13

CHAPTER II

IN VITRO PROPAGATION

2.1 Introduction

Tissue culture technology has proved to be a versatile tool for propagating elite

clones and the screening for useful variants. Plant propagation through culture

technology has emerged from a century old concept, i.e. the totipotency of cells

established by the German scientist Haberlandt (1902). Experiments of Laibach

(1929), White (1934), van Overbeek et al. (1941), Loo (1945) and Murashige and

Skoog (1962) led the way to lay a strong foundation, which brought the technology

and its versatile application to the forefront especially in the field of agriculture and

horticulture. The successful application of plant tissue culture technology for plant

improvement is based upon the mass regeneration of plants from cultured cells or

tissues.

2.1.1. Direct organogenesis

Micropropagation is one of the reliable methods of in vitro studies by

which large number of pathogen free plants can be produced. Micropropagation is the

true-to-type propagation of a selected genotype using in vitro culture technique

(Debergh and Read, 1991). Murashige (1974) has developed the concept of three

developmental stages in micropropagation. They are explant establishment,

multiplication of propagules and rooting and hardening for planting in soil. It has been

further expanded and is now accepted that there are five stages critical for successful

micropropagation. They are preparative stage to minimize contamination, initiation of

culture, multiplication, elongation and root development and transfer to green house

conditions (Debergh and Read, 1991).

Through this technology (Musa sapientum cv.) from a single shoot tip or

axillary bud, a large quantity of uniform and disease free plants with good genetic

potential can be produced within a short time (Vuylsteke and Langhe, 1985 and Akbar

and Roy, 2006). There are many factors contributing to the development of a

successful micropropagation protocol for commercial multiplication. Various

parameters like growing mother plant, choice and nature of explant, phenolic exudates

14

and presence of endogeneous bacteria will influence the establishment of axenic

cultures (Debergh and Macne, 1981; Debergh and Read, 1991).

The survival, multiplication and field establishment of cultures depend upon a

variety of factors such as origin of cultures, physiological stages of explant,

endogenous hormone level and culture environment like nutrient medias, photoperiod,

CO2 concentration, temperature, etc. Each species is unique in these requirements

(Khanna, 1999). Clonal propagation of Curculigo orchioides, for commercial

purposes requires a simple, economical, reproducible and rapid multiplication

protocol which was standardized through in vitro technique using shoot tip and

rhizome disc explants (Nagesh, 2008). Direct plant regeneration from leaf explant of

Enicostemma axillare was obtained using different concentrations of BAP with stable

concentration of KN (Jeyachandran et al., 2005).

2.1.2. Indirect organogenesis

An efficient in vitro dedifferentiation and plant regeneration from callus

culture is needed for mass propagation and several in vitro manipulation studies

including genetic transformation, where direct regeneration may not be effective

(Faisal et al., 2006). The development of reliable tissue culture methods for the

production and maintenance of callus and regeneration of plants were imperative for

the successful application of tissue culture to crop improvement was proposed by

Muthuramu et al. (2007). An efficient in vitro plant regeneration system is a basic

necessity for the culture of callus tissue which provides an important technique which

can be preliminary to the regeneration of whole plant and established plantlets either

directly or via callus culture from leaf and node explants has been reported (Rathod et

al., 2008). In many plants there are reports of successful callus induction and plantlets

regeneration: Ocimum basilicum (Sudhakaran and Sivasankari, 2003). Curcuma

amada (Prakash et al., 2004), Cucumis sativus (Filifecki et al., 2005), Tylophora

indica (Faisal and Anis, 2005), Camellia sinensis (Aoshima, 2005), Pogostemon

cablin (Parida et al., 2005), Sorghum bicolor (Baskaran et al., 2006), Gymnema

sylvestre (Gopi and Vatsala, 2006), Prunus avium (Feeney et al., 2007), Echinacea

purpurea (Jones et al., 2007), Campanula carpatica (Sriskandarajah et al., 2008),

Sarcostemma brevistigma (Thomas and Shankar, 2009), Phaseolus valgaris (Arellano

et al., 2009) and Ocimum sanctum (Shilpa et al., 2010).

15

2.1.3. Somatic Embryogenesis

Somatic embryogenesis represents a simple and very efficient alternative

means of regenerating a large numbers of intact plant through tissue culture

(Zimmerman, 1993). Somatic embryogenesis is an ideal method of mass propagation

of plants possessing the same phenotypic and genotypic characters and for the

production of transgenic plants and mutagenesis (Ammirato, 1983). The development

of embryos from somatic cells of any explants through embryological status

stimulating zygotic embryos is called somatic embryos or embryoids and this process

is called Somatic Embryogenesis. The induction and regeneration of somatic embryos

are dependent on culture conditions; such as the composition of the medium, the

genotype and the explants source (Fuentes et al., 2000) and also the factors involved

in cultural conditions such as pH, sugar and nitrogen level can affect the generation of

embryogenic tissue (Tautorus et al., 1991).

The choice of the explants is a critical factor that determines the success of

most tissue culture experiments. Leaves, cotyledon, petiole and internode were proved

to be the most suitable explants for somatic embryogenesis initiation (Kumar et al.,

1994; Senaratna et al., 1995). Sharma and Millam (2004) defined the development of

a bipolar structure with both root and shoot poles from any sporophytic part of the

plant occurring through the same key stages of embryo development. Zygotic

embryogenesis (ie. globular, heart and torpedo stages) was yet another way of

exhibiting totipotency by plant cell, whereby they first dedifferentiate and then

redetermine towards the embryogenic pathway as has been reported in many

cases.The advantage of somatic embryogenesis was the simultaneous development of

root and shoot systems.

Somatic embryogenic cell can act independently from neighboring Cells and

undergo somatic embryogenesis, or they can continue to differentiate in to secondary

embryogenesis (Raemakers et al., 1995). Propagation through somatic embryogenesis,

however, may have advantages over organogenesis, particularly direct somatic

embryogenesis, because it can potentially scale–up propagation using bioreactors and

produce synthetic seeds (Rani and Raina, 2000).

16

2.2. REVIEW OF LITERATURE

2.2.1. Direct organogenesis

Micropropagation is the true to type propagation of selected genotype using in

vitro culture technique. This technique provides a rapid reliable system for a

production of large number of genetically uniform and disease free plantlets

(Alexanderova et al., 1996). Micropropagation of Agarita (Berberis trifoliata Moric)

was studied using actively growing apical and axillary shoot explants on MS medium

fortified with 11.1µM BA (Wayne, 1996).

Multiple shoots were produced from Capsicum annuum cultured on MS

medium with BAP or Kinetin or with IAA in different combinations. BAP proved to

be superior to kinetin in the production of multiple shoots on MS + BAP (5.0 mg/l)

(Mirza and Narkhede, 1996). Sardana et al. (1998) reported that simple, rapid,

efficient and reproducible protocol for direct regeneration was observed from shoot-

tips of Jatropha curcas on a combination of GA3 (3.0 mg/l) and IAA (3.0 mg/l) on

Murashig and Skoog’s medium. Plantlets were acclimatized and successfully

transferred to pots and finally to the field.

Peralek-Kozlina (1998) achieved the highest multiplication rate on ½ MS

medium supplemented with 0.5 µM BA. The influence of cytokinins, which

stimulates proliferation of axillary shoot from lateral buds and partial induction of

callus, was significant for Centaurea ragusina shoot growth and multiplication.

Multiple shoots were induced from shoot tip explants of in vitro grown seedlings of

Macrotyloma uniflorum on MS medium supplemented with Adenine sulphate (Ads)

and BAP individually and in different combinations and concentrations. Seven to

eight shoots were obtained on a medium supplemented with 2.0 mg/l Ads, 0.75 mg/l

BAP and 0.25 mg/l GA3. The plantlets were successfully transferred to the rooting

media containing 1.75 smg/l IBA (Mohamed et al., 1998).

A reliable procedure for multiple shoot induction and plantlet regeneration was

developed with apical buds collected from 7 to 8 year old trees of Ficus carica L.

using MS medium supplemented with 2.0 mg/l BAP and 0.2 mg/l NAA. The in vitro

regenerated shoots were further multiplied on MS medium supplemented with 2.0

mg/l BAP and 0.2 mg/l NAA and an average multiplication rate of four per subculture

17

was established with 90% success. Excised shoots were rooted in liquid in half

strength MS medium supplemented with 2.0 mg/l IBA and 0.2% activated charcoal

(Kumar et al., 1998).

Rao et al. (1998) have evolved an in vitro propagation protocol for Excoecaria

agallocha L. a mangrove species. Nodal segments were used for axillary shoot

proliferation and the optimum axillary sprouting was seen on a newly formulated

medium containing BA, Zeatin and IBA in concentration of 13.3 µM, 4.65 µM and

1.23 µM respectively. Rooting was achieved by growing shoot in the new medium of

0.23 µM IBA. Regenerated plants were successfully acclimatized to the natural

environment and about 85% plantlets survived under ex vitro condition.

The establishment of a micropropagation protocol for Pothomorphe umbellata

was carried out using leaf segments cultured on ½ strength MS medium supplemented

with 0.5 mg/l 6-benzyl adenine, 0.1 mg/l gibberlic acid added with 10g/l sucrose.

Rooting was achieved using MS medium devoid of growth regulators (Pereira et al.,

2000). Effective protocol for mass propagation of Bacopa monnieri an important

medicinal plant, was developed using shoot tips and nodal segments as explants by

Tejavathi et al. (2001). The explants were cultured on MS medium supplemented with

various auxins (IAA, IBA, NAA and 2, 4-D) and cytokinins (KN, BA and 2-ip).

Multiple shoots were obtained on MS medium supplemented with auxins and

cytokinins with or without coconut milk. Maximum number of plants were obtained

on MS medium containing KN/2-ip (0.1 mg/l) and kinetin (1 mg/l) in shoot tip and

nodal culture. Regenerated plantlets were transferred to soil after a very brief period of

hardening.

Shoot tip of Vigna mungo gave the best response with regard to offshoot

formation from the explants on MS medium supplemented with 0.5 mg/l BAP, 0.5

mg/l 2-ip and 0.1 mg/l NAA. The combination induced differentiation of an average

of 10 shoots in shoot tip explants. The isolated shoots rooted on 0.5 mg/l IAA resulted

in the formation of complete plantlets of an average height of 15cm in 20 days

(Agnihotri et al., 2001).

Thiruvengadam and Jayabalan (2001) achieved an in vitro plant regeneration

and flowering from Vitex negundo on MS medium containing auxin and cytokinins.

18

The highest percentage (95.3%) of flowering was noticed from nodal explants

cultured on MS medium with BAP (2.0 mg/l) and NAA (0.1 mg/l) combination.

Maximum number of flower buds was obtained from nodal explants. The regenerated

shoots were transferred on to MS medium fortified with BAP (0.1 mg/l) and NAA

(0.5 mg/l) for flower maturation and root induction.

A method for in vitro production of Jatropha curcas by in vitro production of

multiple shoot from nodal segments was developed by Rojore et al. (2002). The nodal

segments were cultured on MS basal medium supplemented with cytokinins such as

KN, BAP and auxin viz. IAA, IBA and NAA. Multiple shoots were obtained on MS

medium fortified with IAA at 2.0 mg/l and 1.5 mg/l IBA. However addition of various

additive, viz. ascorbic acid (10.0 mg/l), plus citric acid (50.0 mg/l), plus adinine

sulphate (25.0 mg/l), plus glutamic acid (100.0 mg/l) showed a synergistic effect in

shoot proliferation and its development. The best rooting was achieved on ½ strength

MS medium with NAA (5.0 mg/l). The plantlets were transferred for primary

hardening in a sterilized mixture of soil and vermiculate.

Catapan et al. (2002) reported maximum number of shoot multiplication on

Phyllanthus urinaria on MS media supplemented with 5.0 µM KIN. Das and Rout

(2002) achieved direct plant regeneration from leaf explants of Plumbago rosea and

P. zeyanica on MS medium supplemented with BAP (6.7 µM/l) IAA (1.4 µM/l), Ads

(370 µM/l) and 3% w/v sucrose. The semi-mature leaves in similar medium produced

more shoot buds compared to the younger leaves. More than 85% of the semi-mature

explants produced, shoot buds per leaf explants within 4 weeks of cultures. Shoots

rooted on half strength MS basal medium supplemented with IBA (1.2 µM/l) and 2%

sucrose. Approximately 90% of the in vitro raised plantlets survived in the green

house.

An efficient protocol has been achieved for in vitro production of Datura metel

from stem and leaf explants excised from in vitro source and cultured on MS medium

supplemented with NAA, IBA, 2,4-D, BAP and GA3 either singly or in combination.

It was established that continuous illumination of the cultures promoted better and

efficient growth than 12 hours photoperiod maintained cultures (Sood, 2002). Vasanth

et al. (2002) used in vitro raised shoot tip explants from little millet to produce

19

multiple shoots on MS medium containing BAP (0.3 mg/l) and KN (0.2 mg/l).

Multiple shoots were transferred to shoot elongation medium containing BAP (0.2

mg/l) and GA3 (0.3 mg/l) for further growth. The highest percentage of rooting was

observed with 0.3 mg/l of NAA and maximum root length was recorded with IBA at

0.3 mg/l.

Tiwari et al. (2002) developed improved micropropagation protocol for

Tectona grandis. Nodal explants placed on MS medium supplemented with 22.2µM

BAP and then serially transferred to fresh medium after 12, 24, 48 and 72 h gave

maximum culture establishment. Casado et al. (2002) reported the micropropagation

of Santolina canescens using shoot tip explants on MS medium with BA and KN. The

best axillary bud proliferation was recorded on MS medium containing 1.33µM BA

plus 0.32 µM NAA. More abundant and stronger roots occurred on media containing

2.68µM NAA.

Axillary buds of Ceropegia candelabrum L. (Asclepiadaceae) were

successfully micropropagated in MS medium supplemented with 8.87 µM BA and

2.46 µM IBA and with high proliferation frequency of multiple shooting (Beena et al.,

2003). The supplementation of the medium with various concentrations of KIN

resulted in mass proliferation from the meristem with a maximum proliferation at 0.25

µM/l in the mass multiplication of Echinops spinosissimus (Murch et al., 2003). The

resulting shoots were also found to have significantly more leaves per shoot than in

other treatments. Rooting was achieved by subculturing on to a medium supplemented

with 0.5 or 1.0 µM/l IBA. Santhi et al. (2003) used nodal segments for in vitro

micropropagation of a medicinally important plant species Enicostema littorale

through in vitro culture multiplication. Multiple shoots were induced from the nodal

explants cultured on MS medium fortified with BA and KN at various concentrations.

The multiple shoots were observed in BA 2.5 mg/l, rooted MS medium containing

NAA 2.0 mg/l, IAA 0.1 mg/l and IBA 0.1 mg/l. The well rooted plants were hardened.

The young stem nodal segment of Sophora flavescens were successfully

established in MS basal medium supplemented with 8.88µM BA and 2.69µM NAA

(Zhao et al., 2003). Efficient protocol for successful plant regeneration from the nodal

explants of Avicennia marina was reported by Babrany and Khayri (2003). Nodal

20

explants were cultured on MS medium supplemented with both 6-furfuryl

aminopurine (KIN) and 6-benzyl aminopurine (BA) at 0, 0.5, 1 or 2 mg/l combined

with 0, 0.01, 0.1 or 1 mg/l indole-3-butric acid (IBA). Single shoots grew from either

one or both of the pre-existing axillary buds in the nodal explant. Nodes cultured on 1

mg/l KIN combined with 0.5 mg/l BA with no IBA, gave the highest percentage of

shoot formation (75%). The addition of IBA was inhibitory to shoot formation,

particularly at concentrations above 0.01 mg/l. Maximum shoot elongation occurred in

response to a combination of 0.01 mg/l IBA, 2 mg/l KIN and 0.5 mg/l BA. Resultant

shoots were rooted on a medium containing either IBA, indole-3-acetic acid (IAA) or

α-Naphthalene acetic acid (NAA) at 0.5, 1.0, 2.0 and 4.0 mg/l.

Haw and Keng (2003) reported a successful protocol for micropropagation of

Spilanthes acmella L. a bio-insecticide plant. The aseptic axillary buds formed

multiple shoots within five weeks when cultured on MS medium supplemented with

2.0 mg/l BA. The addition of indole-3-butric acid (IBA) had no significnt effect on the

multiple shoots formation of this plant.

A reproducible protocol has been developed for Micropropagation of

Asparagus recemosus when shoot tips and nodal segments were cultured on MS

medium containing with different phytohormones (Vijay and Kumar, 2003). Large

number of multiple shoots were observed on various concentrations of KN and BAP.

Growth was observed when NAA at low concentration in the medium promoted the

callusing from the cut ends.

In vitro mass multiplication of plants through seedling culture was established

in Ophiorrhiza mungo by Jose and Satheeshkumar (2004). Maximum number of

adventitious shoots per shoot culture was initiated on MS solid medium supplemented

with BAP (2.23µM) after three weeks. Shoots were further multiplied through

subculture of intact shoots and reculture of nodal segment of aseptic shoots in MS

solid medium containing BAP 0.89µM. Shoot elongation was achieved in the medium

containing GA3 (1.44µM) in two weeks. Rooting was favoured in basal agar medium

supplemented with IBA (12.3µM) plus NAA (1.07µM). The plants were successfully

established (100%) in the pots containing sand.

21

Micropropagation of Paederia foetida L. has been achieved through the

culture of nodal explants. The explants produced shoots on MS medium with 0.8%

agar. Maximum shoot proliferation free from callus and vitrification but with poor

rooting could be obtained in liquid MS medium with PVP (0.8%), NAA (0.5 mg/l)

and BA (2.0 mg/l). The best rooting occurred on semisolid MS medium containing

0.8% agar and 0.5 mg/l IBA (Srivastava and Srivastava, 2004).

Jayachandran et al. (2004) have standardized an efficient and reproducible

protocol for the micropropagation of the recalcitrant crop Cajanus cajan L. (VBNI).

The cytokinins (BAP, KN) increased the organogenic potential of hypocotyl, epicotyl,

cotyledon and leaf explants. Maximum elongation of shoots from all the explants

occurred on MS medium containing GA3 (0.5 mg/l) and BAP (1 mg/l). IBA (0.5 mg/l)

was effective for root induction.

Jain and Chaturvedi (2004) induced prolific differentiation of shoot buds in the

intervenal leaf lamina explants excised from field grown plants of Cajanus cajan var.

bahar. All explants were cultured on filter paper bridge in a nutrient liquid medium

supplemented with 0.25 mg/l each of BAP and KN along with 0.05 mg/l IAA and

40mg/l Ads, and they produced an average 50 shoot buds within 45 days. Well

developed shoots had rooted for 100% in 0.25 mg/l in any of the three auxins used viz.

IAA, IBA and NAA.

A protocol was standardized for rapid and large-scale in vitro propagation of

Anethum graveolens by enhanced axillary shoot induction that was dependent on BAP

supply. The synergistic combination of 0.5 mg/l BAP and 0.1 mg/l IBA induced 100%

shoot formation as well as shoot number (6.6 ± 0.48 per explant). Sub-culturing of

shoot tips of in vitro plants on multiplication medium enabled continuous production

of healthy shoots with similar frequency. Rooting of shoots was achieved on a

medium with 1 mg/l IBA and 0.5 mg/l KN. (Sharma et al., 2004a).

Micropropagation of Aloe barbadensis was obtained from shoot tip explants

on MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l) (Baksha et

al., 2005). The best and rapid regeneration was observed on MS medium augmented

with 2 mg/l BAP + 0.5 mg/l NAA. This treatment yielded the highest number (75) of

regenerated shoots with ten shoots per culture. The shoot tip explants initially

22

produced three-five shoots within three-four weeks after inoculation. Sub-culture in

the same medium yielded a cluster of eight-ten shoots per explant. About 95% rooting

was obtained from micro shoots cultured on half strength MS supplemented with

NAA (0.5 mg/l). Well developed rooted plantlets were successfully transferred to the

soil with 70% survival.

Nodal segments of Clitoria ternatea were successfully multiplied (85.6%) in

semisolid MS medium supplemented with 8.9µM BA and 1.34µM NAA (Rout, 2005).

Higher percentage of multiple shoots were obtained from the nodal segments of

Bacopa monnieri in the MS medium supplemented with 2.0 mg/l BAP were reported

by Mohapatra and Rath (2005). In vitro regeneration and multiple shoot induction

from cotyledonary nodes in Gossypium hirsutum L. was evolved by Saeed Rauf et al.

(2005). Maximum number of shoots (3.43 shoot/explants) when cultured on MS

supplemented with 0.25 mg/l KN. Highest percentage of root development and root

length was obtained when shoots were cultured on MS supplemented with 0.5 mg/l

NAA and 0.1 mg/l KN.

Gayatri et al. (2006) reported that the leaf segments of Eryngium foetidum

produced high frequency of multiple shoots in MS medium fortified with BAP (1.5

mg/l) and polyvinyl pyrropidone (250 mg/l). High frequency direct organogenesis of

shoots was achieved on Solanum melongena L. from cotyledonary leaf in MS medium

supplemented with 1.0 mg/l BAP and 1.0 mg/l KIN as was reported by Sarker et al.

(2006). MS medium supplemented with NAA (1µM) and BA (2µM) enabled an

efficient proliferation system (Filho et al., 2005).

Huda and Sikhar (2006) observed growth of meristem on semisolid MS

medium supplemented with 0.05 mg/l KN and 0.1 mg/l GA3. Shoot initiation with

elongation was obtained in MS medium supplemented with 1.0 mg/l BA + 0.1 mg/l

IBA + 0.3 mg/l GA3. Rooting was observed in MS medium fortified with 0.5 mg/l BA

and 0.1 mg/l NAA. In vitro plantlets were successfully planted in soil through gradual

acclimatization.

In vitro micropropagation protocol for Mecardonia tenella was achieved by

Lilliana et al. (2006). The nodal segment of M. tenella were cultured on a medium

supplemented with BAP. The best results were obtained in the treatment containing

23

0.25 and 0.5 mg/l BAP with a multiplication rate of 32 shoots per explants.

Girija et al. (2006) reported direct multiple shoot regeneration from shoot tip and

nodal explants of Ocimum sanctum L. a medicinal herb. Maximum number of

multiple shoots was obtained on MS medium supplemented with BAP (1.0 mg/l) and

KIN (2.0 mg/l). Regenerated shoots were rooted on MS liquid medium containing

NAA (1.0 mg/l).

A rapid and highly effective method for micropropagation from nodal

segments and shoot-tip explants were established for Coleus blumei Benth. Nodal

segments and shoot tips were inoculated on MS medium containing different

combinations of BA with IAA, IBA or NAA. High frequency shoot induction from

both explants was achieved on the medium containing BA (2 mg/l) and NAA (1 mg/l).

Multiple shoots rooted best on MS medium supplemented with IBA (2 mg/l)

(Rani et al., 2006).

In vitro micropropagation of endangered ornamental plant Neotchihatchewia

isatidea was achived by Ahmet Gumuscu et al. (2008). Immature embryos of

N. isatidea were cultured for initiation on MS medium supplemented with

N6-benzylamino-purine (BAP) and a-naphthaleneacetic acid (NAA). Shoot primordia

were visible within 5 - 6 weeks and the shoot primordia later developed into normal

shoots 10 - 12 weeks after the culture initiation on calli developed from immature

embryos. Shoot tips were also excised from developed plantlets for direct shoot

organogenesis and cultured on MS shoot induction medium supplemented with BAP

(0.5, 1.0 and 2.0 mg/l), kinetin (KIN) (0.5, 1.0 and 2.0 mg/l) and thidiazuron (TDZ)

(0.05, 0.10 and 0.50 mg/l). Direct multiple shoots from shoot tips developed in most

media tested. High shoot multiplication (3.73), high rooting (53 %) number of root per

shoot (3.66) and survival ratio (46.6 %) were achieved.

A rapid and efficient plant regeneration protocol for a wide range of alfalfa

genotype via direct organogenesis was developed by Li et al. (2009). Through a

successive excision of the newly developed apical and axillary shoots, a lot of

adventitious buds were directly induced from the cotyledonary nodes when hypocotyls

of explants were vertically inserted into a modified MS medium supplemented with

0.025mg dm-3

thidiazuron and 3mg dm-3

AgNo3 when the lower part of shoots excised

24

from explants were immersed into the liquid medium with 1.0mg dm-3

α-naphthalene

acetic acid for 2 min. and then transferred to hormone free half strength MS medium.

Over 83.3% of the shoots developed roots, and all plantlets could acclimatized and

established in soil.

An efficient system was developed for direct plant regeneration from in vitro

derived leaf explants of Pistacia vera (Tilkat and Onay, 2009). The best medium for

shoot induction was a MS medium with 1 mg/l IAA and 2 mg/l BA. Numerous shoot

primordia developed within 2-3 weeks on the leaf margin and the midrib region,

without any callus phase. The shoot clumps were separated from the leaf explants and

transferred to a MS medium supplemented with 1 mg/l BA, resulting in a

differentiation of the shoot initials into well developed shoots. The elongated shoots

were rooted on a full strength MS basal medium supplemented with 2 mg/l of indole-

3-butyric acid. The rooted plants were transferred to soil with an 80% success rate.

Induction of high-frequency shoot regeneration using nodal segment

containing axillary buds from a 1 year old mother plants of Cannabis sativa was

achieved on MS medium containing 0.5µM thidiazuron which slightly increased shoot

growth. Elongated shoots when transferred to half strength MS medium supplemented

with 500 mg/l activated charcoal and 2.5 µM indole-3-butyric acid resulted in 95%

rooting. The rooted plants were successfully acclimatized (Hemant Lata et al., 2009).

Shameer et al. (2009) conducted in vitro studies employing explants from

node, internode, petiole, shoot bud and leaf lamina of Beleperone plumbaginifolia.

MS medium fortified with 6.6µM BA enabled the proliferation of axillary apical buds.

MS medium with 5.37µM NAA and 2.22µM BA was found superior for shoot

induction from nodal explants. Half-strength MS medium with 5.37µM NAA induced

adventitious roots and 85% plantlets survived when transferred in the field conditions.

Kone Mongomake et al. (2009) studied in vitro regeneration system via direct

organogenesis in Bambara groundnut using hypocotyls and epicotyle cuttings. Basal

MS medium supplemented with BAP (2 mg/l) gave the highest response (73.33-

97.77%) with the regeneration of 3.7 shoots per explants with hypocotyl and 5.8

shoots per explants with epicotyl. The regenerated shoots were readily elongated on

the same medium as used for induction and rooted on half-strength MS basal medium

25

without any growth regulators. 62% of the plantlets were successfully acclimatized

and potted plants were established in soil with 73% of survival rate.

An efficient protocol for in vitro propagation of Digitalis trojana Ivan was

developed via adventitious shoot regeneration. Leaf explants were cultured on MS

which were supplemented with different concentrations of NAA and BAP. The best

shoot proliferation was observed among explants cultured on MS medium with

0.1 mg/l NAA + 3.0 mg/ml BAP. Regenerated shoots were multiplicated by

subculture. Then they were cultured on MS with 0.1% (w/v) activated charcoal for

root formation. All of the in vitro regenerated plantlets were successfully acclimatized

ex vitro and then grown healthy (Nursen and Cuneyt, 2010).

The use of apical meristem culture for simultaneous virus elimination and

shoot proliferation in sugarcane was assesed by van Ramgareeb et al. (2010). Shoot

induction and proliferation via direct organogenesis were achieved on Murashige and

Skoog nutrient medium supplemented with 0.1 mg/l BA and 0.015 mg/l KIN.

Approximately 1,300 shoots were propagated from a single 2 mm meristem in 11

weeks.

2.2.2. Indirect organogenesis

In vitro studies of Solanum trilobatum L. were conducted with explants of leaf

and stem on MS medium containing IAA and BAP/kinetin combinations (Arulmozhi

and Ramanujan, 1997). Shoot bud formation was observed from the leaf callus on MS

medium supplemented with two types of cytokinins viz. BAP and kinetin. Of the two

cytokinins, BAP was found beneficial to regenerating about 70 shoots/culture at 7.5

mg/l level.

Sreelekha and Ramanujan (1997) showed callus formation from foliar explants

of Solanum nigrum L. on MS medium with different levels of IAA and cytokinins.

Arumugham and Rao (1998) observed callus initiation and proliferation from

immature leaflet explants of Aegle marmelos when treated in MS medium with B5

vitamins and different concentrations of 2,4-D(0.5 mg/l) NAA (0.1 mg/l) KN

(0.1 mg/l) and IAA (0.5 mg/l). Plantlets regenerated at 0.1 mg/l NAA + 0.01 mg/l

BAP + 0.1 mg/l kinetin.

26

Callus formation from leaf explants of Enicostema axillare on MS medium

with 8.9µM BA and 0.54µM NAA produced light green callus on the cut surfaces

along the midrib region and at the petiole base (Sudhersan, 1998). Green compact and

hard calli from leaf explants of Datura metel L. was introduced on MS medium with

various concentration of BAP. Maximum callus induction and plant regeneration was

observed at 2.0 mg/l BAP (Muthukumar and Arokiasamy, 1998).

Natarajan et al. (1999) demonstrated regeneration of plantlets from the callus

of stem explants of Hybanthus enneaspermus. Higher frequency of callus proliferation

was obtained from medium containing 2.0 mg/l (2, 4 D) and 0.5 mg/l (BAP). Green

compact calli were obtained after 15 days of explant culture. The optimum callus

growth (96.3%) was obtained from medium containing 2, 4-D (2.0 mg/l) and BAP

(0.5 mg/l). Optimal plantlet regeneration was initiated after two weeks. Maximum

number of plantlets were obtained in medium containing 5.0 mg/l BAP. The

regenerated shoots were rooted on MS medium supplemented with 2.0 mg/l IBA. The

rooted plants were transferred to the field after successful hardening in pots containing

vermiculite.

Green nodular structures developed into dark green shoots over the entire

surface of leaf explants of Plumbago zeylanica when calluses were cultured in the

medium supplemented with BA and IAA. Optimum shoot bud regeneration was in

medium having 4.44µM BA, 1.42µM IAA and 3% sucrose. They found the

percentage of shoot bud regeneration to be 82.3% and the frequency of regenerated

shoots per culture varied from 15.2-52.8% (Rout et al., 1999).

Baburaj et al. (2000) reported the regeneration of about 50 shoot buds from

leaf callus of Clerodendrum inerme on MS medium containing NAA (0.5 mg/l) and

BA (20 mg/l). High frequency shoot organogenesis and plant establishment were

achieved in Coleus forskohlii from leaf derived callus (Sairam Reddy et al., 2001).

Optimal callus was developed from mature leaves on MS medium supplemented with

2.4µM kinetin alone. Shoots were regenerated from the callus on MS medium

supplemented with 4.6µM kinetin and 0.54µM NAA. Regenerated shootlets were

rooted spontaneously on half-strength MS medium devoid of growth regulators. The

in vitro raised plants were established successfully in soil.

27

Muthu Thiruvengadam and Jayabalan (2001) achieved plant regeneration from

internode derived callus cultures of Vitex negundo with explants cultured on MS

medium containing with NAA and BAP combinations. The highest percentage of

callus induction and proliferation was observed on MS + NAA (1.5 mg/l) and BAP

(0.5 mg/l). Rooting of regenerated shoots occurred when transferred to MS + IBA

(1.0 mg/l).

Organogenic callus produced adventitious buds in MS medium augmented

with 13.32µM/l BAP with 2.69µM/l of NAA from the leaf callus of Anisomeles indica

L. obtained by the addition of 9.04µM/l 2,4-D (John Britto et al., 2002). Efficient

plant regeneration was achieved by Koroch et al. (2002) via organogenesis from leaf

callus culture of Echinacea purpurea cultured on MS medium supplemented with

BAP (4.44µM) and NAA (0.054µM). This was most effective providing the highest

shoot regeneration frequencies (100%) associated with a high number of shoots per

explant. Plantlets were rooted on MS + different combinations of IBA.

Induction of callus was achieved from immature leaves of Agave sisalana on

medium supplemented with 9.0µM 2, 4-D and 4.6µM KIN. The calli were regenerated

on MS medium containing BA (8.9-44.0µM) alone, the micro shoots were

subsequently transferred into the medium fortified with BA (2.22µM) and GA3

(1.49µM) for further growth and finally the rooted plants were acclimatized in to soil

(Hazra et al., 2002). Sharma and Wakblu (2003) induced callus from the petiole

explants of Heracleum candicans on MS medium fortified with BAP and 2, 4-D (0.5

mg/l each). Maximum shoot differentiation from callus occurred on MS medium

containing 1 mg/l BAP and 0.2 mg/l NAA. The regenerated shoots were rooted on MS

medium supplemented with1 mg/l IBA and hardened successfully.

Regeneration of multiple shoots via callus induction and organogenesis was

achieved from cotyledon explants of Chick pea (Cicer arietinum L.). Callus induction

and shoot regeneration at various frequencies were observed using different

concentrations and combinations of growth on MS+3.0 mg/l 2, 4-D+3.0 mg/l BAP.

The maximum percentage (40%) of shoot bud formation was obtained on MS medium

fortified with 2.0 mg/l BAP and 0.5 mg/l NAA. The regenerated shoots developed

highest percentage (77%) roots on ½ MS basal medium containing 1.0 mg/l IBA.

28

Regenerated plants were successfully established in soil after acclimatization

(Huda et al., 2003).

Callus induction and plantlet regeneration from petiole explants in ornamental

Alocasia micholitziania were reported by Nguyen et al. (2003). The highest

percentage (71%) of explants inducing callus was obtained on MS medium

supplemented with 0.5µM 2, 4-D and 0.5 µM kinetin in the dark after four months of

culture. Shoots were regenerated under light conditions when 0.5µM BA was added to

MS medium. The callus derived shoots rooted on hormone free MS medium and

within 4 weeks the plantlets were ready for acclimatization.

A high frequency and rapid regenerated protocol via callus and directly from

various explants was developed in Withania somnifera. Callus was initiated from

internodal segment, leaf, root and petiole explants on MS and B5 media supplemented

with 2, 4-D (0.5–3.0 mg/l) and NAA (0.5–3.0 mg/l) either alone or along with KIN

(0.5 -1.0 mg/l). Regeneration was observed from callus of all the explants except roots

on MS medium fortified with BAP (0.5–1.0 mg/l) or in combination with IAA (0.5

mg/l) (Govindraju et al., 2003).

Leaf explants of Andrographis alfalfa proliferated into luxuriant callus mass

on MS medium fortified with 7µM NAA. Interaction of the same hormones at the

concentrations of 1 to 4µM BAP and 0.5 to 1 µM NAA induced differentiation of

shoot buds from the calli. Caulogenesis was optimized (15.05 shoots) at the

concentration of 2µM BAP and 0.5µM NAA was reported by Nagaraja et al. (2003).

Indirect regeneration of a medicinally valuable Curcuma amada Roxb. using

leaf-sheath explants was reported by sheath explants was reported by Prakash et al.

(2004). The semi friable callus obtained from leaf-sheath explants on MS medium

with 9.0 µM of NAA and with 9.0µM 2, 4–D were transferred to 8.88 µM of BA and

2.7µM of NAA containing medium and they produced optimum shoot initiation and

development. The regenerated plantlets were transferred to the field.

Regeneration and multiple shoots via callus induction and organogenesis were

achieved in Elaeocarpus robustus (Rahman et al., 2004). Callus induction and shoot

buds regeneration were obtained from internode explants. The best organogenic callus

was found on modified MS medium supplemented with 0.5 mg/l BA + 0.5 mg/l 2, 4-

29

D. Development of adventitious shoots occurred when the calli were subcultured on

modified MS medium supplemented with BA and NAA. Maximum frequency (80%)

of calli induced adventitious shoots with the highest number of 14.05 ± 1.56 shoots

per callus were obtained when the medium was fortified with 1.0 mg/l BA + 0.1 mg/l

NAA. Pantlets developed roots when in vitro developed micro cutting were implanted

on modified MS medium with 0.2 mg/l of IBA.

A high frequency shoot organogenesis and plant establishment from stem

derived callus of Tylophora indica (Burm.f.) Merr. was achieved by Faisal and Anis

(2005). Callus was developed on medium supplemented with 10µM and 2, 4, 5–

trichloropheroxy acetic acid. The highest rate (80%) of shoot multiplication was

achieved on MS medium containing 5.0µM KN. The developed shoots, rooted best on

half strength MS medium supplemented with 0.5µM IBA. The in vitro raised plantlets

with well developed shoots and roots were acclimatized successfully and grown in

green house.

Dhar and Joshi (2005) have reported the callogenic property of explants

namely hypocotyle, root, cotyledon and leaf from Saussurea obvallata. Leaves were

the best source of explants both for callogenesis and shoot regeneration. Callus

induction and organogenesis from seedling explants of Pseudarthria viscida were

reported by Deepa et al. (2006). Cotyledon and shoot tip explants induced profuse

callusing on MS Medium supplemented with 2, 4-D (1.5-2 mg/l) and BAP (1-1.5

mg/l). Shoot regeneration was achieved on both half and full strength MS medium

supplemented with BAP (2 mg/l) and shoot elongation, when GA3 (1 mg/l) was

added. Roots were induced when shoots were transferred to half strength MS medium

fortified with IBA (1 mg/l).

An efficient and reproducible plant regeneration system was developed from

cells or tissues of agronomically important Indian species Sorghum bicolor (Baskaran

et al., 2006) by using transverse thin cell layers. The calli were cultured on MS

medium supplemented with 2.2-17.8µM/l BAP, in combination with 2.3µM 2, 4-D or

2.7µM NAA. Highly efficient differentiations of multiple shoot buds were initiated

within 4 weeks of culture. Root induction was achieved on half strength MS medium

containing IAA (28.5-29µM). Rooted plants were successfully acclimatized.

30

The calli from rhizome segments of Ruscus aculeatus induced shootlets on MS

medium supplemented with 0.5mg dm-3

2, 4-dichlorophenoxy acetic acid and 1 mg

dm-3

KN (Moyano et al., 2006). Chitra and Komalam (2007) achieved in vitro

propagation of endangered Nilgirianthus ciliatus. Leaf, stem–nodal and inter nodal

segments were used as explants. Leaf explants were found to be the best suited for

callus induction and subsequent organogenesis on MS medium with 0.5 mg/l BA.

Maximum shoot regeneration from inter nodal callus was obtained on medium with

0.5 mg/l IAA and 5 mg/l BA. The in vitro shoots successfully rooted in MS medium

supplemented with various concentrations of IAA (0.5–5 mg/l) and IBA (0.5–5 mg/l)

separately. 75% of rooted plantlets survived in the mist chamber after hardening.

Debnath et al. (2007) reported clonal propagation of Chlorophytum

bosvellianum an endangered medicinal plant. Callus induction was observed when

short segments of inflorescence axis bearing flower bud were innoculated on MS

medium supplemented with concentrations and combinations of 2, 4-D and BA.

Maximum callusing (100%) was obtained on MS medium supplemented with a

combination of 2 mg/l in 2, 4-D and 0.2 mg/l BA. The calli, when subcultured on MS

medium supplemented with 4 mg/l BA showed multiple shoot proliferation. These

shoots were rooted best (87%) on MS medium containing 2 mg/l IBA.

An efficient method for totipotent callus formation and whole plant

regeneration has been developed for chicory (Cychorium intybus L.) Velayuthan et al.

(2007). Totipotent calli of chicory were induced from cotyledon, leaf, hypocotyls and

root explants on MS medium supplemented with different concentrations of IAA,

IBA, NAA and 2, 4-D at 0.5–AµM in combination with BAP (2µM). These calli were

transferred to shoot regeneration medium containing MS basal medium with different

concentrations of combinations of BAP, KIN and IAA. Maximum number of shoots

was obtained on MS medium with BAP (4µM) + IAA (1µM). The shoots were rooted

on MS medium supplemented with IAA, IBA and NAA .

Saima Malik et al. (2007) conducted a study to analyze the effect of plant

growth regulators on callogenesis and direct and indirect organogenesis of Momordica

charantia. Callus cultures were induced from leaf, stem and cotyledonary explants of

M. charantia, at different auxin and cytokinin concentrations either in single or in

31

combination in MS medium. The best callogenic response was observed from all three

explants (leaf, stem and cotyledon) on MS medium supplemented with 1.0 and 1.5 mg

LG 1 BAP with 1.5 mg LG 1 NAA and 1.0 mg LG 1 2,4-D, respectively. The callus

produced was hard, green and compact. Best shooting was observed at 1.0 mg LG 1

BAP + 0.1 TDZ and 1.5 mg LG 1 BAP + 0.2 mg LG 1 NAA from shoot tip and

cotyledonary node explants. Root formation was achieved when generated shoots

were transferred to MS medium both full and half strength supplemented with

different auxin concentrations.

A reliable and reproducible regeneration system was established in maize

genotype using shoot apices as explants. The most effective combination for calli

induction was the modified MS media containing 26.64 µM BAP and 296 µM

adenine. Calli were maintained on MS media with 9 µM 2, 4-D and 4.44 µM BAP for

calli proliferation. Shoots were regenerated from organogenic calli after 4-6 weeks

depending on the genotype and the presence or absence of adenine, with plant

regeneration varying between 29-55%. Root induction was promoted using MS media

supplemented with 1.97 and 2.95 µM Indole-3-butyric acid (Muoma et al., 2008).

Anisuzzaman et al. (2008) developed a viable protocol for indirect shoot

organogenesis of okra. Morphogenic callus induction was observed in the highest

frequency from hypocotyl explants by culturing in MS medium supplemented with 2.0

mg L-1 NAA plus 0.5 mg L-1 TDZ. The highest percentage of shoot regeneration and

highest mean number of shoot per callus mass was obtained with 2.0 mg L-1 BAP

plus 0.1 mg L-1 IBA. Root formation was observed from callus medium containing

1.5 mg L-1 NAA. About 80% of regenerated plantlets survived and showed new leaf

development under ex vitro condition.

Baskaran et al. (2009) developed an effective protocol for in vitro regeneration

of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture

systems. Organogenesis was achieved from liquid culture calluses derived from leaf

and petiole explants of mature plants. Organogenic calluses were induced from both

leaf and petiole explants on MS liquid medium containing 6.0 µM 2, 4-dichloro-

phenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0

µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration

32

was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v

coconut water and 0.06 mM glutamine from leaf-derived calluses. Elongation of

shoots occurred in MS medium with 2.0 µM gibberellic acid (GA3). Regenerated

shoots rooted and hardened when they were transferred to 1/2-MS medium

supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil,

vermiculate and sand (2:1:1) mixture. A system of indirect organogenesis for the

multiple buds production from internode stem sections in Diacol Capiro variety of

potato was established (Yaya-lancheros, 2009). Explants on MS medium with zeatine

riboside (ZR) 2 mg/l, naphtalenacetic acid (NAA) 0.02 mg/l and giberelic acid (GA3)

0.02 mg/l, produced plants ranging between 7 to 9 weeks with 80-100% effectiveness.

A simple protocol was established for indirect shoot organogenesis and plant

regeneration of Cancer Bush (Sutherlandia frutescens L.) using rachis and stem

segments. Different concentrations (0.0-68.08 µmol l-1

) of thidiazuron (TDZ) were

used for callus induction and shoot organogenesis. The highest percentage of callus

formation (97.5%) and the highest percentage of explants forming shoots (88.8%)

were obtained from rachis explants cultured onto Murashige and Skoog (MS) medium

supplemented with 45.41 µmol l-1

TDZ. Shoot clusters were further developed and

grown in MS hormone-free medium. Individual shoots were rooted on different

concentrations and combinations of MS salt strength and IBA. Half-strength MS salt

medium supplemented with 24.6 µmol l-1

IBA was optimal for root induction in which

78% of shoots were rooted. The in vitro plants were successfully acclimatized in a

growth chamber with a 90% survival rate (Yaser Hassan Dewir et al., 2010).

2.2.3. Direct somatic embryogenesis

Pillon et al. (1996) have described direct development of somatic embryos in

cultured zygotic embryos in Arabidopsis. Hypocotyl segments of Eleutherococcus

senticosus cultured on MS medium with 1.0 mg/l 2, 4-D produced somatic embryos

directly from the surface of explants without intervening callus formation (Cho et al.,

1999). Direct somatic embryogenesis was indused from leaf of a Chicorium hybrid

(Chicorium intybus L. var. sativum × Chicorium endive L. var. latifolia) (Bellettre et

al., 1999). Segments from young leaves of an Orchid (Oncidium gower Ramasey)

produced clusters of somatic embryos directly from epidermal and mesophill cells of

leaf tips and wound surfaces without an intervening callus when cultured on a gelrite

33

gelled half MS medium supplemented with a low dosage (0.3-1 mg/l) thidiazuron

(Chen et al., 1999).

Hamama et al. (2001) developed a protocoal for the induction, maturation and

germination of somatic embyos from leaf tissues of Jojoba. Explants were placed on

their adaxial sides in petri dishes and maintained in darkness on half strength MS

medium. Combinations of 2, 4-D (1.35-4.52 µM) with BAP (1.33-4.43 µM) and 2

synthetic cytokinins N-(2-cholro 4 pyride)-N’-phenylurea (1.21-4.03 µM)or (E) -6-

[3-(trifluromethyle ) - but – 2 enylamino] purine (1.11-3.71 µM resulted information

of embryogenic cultures and somatic embryos. Afer two 30 day subcultures,

embryogenic cultures were transferred on to MS/2 medium supplemented with

different auxins and cytokinins. Somatic maturation, germination and plantlet

formation were achieved using NAA (3.75) or IBA (3.44 µM) or in combination with

BA (0.44 or 1.33 µM) or F3ip (0.37 or 1.11 µM).

The leaf explants of Ostericum koreanum supplemented with 5.37µM NAA

and 0.44µM BA in MS medium induced somatic embryos and embryos were matured

up to the third subculture. These mature embryos were germinated in hormone free

medium (Cho et al., 2003). Ipekei and Gozukirmizi (2003) developed a reproducible

system for efficient direct somatic embryogenesis from leaf and internodal explants of

Paulownia elongata. The highest induction frequencies of somatic embryos were

obtained on MS medium supplemented with 3% sucrose, 0.6% phytagel, 500 mg/1+

casein hydrolysate and 10 mg/I TD2. Somatic embryos were induced from leaf

(69.8%) and internode (58.5%) explants after 7 days. Subsequent withdrawal of TD2

from the induction medium resulted in the maturation and growth of the embryos into

plantlets on MS basal media.

Zhang et al. (2005) reported that somatic embryos were directly formed at cut

edges or on the surface of leaf explants, around cut edges or along side surface of

petiole and stem explants of “Golden pathos”. Somatic embryos were produced on

MS medium containing 2.0 mg/l KN and 0.5 mg/l 2,4-D from leaf and petiole

explants. MS medium supplemented with 2.0 mg/l CPPU and 0.5 mg/l 2,4-D from

petiole and stem explants, and 2.0 mg/l TD2 and 0.2 mg/l or 0.5 mg/l from explants.

Somatic embryos matured and grew into multiple buds, shoots or even plantlets after

34

2-3 months on the initial culture medium. Germination was optional on MS medium

containing either 2 mg/l BA and 0.2 mg/l NAA or 2 mg/l zeatin and 0.2 mg/l NAA.

Shoot elongated better and roots developed well on MS medium with no growth

regulators. The regenerated plants grew vigourously after transplanting to a soil –less

container substrate in a shaded green house.

Direct somatic embryogenesis and plant regeneration from ray florets of

Chrysanthemum were reported by Mandal Datta (2005). Somatic embryos developed

asynchronously on the adaxial surface of ray floret cultured on MS medium

supplemented with 2, 4-D and BA. Precious germination of somatic embryos was

noticed on the same medium. Germinated somatic embryos with shoots were

transferred to growth regulator free MS medium where roots were formed.

Acclimatized plants were transferred to soil and produced true –type flowers.

A simple and effective method of regenerating Syngonium podophyllum

‘variegatum’ via direct somatic embryogenesis has been established on MS medium

supplemented with N- (2 chloro – 4- pyridyal) -N – phenyel urea (CPPU) or N phenyl

– N’- 1,2,3, thiadiazol -5- ylurea (TDZ) with either NAA or 2, 4-D. The frequency of

petiole explants with somatic embryos was produced as high as 8.6% when cultured

on medium containing 2.5 mg/l TDZ with 0.5 mg/l NAA. Upto 85% of somatic

embryos were able to germinate after transferring on to medium containing 2.0 mg/l

BA and 0.2 mg/l NAA (Zhang et al., 2006).

Rhimi et al. (2006) observed highest frequency of embryogenesis from

cotyledon and zygotic embryos within 3 weeks of subculture in two varieties of

Cucumis melo. The somatic embryogenesis of Pinus pinaster from mature zygotic

embryos was the highest on basal medium with 13.6 µM 2, 4-D and 4.4 µM 6-

benzylamino purine, supplemented with L-glutamine and Casein hydrolysate (Walter

et al., 2006).

You et al. (2007) established the protocol for plant regeneration of Panax

japonicas via Direct somatic embryogenesis. Somatic embryos were directly obtained

from the segments of zygotic embryos on MS medium with 4.4 µM 2, 4-D. Thereafter

somatic embryos were produced by repetitive secondary somatic embryogenesis. A

coffee (Coffea arabica L. cvs. Caturra and Catuai) plant regeneration protocol via

35

direct somatic embryogenesis was established by Gatica-Arias et al. (2008). Somatic

embryogenesis and subsequent plant regeneration were established from hypocotyls

and internode explants collected from in vitro grown seedling and in vitro -

proliferated shoots of Phellodendron amurense Rupr. (Azad et al., 2009).

Shweta and Sulekha (2009) designed an experiment to test mature zygotic

embryo axis derived plumule with three meristems for somatic embryogenesis in

peanut. They induced somatic embryos from the determined organogenic buds of the

axillary meristem, by culturing the nodal explant vertically on embryo induction

medium. Sridevi and Lundquist (2009) reported induction of direct somatic

embryogenesis in juvenile and adult tissues of Seabuckthorn (Hippophae ramnoides

L.) in MS-basal medium supplemented with 2.0 µM N (2-chloro-4 pyridyl-) N’-

phenyleurea (CPPU), 0.5 µM NAA and varying concentration of BA. The most

effective medium of inducing somatic embryogenesis in Juvenile explants containing

half strength MS salts and 2.2 µM BA and full strength MS salts and 13.2 µM BA for

adult explants.

Somatic embryogenesis was achieved for Chamelaucin uncinatum and C.

repens by Ratanasanobon and Seaton (2010). Somatic embryos from young leaves of

C. uncinatum and C. repens were induced in vitro on MS agar medium containing 20

g/l sucrose and 2,4-D. Somatic embryo cultures maintained on MS medium

supplemented with 0.1 µM 2,4-D were induced to develop into plantlets after transfer

to a hormone – free medium under light.

A protocol for plant regeneration via somatic embryogenesis was developed in

two Chick-pea (Cicer arietinum L.) cultivars 1 CCV-10 and Annigeri (Kiran et al.,

2010). Somatic embryos were induced from immature cotyledons on MS medium

supplemented with different concentrations of 2, 4-dichloro phenoxyacetic acid (2,4,5-

T), α-naphthaleneacetic acid (NAA) and picloram alone or in combination with 0.5 –

2.0 mg dm-3

N6 – benzyl aminopurine (BA) or kinetin (Kin). The well formed,

cotyledonary shaped embryos germinated into plantlets with 36.6% frequency on MS

medium supplemented with 2.0 mg dm-3

BA +0.5 mg dm-3

ABA. Regenerated plants

were transferred to soil and grown to maturity.

36

2.2.4. Indirect Somatic Embryogenesis

Somatic embryogenesis and whole plant regeneration were achieved in callus

cultures derived from hypocotyle, cotyledon and leaf explants excised from seedling

of Gymnema sylvestre (Ashok kumar et al., 2002). Embryogenic callus was induced

on MS medium containing 2, 4-D (0.5-5.0 µM) BA (0.5-2.0 µM) and 2% (w/v)

sucrose in 6-8 weeks of culture. Globular / heart stage embryos developed in induction

medium. These embryos produced torpedo and cotyledon stage embryos upon

subculturing on embryo maturation medium EM8 (medium containing MS salt, B5

vitamins, 0.5 µM BA and 2% sucrose). Embryo germination and plantlet formation

was achieved by subculturing mature embryos on fresh EM8 medium. The plantlets

were acclimatized in the greenhouse.

Nayak (2002) reported that embryogenic callus cultures of Jumrosa, a hybrid

Cymbopogon were established from nodal explants on MS medium supplemented

with 3 mg/l 2,4-D, 0.5 mg/l KN and 0.2 mg/l NAA. The calli differentiated into

numerous somatic embryos with clear stages of ontogenic development upon

subculture on MS medium supplemented with 1-5 mg/l BA and 0.1 -1.0 mg/l NAA.

Number of somatic embryos produced the best combination of MS basal medium

containing 3 mg/l BA and 0.5 mg/l NAA. A maximum number of (57.4 ± 1.2)

plantlets could be obtained from approximately 50 gm callus with in 90 days of

culture.

Jayatichowdhury et al. (2003) analysed the plant regeneration and bulblet

formation were obtained through somatic embryogenesis in garlic. Leaf bases

measuring 2 to 3 mm were excised and cultured on MS medium containing 2, 4-D

alone or in combination with IAA or NAA for callus induction and embryo formation.

The maximum frequency (70%) of embryogenic callus was obtained in medium

supplemented with 1.5 mg/l 2, 4-D and 1.0 mg/l IAA. After 8 weeks the calli with

developing embryos were transferred on MS medium fortified with different

concentrations (2-8 mg/l) of kinetin for plant regeneration. The highest percentage

(64%) of calli induced plantlet formation and maximum number (6.8) of plantlets per

callus were on to medium supplemented with 6 mg/l kinetin. Increased use of sucrose

enhanced the bulblet formation.

37

High frequency somatic embryogenesis and plant regeneration in petiole and

leaf explant cultures and petiole derived embryogenic cell suspension cultures of

Hylomecon vernalis max were described by Kim et al. (2003). Petiole explants

formed embryogenic calluses at frequency of 53% when cultured on B5 medium

supplemented with 13.6 µM 2,4-D alone. Leaf explants formed embryogenic calluses

at a frequency of 21% when cultured at a combination of 4.52 µM 2, 4-D and 2.22 µM

BA. Cell suspension cultures were established with petiole-derived embryogenic

calluses using liquid B5 medium with 4.52 µM 2, 4-D. Upon planting on to B5 basal

medium, cell suspension cultures produced numerous somatic embryos, which then

developed into plantlets. Regenerated plantlets were transplanted to potting soil and

grown to maturity in a green house.

Yang et al. (2003) established a protocol for large-scale propagation of

Phragmites communis Trin. by somatic emgryogenesis. Stem segment explants

produced hard white callus on the semi-solid MS medium supplemented with 9.05 µM

2, 4-D for 4 weeks. Then the callus was transferred to MS medium supplemented with

4.52 µM 2, 4-D, yellow embryogenic callus with some nodular structures was formed.

When the embryogenic callus was transferred to MS medium supplemented with 0.45

µM 2, 4-D, differentiation was initiated to form small green islands on the surface of

the callus after two weeks in culture. Within 4 weeks a large number of somatic

embryos were formed with a frequency of 86.7%. Six weeks later, they developed into

strong plantlets. These plants were cultured on liquid ½ MS medium with 2.69 µM

NAA added 2.46 µM IBA roots developed. The rooted plants were transferred to soil

with over 85% survival.

Kim et al. (2004) described the culture conditions for plant regeneration in

immature zygotic embryo-derived embryogenic suspension cultures of Catharanthus

roseus. Immature zygotic embryos formed off-white, friable calluses on MS medium

supplemented with 4.52 µM 2, 4-D after 8 weeks of culture. After a second subculture

using MS basal medium at 4 week intervals off-white friable calluses formed

yellowish compact embryogenic calluses, upon transfer to MS basal medium,

embryogenic calluses give rise to numerous somatic embryos. Cell suspention cultures

were estsblished with embryogenic calluses using liquid MS medium supplemented

38

with 4.52 µM 2, 4-D. Embryogenic cell clumps from cell suspension cultures

developed into plantlets when planted on to MS basal medium.

Eisa et al. (2005) developed a protocol for a high frequency somatic

embryogenesis for the production of virus free plants, in which embryogenic callus

was derived from non embryogenic explants. George et al. (2005) evolved a protocol

for direct shoot organogenesis and somatic embryogenesis of Centella asiatica using

leaf and internode derived calluses on MS medium having BA (0.5mg/l) in

combination with NAA (0.5mg/l) respectively for direct regeneration. Somatic

embryos were obtained from MS medium containing 2,4-D (0.5mg/l)+KN(0.25 mg/l).

Somatic embryogenesis from callus drived from leaf and nodal segments of

strawberry were reported by Biswas et al. (2007). The highest percent of cultures with

somatic embryos was achieved on MS medium supplemented with 1.0 mg/l BAP and

50% proline. Regenerated plants were successfully transferred to soil and showed

normal morphology. Embryogenic callus in Catharanthus roseus was intiated from

hypocotyls on MS medium with 1.0-2.0 mg/l of 2, 4-D or Chlophenoxyacetic acid

(CPA). Numerous somatic embryos were induced from primary callus on MS medium

supplemented with NAA within two weeks of culture. Embryo proliferation was much

faster on medium supplemented with BAP. After transfer to medium with GA3 1.0

mg/l mature green embryos were developed and germinated well into plantlets on MS

liquid medium with 0.5 mg/lBAP (Junaid et al., 2007).

A highly reproducible system for efficient plant regeneration from protoplast

via somatic embryogenesis was developed by Wang et al. (2008). Callus forming

frequency (82.86%) was obtained in protoplast cultures from suspension culture cells

in medium with 0.45µM KN, 1.5% glucose and 1.5% maltose. The calli from

protoplast culture were transferred to somatic induction medium and 12.7% of normal

plantlets were obtained on medium contained 3% maltose or of each sucrose +

maltose+ glucose, 2.46µM IBA and 0.93µM KN.

Kim et al. (2009) described culture conditions for high frequency plant

regeneration from zygotic embryo explants via somatic embryogenesis in Rugosa

rose. Mature zygotic embryo, cotyledon and radicle explants formed embryogenic

calluses at frequencies of 3.8, 6.7 and 88% when cultured on ½ strength MS medium

39

supplemented with 2.26, 9.05 and 9.05 µM 2,4-D respectively. Embryogenic calluses

produced numerous somatic embryos, which then developed into plantlets on ½

strength MS without growth regulators. Regenerated plantlets were grown to whole

plants in a growth chamber.

Siva et al. (2009) developed a successful protocol for induction of somatic

embryogenesis and organogenesis in Oldenlandia umbellata L. Emerging young

leaves, shoot apices and stems were used as explants. For rapid embryogenic callus

induction MS medium supplemented with 0.2 mg/l NAA 0.5 mg/l BA and 0.1 %

coconut milk induced the highest frequency (95.86%). Shoot developed upon transfer

of embryogenic calli to MS medium containing 1.5 mg/l BA, 0.3 mg/l NAA and 1%

CM. For root induction 0.3 mg/l NAA and 1.0% CM promoted highest and earliest

rooting.

Callus was initiated three different “esculenta” taro cultivars by culturing corm

slices in the dark on half- strength MS medium supplemented with 2.0 mg/l 2,4-D for

20 days followed by sub-culture of all corn slices to half-strength MS medium

containing 1.0 mg/l thidiazuron (TDZ). Somatic embryos formed when callus was

transferred to hormone free medium ~72% of the embryos germinated into plantlets

on this medium. Plants derived from somatic embryos appeared phenotypically

normal following 2 months of growths in a glass house (Pradeep et al., 2009).

An effective protocol was developed for in vitro propagation of Psoralea

corylifolia via somatic embryogenesis in cell suspension culture (Baskaran and

Jayabalan, 2009). Embryogenic callus was obtained on MS medium supplemented

with 6 µM NAA and 30 µM glutamine from transverse TCLs from 10 day old

hypocotyle explants with 96.4% frequency. Embryogenic callus produced a higher

number of somatic embryos on MS medium containing 30g/l sucrose, 1 µM NAA, 4

µM BA, 15 µM glutamine and 2 µM absicisic acid after 4 weeks of culture. Somatic

embryos successfully germinated on half MS medium containing 20g/l sucrose, 8g/l

agar and supplemented with 2µM BA, 1µM ABA and 2µM gibrellic acid within two

weeks of culture. Somatic embryos developed into normal plants which hardened with

100% efficiency in soil in a growth chamber.

40

Harini and Sathyanarayana (2009) reported the induction of somatic embryos

in Mucuna pruriens. Different explants cultured on MS medium supplemented with

11.31 µM 2, 4-D produced golden yellow embryogenic callus that induced

synchronized embryo development on MS basal liquid medium. The pre-embryogenic

mass progressively developed to globular, heart, torpedo and cotyledentory shaped

embryos.

Vibha et al. (2009) reported a rapid and reliable protocol for high fidelity

regeneration of Mucuna pruriens via somatic embryogenesis. Embryogenic callus was

induced from cotyledon segments of in vitro grown seedlings on MS medium

supplemented with 6.7 µM 2,4-D. High frequency somatic embryogenesis was

achieved after transfer of embryogenic callus clumps to MS medium supplemented

with 2.3 µM kinetin and 5.4 µM NAA supplemented with 13.6 µM adenine sulphate.

Mature somatic embryos were converted to plantlets on half strength MS basal

medium with 90% survival rate in the field condition.

A protocol for somatic embryogenesis in Azadirachta indica has been

standardized using in vivo leaflets (Shekhawat et al., 2009). Embyogenic calli were

induced on MS medium supplemented with 1.5 mg dm-3

kinetin and 1.5 mg dm-3

indole-3 acetic acid and subsequently all stages of somatic embryos (globular, cordate,

torpido and cotyledonary) were observed. Maturation of these embryos was

accomplished with the same growth regulators after three subcultures. The matured

somatic embryos were transferred on to half strength MS medium devoid of growth

regulators for their germination (82%) plantlets were acclimatized in the field with

survival rate of 80-83.5%.

Embyogenic calli could be induced from the leaf, petiole and root pieces of

Hyoscyamus niger using different induction medium. Small globular somatic embryos

could only be produced from the leaf explants cultured on MS medium supplemented

with 6mg/l picloram. The globular embryos increased in size and developed into

torpedo, heart shaped and bipolar embryos accompanied by root formation after

cultured on to MS basal medium for one week then followed by two weeks of culture

in the maturation medium(MS + 1.0 mg/l BA) (Chan Lai Keng et al., 2009).

41

Naik and Murthy (2010) achieved somatic embryogenesis from cell

suspension cultures of Niger (Guizotia abyssinica). Initially friable ebryogenic

calluses were induced from cotyledonary leaves of niger on MS medium containing 5

µM 2,4-D and 0.5 µM kinetin. Cell suspension cultures were established by using

embryogenic calluses in MS liquid medium containing 5 µM 2,4-D and 0.5 µM KIN.

Initiation of somatic embryogenesis and development up to globular states from

embryogenic cell clumps occurred in the liquid medium itself. Thereafter

embryogenic cell aggregates were transferred to MS agar medium supplemented with

3 µM KIN for embryo differentiation, were as maturation of somatic embryos

occurred in MS agar medium containing 10 µM abscisic acid.

Somatic embryogenesis and regeneration from shoot primodia of Crocus

heuftelianus were reported by Zita Demeter et al. (2010). They induced an

embryogenic callus line on basal MS medium supplemented with Gamborg’s

vitamins, 2% (w/v) sucrose, 10 mg/l NAA and 1 mg/l BA. Globular stage embryos

developed on this medium for obtaining mature embryos and plant regeneration,

firstly a decrease of auxin/cytokinin concentration and ratio, then secondly a decrease

in the strength of culture medium and the concentration of carbon source was used.

Robert Konieczny (2010) developed a plant regeneration protocol for

Trifolium nigrescens via somatic embryogenesis. Immature Zygotic Embryos at

torpedo (TZE) and Cotyledonary Stage Embryos (CSE) were cultured on media with

different auxins and cytokinins at different concentration embryogenic callus occurred

on CSE after 10 days on MS medium supplemented with 6-furfurylaminopurine

(kinetin) or N6- [2-isopentenyl] adenine (2ip) along with 2,4-D. Plant regeneration

was achieved after transfer of somatic embryos or embryo-derived first shoots to

medium without plant growth regulators.

Mi-Suk Seo et al. (2010) developed a rapid and efficient shoot regeneration

system for Panicum spp. by adjusting the regeneration medium and studying the

response of different genotypes and the influence of explant type (mature seed,

immature embryo and shoot apex). The highest frequency of shoot regeneration was

obtained on MS medim supplemented with 30 gL-1 multose and 1 mg L-1 N-phenyl –

N’–[C 1,2,3-thidiazol -5-gl)urea]. The callus formed green spots after 1 week of

culture and showed primarary green shoots after 2 weeks.

42

2.3. MATERIALS AND METHODS

2.3.1. Plant material (Source of the explants)

Healthy plants and fresh rhizomes of Kaempferia galanga L. were procured

from the herbal garden of Kerala agricultural university, Vellanikkara, Trichur Dt.,

Kerala. The plants were identified at Rapinat Herbarium and Centre for Molecular

Systematics, St. Joseph’s College, Tiruchirappalli, S. India.

2.3.2. Preparation of plant material

Planting beds were prepared with river sand and soil. The rhizomes with 3-4

healthy shoot buds were planted on these beds and were frequently watered and

covered with leaves for shade and also to retain moisture. On sprouting, the rhizomes

were transplanted to garden pots containing sterile garden soil and sand (1:1).

The pots were maintained in the green house of Rapinat Herbarium, Tiruchirappalli.

2.3.3. Direct organogenesis

2.3.3.1. Selection of explants

Both in vivo and in vitro plants were used for this study. Rhizomes and young

sprouting vegetative buds collected from 1 to 5 months old healthy green house.

Grown plants and shoot tip, leaf-sheath from in vitro derived plants were used as

explants.

2.3.3.2. Sterilization of explants

The excised in vivo explants (rhizome bud and rhizome segments) were

thoroughly washed with running tap water for 10-15 minutes. Then the explants were

washed with detergent (Teepol 5%v/v) solution for 5 minutes, Fungicide (Bavistine

2%w/v) treatment for 3 minutes followed by soaking in 70% (v/v) ethanol for 30

seconds and finally disinfected with 0.1%(w/v) HgCl2 for 2 minutes and rinsed with

sterile distilled water five times.

2.3.3.3. Sterilization of Glasswares

The glasswares include culture tubes, conical flask, beakers, petridishes,

pipettes, standard flasks, and measuring cylinders etc. The glasswares were first

soaked in sulphuric acid for four hours and then washed well under a jet of tap water.

Then they were soaked in detergent solution for 30 minutes and washed thoroughly

using running tap water to remove the trace of detergent. Finally, they were rinsed

with sterile distilled water, dried in a hot air oven and autoclaved at 1210C for 20

minutes.

43

2.3.3.4. Culture medium

MS (Murashige and Skoog, 1962) basal medium (Table - 2.1) containing MS

vitamins was employed for the rhizome bud, rhizome disc, shoot tip and leaf-sheath

culture. Sucrose 3% was used as carbohydrate source in the media. Growth hormones

(BAP, KN, NAA and GA3) were added individually as well as with combination at

different concentration to the medium. Cytokinins (BAPand KN) were incorporated in

the range of 0.5-5 mg/l and GA3 0.1-2.5 mg/l, Auxin (NAA, IBA and IAA) 0.1-2.5

mg/l. The pH of media was adjusted to 5.6-5.8 prior to the addition of 0.8 (w/v) agar

and autoclaving at 1210C and 1.06 kg cm

-2 pressure for 20 min. The cultures were

incubated at 25± 20C under 16 hours photoperiod (irradiance of 1000 lux) and relative

humidity of 60%.

2.3.3.5. Inoculation of explants

Rhizome buds, rhizome discs were excised from in vivo seedlings and leaf-

sheaths and shoot tips were collected from in vitro plantlets. All the explants were

placed both in vertical and horizontal position. Each treatment consisted of at least 7

explants and the experiments were repeated five times. The culture vessels were then

incubated at 25± 20C under 16 hours Photoperiod of 40 µ mol m

-2s

-1, provided by

cool-white fluorescent lamps.

2.3.3.6. Shoot Proliferation and multiplication

Different explants were cultured on MS basal medium containing 3% (w/v)

sucrose, 0.8% (w/v) agar and various concentrations of BAP, KN (0.5-3.0 mg/l) alone

or in combination with auxin (0.1-2.5 mg/l) and were used for shoot proliferation.

After two weeks the clumps of shoots were subcultured on MS medium containing

3% (w/v) sucrose and 0.8% agar (w/v) with suitable growth regulators for

multiplication and maturation of the shoots. The cultures were incubated at 25± 20C

under 16/8 hours light/dark photoperiod. After two weeks, shoots longer than 3.0 cm

were counted and transferred to rooting medium.

2.3.3.7. Root Induction and Transplantation

Shoots without roots were excised and transferred to MS basal medium

containing 3% (w/v) sucrose, 0.8% (w/v) agar and different concentration of NAA,

IBA, and IAA (0.1-2.5 mg/l) alone or in combination of cytokinins such as KN, BAP

(0.1-2.5 mg/l) for induction. The number of roots in each plantlets was counted.

44

Plantlets with well developed roots were removed from the culture tubes and after

washing their roots in running tap water, they were grown in the mixture of sand,

garden soil and farmyard manure in the ratio of 1:2:1: in the paper cups for a month

and subsequently transferred to pots. Potted plants were covered with transparent

polythene wraps to high humidity and watered every three days with half strength MS

salt solution free of sucrose for two weeks.

2.3.3.8. Acclimatization and Hardening of Regenerants

Plants raised through tissue culture, need gradual acclimatization for their

survival in the natural condition, instead of transferring directly to the pots. Plantlets

were left for three weeks in paper cups at the controlled temperature (25±20C) with

60% relative humidity. The survival percentage was observed in all the explants. The

well grown plants were transferred to bigger pots containing soil mixture and placed

in green house. Later the plants were established in the field. The plants growing in

the field were observed for their further growth and survival. Samples were

photographed at different stages of growth period.

2.3.3.9. Statistical Analysis

The cultures were examined periodically and the morphological changes were

recorded on the basis of visual observation. Each treatment consisted of at least 7

explants and the experiment was repeated five times. A complete randomized design

was used in all experiments and analysis of variance and mean separations were

carried out using Duncan’s Multiple Range Test (DMRT). Significance was

determined at the 5% level (Gomez and Gomez, 1976).

2.3.4. Indirect Organogenesis

2.3.4.1. Source of explants

In vivo and In vitro plants of Kaempferia galanga were used as the source of

explants. Rhizome buds and rhizome discs are collected from 1 to 5 months old

healthy green house grown plants. Leaf and leaf-sheaths were collected from in vitro

derived plants.

2.3.4.2. Sterilization of explants

The excised in vivo explants (rhizome buds and rhizome discs) were

thoroughly washed with running tap water for 10-15 minutes. Thereafter the explants

45

were washed with detergent (Teepol 5%v/v) solution for 5 minutes, Fungicide

(Bavistine 2%w/v) treatment for 3 minutes then soaked in 70%(v/v) ethanol for 30

seconds and finally disinfected with 0.1%(w/v) HgCl2 for 2 min. and rinsed with

sterile distilled water five times.

2.3.4.3. Medium and culture conditions

MS (Murashige and Skoog, 1962) basal medium containing MS vitamins,

0.8% agar, and 30 g/l sucrose was used. Cytokinin (BAP +KN) and Auxins (2, 4-D,

NAA,) were tested alone or in combinations. The combinations of 2, 4-D+ BAP, 2, 4-

D+KN, NAA+BAP and NAA+KN were also tested for callogensis from the explants.

The pH of the media was adjusted to 5.6-5.8, prior to the addition of 0.8% (w/v) agar

and autoclaving at 1210C and 1.06 Kg cm

-2 pressure for 20 min. The cultures were

incubated at 25±20C under 16 hours photoperiod (irradiance of 1000 Lux) and relative

humidity of 60%.

2.3.4.4. Incubation of explants

All explants such as rhizome bud, rhizome disc, leaf and leaf-sheath were

cultured in both vertical and horizontal position. Plant growth regulators (2, 4 D,

NAA, BAP and KN) were added individually as well as with combination at different

concentrations to the medium. Each treatment consisted of 7 explants and the

experiments were repeated five times. The cultures were kept at 25±20C under 16

hours photoperiod with cool white fluorescent lamps.

2.3.4.5. Callus Induction

Different explants were cultured on MS basal medium containing 3% (w/v)

sucrose, 0.8% (w/v) agar and various concentrations of auxins (2,4-D, NAA) alone or

in combination with cytokinins (BAP, KN) for optimal callus induction. In the present

investigation data were recorded at 20 and 30 days after inoculation of explants on the

regeneration medium. The calli were graded according to their colour in a symbol of

B- brown, G- green, W- white, Y- yellow, PY- pale yellow, PG- pale green, GY-

greenish yellow, GW- greenish white, GB- greenish brown, WB- white brown. The

nature of callus was measured by the callus compactness and graded into three

categories: friable (F), semi-friable (SF), mucilaginous (MU) and compact (C).

Abundance of callus was measured by a transparent measuring ruler and graded

46

according to their length: large (L) = 20 mm and above, medium (M) = 10 to 20 mm

and small (S) = 10 mm below. The calli were subcultured and maintained in vitro

conditions. The effects of these quantitative characters with duration of the time for

regeneration were estimated in percentage.

2.3.4.6. Shoot bud Regeneration and Multiplication

Well developed calli were transferred to regeneration medium. The nutrient

medium consisted of Murashige and Skoog (1962) salt and vitamins and 3% (w/v)

sucrose and 8 g/l (w/v) agar strengthened with different concentrations of Auxins

(2, 4-D, NAA) and combination of cytokinins (BAP and KN). The pH was adjusted to

5.6 to 5.8 prior to autoclaving. The cultures were incubated at 25±20C, 16 hours

Photoperiod provided by cool white fluorescent tubes. Data on percentage of calli

forming shoots and their number were recorded after 4 weeks of inoculation.

2.3.4.7. Shoot Elongation

Proliferated multiple shoots were divided into small clusters of 2-3 shoots.

They were sub-cultured on shoot elongation medium containing GA3 (0.1 - 2.5 mg/l)

alone or in combination with cytokinins BAP/KN (0.1 - 2.5 mg/l) or auxin NAA

(0.1 - 2.5 mg/l). The cultures were inoculated at 25 ± 20C under 16/8 hours light/dark

photoperiod. After two weeks, shoots longer than 5.0 cm were counted and transferred

to rooting medium.

2.3.4.8. Rooting Induction and acclimatization

Excised plantlets 5 cm in length were transferred to MS medium containing

3 %( w/v) sucrose, 8g/l (w/v) agar, different contractions of Auxin (IBA, NAA and

IAA) for root initiation. Rooted plantlets from different treatments were removed from

the culture tubes, washed off the medium in running tap water and transplanted in

cups containing sand, garden soil and farmyard manure in the ratio 1:2:1. Half

strength MS medium was applied to moisten the soil, which was then covered with

polythene bags. Cups were placed under 1000 lux light at 16 hours photoperiod for

one week. After three to four days, holes were made in the polythene wraps to

gradually expose them to the external environment. After two weeks they were

transferred to larger pots containing 50% sand and 50% farmyard manure and shifted

to green house.

47

2.3.4.9. Statistical Analysis

Each treatment consisted of at least 7 explants and the experiment was

repeated five times. A complete randomized design was used in all experiments and

analysis of variance and means separation were carried out using Duncan’s Multiple

Range Test (DMRT). Significance was determined at the 5% level (Gomez and

Gomez, 1976).

2.3.5. Somatic Embryogenesis

2.3.5.1. Explant Selection

Rhizome bud explants collected from in vivo plants and leaf and leaf-sheath

collected from in vitro raised seedling were used for preset investigation.

2.3.5.2. Direct somatic embryogenesis

The medium consisted of semi-solid MS medium consisting of MS mineral

salt, vitamins, 0.6% agar, 30g/l sucrose, auxins and cytokinines. The pH was adjusted

to 5.8 before autoclaving at 1210C for 20 min. Leaves were cut into 1.2-1.5 cm

squares, leaf-sheath and rhizome buds were cut into 1cm long segments in sterile petri

dishes. The explants were placed in 25 × 150 mm tubes containing 15 ml semisolid

MS medium. The cultures were kept in dark for one week. After two weeks of

incubation, the cultures were transferred to fresh media with the same composition.

2.3.5.3. Indirect somatic embryogenesis

2.3.5.3.1. Callus Induction

Semi-solid MS basal medium containing MS vitamins, 0.6% agar, 30 g/l

sucrose and 2, 4, D (0.1-2.5 mg/l) and cytokinin (0.1-2.0 mg/l) were used as inoculum

and explants placed horizontally on the medium. Leaves were cut into 1.2-1.5 cm

squares, leaf-sheath and rhizome buds were cut into 1cm long segments in sterile

petridishes. These leaf segments were placed with either the abaxial or adaxial surface

down in 25×150 mm tubes containing 15 ml semi-solid MS medium. The callus

cultures were maintained at 25±20C under 16 hours photoperiod in 80µE m

-2 S

-1 light

intensity (TL 40w/54w cool white fluorescent lamps). Calli obtained were subcultured

after 15-21 days either in the same medium for callus via. somatic embyogenesis or

transferred to the liquid medium to establish cell suspension for further experiments.

48

2.3.5.3.2. Suspension culture

Cell suspension culture was initiated by transferring 15-21 days old greenish

white friable calli (1g fresh mass) from semisolid MS medium. They were aseptically

transferred to 150 ml Erienmeyer flask containing 50 ml of MS liquid medium per

flask, with (0.1-2.5 mg/l) NAA individually or along with BAP or KN (0.15- 2.5

mg/l), 3% sucrose and pH was adjusted to 5.8 before autoclaving at 1210C for 15 min.

The suspension was agitated on a Gyratory shaker (100-110rpm). Cultures were

maintained at light intensity of 1.5µMm-2

S-2

16h photoperiod and 25±20C. Further

subcultures involved the replacement of 10 ml of the cell suspension with fresh

medium of the same formulation at 6 day intervals. Cultures were filtered through

125µM stainless steel sieves to separate individual cells and small cell clumps.

Samples of suspension cultures were taken at random at the end of 21 days of