands showing the genetic homogeneity in plants

regenerated via organogenesis and somatic embryogenesis. The polymorphic amplification

products were produced by primers MAP 08, MAP 09, MAP 10, MAP 16 and MAP 18.

Thus, about 84.33% of monomorphism was exhibited by the randomly selected plants of

C. borivilianum regenerated in vitro through organogenesis or embryogenesis in the

present study (Table-48). The number of scorable bands amplified with a given primer

ranged from 2 (MAP 02 and MAP 12) to 8 (MAP 08). The size of amplified fragments

ranged from approximately 0.2 kb to 2.7 kb amongst different primers tested in the present

study.

Table-48: Monomorphic and polymorphic bands as observed with different MAP

primers

Primers Total number of bands

Monomorphism Polymorphism Number of

bands Percentage

(%) Number of

bands Percentage

(%) MAP02 MAP08 MAP09 MAP10 MAP12 MAP14 MAP16 MAP17 MAP18 MAP20

2 8 5 5 2 3 4 6 6 6

2 4 4 4 2 3 2 6 5 6

100.00 50.00 80.00 80.00

100.00 100.00

50.00 100.00

83.33 100.00

- 4 1 1 - - 2 - 1 -

- 50.00 20.00 20.00

- -

50.00 -

16.67 -

Total 47 38 84.33 9 15.67

179

The similarity matrix based on Nei and Li’s (1979) principle was used to generate

a graphic phenogram through UPGMA using NTSYSpc version 2.02j to find out the

similarity between the plants of C. borivilianum regenerated through organogenesis or

embryogenesis along with control parent plant. The similarity coefficients of all the 18

sample plants are presented in Table-49. Similarity coefficients among the 18 randomly

selected sample plants ranged from 86% to 100%. The most diverse pair were sample 10

and 17 showing 86% similarity. Another diverse pair of sample 1 and 13 showed 89%

similarity. The rest of the plants showed much narrower variability with similarity

coefficients of 90% to 100%. The similarity coefficients in plants regenerated through

organogenesis ranged from 93% to 100% while in the plants regenerated through somatic

embryogenesis, similarity coefficient varied from 86% to 100%. Thus the similarity

coefficients obtained in plants regenerated through organogenesis or embryogenesis were

comparable to each other.

Table-49: Similarity coefficients of 18 randomly selected C. borivilianum plants

regenerated in vitro through organogenesis or somatic embryogenesis along with

parent plant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1 1

2 0.96 1

3 0.95 1 1

4 0.95 0.97 1 1

5 0.96 0.97 1 1 1

6 0.93 0.96 0.96 0.97 0.96 1

7 0.93 0.98 0.97 0.97 0.97 0.96 1

8 0.94 0.97 0.96 0.97 0.98 0.97 1 1

9 0.93 0.96 0.95 0.96 0.97 0.96 1 1 1

10 0.93 0.96 0.95 0.97 0.96 0.94 1 0.97 0.97 1

11 0.92 0.97 0.97 0.97 0.96 0.95 0.97 0.96 0.96 1 1

12 0.92 0.97 0.97 0.94 0.94 0.93 0.94 0.94 0.95 0.96 0.98 1

13 0.89 0.95 0.97 0.97 0.95 0.95 1 0.96 0.96 0.96 0.97 0.98 1

14 0.9 0.96 0.96 0.97 0.97 0.95 0.96 0.97 0.97 0.95 0.98 0.97 0.98 1

15 0.9 0.95 0.93 0.95 0.96 0.94 0.97 0.95 0.95 0.96 0.97 0.99 0.96 0.97 1

16 0.91 0.92 0.92 0.97 0.97 0.93 0.93 0.97 0.93 0.93 0.98 0.97 0.96 0.95 0.97 1

17 0.93 0.96 0.96 0.97 0.96 0.94 0.93 0.97 0.98 0.86 0.96 0.96 0.96 0.96 0.95 0.95 1

18 0.91 0.93 0.93 0.94 0.94 0.94 0.92 0.98 0.93 0.92 0.98 0.95 0.94 0.95 0.96 0.96 0.98 1

Sample1: Parent plant (control); sample 2-10: plants regenerated through organogensis; sample 11-18: plants

regenerated through somatic embryogenesis

In order to analyze the relatedness among all the in vitro raised plants along with

control plant, the UPGMA based dendrogram was constructed using paired matrix values.

180

As it is evident from dendrogram (Fig.-27), in vitro raised plant numbered 1

(regenerated through organogenesis) constituted a separate major cluster from the other in

vitro raised plants. In general the average matrix of indices calculated on the basis of

analysis of 20 different decamer primers vs. plants regenerated either through

organogenesis or somatic embryogenesis showed that all sampled plants had a narrow

genetic base. However, the unique polymorphic profiles of DNA markers were able to

distinguish between plants of different origin.

Coefficient0.80 0.85 0.90 0.95 1.00

1

2

3

4

5

7

8

9

10

11

13

14

6

12

15

16

17

18

Fig 27: UPGMA based dendrogram showing the relationship among 18 in vitro raised plants of C.borivilianum regenerated through organogenesis or embryogenesis along with parent plant

181

3.5 DISCUSSION

Mass propagation via tissue culture (micropropagation, clonal propagation) of selected,

improved material is advantageous over conventional methods of vegetative propagation

(cutting, grafting). Plants can be produced by tissue culture through one of two different

pathways: organogenesis or somatic embryogenesis. Superiority of somatic embryogenesis

over organogenesis for large-scale multiplication of some plant species has been observed.

It has several advantages compared to other propagation methods. The mass propagation

of plants in a relatively shorter time through multiplication of embryogenic propagules is

the commercially attractive application of somatic embryogenesis (Merkle et al., 1990).

Somatic embryogenesis offers a possibility of developing scale-up technology as

compared to organogenesis. Somatic embryos are small and can be adequately handled in

scale-up procedures with low labour inputs since embryos can be grown individually and

freely floating in liquid medium. Somatic embryogenesis has the advantage over axillary

or adventitious shoot propagation as embryos are bipolar, bearing both root and shoot

apices which are necessary for complete plant development. Because of simultaneous

development of both the shoot and root meristems and its single cell origin, propagation

via somatic embryogenesis results in uniform plant regeneration, a process involving less

subculturing of tissue resulting in time and cost-efficiency.

The high volume multiplication of embryogenic propagules can be utilized directly

in various studies as in regeneration of genetically transformed plants (Litz and Gray,

1995; Vicient and Martinez, 1998). Many have since investigated the nature of somatic

embryogenesis aimed to develop its potential for applied uses like clonal

micropropagation (Halperin, 1995; Krikorian and Simola, 1999). However, the potential

of somatic embryogenesis in many plant species is yet to be explored due to low

germination rate of somatic embryos and induction of somatic embryogenesis with a low

frequency and in limited number of explants.

Chlorophytum borivilianum (family-Liliaceae) commonly known as safed musli,

possesses several pharmacologically important steroids and saponins (Tandon et al., 1992;

Tandon and Shukla, 1993). Dried roots are used as tonic in various Ayurvedic

formulations and are exported from India in substantial amount. Conventionally this plant

is propagated by division of stocks or seeds. Low seed set, poor seed viability and

182

germination (Bordia, 1991) and decay of tubers used for vegetative propagation during

storage affect efficiency of conventional methods of large-scale propagation of C.

borivilianum. The immediate task is to conserve and multiply the plant in bulk amount

required for its domestication to meet the present demand. The present work describes an

improved method for large-scale rapid multiplication of C. borivilianum through high

frequency somatic embryogenesis. Attempts have been made to standardize various

culture conditions of somatic embryogenesis employing liquid culture medium in C.

borivilianum to use the protocol for large-scale multiplication. For this various aspects

related to induction, development, maturation and germination of somatic embryos were

first studied in semi-solid medium before investigating them in liquid medium in the

present investigation. Histolgical studies were also carried out to ascertain the

embryogenic nature of differentiating structures. Subsequently, plants regenerated through

somatic embryogenesis were established in soil and genetic fidelity of these plants was

analyzed for confirming their true-to-type nature and the level of genetic similarity

between plants regenerated through organogenesis and embryogenesis was compared

through PCR-based Random Amplified Polymorphic DNA (RAPD) analysis approach.

In the present study two different explants i.e. leaf segments and shoot bases

obtained from the sterile in vitro grown plants of C. borivilianum and seedlings

obtained by germinating seeds on MS basal medium supplemented with 20 mgl-1 GA3

were tested to find out suitable explant for obtaining optimal callus induction. It was

observed that the type of explant has a significant effect on the callus induction and

growth response. Amongst different explants tested in the present study, leaf explants

were non-responsive.

Type and concentrations of phytohormones tested in the present study were

also important as far as frequency of callus induction and growth was concerned.

Different auxins i.e. IAA, IBA, NAA, 2,4-D or P added to MS basal medium and TDZ

either individually or its combinations with AS could not evoke callus induction from

leaf explants. Shoot base explants were also not much responsive and did not induced

183

callus formation on media supplemented with auxins, IAA or IBA but NAA (0.05-2.0

mgl-1) provoked callus induction response and optimal growth of callus was obtained

on 2.0 mgl-1 level. While 2-4,D induced callus formation only at 0.05 mgl-1 level in

cultured shoot bases but the growth of callus induced at 0.05 mgl-1 level of 2,4-D was

lower than that obtained on 2.0 mgl-1 of NAA. Similarly, TDZ was added to MS basal

medium to test its efficacy on callus induction in both the cultured explants i.e. leaf

segments and shoot bases. A light yellow to yellowish-green callus was observed in

cultured shoots on most of the concentrations in the range of 0.05-6.0 mgl-1. Although

callus induction was observed on all the levels of NAA tested but rooting was

observed in calli induced on NAA supplemented media and therefore NAA was not

considered to be optimal for further studies. In an effort to further enhance callus

induction response, Kn or AS was supplemented to TDZ containing medium (5.0

mgl-1). Addition of AS (10.0-30.0 mgl-1) increased callus-inducing efficiency of

cultured shoot bases only marginally. On the other hand addition of Kn had no

beneficial effect on callus induction response in case of shoot base explants.

Two different basal media, MS and B5, each supplemented with different

combinations of 2,4-D and Kn were tested for induction of somatic embryogenesis in

seedling explants of C. borivilianum. Supplementation of MS basal medium with GA3 (20

mgl-1) increased seed germination frequency from 10% to 40%.

In the present study the acquisition of embryogenic potential in seedling

explants is manifested through a callus phase (indirect embryogenesis). It was

observed that MS medium supplemented with 2,4-D and Kn exhibited better

response than B5 medium and moderate to good callus induction frequency was

observed on MS medium containing 0.25 mgl-1 of Kn and 0.25-0.50 mgl-1 of 2,4-D.

Induction of somatic embryogenesis was observed during regular subculturing of

calli maintained on MS basal medium supplemented with different concentrations

and combinations of 2,4-D and Kn and small rounded globular shaped somatic

embryos were observed regularly at yellowish-white callus in different frequencies at

different media combinations. Observations revealed that higher number of somatic

embryos were observed on MS basal medium containing 0.25-0.50 mgl-1 2,4–D and

0.25 mgl-1 Kn. MS or B5 basal medium without any phytohormones exhibited poor

somatic embryogenesis response and the number of somatic embryos induced was

lower than that observed on either basal media supplemented with different

concentrations and combinations of 2,4-D and Kn tested in the present study.

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Amongst different concentrations of 2,4-D tested, 0.25 mgl-1 was preffered over 0.50

mgl-1 because higher concentrations of 2,4-D were found to be inhibitory for somatic

embryogenesis although cultures maintained at higher concentrations of 2,4-D were

slightly more friable than the culures grown at lower concentrations.

Induction and expression of somatic embryogenesis might be triggered by

different factors and the choice of explant is a critical factor for embryogenic callus

induction. Unlike dicots, the vegetative parts of a monocot plant do not readily

proliferate in cultures. Usually explants are best taken from embryogenic or

meristematic tissues (young inflorescences, leaves and seedlings). The embryogenic

cultures from callus induced in leaflets of in vitro-grown plants of M. truncatula were

used to establish suspension cultures (Duque et al., 2006). Embryogenic callus

derived from mature seeds of dune reed (Phragmites communis) was used to establish

suspension culture (Wang et al., 2001). In the present study also seedling explants

were most responsive and were used for initiation and establishment of embryogenic

tissue while the explants obtained from leaves were non-responsive. Previously Arora

(1999) reported that in case of leaf explants obtained from in vitro maintained

shoots/plantlets of C. borivilianum, explant response was erratic and low and

associated with slow callus growth. Little callus produced on certain treatments did

not survive during further subculturing. Callus produced on high concentration of

TDZ from a few explants grew and produced embryos. However, leaf explants could

not form a system for continuous production of somatic embryos. Joshi et al. (2003)

obtained fluffy unorganized, shiny mass of callus from young shoot bases in

Chlorophytum on MS medium containing 5.0 mgl-1 BAP and subsequently shoots

regenerated from this callus mass when subcultured at fresh medium.

The nature of basal medium providing different levels of nutrition can also effect

the frequency of somatic embryogenesis. Varisai et al., 2004 observed that amongst MS

and B5 basal media tested for somatic embryogenesis only MS medium was responsive in

terms of somatic embryogenesis. In the present study also observations revealed that MS

medium providing high nutrition level was found to be more efficient than B5 medium for

somatic embryogenesis. While Jain et al. (1997) reported induction of somatic embryos in

C. borivilianum from seedling derived callus on B5 medium supplemented with 2,4-D. In

monocots, in most of the cases primary embryogenesis was induced on auxin-

supplemented media. Among the different auxin analogues used to induce somatic

embryogenesis, 2,4-D is by far the most efficient (Varisai et al., 2004) and therefore, this

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synthetic growth regulator is used in the majority of embryogenic cell and tissue culture

systems. The influence of exogenously applied auxins particularly 2,4-D on the induction

of somatic embryogenesis is well documented (Dudits et al., 1991; Yeung, 1995; Feher et

al., 2003). It can be suggested that 2,4-D above a certain concentration has a dual effect in

these cultures, as an auxin (directly or through endogenous IAA metabolism) and as a

stressor (Feher et al., 2002).

In some of the cases where the exogenous application of auxins has proved to be

efficient treatment for the induction of somatic embryogenesis, further development of the

existing somatic embryos can be achieved by reducing or removing auxin from the culture

media. Rangaswamy (1986) observed that optimal development or histo-differentiation

requires removal or reduction of the auxin used for embryo induction. In the present study

optimal somatic embryogenesis was observed on reducing the concentration of 2,4-D

which was initially used for induction of embryogenenic callus. Contrary to this induction

and development of embryos upto the torpedo-cotyledonary stages was achieved on the

same auxin medium indicating the relative insensitivity to inductive hormone in

Macrotyloma uniflorum (Varisai et al., 2004).

Although the addition of cytokinins as the sole plant growth regulator has proved

to be effective in inducing somatic embryogenesis (Sagare et al., 2000), in most of the

cases, the effect of these additives is not universal, and many times their addition should

often be coupled with that of auxins to obtain the desired effect (Merkle et al., 1995).

Consistent with the observations in the present study, Duque et al. (2006) also observed

that cells acquire embryogenic competence when growing in the maintenance medium

which is supplemented with 2,4-D and Kn while Varisai et al. (2004) reported that

addition of BAP and/or Kn to the 2,4-D medium suppressed somatic embryogenesis and

produced greenish compact callus.

Attempts have also been made to propagate Chlorophytum through somatic

embryogenesis by various workers (Kukda et al., 1994; Purohit et al., 1994b; Jain et al.,

1997; Arora et al., 1999 and Joshi et al., 2003) where various factors influencing somatic

embryogenesis were studied. Embryogenic callus in C. borivilianum was induced on MS

medium containing 1.0 mgl-1 2,4-D and maturation of somatic embryos was observed on

MS medium supplemented with 0.1 mgl-1 2,4-D. Conversion of these embryos into

plantlets was observed on plant growth regulator free basal MS medium (Kukda et al.,

1994). Purohit et al. (1994b) also reported somatic embryogenesis and plant regeneration

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in Chlorophytum. Callus was induced on MS medium containing 1.0 mgl-1 2,4-D.

Subsequently by subculturing on MS medium supplemented with 0.5 mgl-1 2,4-D, yellow,

compact, hard, nodular, shiny somatic embryo developed. Embryogenic cultures were

maintained by repeated subculturing every four weeks on MS medium containing 0.25

mgl-1 2,4-D. Jain et al. (1997) reported induction of somatic embryos in C. borivilianum

from seedling derived callus on B5 medium supplemented with 0.5 mgl-1 2,4-D. Arora et

al. (1999) reported multiplication through somatic embryogenesis in callus cultures

obtained from seedling explants. Somatic embryos were obtained on MS medium

containing 2,4-D and Kn. In the present studies also induction of somatic embryogenesis

was observed on Kn and 2,4-D supplemented medium thereby indicating the importance

of exogenous phytohormones in regulating embryogenesis in seedling derived callus of C.

borivilianum.

Optimizing medium for enhancing growth of embryogenic callus and induction of

somatic embryos in seedling derived callus was necessary. Therefore, manipulations in

MS medium constituents were made and experiments were conducted to study effect of

different levels of inorganic nitrogen sources i.e. NH4NO3 and KNO3 in the MS basal

medium containing 0.25 mgl-1 each of 2,4–D and Kn on somatic embryogenesis because

nitrogen levels have been shown to affect somatic embryogenesis and optimum levels

need to be worked out for different species/genotypes for obtaining desired response. In

the present study, observations revealed that maximum number of somatic embryos

(19/inoculum) were obtained on MS medium containing 143 mgl-1 NH4NO3 and 1083 mgl-

1 KNO3. It was observed that high levels of NH4NO3 or KNO3 were not beneficial for

somatic embryogenesis and the growth of the callus was reduced at high nitrogen levels.

Levels of NH4NO3 and KNO3 optimal for somatic embryogenesis were lower than in

normal strength MS medium (1650 mgl-1 NH4NO3 and 1900 mgl-1

KNO3). The

concentrations and combinations of KNO3 and (NH4)2SO4 in the medium had shown a

marked effect on the somatic embryogenesis in seedling derived callus of C. borivilianum

(Arora et al., 1999). The relevance of nitrogen for induction of somatic embryogenesis

through either NH4NO3 and/or KNO3 in the medium has also been demonstrated in other

plant species (Feng and Ouyang, 1988; Choi et al., 2003).

In the present study it was observed that while lower levels of Kn or TDZ were

more effective for somatic embryogenesis, 2-iP supplemented media showed optimal

response at higher levels. The maximum number of somatic embryos (23/inoculum) were

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obtained on modified MS medium (medium containing 143 mgl-1 NH4NO3 and 1083 mgl-1

KNO3) supplemented with 0.25 mgl-1 2,4–D and 1.5 mgl-1 2-iP followed by induction of

an average number of 21.6 somatic embryos at modified MS medium supplemented with

0.1 mgl-1 TDZ. To further enhance the somatic embryogenesis, two amino acids viz.

glutamine and proline were added to the control medium (modified MS medium

containing 0.25 mgl-1 2,4-D and 1.5 mgl-1 2-iP) and were observed to have a stimulatory

effect on the growth of cultures and somatic embryogenesis (average number of somatic

embryos/inoculum,) although glutamine was less responsive than proline. The medium

fortified with 100 mgl-1 proline gave best response both in terms of somatic

embryogenesis (an average number of 43.2 somatic embryos/inoculum) and culture

growth (an average callus f. wt. of 0.34 g) and compared to the control, 1.88-fold higher

response in terms of average number of somatic embryos was obtained on medium

supplemented with 100 mgl-1 proline.

Amino acids have been reported to affect somatic embryogenesis in many plant

species. In alfalfa amino acid enrichment of the medium increased the number of embryos

regenerated (Skokut et al., 1985). Proline is known to enhance somatic embryogenesis in

maize (Armstrong and Green, 1985) and pollen embryogenesis in cereals (Sozinov et al.,

1981). As observed in these studies, in the present study also proline exerted a positive

influence on somatic embryogenesis compared to glutamine while in the studies of Varisai

et al. (2004), glutamine enhanced the induction, growth and maturation of somatic

embryos in Macrotyloma uniflorum (Varisai et al., 2004). However, addition of proline or

alanine reduced the frequency of somatic embryo induction and maturation. This

suggested that the exogenous supply of amino acids play a pivotal role in the induction

and physiological maturity of somatic embryos.

In somatic embryos cultured on modified MS medium supplemented with 0.25

mgl-1 2,4-D, 1.5 mgl-1 2iP and 100 mgl-1 proline, 30% germination response was

observed on subculturing to MS medium supplemented with 3.5 mgl-1 BAP while

higher concentrations (>4.5 mgl-1) of BAP were inhibitory for germination. Kukda et

al. (1994) observed that somatic embryos germinated into plantlets on plant growth

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regulator free basal MS medium. Similarly auxin free MS medium was employed for

germination of embryos and only 20% germination was reported (Purohit et

al.,1994b). Arora et al. (1999) also reported multiplication through somatic

embryogenesis in callus cultures obtained from seedling explants and MS medium

supplemented with BAP was employed for germination of somatic embryos. While

Jain et al. (1997) reported plantlet formation on B5 medium without addition of any

growth regulators to the culture medium.

Addition of BAP to MS medium was found to be supportive for the regeneration of

plantlets from two cell lines of dune reed (Wang et al., 2001). Addition of cytokinins to

MS medium enhanced regeneration of plantlets from somatic embryos and the maximum

rate was observed with Kn supplemented medium in Lilium x formolongi Hort. (Ho et al.,

2006). On the other hand Varisai et al. (2004) reported very low (5%) germination rate on

MS medium without phytohormones. In present study also lowest germination frequency

(3%) was observed on MS medium without any phytohormones compared to MS medium

supplemented with BAP.

Although somatic embryogenesis has been observed in the present study but

embryo maturation rate was low therefore experiments were performed for testing

effect of types and levels of different carbohydrate sources on somatic embryo

maturation and germination, not reported earlier in C. borivilianum. Sucrose/glucose

employed as carbohydrate source produced highest average number of total somatic

embryos (44/41) followed by maltose, mannose, fructose or cellobiose. The number of

total somatic embryos was low in xylose or rhamnose and somatic embryogenesis was

not observed in cultures inoculated on media containing arabinose, galactose, lactose

or soluble starch. The average number of mature (cotyledonary stage) embryos was

highest in sucrose (17) followed by glucose (14) and maltose (6). Somatic

embryogenesis was completely inhibited on control medium (without any

carbohydrate source). The embryo maturation was low in fructose, cellobiose or

mannose and was not observed altogether on media supplemented with rhamnose or

xylose. Therefore, these carbohydrate sources were inhibitory for somatic embryo

development. Although callus growth was higher on medium supplemented with

maltose than medium supplemented with sucrose or glucose but number of total

somatic embryos and mature embryos were lower indicating that medium

supplemented with sucrose or glucose were more supportive for embryogenic callus

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growth and subsequently for induction and development of somatic embryos as

compared to medium supplemented with maltose.

During the study it was also observed that the cultures previously incubated on

sucrose or glucose containing media supported higher rate of germination of somatic

embryos compared to cultures raised on other carbohydrate sources. Amongst these two

carbohydrate sources, germination frequency was higher (30%) on sucrose than on

glucose (20%) followed by maltose (15%). The frequency of somatic embryo germination

decreased considerably on control medium which was devoid of any carbohydrate source.

The lowest germination frequency (5%) was observed on fructose or cellobiose

supplemented media.

Amongst various sucrose levels tested in the present study (1-11%), highest

number of cotyledonary stage embryos was observed at 3% sucrose level (control

medium). Media supplemented with >7% sucrose level were inhibitory to the development

of somatic embryos. 3% sucrose level besides supporting the embryo production also

supported highest germination frequency (30%). The lowest germination frequency (10%)

was observed on medium supplemented with 1% sucrose level. Sucrose levels above 5%

completely inhibited not only the germination of embryos but also somatic embryo

development and maturation.

The types and levels of carbohydrates can play vital role in somatic

embryogenesis. The type of carbon source has been found to affect the initiation of

somatic embryos in horsegram (Macrotyloma uniflorum), with sucrose being more

effective than glucose and fructose. Sucrose at 3% level was found to be the most

effective carbohydrate for the induction and further development of the somatic

embryos (Varisai et al., 2004). Consistent with these observations, in the present

study also sucrose and glucose at 3% concentration gave best response in terms of

induction and development of somatic embryos followed by maltose. In alfalfa

(Medicago sativa) maltose is reported to be supportive for somatic embryogenesis

(Strickland et al., 1987) while it was completely ineffective for somatic

embryogenesis in horsegram (Varisai et al., 2004), peanut and soyabean (Eapen and

George, 1993).

In nature carbohydrate is translocated within the plant as sucrose and the

tissue may have the inherent capacity for uptake, transport and utilization of

sucrose. In C. borivilianum sucrose was found to be best for induction of somatic

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embryogenesis and maturation of embryos whereas carbohydrates other than

sucrose, glucose or maltose inhibited embryo maturation either partially or

completely. The inhibitory effect of these carbohydrates may be due to lack of proper

uptake and transport or subsequent utilization by the tissue. Fructose and soluble

starch supported embryogenesis in Medicago sativa (Strickland et al., 1987), but they

had an inhibitory effect on somatic embryogenesis in finger millet (Eapen and

George, 1990). Sorbitol or mannitol in combination with sucrose was found to be

beneficial for inducing differentiation in long term cultures of rice (Kavi Kishor,

1987). Eapen and George (1990) observed that supplementation of the medium with

osmotic agents such as mannitol or sorbitol reduced the embryo germination

frequency in finger millet. In the present study also on media supplemented with

plasmolyzing osmotica viz. D-mannitol or D-sorbitol, somatic embryogenesis and

germination of embryos was completely inhibited.

Non-plasmolyzing osmoticum viz. polyethylene glycol (PEG) at 3% level was

found suitable for embryo maturation and highest number of embryos (32)

developed to cotyledonary stage at this level exhibiting 1.88-fold increase in number

of cotyledonary stage embryos compared to control medium. The number of total

somatic embryos was also highest (56) at this level. Polyethylene glycol (1-3%)

improved germination frequencies without limiting maturation. Germination

percentage of somatic embryos increased to 50% at 3% PEG level compared to 30%

in control medium.

Synthesis and accumulation of storage compounds especially storage proteins and

acquisition of desiccation tolerance determine the ability of embryos to germinate and

convert into plants (Kermode, 1995). The importance of water relations in controlling

embryo maturation proposed by Fischer et al. (1987), has been supported by results from

both embryo culture experiments (Xu et al., 1990) and in situ studies (Saab and Obendorf,

1989). Attempts to simulate the in vivo environment through modification of the

maturation media used for somatic embryos increased storage compound levels and

desiccation tolerance (Finkelstein and Crouch, 1986; Xu et al., 1990). Studies in alfalfa

(Xu et al., 1990) and rapeseed (Finkelstein and Crouch, 1986) have shown that embryo

191

maturation is frequently associated with a low osmotic potential in tissue or medium

surrounding the embryos.

Plasmolyzing osmotica such as sugar alcohols (mannitol, sorbitol etc.) readily pass

through the cell wall and cause temporary plasmolysis untill their movement into the

cytosol leads to osmotic recovery (Attree and Fowke, 1993). In contrast, PEG molecules

(non-plasmolyzing osmoticum) are too large to move through the cell wall and do not

cause plasmolysis. Non-plasmolyzing osmotica are more effective in promoting somatic

embryo maturation (Attre et al., 1991). In the present study PEG was effective in

promoting embryo maturation in C. borivilianum while mannitol and sorbitol were

inhibitory for embryo maturation. Supplementation of maturation medium with 5% PEG

or 3% sorbitol improved germination frequencies in soybean (Glycine max) while 3%

mannitol did not improve embryo maturation and germination (Walker and Parrot, 2001).

At the beginning of desiccation, most embryos have changed colour from dark-

green to yellowish-green indicating physiological maturity and quiescence (Saab and

Obendorf, 1989; Komatsuda et al., 1992). Somewhat similar to these observations, in the

present study also, the embryos cultured on PEG containing media became whitish to

yellowish-white in colour as compared to control medium (without PEG) on which most

of the embryos were yellowish to yellowish-green in colour indicating their advancement

towards physiological maturity.

ABA was not much effective for maturation and germination of embryos and

amongst different concentrations tested, ABA was responsive only at 0.26 mgl-1 level

followed by 0.13 mg l-1. But the number of cotyledonary stage embryos on these levels of

ABA was lower than observed on control medium. On other ABA concentrations embryos

became brown and necrotic and did not develop further.

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Endogenous levels of ABA affect initiation of embryogenic cultures in some

monocots (Bhaskaran and Smith, 1990). It has been observed in some cases that ABA

promotes maturation (Black, 1991) and reduces the frequency of abnormal morphogenesis

(Etienne et al., 1993). Addition of ABA (1.0 µM) to the culture medium significantly

promoted somatic embryo maturation in Quercus suber but no significant differences in

the germination of mature embryos were observed among different treatments (Garcia-

Martin et al., 2005). On the contrary in the present studies ABA was not effective for

maturation and subsequently for germination of embryos.

The availability of mineral nutrients to the cultured tissue during culture period

depends on the physical state of culture medium (whether agar-gelled or liquid). Other

factors such as pH also determine the rate of absorption of the various nutritional

constituents (Debergh et al., 1994).

In the present investigation better growth and maturation of somatic embryos was

observed on liquid culture medium as compared to semi-solid medium. In liquid culture

medium an average number of 66 somatic embryos were induced compared to 54 on semi-

solid medium. The number of globular and cotyledonary stage somatic embryos was

higher in liquid medium (20 and 32 respectively) as compared to semi-solid medium (16

and 26 respectively). Therefore liquid medium is more effective for induction and

proliferation of globular stage somatic embryos and embryo maturation than semi-solid

medium and compared to semi-solid medium, 1.06-fold increment in number of

germinated embryos was observed in liquid culture medium.

Somatic embryogenesis using liquid medium has not been reported so far in

C. borivilianum as a regeneration system. The aim in the present study was to develop

an efficient, rapid regeneration method for mass propagation which is easy to scale-

up in bioreactors using liquid medium. For large-scale propagation of plants, the

development of somatic embryos in liquid medium has a number of advantages.

Besides this, somatic embryos could also be used as target materials for in vitro

selection of mutants and production of transgenic plants (Finer and McMullen,

1991). The advantage of developing somatic embryo cultures in liquid medium is

constant availability of embryogenic-comptent cells. Initiation and/or establishment

of cell suspension cultures have been reported in many plant species where effects of

different factors such as inoculum density, pH levels etc. were studied on induction,

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growth and development of somatic embryos in liquid medium (Wang et al., 2001;

Kosky et al., 2002; Ho et al., 2006)

Results in the present study revealed that initial inoculum of 0.4 g/40 ml of

culture medium produced an average number of total 79 somatic embryos. The

number of cotyledonary embryos (38) was also higher at this inoculum density

compared to other inoculum densities. At inoculum densities beyond 0.6 g/40 ml of

culture medium, inhibition in somatic embryo development and maturation was

observed. An inoculum density of 3.0 g/20 ml of culture medium combined with 30 gl-

1 sucrose showed a 2-fold increase in growth of cell clumps in Lilium x formolongi cv.

Nokirula (Ho et al., 2006). High inoculum densities (3.5 and 4.0 g/20 ml) were

unfavourable for the growth. In the present study the best growth of embryogenic

cell clumps was found at 0.4 g/40 ml of culture medium and compared to control

medium, 1.14-fold increase in fresh weight of embryogenic cell aggregates was

obtained in case of banana hybrid cultivar FHIA-18 (AAAB), an inoculum density of

0.6 g/25 ml of culture medium was found to be best for somatic embryo growth and

muliplication (Kosky et al., 2002).

In the present study at 0.6, 0.8 or 1.0 g/40 ml of culture medium inoculum

densities, the number of globular embryos was more than cotyledonary embryos

while the reverse was true in case of inoculum densities less than 0.6 g/40 ml of

culture medium. Therefore, it seems that the development and further maturation of

somatic embryos is impeded at higher inoculum densities (>0.6 g/40 ml of culture

medium).

In embryogenic suspension cultures, cell density could be an important factor

that affects somatic embryogenesis. While a high cell density is required for the

formation of embryogenic cell clusters from single cells (Nomura and Komamine,

1985), a relatively lower cell density favours the development of embryos from

embryogenic cells (Fujimura and Komamine, 1979). This may be due to the secretion

of proteins and/or other cellular factors into the culture medium. Secreted

(extracellular) and constitutive (intracellular) pr oteins are considered to play very

important role in induction of somatic embryogenesis (Gavish et al., 1992).

It was observed that 5.86 pH level (control) was better than other pH levels tested

in the present study. pH levels in the range of 4.86-6.86 were observed to be favourable

for the development and maturation of somatic embryos while acidic (pH level 3.86) or

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alkaline (pH level 7.86) media were favourable for differentiation of somatic embryos

only up to the globular stage and suppressed their further development. It was observed

that pH levels 4.86 to 6.86 were supportive for germination of embryos. These levels were

also supportive for somatic embryo maturation. The best germination response (57.5%)

was obtained at 5.86 pH level (control medium) followed by 6.86 pH level (38%). At pH

levels lower than 4.86 (acidic media) or higher than 6.86 (alkaline media), germination

was completely inhibited. Somewhat similar observations were also noted in alfalfa where

a higher rate of embryo production was observed at a constant pH of 5.5 than at a non-

buffered medium or at lower pH levels (Stuart et al., 1987). The changes in carrot embryo

development were associated with sugar uptake and ammonium depletion and can be

attributed to enzyme and metabolic activity at an optimal pH (Jay et al., 1994). It appears

that pH requirements are species specific and developmental stage dependent. Kosky et al.

(2002) also observed that acidic pH level 3.8 resulted in more uniformity (87.2% embryos

were in globular stage) in embryo development up to initial stages confirming the pH

control on synchronization of embryo development. Smith and Krikorian (1990a, b) also

reported that at lower pH level embryo development stopped at the pre-globular stage. In

chickpea also low pH (4.5) suppressed embryo maturation. In the present study

observations of the effect of very high or low pH on inhibition of embryo development

may be subsequently utilized for synchronization of somatic embryos to the initial stages

of embryo development thus preventing asynchronous development of somatic embryos.

The homogenity thus obtained in embryo development will make it convenient to handle

cultures in propagation through somatic embryogenesis at large-scales

Casein acid hydrolysate (CH) did not have any stimulatory effect on somatic

embryogenesis especially on embryo maturation in C. borivilianum. Germination of

somatic embryos was completely inhibited at different levels of casein acid hydrolysate. In

liquid culture having different CH levels development of root like structures from cell

aggregates was observed indicating that these cultures may have become non-

embryogenic. CH has been commonly added to tissue cultures of several members of

family Poaceae but growth stimulation was not always reported. However, casamino acids

(vitamin free product of casein hydrolysate) stimulated the growth of friable embryogenic

Z. mays callus (Green et al., 1983). It has been added to culture media in several studies on

somatic embryogenesis in this family (Ozias-Akins and Vasil, 1983). CH inhibited embryo

growth in finger millet (Eapen and George, 1990) as also observed in the present study.

This is in contrast with the work in barley where these substances improved embryo

195

development (Luhrs and Lorz, 1987). Root development was stimulated by the prescence

of CH (Gray and Conger, 1985) as also observed in the present study.

Somatic embryogenesis is closely similar to zygotic embryogenesis (Yeung, 1995;

Thorpe and Stasolla, 2001). It is also assumed that it passes through the same

characteristic stages (Gray et al., 1995; Toonen and de Vries, 1996). The attainment of a

globular appearance is regarded as one of the first key features of somatic embryo

development. In histological studies, somatic embryos of C. borivilianum showed pattern

of development similar to zygotic embryos as has been observed earlier (Mathe et al.,

2000; Ma and Xu, 2002; Sharma and Millam, 2004). Based upon these histological and

morphological observations it was concluded that the stages of somatic embryos observed

in the present study are consistent with the general definition of development of somatic

embryogenesis.

In the present study a procedure for plant multiplication through somatic

embryogenesis pathway using seedling explants has been standardized and

subsequently genetic fidelity of plants regenerated through this pathway has been

assessed and compared with plants regenerated through organogenesis by PCR-

based RAPD approach. For evaluating genetic variations in plants regenerated

through somatic embryogbnesis, RAPD and RFLP techniques have been used as

powerful tools to study variations among in vitro regenerated plants (Isabel et al.,

1993; Jayanthi and Mandal, 2001). However, the use of RAPD markers is more

advantageous than RFLP markers because the large number of samples can be

analyzed economically and quickly, the specific DNA fingerprints obtained are

independent of ontogenic expression and most of the genome can be sampled with a

potentially unlimited number of markers. In the present investigation, a set of 20

primers (MAP 01 to MAP 20) were used to study DNA polymorphism for

determining the similarity index among the 17 randomly selected plants of C.

borivilianum regenerated through organogenesis or embryogenesis along with the

parent plant. Similarity coefficients among the 18 randomly selected sample plants

ranged from 86% to 100% (93% to 100% in plants regenerated through

organogenesis and 86% to 100% in the plants regenerated through somatic

embryogenesis). Most of the plants showed much narrower variability with similarity

coefficients of 90% to 100%. In general the average matrix of indices calculated on

the basis of analysis of 20 different decamer primers vs. plants regenerated either

through organogenesis or somatic embryogenesis showed that all sampled plants had

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a narrow genetic base. However, the unique polymorphic profiles of DNA markers

were able to distinguish between plants of different origin.

All the responsive primers tested in the present investigation produced

monomorphic bands showing the genetic homogeneity and the similarity coefficients in

plants regenerated through organogenesis or embryogenesis were comparable to each

other. Therefore, little variation was observed in plants regenerated through somatic

embryogenesis compared to plants regenerated via organogenesis illustrating the

suitability of protocol developed for micropropagation of C. borivilianum via somatic

embryogenesis. The results observed in the present study were further substantiated by the

studies of Bennici et al. (2004). Somaclonal variations were not detected amongst the

Foeniculum vulgare Mill. plants of organogenic and embryogenic origin and in parent

plant. The genetic stability of in vitro raised plantlets of various species regenerated via

somatic embryos (Leroy et al., 2000; Sanchez et al., 2003; Bennici et al, 2004) and via

axillary buds (Shu et al., 2003; Martins et al., 2004) has been reported previously. In

contrast, variations in plantlets regenerated via organogenesis or somatic embryos were

also observed (Kiss et al., 2001; Gesteira et al., 2002). Thus, based on RAPD analysis it

can be concluded that genetic uniformity of plants regenerated in vitro in the present study

is not influenced by the different growth regulator used or by the mode of regeneration

(organogenic or embryogenic). The results are of interest because some earlier reports

mentioned that the hormone type and ratio may influence genomic stability during the

culture (Bogani et al., 1996). Bennici et al. (2004) stated that the lack of variations in their

Foeniculum vulgare regenerants may be due to developmental constraints which exert

selection against variant cells during regeneration.

The reproducibility of the amplification results obtained with 15 of the initial 20

primers corroborate the utility of modified CTAB method (Khanuja et al., 1999) used to

isolalate DNA. The genetic similarity observed in the plants regenerated via somatic

embryogenesis suggested that this regeneration mode in C. borivilianum can be adopted

for large-scale propagation. The observations of monomorphism between plants

regenerated through somatic embryogenesis and organogenesis lead to the conclusion that

C. borivilianum plants regenerated by different modes in this study are likely to be

genetically true to the plant of origin.

Thus, in the present study an efficient procedure for high frequency plant

multiplication employing somatic embryogenesis pathway has been standardized using

197

seedling explants by studying induction and development of somatic embryos in seedling

derived callus (indirect embryogenesis) and germination of somatic embryos (Fig.-28)

with the intentions to use the protocol for large scale multiplication of C. borivillianum

employing liquid culture medium for commercial applications.

Fig-28: Flow-chart for micropropagation of C. borivilianum via somatic embryogenesis: Seeds from

mother plants are surface-sterilized and kept for germination on MS basal medium supplemented

with 20 mgl-1 GA3; Stage 1: Germinated seedlings are cultured on MS basal medium containing 0.25

mgl-1 2,4-D, 0.25 mgl-1 Kn and 3% sucrose for embryogenic callus induction (Later somatic embryos

induced from embryogenic callus on same medium); Stage 2: Globular and/or heart shaped embryos

are transferred on modified MS medium (143 mgl-1 NH4NO3 and 1083 mgl-1 KNO3) containing 0.25

mgl-1 2,4-D, 1.5 mgl-1 2-iP, 100 mgl-1 proline, 3% polyethylene glycol and 3% sucrose for their

maturation; Stage 3: Mature embryos are cultured on MS medium containing 3.5 mgl-1 BAP and 3%

sucrose for germination and plantlet development; Stage 4: Plants with developed root system are

transplanted into soil.