7

REVIEW OF LITERATURE

Due to rapid deforestation and depletion of genetic stocks, concerted efforts must be made to

evolve new methods for mass propagation and production of plant species which are high

yielding, resistant to pest and disease associated with increased photosynthetic efficiency.

Conventional breeding is rather slow and less productive and cannot be used efficiently for

the mass multiplication and genetic improvement of trees. To circumvent this, plant tissue

culture and genetic transformation methods offer an important option for effective

multiplication and improvement of plant species. The technology of plant tissue culture offers

advantages over conventional methods of propagation for a rapid and large scale

multiplication of important plants under in vitro conditions irrespective of season with

conservation of space and time (Nehra and Kartha, 1994; Rao et al., 1997). This technique

provides rapid multiplication of selected superior varieties. During the past decade, major

advances have been made in this field and now it has become an industrial technology. An

overview of work carried out earlier on the different plant species have been given in the

following sections.

2.1 Micropropagation

In nature, the methods of plant propagation may be either asexual or sexual. Sexually

propagated plants demonstrate a high amount of heterogeneity since their seed progeny are

not true-to-type unless they have been derived from inbred lines. Asexual reproduction, on

the other hand, gives rise to plants which are genetically identical to the parent plant and thus

permits perpetuation of the unique characters of the cultivars. Multiplication of genetically

identical copies of a cultivar by asexual reproduction is called clonal propagation. When

clonal propagation is through tissue culture, it is popularly called micropropagation.

Advanced biotechnological methods of culturing plant cells and tissues should provide new

means for conserving and rapidly propagating valuable, rare and endangered forest tree

species. The application of micropropagation techniques as an alternative mean of asexual

propagation of important plant species has increased the interest of workers in various fields.

There is also a conservation use for those species that are at risk, rare, endangered or of

special cultural, economic or ecological value (Benson, 2003). The potential benefits of

micropropagation of elite genotypes for production of clonal planting stock for

8

afforestation/reforestation have long been recognized (Winton, 1971; Whitehead and Giles,

1977). In general woody trees are difficult to regenerate under in vitro conditions.

The difficult-to-root recalcitrant species for which there is a serious dearth of planting

material are multiplied through tissue culture and made available for the afforestation

programme. Plant Tissue Culture is a technique by which any plant part can be cultured on a

nutrient medium under sterile conditions with the purpose of obtaining growth. In vitro

methods can be used to produce, maintain, multiply and transport pathogen free plants safely

and economically. This technology is being extensively used for large-scale production of

elite planting material of desired characteristics.

The pioneering experiments were initiated by the father of tissue culture

Gottlieb Haberlandt in 1898, chose single cell, isolated from the palisade tissue of

leaves, epidermis and epidermal hairs of different plants. He grew them on Knop's

(1865) salt solution with sucrose and observed obvious growth in the palisade cells

but could not succeed because of handling with highly differentiated cells and lack

of proper techniques. Hanning (1904) successfully cultured embryos of Raphanus

sativus, Raphanus landra, Raphanus caudatus and Cochlearia danica on Tollen's

medium and obtained transplantable seedlings. The first commercial use of plant

tissue culture on artificial media was in the germination and growth of orchid

plants, in the 1920’s. White (1934) was the first to achieve success in organ cultures of

tomato root tips. The first calli from cambial explants of angiosperms and conifers

trees was observed by Gautheret (1934). Gautheret, White and Nobecourt (1939) laid the

foundation for further work in the field of plant tissue culture. The tissue culture media,

presently in use were modifications of those established by these three pioneers. Ball (1946)

developed the first whole plants through tissue culture from shoot tips of Lupinus and

Tropaeolum. There was a great deal of research in 1950-60, but it was only after the

development of a reliable artificial medium (Murashige & Skoog, 1962) that plant

tissue culture really ‘took off’ commercially. Morel (1950) successfully cultured

monocot plant tissue with the help of coconut milk. Miller et al., (1955) discovered

and isolated 6-furfuryl amino purine and named it as Kinetin. Torrey (1957) and Muir et

al., (1958) demonstrated the single cell proliferation into callus. The shoot and root

initiation in cultured callus can be regulated by varying ratio of auxins and

cytokinins in the medium (Skoog and Miller, 1957). The first successful report on

the formation of somatic embryos from carrot tissue was achieved by Steward

(1958) and Reniert (1959). Some other techniques which controls plant

9

regeneration and morphogenesis in culture included: production of virus free plants

(Morel, 1960), haploid plant production (Guha and Maheswari, 1964), production

of secondary metabolites (Kaul and Staba, 1967), protoplasts fusion by

polyethylene glycol (Kao et al., 1974). The first complete plants from tissue

culture of tree species were regenerated by Winton (1968) from leaf explant of

Populus trichocarpa. Micropropagation by the method of multiple shoot production

directly from seeds has been reported in many herbaceous and woody species (Rodriguez,

1982).

Plant tissue culture is an important alternative to conventional method of vegetative

propagation mainly for raising of elite and rare species (Rao et al., 1996). Plant tissue culture

provides a best tool for large scale production of propagules especially in case of endangered

medicinally important plant species where explant material is available in a very small

quantity. Micropropagation of mature trees employing vegetative explants has been a difficult

task and lagging behind that of herbaceous plant due to various factors, like juvenility,

maturity, inherent slow growing habit, exogenous and endogenous infection, presence of

phenolic compounds, long complex life cycles and great genetic variations (Bonga and

Durzan, 1986; Durzan, 1985; Zimmermen, 1985; Bajaj, 1991). During the last few years,

micropropagation techniques have been used for the rapid and large scale propagation of a

number of fruit and forest trees (Hutchinson and Zimmerman, 1987). Several woody species

such as poplars, wild cherry, eucalyptus, red wood, radiate pine and teak are at present

commercially micropropagated (Thorpe, 1990; Bajaj, 1997). In vitro propagation of forest

tree species is an effective way to capture genetic gain and produce large amounts of plant

material.

It is well established that in vitro propagation of plant species is influenced by several

factors, like genotype, age and source of initial tissue/organ which in turn are related to their

endogenous hormonal status (George, 1993). Tissue culture techniques are also used for

virus eradication, genetic manipulation, somatic hybridization and other procedures

that benefit propagation, plant improvement and basic research. A large number of

horticultural plantations, forest species, important fruit trees and medicinal plant are being

propagated in vitro on commercial scale (Bhojwani and Arumugam, 1993). There are number

of factors that affect the success of in vitro regeneration are discussed here.

10

2.1.1 Selection of explants

The manipulability of organ formation in tissue culture is often determined by the choice of

the explants. While it is generally accepted that totipotentiality is characteristic of all plant

cells. Several plant parts can be used as explants to initiate a plant tissue culture. However,

each plant organ differs in its rate of growth and regeneration because the cells in that organ

exist in a particular developmental stage. Organs also differ in their metabolic activity and

capacity to transport and utilize growth regulators. Generally, meristematic tissues such as the

root and stem tips and auxiliary buds are good explants because they show the most rapid rate

of cell division. Also, these tissues have a greater ability for the uptake and concentration of

growth regulators. The regeneration potential of explants is attributed by the physiological

state, age and cellular differentiation among the constituent cells (Murashige, 1974).

The influence of plant material on the growth and development in tissue culture are related to

many factors such as genotype, age of the plant, age of the tissue or organ, physiological state

of the explants, the state of health of the plant, effect of season throughout the year such as

winter and summer, growth condition such as photoperiod, position of explant within the

plant, size of the explants, wound surface area, method of inoculation, etc. The use of young

and meristematic tissue as in many cases enabled raising of regenerative cultures when

mature and differentiated explants failed to give such response. Explants cells already in an

active process of division contribute to increase in the adventitious bud induction on the

explants (Mohammed et al., 1992: Thome et al., 1995). Juvenility is one of the most

important factor influencing the in vitro response of many woody species (Bonga, 1987).

Nodal explants from mature plants have been used for regeneration in many

tree species like as: Prosopis juliflora (Nandwani and Ramawat, 1991), Dalbergia sissoo

(Gulati and Jaiwal, 1996), Ficus religiosa (Deshpande et al., 1998), Sterculia urens

(Sunnichan et al., 1998), Madhuca latifolia (Bansal and Chibbar, 2000), Melia azedarach

(Shahzad and Siddique, 2001), Toona ciliata (Mroginski et al., 2003), Belanites

aegyptica (Ndoye et al., 2003), Michelia champaca (Iyer et al., 2005), Cassia

angustifolia (Siddique and Anis, 2007), Searsia dentata (Prakash and Staden, 2008), Morus

alba (Balakrishnan et al., 2009), Balanites aegyptiaca (Siddique and Anis, 2009), Arbutus

unedo (Gomes and Canhoto, 2009), Azadirachta indica (Arora et al., 2010), Spondias

mangifera (Tripathi and Kumari, 2010), Acacia auriculiformis (Girijashanker, 2011)

Dalbergia sissoo (Ali et al., 2012).

11

Mehra and Cheema (1980) reported multiple shoots induction from nodal segments

of Populus. Sharma et al., (1984) established callus cultures on modified MS

medium from axillary buds and shoot tips of Phoenix dactylifera. Rai and

Jagdishchandra (1987) induced multiple shoots directly from seeds and seedling

explants of Cinnamomum zeylanicum. Different explants viz. stem, root, leaf and

hypocotyl segments and axillary buds were used for regeneration in Albizzia

lebbeck (Arya et al., 1978; Bhargava et al., 1981). Sharma et al., (1988) reported

the somatic embryogenesis and plant regeneration from shoot tip derived callus of

Phoenix sylvestris. Gharyal and Maheshwari (1990) observed callus formation from

stem and petiole explants of Cassia fistula and Cassia siama and its differentiation

in to shoot buds. Nandwani and Ramawat (1991) reported plantlets regeneration

from nodal segments of Prosopis juliflora.

Rout and Das (1993) reported multiple shoots induction from apical and

axillary meristem explants derived from seedlings of Madhuca longifolia. Perez-

Parron et al., (1994) used shoot tips and nodal segments of Fraxinus angustifolia

for direct regeneration. Mohan et al., (1995) achieved regeneration from hypocotyl

and cotyledonary node explants of Moringa pterygosperma. Purohit and Dave

(1996) reported multiplication of Sterculia urens through cotyledonary node

segments. Micropropagation protocols for Cinnamomum camphora were developed by

Babu et al., (1997) and Huang et al., (1998). Ajithkumar and Seeni (1998) observed direct

organogenesis from nodal, leaf, shoot tip and internode segments of Aegle marmelos. The

protocol for micropropagation of Lagerstroemia reginae using shoot bud culture was

developed by Sumana and Kaveriappa (2000). Sheeja et al., (2000) reported

micropropagation of Cinnamomum verum from mature nodal segments. Indirect

organogenesis from nodal explants of Melia azedarach was reported by Shahzad and

Siddiqui (2001). Multiple shoots were initiated from cotyledonary node segments of Acacia

catechu (Sahni and Gupta, 2002). Romano et al., (2002) has developed an in vitro

propagation protocol based on axillary bud proliferation for mature female trees of Ceratonia

siliqua in MS medium supplemented with BAP or Zeatin. Martin (2003) developed a

protocol for in vitro regeneration of rare woody aromatic medicinal plant-Rotula

aquatica by means of direct and indirect organogenesis. Shou et al., (2005) developed

a protocol for micropropagation of Cinnamomum camphora by the formation of adventitious

shoots from the delicate caudex. Iyer et al., (2005) reported multiple shoots from nodal

12

explants of Michelia champaca. Pandey et al., (2006) developed a protocol for the

regeneration of complete plantlets from nodal explants of Terminalia arjuna.

Pasqual and Ferreira (2007) developed pathogen free plantlets of Ficus carica

through micropropagation. Multiple shoots were produced from mature nodal

segments of Syzigium cumini (Remashree et al., 2007). Soulange et al., (2007)

reported multiple shoot induction in Cinnamomum camphora. Sujatha and Hazra, (2007)

used MS basal medium for induction of multiple shoots from mature-tree-derived

axillary meristems of Pongamia pinnata. Khalafalla and Daffalla (2008) reported

multiple shoots from cotyledonary node segments of Acacia senegal.

Micropropagation protocol has been developed by different workers for efficient plantlet

regeneration through nodal segments of different plants viz. Eucalyptus polybractea

(Goodger et al., 2008) Phoenix dactylifera (Aslam and Khan, 2009), Melia azedarach

(Hussain and Anis, 2009), Arbutus unedo (Gomes and Canhoto (2009), Oroxylum indicum

(Gokhale and Bansal, 2009). Mostafa et al., (2010) developed a protocol for

micropropagation of Arbutus andrachne using explants from seedlings. Similarly, Tripathi

and Kumari (2010) developed an efficient in vitro regeneration protocol for Spondias

mangifera using cotyledonary nodes. Micropropagation from nodal explants from epicormic

shoots of Dalbergia sissoo was developed by Thirunavoukkarasu et al., (2010). Tyagi et al.,

(2010) reported the in vitro regeneration of Crataeva adansonii through nodal segments.

Regeneration of Gomortega keule through zygotic embryos was reported by Mun˜oz-Concha

and Davey (2011). Khalafalla et al., (2011) also reported the intact embryo cultures of

Boscia senegalensis. Clonal propagation of Acacia auriculiformis through axillary

buds was reported by Girijashankar (2011). An efficient and improved in vitro

propagation method for Terminalia catappa has been developed from nodal explants

(Phulwaria et al., 2012). Mehta et al., (2012) developed a protocol for rapid

micropropagation and callus induction of Terminalia bellerica.

Seasonal changes greatly influence explants establishment (Siril and Dhar, 1997).

Seasonal conditions at the time of explants collection may influence the in vitro growth of

explants, phenolics exudation and degree of contamination. Most vegetative propagation

techniques relaying on morphogenetic process are conditioned by the season (Hartman and

Kester, 1986). There is increasing evidence that seasonal differences influence the regulation

of cell cycle and this can affect morphogenetic processes (Anderson et al., 2001). Gupta et

al., (1980) noticed the seasonal effects on regeneration of Tactona grandis. The nodal

13

segments of Eucalyptus tereticornis collected during July to September were more responsive

because of negligible phenolic exudation from explants as compared to that collected in

October-November and May-June due to high amount of phenolic exudation (Das and Mitra,

1990). Excellent regeneration has been reported in plant species during spring season

(March-May) when reserve food material (carbohydrate) is made available and helps plants

sprout and bloom (Bhatt and Todaria, 1990). The nodal explants harvested during the months

of March-April and August-October was found to be the best for cultures establishment of

Capparis decidua (Deora and Shekhawat, 1995). Bansal and Chibbar (2000) observed best

response from nodal segments of Madhuca latifolia in the month of May. The collection of

explants during a relatively milder weather condition (December to March) was best for

promoting survival of explants in Acacia sinuata (Vengadesan et al., 2003). Sharma et al.,

(2003) also observed best explants response in the month of October and November with

maximum number of shoot buds in Crataeva adansonii. The best shoot initiation response

was reported from November to February when the trees produced fresh sprouts; the shoot

initiation was rare in explants collected during other periods in Callophyllum apetalum (Nair

and Seeni, 2003). Nodal explants of Myrica esculenta collected during winter (November-

December) gave the maximum response (Bhatt and Dhar, 2004). The nodal segments of

Wrightia tinctoria collected during March-June from young lateral branches showed

maximum bud break response (Purohit and Kukda, 2004). In Holarrhena antidysenterica,

nodal explants showed maximal morphogenic response from May to July, and declined in

subsequent months till dropping to zero from October to February (Kumar et al., 2005).

Singh and Goyal (2007) observed that the season between August-October was best for

explant collection in Salvadora oleoides. Pati et al., (2008) observed that nodal explants of

Aegle marmelos excised during September-October was found ideal because most of the

explants shows bud break whereas, bud break frequency reduced in other months. The

cultures of Melia azedarach initiated during March exhibited the best response not

only in terms of the frequency of bud break but also in shoot vigor (Husain and Anis,

2009). The explants of Maerua oblongifolia collected during the months of July-August

responded best in vitro as compared to explants harvested in any other months of the year

(Rathore and Shekhawat, 2011). The nodal, inter-nodal segments and shoot apices of Ficus

religiosa collected in May and June gave maximum response (Siwach et al., 2011).

The variation in regenerative behaviour among explants is sometimes

attributable to the age of the tissue or organ and the extent to which constituent

14

cells are differentiated. Thorpe and Biondi (1984) also reported that the

physiological state of the tree has been known to influence the behaviour of

explant in culture. The capacity for clonal propagation is closely linked with the

genetic and physiological factors that control the transition from juvenile to mature

growth in trees (Bonga, 1982). In general, juvenile tissue will better respond to in

vitro treatments.

Explants from mature trees of Eucalyptus citriodora required pre-treatment

for induction of shoot buds but explants from seedlings did not require any pre-

treatment (Gupta et al., 1981). Gulati and Jaiwal (1996) reported that nodal

explants from coppied shoots of mature Dalbergia sissoo exhibited least phenolic

exudation and responded better shoot regeneration than the explants obtained from

mature tree. Philomina and Rao (1999) reported multiple shoots from seed culture

of Sapindus mukorossi. Jha et al., (2002) observed the adventitious shoots formation from

the cotyledonary node explants of Sesbania rostrata. Axillary buds of Crataeva

adansonii taken from the root stock growths proliferated better in comparison to

buds taken from the mature tree (Sharma et al., 2003). Chowdhary et al., (2004)

reported that the nodal explants of Dendrocalamus strictus taken from the 1st and

3rd

positions from base of the secondary branches showed better response

compared to the 4th to 6th positions. This was due to differences in the

physiological states of the two explants. Shoot cultures of Toona ciliata has been

established from nodal segments taken from 2 years and 10 years old trees but the

successful rooting was achieved only from the nodal segments taken from 2 year

old tree (Mroginski et al., 2003). This loss of rooting ability has also observed in

several other woody species when the explants came from adult plants (Monteuuis,

1987; Capuana and Gianini, 1997). Nodal segments taken from 11-15th position

proved to the best explants for regeneration of Aegle marmelos (Pati et al., 2008).

Cotyledonary node explants of Acacia senegal cultured on induction medium containing

growth regulators gave the highest in number and longest in vitro regenerated shoot

compared to those induced from nodal segment cultured on the same media (Khalafalla and

Daffalla, 2008). The morphogenetic capacity of cotyledonary node explants of

Parapiptadenia rigida was superior to that of nodal segments (Kielse et al., 2009). In

Sapindus trifoliatus, the 4-week-old seedling explants showed maximum shoot proliferation

as compared to 1, 2, and 3 or 5week-old seedling explants (Asthana et al., 2011). The mature

15

nodal explants of Maerua oblongifolia selected from the adult plants showed poor response

as compared to nodal stem segments prepared from fresh shoots sprouts (Rathore and

Shekhawat, 2011). Explant type has been shown to effect multiple shoot induction in a

number of trees including Dalbergia sisso (Pradhan et al., 1998), Albizia lebbeck (Mamun et

al., 2004), Pterocarpus marsupium (Anis et al., 2005) and Albizia odoratissima (Rajeswari

and Paliwal, 2006).

The orientation of explants also plays an important role in regeneration potential.

The horizontal position of the explants has been reported to promote adventitious

shoot formation in many higher plants (Frett and Smagula, 1983; Pierik, 1987).

McClelland and Smith (1990) reported that the horizontal orientation of explants

produced the more shoots per explants in woody species viz. Amefanchier spicata,

Acer rubrun, Border forsythia, Betula nigra. Similar observations on the influence

of explant orientation have also been made for other tree species including Pyrus

communis (Lane, 1979) and Tamarindus (Jaiwal and Gulati, 1991). The horizontal

orientation of nodal explants of Fraxinus angustifolia reported the highest

multiplication rate (Perez-Parron et al., 1994). The orientation of disc in the floral stem

was the most important factor affecting shoot regeneration in Crinum macowanii (Slabbert et

al., (1995). Bhuyan et al., (1997) reported the maximum shoot proliferation in Murraya

koenigii, when the shoot-forming region was in direct contact with the medium surface or

slightly embedded into the medium. Similarly, Bansal and Chibbar (2000) also observed that

vertically placed nodal segments of Madhuca latifolia differentiated more shoots than

explants placed horizontally. Sometimes the vertically placed nodal explants differentiated

more shoots than explants placed horizontally. This could possibly be due to the influence on

polarity of growth regulator transport and directed supply of nutrients (Durzan, 1984).

Shimada et al., (2007) reported that the frequency of adventitious bud formation in Begonia

was dependent on the position of leaf explants on the medium. The abaxial orientation of leaf

explants of Bacopa monnieri on medium can induce fast shoot bud regeneration (Joshi et al.,

2010).

There is great importance of the size of explants to be cultured. Larger the explants,

poor will be the response. The small explants are more easily directed by the substances

contained in the medium. Okazova et al., (1967) reported that small explants are more likely

to form callus while larger explants maintain greater morphogenetic potentiality. This may be

due to the available food reserves and growth regulators which proved useful in the initiation

16

of new growth (Anderson, 1980). Pati et al., (2008) also reported that 3 cm long nodes having

one axillary bud gave quicker bud induction in Aegle marmelos.

Sometimes, the reproductive parts were also used for the regeneration. Regeneration

through inflorescence calli was observed in Aerva tomentosa (Murgai, 1959). Vazquez and

Short, (1978) reported the callus induction from floral parts of African violet. Immature

inflorescences of ginger resulted in the conversion of floral buds into plantlets directly

without intervening callus phase when cultured on MS medium (Babu et al., 1992). Zhong et

al., (1993) developed the regeneration protocol from inflorescence pieces of Beta maritima.

The highest numbers of shoots were regenerated from immature floral stems of Crinum

macowanii (Slabbert et al., 1995). Hannweg et al., (1996) cultured the pieces of inflorescence

stems of Bowiea volubilis. Nirmal and Sehgal (1999) has been developed the protocol for the

micropropagation of Ocimum sanctum through young inflorescence explants. Salvi et al.,

(2000) observed the shoot proliferation from immature inflorescence of Curcum longa. Tyagi

et al., (2005) induced, direct somatic embryogenesis from mature zygotic embryos of

Capparis decidua. Asnita and Norzulaani (2006) reported shoot-like structures from the male

inflorescence of Musa acuminata cultured on MS medium supplemented with BAP.

Shankramurthy and Ksishna (2006) observed the luxuriant mass of callus on MS medium

supplemented with IBA and Kinetin from the immature ovaries of the inflorescence segments

of Embelia ribes. Sharma and Mohan (2006) developed a novel method of shoot regeneration

of Chlorophytum borivilianum from immature floral buds. Inflorescence apices are suitable

explants for the rapid in vitro propagation of Musa species (Resmi and Nair, 2007). Nirmal

and Sehgal (2010) reported the regeneration of Ocimum sanctum using young inflorescences

of mature plants.

2.1.2 Culture media

Growth and morphogenesis of plant tissues in vitro are largely governed by the composition

of the culture media. Although the basic requirements of the cultured plant tissues are similar

to those of whole plants, in practice, nutritional components promoting optimal growth of a

tissue under laboratory conditions may vary with respect to the particular species. Media

compositions are therefore formulated considering specific requirements of a particular

culture system and a number of media have been devised for specific tissues and organs. In

vitro growth of plants is largely determined by the composition of the culture medium. The

importance of nutrition in plant tissue culture is initially reported by Gautheret (1955). The

main components of most plants tissue culture media are mineral salts, sugar as carbon

17

source and water. Other components may include organic supplements, growth regulators and

a gelling agent (Gamborg et al., 1968; Gamborg and Phillips, 1995). Although, the amounts

of the various ingredients in the medium vary for different stages of culture and plant species.

The basic MS (Murashige and Skoog, 1962) and LS (Linsmaier and Skoog, 1965) are most

widely used media. During the past decades, many types of media have been developed in

plant tissue culture (Street and Shillito, 1977; Pierik, 1989; Torres, 1989). Media

compositions have been formulated for the specific plants and tissues (Nitsch and Nitsch,

1969). Some tissues respond much better on solid media while others on liquid media. As

such, no single medium can be suggested as being entirely satisfactory for all types of plant

tissues and organs. Different culture media are proposed by the different scientists from time

to time are varies in salt concentrations from each other’s. Some of the earliest plant tissue

culture media were developed by White (1943) and Gautheret (1939). All subsequent media

formulations are based on White’s and Gautheret’s media. Humidity in the culture vessel and

osmotic potential of the medium affects the growth and development of plantlets in vitro in

different ways (Brown et al., 1979; Ziv et al., 1983).

Some common media used to fulfill the requirements of cultured tissue are MS (Murashigue

and Skoog, 1962), Gamborg (1968), Nitsch and Nitsch (1969), Gressholf and Doy (1972),

Eeuwens (1976), Llyod and McCown (1980), Branton and Blake (1983). Murashique and

Skoog (1962) is the most widely used medium, especially in procedures where plant

regeneration is the main objective.

There are some examples where modified MS medium have also been used viz.

Moringa pterygosperma (Mohan et al., 1995), Hovenia dulcis (Echeverrigaray et

al., 1998), Lagerstroemia reginae (Sumana and Kaveriappa, 2000), Bambusa

vulgaris (Ndiaye et al., 2006). Adventitious shoots were induced from the hypocotyl

explants of Sesbania rostrata on Nitsch’s medium (Nitsch, 1969). Mukhopadhyay and

MohanRam (1981) used Gamborg's B5 medium for the multiplication of Dalbergia

sissoo. Whereas Datta and Datta (1983) obtained multiple shoots from nodal

explants of Dalbergia sissoo on MS medium supplemented with vitamins of

Gamborg's B5 medium and NAA. Reddy et al., (1987) also used MS and B5 media

for callus initiation in Ricinus communis. Muralidharan and Mascarenhas (1987)

reported somatic embryogenesis in Eucalyptus citriodora on semisolid agar based B5 medium

supplemented with NAA and increased sucrose concentration (5%). Dewan et al., (1992) also

observed the higher number of shoots (6.3) on B5 medium in Acacia nilotica. Sarasan et

al., (1994) used MS and B5 medium for induction of callus and somatic

18

embryogenesis in Hemidesmus indicus. Agretious et al., (1996) was observed

better shoot multiplication on MS medium as compared on B5 and White medium

for regeneration in Alpinia calcarata. Babu et al., (2000) cultured nodal explants of

Murraya koengii for regeneration on Woody plant basal medium (WPM). Bhargava et al.,

(2003) reported the formation of globular proembryonic mass of callus on MS

medium after 40-50 days of incubation, and then transfer to B5 medium for fragile

snowy callus in Phoenix dactylifera. Sharada et al., (2003) used MS medium for

shoot induction and B5 or WPM medium for root development in Celastrus

paniculatus.

The superiority of WPM medium on the regeneration was reported in different plants by

different workers viz., Annona squamosa (Lemos and Blake, 1996), Prunus armenica

(Tornero et al., 2000), Quercus floribunda (Purohit et al., 2002) Camellia reticulata (Jose

and Vieitez, 2003), Cinnamomum camphora (Babu et al., 2003) Vitis thunbergii (Lu, 2005),

Garcinia indica (Chabukswar and Deodhar, 2006), Capparis spinosa (Musallam et al., 2011),

Salix tetrasperma (Khan et al., 2011).

The highest frequency of shoot bud proliferation from the cultures of Crataeva

adansonii was observed on MS medium followed by LS medium (Sharma et al.,

2003). Purohit and Kukda (2004) reported the maximum number of shoots on MS

medium followed by SH, WP, B5, and White’s media in Wrightia tinctoria. MS

medium was the most effective for in vitro shoot multiplication from nodal

explants of Holarrhena antidysenterica, amongst the five different basal media assayed

viz. B5, Nitsch, WPM, MS, and Knop’s media (Kumar et al., 2005). WPM medium was

found to be superior to MS medium for the induction of multiple shoots in Tinospora

cordifolia (Raghu et al., 2006). Moreover, shoots of Mucuna pruriens were much longer and

more vigorous and produced more in number on half-strength MS medium than in B5

medium (Faisal et al., 2006). Sotelo and Monza (2007) reported that the shoots of Eucalyptus

maidenii developed on the Quoirin and Lepoivre (1977) basal medium had the best shape,

size and colour. Chorabik (2007) used the two types of media (MS and SH) containing macro

and microelements, enriched with myoinositol glutamine, casein hydrolysate, vitamins and

sucrose for the micropropagation of Abies grandis. Multiple shoots were induced from the

nodal segments of Syzigium cumini inoculated on WPM medium (Remashree et al., 2007).

Tamta et al., (2008) reported the highest numbers of shoots regenerated in Quercus

semecarpifolia on WPM medium. Hazeena and Sulekha (2008) used MS medium for callus

induction and plantlet regeneration using cotyledons explants of Aegle marmelos. Park et al.,

19

(2008) reported adventitious shoot formation in Salix pseudolasiogyne in WPM medium

supplemented with BAP, Zeatin and GA3. Lamrioui et al., (2009) tried MS (1962), Quoirin

and Lepoivre (1977) and Knop (1965) medium for the in vitro germination of Prunus avium.

Thirunavoukkarasu et al., (2010) used MS and WPM medium for plantlet regeneration

through nodal segments of Dalbergia sissoo. MS and WPM medium with different

combinations of cytokinins and auxins were tried for different phases of micropropagation of

Fraxinus micrantha (Bisht et al., 2011). The plantlets of Punica granatum regenerated on MS

medium were found to have better survival compared to WPM medium (Patil et al., 2011).

Bhore and Preveena (2011) used MS, N6 and B5 media to initiate in vitro cultures of

immature zygotic embryos of Mimusops elengi. The best in vitro root development in

Warburgia ugandensis was observed on half strength WPM medium (Kuria et al., 2012).

Plant cells and tissues in culture medium lack autotrophic ability and therefore need external

carbon for energy. The most preferred carbon energy source in plant tissue culture is sucrose.

It is generally used at a concentration of 2-5% while autoclaving the medium sucrose is

converted to glucose and fructose. Sucrose plays an important role in vascular tissue

differentiation. Maltose, galactose, lactose and mannose are the other sources of carbon.

Other carbohydrates may be used occasionally, but none has shown consistent superiority

over sucrose. Ramawat and Arya (1977) studied the effect of carbohydrate on callus culture

of Ephedra gerardiana and Ephedra foliata. Muralidharan and Mascarenhas (1987) reported

somatic embryogenesis in Eucalyptus citriodora on semisolid B5 medium supplemented with

increased sucrose concentration (5%). MS medium supplemented with 2% sucrose was

optimal for culturing of shoot tips of Tamarindus indica (Kopp and Nataraja, 1990). The

supplementation 2-6% sucrose in MS medium supported best root development in Eucalyptus

sideroxylon (Cheng et al., 1992). Marino et al., (1993) reported in vitro proliferation and

rooting capacity of Prunus armeniac on modified MS medium enriched with varying growth

regulator concentrations and sucrose (58.4 mM) or sorbitol (116.8 mM) as main carbon

energy sources. Bennett et al., (1994) reported that 2% sucrose is sufficient for multiplication

and rooting in Eucalyptus globulus. Franca et al., (1995) found that 3% sucrose is effective

for shoot initiation from cotyledonary node of Stryhnodendron polyphythum.

Supplementation of sucrose (3%) and glucose (4%) were the best carbon sources for

proliferation and rooting phases of Quercus suber (Romano et al., 1995). Kumari et al.,

(1998) observed that 2% sucrose has been found more effective for the development of

globular embryos in Terminalia arjuna. Chetia and Handique (2000) found that 3 per cent

sucrose is required for multiple shoot induction in Plumbago indica. Deb (2001) reported the

20

germination of somatic embryos of Melia azedarach on MS medium containing 2 per cent

sucrose. WPM medium supplemented with 1.5% sucrose was optimal for shoot proliferation

from terminal axillary buds of Alnus nepalensis (Thakur et al., 2001). Shahzad and Siddique

(2001) reported that 2-3% sucrose was required for callus induction as well as for shoot

proliferation in Melia azedarach. Among the different carbon sources, i.e., fructose,

galactose, maltose, mannose, and sucrose at 3% (w/v), sucrose supported the best caulogenic

response in Sesbania rostrata (Jha et al., 2002). The medium containing 88 mM of sucrose

was used for the multiplication of Tectona grandis (Gangopadhyay et al., (2003). The highest

number of somatic embryos in Larix leptolepis was noticed on medium containing 0.2M

maltose (Kim and Moon, 2007). Parkash and Staden (2008) reported the induction of

maximum number of shoots from nodal explants of Searsia dentata on MS medium

containing 3% sucrose. The medium containing 4.0% sucrose significantly increased the

number of secondary embryos in Juglans regia (Vahdati et al., 2008). Jain et al., (2008)

reported that, 3 per cent sucrose was preferred carbon source both in terms of growth and

preventing shoot tip necrosis compared to glucose, maltose and fructose at equimolar

concentrations in Harpagophytum procumbens. The supplementation of 2% sucrose was

efficient in plantlet regeneration from the cotyledonary nodes explants of Azadirachta indica

(Reddy et al., 2006), Holarrhena antidysenterica (Mallikarjuna and Rajendrudu, 2009) and

Eucalyptus camaldulensis (Dibax et al., 2010). The best shoot multiplication in Amygdalus

communis was obtained on MS media containing 30 g l-1 sucrose (Akbas et al., 2009).

Plantlets of Simmondsia chinensis regenerated on 0.5% sucrose formed fine and thick roots

(Mills et al., 2009). The MS medium supplemented with higher levels of sucrose (4%)

showed significantly lower frequency of mature somatic embryos in Eucalyptus

camaldulensis, compared to basal medium containing lower concentrations of sucrose

(Prakash and Gurumurthi, 2010). MS medium supplemented with 1.0% sucrose induced the

highest number of somatic embryos in Schisandra chinensis (Chen et al., 2010). Chaari-

Rkhis et al., (2011) used 30 g l-l of mannitol for regeneration in Olea europaea. Gadidasu et

al., (2011) reported the supplementation of 2 % sucrose resulted best organogenesis in

Streblus asper.

Growth additives such as activated charcoal, silver nitrate, silver thiosulphate, ascorbic acid,

jasmonic acid and polyamines cannot strictly be defined as plant growth regulators but they

exert growth modulating effects and may play a novel mean of overcoming recalcitrance

problems of woody plants (Gaspar et al., 1996; Benson, 2000).

21

Addition of yeast extract to MS medium supplemented with NAA and Kn increased

the number of differentiated roots in Dalbergia lanceolaria (Anand and Bir, 1984).

Lakshmi Sita and Shobha Rani (1985) obtained multiple shoots in Eucalyptus grandis on MS

medium supplemented with additional thiamine. Mittal et al., (1989) obtained multiple shoots

from axillary buds of Accacia auriculiformis on Gamborg’s (B5) basal medium supplemented

with coconut milk and BAP. Sita and Swami (1992) also supplemented growth adjuvants like

coconut milk, casein hydrolysate and adenine sulphates to the media for direct organogenesis

and somatic embryogenesis in Dalbergia latifolia. Positive effect of CM in nutrient medium

was developed in Hemidesmus indicus (Sarasan et al., 1994), Elaeocarpus robustus (Roy et

al., 1998). Multiple shoot induction was reported on MS medium supplemented with coconut

milk (5-15%) in Alpinia galanga (Mustafa and Hariharan, 1997) and in Holostemma ada-

kodein (Martin, 2002). Desphande et al., (1998) observed that the MS medium supplemented

with 1-2 mg l-l of adenine sulphate is sufficient for but break and multiple shoot induction in

Ficus riligiosa. Deb (2001) used casein hydrolysate (200 mg l-l) for the induction of

embryogenic callus from imbibed seeds of Melia azedarach. Kaur et al., (1996) reported

maximum shoot bud induction from the cotyledonary nodal explants of Acacia senegal on

MS medium supplemented with adenine sulphate (25.0 mg l-1), ascorbic acid (10.0 mg l-1)

and glutamine (146.0 mg l-1

). Gangopadhyay et al., (2003) used MS medium supplemented

with 0.27 mM adenine sulphate for the multiplication of Tectona grandis. Shrivastava et al.,

(2006) reported shoot differentiation from the cotyledonary nodes and leaves segments of

Cassia senna when cultured on MS medium supplemented with BA, adenine sulphate as well

as complex nitrogenous supplement namely coconut milk (CM). Maximum shoot

proliferation was achieved from nodal explants on MS medium supplemented 135.7 mM

adenine sulfate (Vengadesan et al., 2003). Reddy et al., (2006) obtained the maximum shoot

proliferation from nodal explant of Azadirachta indica inoculated on MS medium

supplemented with 40 mg l-l adenine sulphate, 100 mg l-l glutamine, 10 mg l-l thamine HCl.

Addition of CH or CM induced the development of shoots with profuse callus in Holarrhena

antidysenterica (Mallikarjuna and Rajendrudu, 2009). The addition of casein hydrolysate

significantly increased the number of shoots per explants in Crataeva nurvala (Babbar et al.,

2009), Pongamia pinnata (Belide et al., 2010). Negi et al., (2011) observed enhanced shoot

growth in Cassia auriculata by adding adenine sulphate (25.0 mg l-1), ascorbic acid (20.0 mg

l-1) and L-glutamine (150.0 mg l-1).

Activated charcoal is commonly used in tissue culture media to improve cell

growth and development (Pan and Staden, 1998; Thomas, 2008). The beneficial

22

effects of AC may be attributed to its irreversible adsorption of inhibitory

compounds in the culture medium and substantially reduce the toxic metabolites,

phenolic exudation and exudate accumulation (Fridberg et al., 1978; Thomas,

2008). Rahman et al., (1993) studied the effects of activated charcoal (0.3 per cent w/v) on

shoot elongation in Caesalpinia pulcherrima. Gulati and Jaiwal (1996) used 1% activated

charcoal in medium for root induction in Dalbergia sissoo. Deshpande et al., (1998) also

observed the effects of activated charcoal (0.3 per cent w/v) on shoot elongation in Ficus

religiosa. Mao et al., (2000) also reported the effect of activated charcoal on in vitro

root induction of Litsea cubeba and Sharda et al., (2003) in Celastrus paniculatus.

Similarly, Dibax et al., (2005) found that in addition to suppressing phenolics and

thus browning, adding activated charcoal to the culture medium for regenerating

Eucalyptus enhanced the elongation of shoots and made the leaves dark green and

vigorous. Beneficial effects of activated charcoal were also found on multiple

shoot induction from nodal explants of Wattakaka volubilis (Chakradhar and

Pullaiah, 2006). Agarwal and Kanwar (2007) reported maximum root development

in Morus alba on MS basal medium supplemented with 0.005g l-1 activated

charcoal. Khaled et al., (2009) found that the absorption of inhibitory compounds

form medium or explants by AC resulted in rooting in Ficus anastasia. Addition of

activated charcoal into medium significantly improved the growth of regenerated

shoots of Populus trichocarpa (Kang et al., 2009). A high concentration of activated

charcoal (5% w/v) was optimum for the induction of roots from the regenerated plants of

Commiphora mukul (Kant et al., 2010). In Acacia nilotica, the highest number of shoots

and their elongation was achieved when 200mg l-l activated charcoal was added in

MS medium (Dhabhai and Batra, 2010). Suranthran et al., (2011) reported the best the

growth and development on MS medium supplemented with 2 g l-l AC which significantly

increased plantlet height as well as root length in Elaeis guineensis. The supplementation of

2g l-1

activated charcoal in the culture medium is optimum for shoot elongation in Ocotea

porosa (Pelegrini et al., 2011). Highest root development response of regenerated shoots of

Morus macroura was observed in half strength MS medium supplemented with 4 µM IBA

and 0.1% activated charcoal (Akram and Aftab, 2012).

Gelling and solidifying agents are commonly used for preparing semisolid or solid tissue

culture media. Agar-agar is the most commonly used at the concentration of 0.8% in culture

medium. Use of high concentration of agar makes the medium hard and prevents the

diffusion of nutrients into tissues. In employing a gel medium it is significant to

23

consider both the gel concentration and the quality of the gelling agent. High

concentration of agar, resulting in an excessively hard gel, can inhibit growth of

excised plant tissues. The required concentration of agar should be established

systematically by considering specific needs of each case. Yamuna et al., (1993)

used 6 % agar during regeneration of Cephaelis ipecacuanha. Echeverrigaray et

al., (1998) used MS medium supplemented with 0.7 % agar for the regeneration of

Hovenia dulcis. Naik et al., (2000) used 6% agar-agar during the micropropagation of

Punica granatum. Mroginski et al., (2003) added 0.7% agar to solidify the media used for

micropropagation of Toona ciliata. Uddin et al., (2005) used 5.0 g l-l agar for the

solidification of media used for the micropropagation of Peltophorum pterocarpum. MS

medium solidified with 10 g l-1 of agar was used for the multiplication of Eucalyptus

maidenii (Sotelo and Monza, 2007). All the media were solidified with 0.7%

bacteriological agar-agar for the regeneration of Stereospermum personatum

(Shukla et al., 2009). Supplementation of 6 g l-l agar was resulted best

solidification response and culture establishment in Eucalyptus camaldulensis (Dibax

et al., 2010). Addition of 0.7% agar-agar to the medium resulted best results in Boscia

senegalensis (Khalafalla et al., 2011). Bisht et al., (2011) used MS and WPM medium

containing 0.4% agar for the culture establishment of Fraxinus micrantha.

Plant cells and tissues require optimum pH for growth and development in cultures. The pH

affects the uptake of ions, hence it must be adjusted in between 5-6.0 by adding 0.1N NaOH

(or) Hcl usually the pH higher than six results in a fairly hard medium whereas pH below five

does not allow satisfactory solidification of medium.

2.1.3 Cultural conditions

Light and temperature are the major environmental factors which effects the

vascular tissue differentiation. Most of the cultures grow well within a wide range

of photoperiods, light intensities and optimal temperature (White and Risser, 1964)

whereas some cultures are temperature sensitive (Staritsky, 1970). Cultures are

usually maintained at a constant temperature around 25±20C and 16 hrs

photoperiod followed by 8hrs dark. It appears that the best light exposure period

for a given tissue culture is dependent upon the intensity of the illumination

employed, and probably other factors. Light intensity has influence on biological

effectiveness of the growth regulators added to the growth medium as well as to

affect the endogenous hormone balance in the tissues. When constant conditions are

satisfactory, it may still be necessary to establish optimum temperatures for specific cases.

24

Nitsch and Nitsch (1967) observed that 9-hr daily exposure period using 7000 lux

intensity, regenerated maximum number of shoots in Plumbgo. Calleberg and

Johansson (1993) studied that the direct regeneration was stimulated when the anther cultures

were incubated at 200C. Gupta et al., (1981) obtained multiple shoots from 20 year

old tree of Eucalyptus citriodora when cultured on MS medium incubated at 150C

in continuous light followed by incubating culture at 250C in 16 hours photoperiod.

A high rate of multiplication in Eucalyptus tereticonis has been achieved on MS medium at a

slightly higher temperature (30-320C) (Das and Mitra, 1990). The cultures of Capparis

decidua were incubated at 28 ± 20C with 60% relative humidity and 35-43 µ mol m-2 s-1

photon flux density for 12 h/d photoperiod (Deora and Shekhawat, 1995). All the cultures of

Triphyophyllum peltatum were kept under a 14/10-h (day/night) photoperiod (Bringmann and

Rischer, 2001). Cultures of Melia azedarch were incubated in light at 12-h photoperiod (Deb,

2001). Rajore et al., (2002) reported the multiple shoot formation from nodal

explants of Jatropha curcas inoculated on MS medium incubated at 25±2°C for 16

hours photoperiod. The cultures of Phoenix dactylifera were shifted from dark to

14hr. photoperiod for callus induction (Bhargava et al., 2003). All the cultures of

Andrographis paniculatus were incubated at 22±2 0C provided with 12-h of photoperiod

(Nagaraja et al., 2003). Best growth in cultures of Capparis decidua was reported at 28±10C

(Tyagi et al., 2005). Raghu et al., (2006) noticed best response in Tinospora cordifolia when

incubated under a 10-h photoperiod. Dibax et al., (2010) maintained the cultures of

Eucalyptus camaldulensis in darkness in the growth chamber for 60 days. Girijashankar

(2011) incubated the cultures of Acacia auriculiformis under 16 h photoperiod and 8 h dark

with light intensity of 50µE/m2/s provided by white fluorescent tube lights and at the

temperature 28±2°C. Negi et al., (2011) applied a light regime of 14 hours with 100µmol m-2

s-1

light intensity provided by cool-white fluorescent tubes at 25± 2ºC followed by 10 hrs.

dark period to the cultures of Cassia auriculata. Different temperature conditions ranging

from 23 to 30°C were provided to the cultures of Dalbergia sissoo, however, optimum results

were obtained at 26±1°C and 16/8 h (light/dark) photoperiod (Ali et al., 2012).

2.1.4 Plant growth regulator

Regulation of developmental process in plant tissue culture generally requires the addition of

plant growth regulators to the medium. Plant growth regulators have a significant influence

on shoot regeneration during the initial induction phase (Matt and Jehle, 2005). The growth,

differentiation and organogenesis of tissues become feasible only on the addition of one or

25

more plant regulators to a medium. The ratio of growth regulators required for root and shoot

induction varies considerably with the tissue, which seems directly correlated to the quantum

of growth regulators synthesized at endogenous levels within the cells of the explants

(Razdan, 2003). The age of the mother plant, the conditions under which it has been growing

and the season at which explants are taken, are influenced by the level of naturally occurring

auxins in them (Cassells, 1979). Skoog and Miller (1957) reported that morphogenesis of in

vitro cultured tissues as well as plant development were regulated by plant growth regulators

especially auxins and cytokinins.

Auxins promote cell division and root differentiation. IBA, NAA, IAA, 2, 4-D, etc. are very

widely used as auxins in micropropagation and are incorporated into nutrient media to

promote the growth of the callus, cell suspensions or organs and to regulate morphogenesis,

especially in conjunction with cytokinin. Cytokinins like BAP, Kinetin, Zeatin, etc. are

responsible for all cell division and shoot differentiation. BAP has been the most effective

cytokinin for shoot tip meristem and bud cultures followed by Kinetin (Murashige, 1974).

Cytokinin has been regularly incorporated into tissue culture for shoot regeneration (George

and Sherrington, 1984). The ratio of the auxin to the cytokinin determining the type

of culture established or regenerated, a high auxin to cytokinin ratio generally

favours root formation, whereas a high cytokinin to auxin ratio favours shoot

formation and intermediate ratio favours callus production.

Sehgal (1975) cultured the leaf and petiole segments of Begonia

semperflorens on modified White's basal medium supplemented with various

growth regulators. Goyal and Arya (1979) observed the regeneration in Posopis cineraria

on MS medium supplemented with different concentrations and combinations of Kinetin,

IAA, IBA and BAP. Nodal explants of Prunus cerasus responded best on MS

medium fortified with BAP, IBA and GA3 (Curovic and Ruzi, 1987). Muralidharan

and Mascarenhas (1987) reported somatic embryogenesis in Eucalyptus citriodora on B5

medium supplemented with NAA. Kopp and Nataraj (1990) regenerated plantlets of

Tamarindus indica on MS medium supplemented with 2.0 mg l-l BAP. Multiple

shoots were obtained from cotyledonary nodes of Dalbergia latifolia on MS

medium fortified with BAP (Sita and Swamy, 1992). Reddy et al., (1998) observed the

maximum number of shoots from mature nodal explants of Gymnema sylvestre, on the

medium containing BAP (5.0 mg l-l) and NAA (0.2 mg l-l). Ajithkumar and Seeni (1998)

also reported that in Aegle marmelos, BAP produced longer shoot than Kinetin.

Kathiravan and Ignacimuthu (1990) reported that the combination of BAP and

26

Kinetin produced maximum number of shoots in Canavalia virosa.

Supplementation of BAP (1.5 mg l-l) + NAA (0.1 mg l-l) have been found to show a

good response of shoot proliferation in Vitex negundo (Thiruvengadam and

Jayabalan, 2001). Multiple shoots were initiated from cotyledonary nodes excised from in

vitro grown seedlings of Acacia catechu on MS medium adjuvanted with 1 to 100 µM of BA

(Sahni and Gupta, 2002). Rathore et al., (2004) noticed the multiple shoot formation from the

nodal explants of Syzygium cuminii on MS medium supplemented with BAP (9.0 µM).

Tayagi et al., (2005) reported that the fortification of 2, 4-D induced callus mediated

embryogenesis in Capparis decidua. Gopi and Vatsala (2006) reported the potential of 2, 4-D

(0.1-5.0 mg l-l) + NAA on callus induction in Gymnema sylvestre. Chabukswar and Deodhar

(2006) reported multiple shoot induction in Garcinia indica in WPM medium supplemented

with 8.9 µM BA and 0.5 µM thidiazuron (TDZ). Singh and Lal (2007) reported that media

supplemented with BAP (1.0 mg l-l) in combination with NAA (2.0 mg l-l) supported hundred

per cent callus induction from hypocotyl and cotyledonary leaf segments of Leucaena

leucocephala. Siddique and Anis, (2007) developed an efficient, rapid and

reproducible plant regeneration protocol using nodal explants of Cassia

angustifolia cultured on MS medium supplemented with BAP and TDZ. The

maximum per cent regeneration in Olea europaea was achieved on the medium

supplemented 2.22 µM BAP (Peixe et al., 2007). Kalimuthu et al., (2007) developed a

regeneration protocol for Jatropha curcas using nodal explants on MS supplemented with

BAP (1.5 mg l-1

), Kn (0.5 mg l-1

) and IAA (0.1 mg l-1

). The maximum numbers of shoots in

Aegle marmelos were obtained on the medium supplemented with 8.84 µM BAP in

combination with 5.7 µM IAA (Pati et al., 2008). The best shoot regeneration from the callus

of Aegle marmelos was obtained on MS medium containing 8.8 µM BA and 2.85 µM IAA

(Hazeena and Sulekha, 2008). The highest shoot regeneration frequency as well as shoot

length was induced from nodal explants of Pterocarpus marsupium on MS medium amended

with 4.0 µM BA, 0.5 µM IAA and 20 µM adenine sulphate (Husain et al., 2008). Nodal

explants of Searsia dentata produced multiple shoots when cultured on MS medium

supplemented with 0-2.5 µM BA (Prakash and Staden, 2008). Best shoot induction response

was observed from the nodal explants of Ficus religiosa on MS basal medium supplemented

with 0.5 mg l-1 BAP + 0.1 mg l-1 IAA (Hassan et al., 2009). High shoot multiplication was

observed from axillary buds of Oroxylum indicum inoculated on MS medium fortified with

4.43 µM BAP (Gokhale and Bansal, 2009) and from axillary buds of Morus alba inoculated

on the medium supplemented with 0.5mg l-1 BAP (Balakrishnan et al., 2009). Prakash and

27

Gurumuurthi (2010) obtained callus from zygotic embryos of Eucalyptus camaldulensis

inoculated on MS medium containing 0.5 mg l-l BAP + 0.1 mg l-l NAA. MS medium

containing 3 mg l-1 BA and 0.05-0.1 mg l-1 NAA was most effective in induction of shoots

from nodal explants and shoot tips of Crataeva adansonii (Tyagi et al., 2010). The nodal

explants of Dalbergia sissoo exhibited maximum response on the MS medium fortified with

6.6 µM BA + 1.14 µM IAA (Thirunavoukkarasu et al., 2010). Okere and Adegeye (2011)

reported the best in vitro regeneration in Khaya grandifoilolia on MS medium supplemented

with 1.0 mg l-l BAP + 0.1 mg l-l NAA and 10 mg l-l of adenine sulphate. Zygotic embryos of

Gomortega keule were cultured on WPM medium supplemented with 0.1 mg l-1 NAA and 1.0

mg l-1 BAP (Mun˜oz-Concha and Davey, 2011). Maximum per cent shoot induction and

multiplication in Maerua oblongifolia was achieved on MS medium containing 2.0 mg l-1

BAP (Rathore and Shekhawat, 2011). The media supplemented with 0.2-2 mg l-1 BAP + 0.1-

1 mg l-1 NAA was sufficient for establishment of the culture of Punica granatum (Patil et al.,

2011). Ali et al., (2012) obtained the best in vitro shoot induction in Dalbergia sissoo on MS

medium containing 1.0 mg l-l BAP + 0.25 mg l

-l NAA. The highest numbers of shoots were

resulted from shoot tip explant of Balanites aegyptiaca on medium supplemented with 1.0

mg l-1 Kinetin + 0.2 mg l-1 NAA (El-Mekawy et al., 2012). Amina et al., (2012) observed

multiple shoots from the nodal explants of Helianthemum lippii on MS medium containing

BAP (0.25- 2.0 mg l-1).

2.1.5 Organogenesis

Organogenesis is the de novo production of plant organs (buds, shoots and roots) from

organized tissues or callus. Organogenesis refers to the development of adventitious organs

or primordia from undifferentiated cell mass in tissue culture by the process of

differentiation. In contrast to axillary bud proliferation, organogenesis proceeds de novo via

organization of meristems. It involves the induction of localized meristematic activity by

treatment with plant growth regulators. This leads to the formation of primordium and

eventually the formation of shoot. Micropropagation may be accomplished by either

organogenesis or somatic embryogenesis. Organogenesis involves differentiation of

microshoots and root at different time periods during plantlets development. Usually

microshoots are induced on tissues in a cytokinin rich medium and subsequently microshoots

are rooted in an auxin-enriched medium to give rise to plantlets. Organogenesis has been

introduced on callus, organ, cell and protoplast cultures in plants. However organogenesis is

greatly influenced by the genotype, physiological state of the explants, age of the explants

28

and the in vitro environment, both the light and temperature and the composition of the

medium, in particular plant growth regulators concentration.

Direct Organogenesis

Propagation through axillary bud multiplication is an easy and safe method for obtaining

uniformity and it also assures the consistent production of true-to- type plants within a short

span of time (George, 1993; Salvi et al., 2001). Direct organogenesis is regarded as the most

reliable method for clonal propagation because it upholds genetic uniformity among the

progenies (Beegam et al., 2007).

The direct organogenesis is reported in different tree species by different workers viz.

Eucalyptus torelliana and Eucalyptus camaldulensis (Gupta et al., 1983), Ficus religiosa

(Deshpande et al., 1998), Camellia sp. (Rajkumar and Marimuthu, 2000), Anacardium

occidentale (Hedge et al., 2000), Morus alba (Anis et al., 2003).

In Prosopis cineraria supplementation of Kinetin in combination with IAA induced

higher rate of shoot multiplication than BAP (Goyal and Arya, 1979; 1984). Direct

adventitious shoot formation in Cinnamomum verum was achieved on MS medium

supplemented with 0.1-1.0 mg l-1 Kinetin and BAP (Rai and Chandra, 1987). Mittal et al.,

(1989) observed the multiple shoots induction from cotyledonary nodes of Acacia

auriculiformis. Inomoto and Kitani (1989) developed a micropropagation protocol through

nodal segments of Cinnamomum aromaticum. Dass and Mitra (1990) reported maximum

number of shoots from nodal explants of Eucalyptus tereticornis inoculated on MS medium

supplemented with BAP (1.0 mg l-l) and NAA (0.1 mg l

-l). Mathew and Hariharan (1990)

also reported the adventitious shoot formation from the nodal segments of Syzygium

aromaticum. Rout and Das (1993) reported the multiple shoots induction from apical and

axillary meristems derived from seedlings of Madhuca longifolia. Singh et al., (1993)

observed direct regeneration from axillary bud of Acacia nilotica on MS and WPM medium

fortified with BAP (1.0 mg l-l). In vitro propagation of Moringa pterygosperma from

cotyledonary explants was achieved by Mohan et al., (1995). High-frequency shoot

proliferation was induced from intact seedlings of Murraya koenigii on MS medium

supplemented with 5.0 mg l-l BA (Bhuyan et al., 1997). Sharma and Padhya (1996) also

found the rapid multiplication of Crataeva nurvala through axillary buds on MS medium

supplemented Kinetin and BAP. Direct adventitious shoot formation in Cinnamomum verum

was achieved in WPM medium supplemented with 0.5-4 mg l-1 BAP and Kinetin (Mini et al.,

1997). Reddy et al., (1998) observed the maximum number of shoots from mature nodal

29

explants of Gymnema sylvestre on the medium containing BAP (5.0 mg l-l) and NAA (0.2 mg

l-l). Similarly, Ajithkumar and Seeni (1998) also achieved rapid clonal multiplication of Aegle

marmelos by enhanced axillary bud proliferation on MS medium supplemented with BAP

(2.5 mg l-l) + IAA (1.0 mg l

-l). Jagadishchandra et al., (1999) obtained multiple shoots from

the axillary buds of Pisonia alba on MS medium supplemented with BAP and Kinetin.

Philomina and Rao (2000) reported multiple shoot induction from apical and axillary

meristems of Sapindus mukorossi. Direct adventitious shoot formation in Cinnamomum

verum was achieved on WPM medium supplemented with 0.5-2 mg l-1 BAP and Kinetin

(Sheeja et al., 2000). Komalavalli and Rao (2000) established the protocol for direct

regeneration of Gymnema sylvestre through nodal segments. Mathew et al., (2001) reported

multiple shoot induction through the nodal explants of Garcinia indica and Garcinia gummi-

gutta. Maximum number of shoots were induced from cotyledonary node explants of Acacia

sinuata on MS medium containing 6.66µM BAP+4.65µM Kn (Vengadesan et al., 2002).

Rajore et al., (2002) reported multiple shoots induction from nodal segments of Jatropha

curcas on MS medium fortified with Kn (2.0 mg l-1

) and IBA (1.5 mg l-l). Shu-Hwa et al.,

(2002) reported the multiple shoot formation from seedling and mature explants of

Cinnamomum kanehirae. A high frequency shoot differentiation from nodal explants of

Morus alba was observed on MS medium supplemented with BAP and Kinetin (Anis et al.,

2003). Ramesh et al., (2005) developed an efficient protocol for the micropropagation of

Terminalia bellirica using cotyledonary nodes. Rathore et al., (2005) reported the higher

shoot multiplication through the nodal shoot segments of Maerua oblongifolia

inoculated on MS medium supplemented with various growth regulators. A rapid

multiplication of Nyctanthes arbour-tristis through in vitro axillary shoot

proliferation was reported by Siddique et al., (2006). An efficient, rapid and

reproducible plant regeneration protocol was successfully developed for Cassia

angustifolia using nodal explants on MS medium supplemented with BAP and TDZ

(Siddique and Anis, 2007). Direct shoot formation was achieved in Cinnamomum

camphora on MS medium supplemented with 1.0 mg l-1 BAP and 0.05-2.5 mg l-1TDZ

(Soulange et al., 2007). The highest frequency of shoot regeneration via direct

organogenesis was obtained from petiole explants of Populus ciliata on MS

medium supplemented with 1.5 mg l-l Kn and 0.1 mg l-l IAA (Thakur et al., 2008).

Shukla et al., (2009) developed the in vitro plantlet regeneration protocol from seedling

explants of Stereospermum personatum. Direct regeneration through nodal segments was

observed in Dalbergia sissoo (Thirunavoukkarasu et al., 2010). Multiple shoots were induced

30

from shoot tip explants derived from seedlings of Pterocarpus santalinus (Balaraju et al.,

2011). Explants taken from the seedlings of Sapindus trifoloatus yielded the maximum shoot

regeneration frequency on full-strength MS medium supplemented with 1.0 mg l-1 BAP

(Asthana et al., 2011). Shoot proliferation and elongation was achieved from shoot tips of

Warburgia ugandensis cultured on full strength MS medium containing 1.13 mg l-1 BAP and

0.11 mg l-1 Kn (Kuria et al., 2012).

Indirect Organogenesis

Callus production can be induced from a number of explants like leaf, roots, flower parts and

parts of seed. Explants like tuber, shoot tips, hypocotyl, leaf and stem have been used to

initiate callus with morphogenic potential (Chang and Chang, 1998; Manju and Subramanian,

1999; Kelkar and Krishnamoorthy, 1998).

Lakshmi (1979) raised plantlets of Eucalyptus citriodora from cotyledonary callus.

Shoot buds and root formation was observed from hypocotyl callus of Broussonetia

kasinoki (Ohyama and Oka, 1980). Regeneration was obtained from callus in Leucaena

leucocephala (Venketeswaran and Romano, 1982) and in Albizzia lebbeck (Upadhyay and

Chandra, 1983). Internodal segments of Dalbergia latifolia produced callus on MS media

containing IAA alone or IAA and IBA in combination (Rao, 1986). Vigorous callus

formation was noticed in Cinnamomum verum on MS medium supplemented with 2, 4-D and

BAP (Rai and Chandra, 1987). Adventitious shoot regeneration was reported through

callus culture in Eucalyptus camaldulensis (Murlidharan and Mascarenhas, 1987).

The regeneration of plantlets was achieved through callus derived from shoot tips and shoot

segments of Dalbergia latifolia on MS medium containing NAA and BAP (Rai and Chandra,

1988). Gharyal and Maheshwari (1990) observed callus induction from stem and petiole

explants of Cassia fistula and Cassia siamea and its differentiation into shoot buds.

Nandwani and Ramawat (1991) reported the callus induction and plantlet formation in

Prosopis juliflora. In vitro callus induction and plantlet regeneration from leaf tissue

of Aralia elata has been observed by Jhang et al., (1993). Remeshree et al., (1994)

developed the protocol for callus induction and differentiation of Aristolochia bracteolata.

Chakravarty and Goswami (1999) reported the callus initiation and regeneration of

plantlets from epicotyl explants of Citrus acida on MS medium containing BAP

and 2, 4-D. Calli were induced from different explants of Acacia mangium on MS basal

medium containing 9.05 µM 2, 4-D and 13.95 µM Kinetin (Xie and Hong, 2001). The

maximum shoots were regenerated when the callus derived from the nodal segments of

31

Kigelia pinnata transferred to the medium containing 3.0 mM TDZ and 0.5 mM NAA

(Thomas and Puthur, 2004). Callus formation and shoot differentiation was observed from all

explant of Saussurea obvallata cultured on MS medium containing BA and NAA (Dhar and

Joshi, 2005). Kumari and Shivanna (2005) established a protocol for callus induction

and in vitro regeneration of plantlets from calli derived different explants of

Desmodium oojeinense. Agrawal and Sardar (2006) described high frequency shoot

regeneration through leaflet and cotyledon derived calli of Cassia angustifolia.

High frequency of adventitious shoots were achieved from zygotic embryos derived

callus of Taxus wallichiana on ½ WPM basal medium supplemented with 2.5 mg l-1

BA (Datta et al., 2006).

The highest plantlet regeneration from callus was obtained on (1/4) MS medium

supplemented with 0.2 mg l-l Kn in Irvingia gabonesis (Fajimi et al., 2007).

Soulange et al., (2007) reported callus induction from leaf explants of Cinnamomum

camphora on MS medium containing 1.0 mg l-1 BAP + 0.005-5 mg l-1 TDZ. The highest

numbers of shoots were regenerated from the callus derived from leaf segments of Prunus

serotina on WPM medium supplanted with 9.08 µ M TDZ and 0.54 µM NAA (Liu and Pijut,

2008). Hasan et al., (2008) observed the callus induction and shoots differentiation in Cassia

obtusifolia on the medium having 2.0 mg l-1

2, 4-D + 0.2 mg l-1

Kn. Callus induction and

multiple shoot induction was observed from nodal segments of Sarcostemma brevistigma

cultured on MS medium supplemented with BA or Kinein alone or in combination with NAA

(Thomos and Shanker, 2009). Maximum per cent callus induction from leaf segments of

Citrus jambhiri was reported on MS medium supplemented with 2, 4-D (4.0 mg l -1) and

maximum regeneration through callus was noticed on medium fortified with 0.5 mg l-1 NAA

+ 1.0 mg l-1

BA (Savita et al., 2010). The highest frequency of callus induction was observed

from the nodal segments of Ficus religiosa on MS medium supplemented with 2.26 µM 2, 4-

D (Siwach et al., 2011). Friable callus derived through immature embryos of Zelkova sinica

regenerate shoots when cultured on WPM containing 5.4 µM NAA + 9.0 or 11.2 µM BA (Jin

et al., 2012). The medium supplemented with 0.10 mg l-1 BAP was found to be the best

concentration for shoot differentiation from the callus induced from axillary bud explants of

Michelia champaca (Abdelmageed et al., 2012).

32

Somatic embryogenesis

Somatic embryogenesis is defined as a process in which a bipolar structure, resembling a

zygotic embryo, develops from a non-zygotic cell without vascular connection with the

original tissue. Somatic embryos are used for studying regulation of embryo development,

but also as a tool for large scale vegetative propagation. Somatic embryogenesis is a multi-

step regeneration process starting with formation of proembryogenic masses, followed by

somatic embryo formation, maturation, desiccation and plant regeneration.

Somatic embryogenesis has been first reported by Steward et al., (1958) in

the cultures of Daucus carota. Muralidharan and Mascarenhas, (1987) reported somatic

embryogenesis from embryos of Eucalyptus citriodora cultured on semisolid agar based B5

medium supplemented with NAA and increased sucrose (5%) concentration. The somatic

embryogenesis was obtained from callus derived from immature embryos of Aesculus

hippocastanum cultured on MS medium supplemented with 3.0 mg l-1 2, 4-D, 1.0 mg l-1 Kn,

250 mg l-1 CH and 250 mg l-1 proline (Radojevic, 1988). Shoots were regenerated from

the hypocotyl derived calli of Albizia richardiana on B5 Medium supplemented BAP

(Tomar and Gupta, 1988). Zygotic embryos of Eucalyptus citriodora cultured on

medium containing 3.0 mg l-l NAA formed somatic embryos (Murlidharan and

Mascarenhas, 1987).

Immature zygotic embryos have been found to be a better initial material for somatic

embryo induction in many species, such as Prunus avium (Garin et al., 1997), Phoenix

canariensis (Huong et al.,1999), Larix leptolepis (Kim et al., 1999), Myrtle communis

(Canhoto et al., 1999) and Cryptomeria japonica (Igasaki et al., 2003).

Inamdar et al., (1990) reported somatic embryogenesis through callus derived from

shoot apices of Crataeva nurvala on MS medium containing 2,4-D. Somatic

embryogenesis has been achieved from callus derived from young leaf of Thevetia

peruviana, on MS medium containing 9 µM 2,4-D and 4.6 µM Kn (Kumar, 1992). The calli

transferred to the liquid medium supplemented with 0.5 mg l-1 IAA, formed somatic embryos

after 4 to 5 weeks incubation in Azadirachta Indica (Su et al., 1997). Somatic embryos

were induced from calli derived from hypocotyl explants of Sterculia urens cultured on MS

medium supplemented with 0.45 µM TDZ (Sunnichan et al., 1998). Somatic embryos

were obtained from hypocotyl of mature embryos of Eucalyptus globulus cultured on media

33

containing a high concentration of picloram or IBA, 2, 4-D (Nugent et al., 2001). Somatic

embryogenesis was induced from juvenile explants of Eucalyptus globulus cultured at 24°C

in darkness on MS medium supplemented with different growth regulator combinations

(Pinto et al., 2002). Bhargava et al., (2003) reported regeneration in Phoenix

dactylifera via somatic embryogenesis from callus transferred on MS and B5

medium supplemented with auxins (2, 4-D/IBA/IAA) alone or in combination with

cytokinins. Callus induced from cotyledon and mature zygotic embryo of Terminalia

chebula, produced somatic embryos on MS basal medium supplemented with 50 g l-1 sucrose

(Anjaneyulu et al., 2004). Plant regeneration via somatic embryogenesis was achieved from

embryogenic callus derived from immature zygotic embryos of Azadirachta indica on MS

medium supplemented with 1.11 µM BA and 4.52-6.78 µM 2, 4-D (Rout, 2005). Somatic

embryos were also induced from calli derived from leaflets of Litchi chinensis on

B5 medium containing 4.52 µM 2,4-D and 9.30 µM Kinetin (Raharjo and Litz, 2007).

Kim et al., (2007) obtained somatic embryos directly from cotyledon explants of

zygotic embryos of Podophyllum peltatum cultured on MS medium supplemented

with NAA. Pinto et al., (2008) developed a reproducible protocol for somatic

embryogenesis from mature zygotic embryos of Eucalyptus globulus. The highest induction

frequencies and rapid maturation of somatic embryos was induced from embryogenic calli

derived from the leaf explants of Phœnix dactylifera on MS medium supplemented with 1.0

mg l-1 ABA (Othmani et al., 2009). Somatic embryos were induced from the callus

transferred into MS liquid medium containing NAA (1.0 mg l-l) and BA (1.0 mg l

-l) in

Gymnema sylvestre (Ahmed et al., 2009). Somatic embryos were induced from the calli

derived from petiole and leaf explants of Olea europaea cultured on MS medium without

growth regulators in the dark (Lopes et al., 2009). MS medium supplemented with 12.25 µM

IBA + 4.56 µM Zeatin was the best medium for somatic embryo induction from petiole

derived calli of Olea europaea (Capelo et al., 2010). Shi et al., (2010) reported an efficient

protocol for somatic embryogenesis in Cinnamomum camphora. Somatic embryogenesis in

Gomortega keule was accomplished on MS medium fortified with 1.0 mg l-l 2,4-D and 1.0

mg l-l 2iP (Mun˜oz-Concha et al., 2011). The highest frequency of somatic embryos

induction in Murraya koenigii was recovered from zygotic embryo derived callus transferred

on medium containing 2.27-9.08 µM TDZ (Paul et al., 2011). Somatic embryos were induced

from immature zygotic embryos derived callus of Picea abies cultured on medium

supplemented with 2, 4-D and a cytokinin (Hakman and Arnold, 2012). Somatic embryos

34

were induced from the filaments of Aesculus hippocastanum when cultured on a medium

fortified with 2.5 µm l-l BAP and 5.0 µm 1-1 2, 4-D (Jörgensen, 2012).

2.1.6 Root Development

The development of a perfect plantlet is incomplete without the regeneration of

roots. The occurrence of a phase of dedifferentiation suggests that cells may produce a root

or shoot apical meristem, depending on the added growth regulator during the induction

phase. Root induction was observed in plant growth regulator free Gamborg's B5

medium as well as on 0.1 mg l-1 IAA supplemented medium in Albizzia lebbeck,

Casia fistula and Cassia siamea (Gharyal and Maheshwari, 1990). Regenerated

shoots of Prosopis cineraria developed roots by pulsing with 100 mg l-1

IBA for 4 h

and then culturing on growth regulator free half strength MS medium (Shekhawat et

al., 1993). In Moringa pterygosperma, half strength MS medium supplemented with

GA3 (0.2 mg l-l) produced roots within 7 days in 25 per cent cultures, however,

better rooting was developed with 0.2 mg l-l IBA (Mohan et al., 1995). Sharma and

Padhya (1996) observed root induction from regenerated shoots of Crataeva

nurvala within 7 days on MS medium fortified with 0.5 µ M NAA. Roots were

developed on excised shoots when they were transferred to half-strength MS containing 1.0

mg l-l IBA in Murraya koenigii (Bhuyan et al., 1997). Mustafa and Hariharan (1997)

noticed best root induction from regenerated shoots of Alpinia galanga on MS

medium supplemented with the combinations of NAA and IBA. MS medium (¼)

strength proved most suitable for root induction in Canavalia virosa (Kathiravan

and Ignacimuthu, 1999). A high frequency root development was observed from the

regenerated shoots of Morus indica cultured on medium fortified with 1.0 mg l-1 2, 4-D

(Chitra and Padmaja, 1999). Best rooting in Eucalyptus tereticornis was obtained on half-

strength, MS medium supplemented with 1.0�mg l-1 IBA (Sharma and Ramamurthy, 2000).

Sheeja et al., (2000) reported the root development in Cinnamomum verum on WPM medium

containing 0.5 mg l-1 each IBA and IAA.

Supplementation IBA (2.0 mg l-1) has been found to be effective for root formation

from the excised shoots of Alnus nepalensis (Thakur et al., 2001). In Cardiospermum

halicacabum roots could also be produced in plant growth regulator free media (Babber et al.,

2001). In vitro regenerated shoots of Acacia sinuata induced roots when transferred to half

strength MS medium supplemented with 7.36 µM IBA (Vengadesan et al., 2002). Joshi and

35

Dhar (2003) observed maximum frequency of root development in Saussurea

obvallata on MS half strength medium fortified with 2.5 µ m IBA. Ndoye et al.,

(2003) reported best root development in regenerated shoots of Belanites

aegyptiaca on higher concentration (20 mg l-l) of IBA or NAA. The best root

development was recorded on MS medium supplemented with 1.0 mg l-l NAA in Morus alba

(Anis et al., 2003). Iyer et al., (2005) observed the roots from regenerated shoots of

Michelia champaca on MS medium supplemented with IBA. Regenerated shoots of

Holarrhena antidysenterica were excised and rooted in auxin free MS basal medium

(Mallikarjuna and Rajendrudu, 2007). Vadodaria et al., (2007) found better root formation

on medium containing NAA (0.1 mg l-l) and 1% sucrose in Glycyrrhiza glabra. Higher

percentage of root induction on regenerated shoots of Populus ciliata was obtained on MS

medium supplemented with 0.1 mg l-l IAA (Thakur et al., 2008). Regenerated shoots of

Oroxylum indicum were rooted on half strength MS medium supplemented with 4.92 µM

IBA (Gokhale and Bansal, 2009). Aslam and Khan (2009) observed a high frequency root

development in Phoenix dactylifera on solid MS medium supplemented with 24.6 µM IBA,

however, the root length was higher in liquid medium. The regenerated shoots of Acacia

nilotica were rooted on half strength MS medium fortified with 0.5 mg l-l IBA (Dhabhai and

Batra, 2010). Root induction was observed from regenerated shoots of Aegle marmelos on

MS medium supplemented with 3000 ppm IBA (Warrier et al., 2010). Best rooting response

was observed on half strength MS medium containing IBA (1.0 µM) in Cassia sophera

(Parveen and Shahzad, 2010). Maximum per cent root induction response was obtained by

placing the regenerated shoots of Sapindus trifoliatus in liquid MS medium supplemented

with 1.0 mg l-1 IBA for 24 h and then transferring to the agar solidified MS medium devoid

of IBA (Asthana et al., 2011). Half strength MS basal without supplemented phytohormones

showed best rooting response when compared to all other treatments evaluated in Acacia

auriculiformis (Girijashankar, 2011). The regenerated shoots of Boswellia serrata were

rooted on the medium gelled with 0.6 mg l-l agar (Suthar et al., 2011). The best in vitro root

induction in Warburgia ugandensis was induced on half strength WPM containing 1.0 mg l- l

NAA (Kuria et al., 2012). The rooting response in Pongamia pinnata was enhanced on half-

strength MS media supplemented with 0.5 mg l-1

IBA (Kesari et al., 2012).

2.1.7 Hardening of the regenerants

The heterotrophic mode of nutrition and poor physiological mechanisms and lack of cuticle

on leaves to control water loss, tender the micro propagation plants vulnerable to the

transplantation, plants are acclimatised in suitable compost mixture (or) soil in pots under

36

controlled conditions of light temperature and humidity. Inside the glasshouse the plants

increase their resistance to moisture stress and disease. The plantlets have to become

autotrophic in contrast to their heterotrophic state induced in micropropagation culture.

Transfer of plantlets to soil is the most critical step in micropropagation. The plantlets are

maintained under highly protected conditions in vitro i.e. high humidity, low irradiance, low

CO2 levels and high sugar content.

It is a general observation that the step of transfer from tissue culture vessels to soil is

often very difficult because the in vitro produced plants are not well adapted to an in vivo

climate. Apart from the many adaptation problems of the leaf and shoot systems, the system

of root regeneration in vitro in agar-gelled media appears to be one of the most vulnerable

one. In many cases even negatively gravitropic roots appear in agar gelled media within glass

vessels. Furthermore, the in vitro formed roots do not function properly (fewer root hairs) in

vivo, are rather weak, and often die; in soil, in vitro formed roots often have to be replaced by

newly formed roots. As a consequence of the non-functional roots, transpiration outside the

glass vessels is too high and can result in the loss of many plants. Plants with a good root

system were transferred to small plastic pots containing vermiculite: perlite (1:1)

within a period of 10 days (Deshpande et al., 1996). Daneil et al., (1999) nourishes

the regenerated plantlets of Naregamia alata in vermiculite with a dilute solution

of nutrients. Regenerated plantlets of Litsea cubeba were transplanted into a

potting mixture of sand, loam and peat (1:1:1) for hardening (Mao et al., 2000).

Regenerated plantlets of Eucalyptus tereticornis were hardened in a non-sterile potting mix

at high humidity (Sharma and Ramamurthy, 2000). Sheeja et al., (2000) have reported 43%

establishment of Cinnamomum verum plantlets in soilrite. Rooted shoots of Holostemma

ada-kodien were transferred directly to small pots filled with sterile soilrite and

sand (1:1) ratio (Martin, 2002). In vitro raised rooted plants of Acacia sinuata were

hardened in a growth chamber at 80% relative humidity under 20µmolm-²s-¹ photon lux for

16 hours photoperiod at 35±2˚C (Vengadesan et al., 2003). The acclimatized plantlets of

Syzygium cuminii were transferred to polybags containing mixture of organic manure, garden

soil and sandy soil (1:1:1) for hardening (Rathore et al., 2004). The rooted plantlets of

Terminalia bellereica were transferred to pots and hardened under greenhouse conditions at

65% relative humadity (Ramesh et al., 2005). The regenerated plantlets of Azadirachta indica

were transferred to poly cup containing soil and vermicompost (3:1) and maintained under

high humidity (Reddy et al., 2006). Sharma et al., (2006) used sterilized vermi-compost and

37

soil mixture (1:3) in pots to acclimatize the regenerated plantlets of Vitex negundo .The

regenerated plantlets of Jatropha curcas were hardened on a mixture of decomposed coir

waste, perlite and organic compost in the ratio of 1:1:1 (Kalimuthu et al., 2007). All in vitro

rooted plants of Olea europaea were transferred into jiffy-pots filled with vermiculite-perlite

3:1 (v/v) substrate (Peixe et al., 2007). Soulange et al., (2007) also reported the establishment

of in vitro developed plantlets in pots containing top soil and compost (2:1).

For acclimatization the regenerated plantlets of Sterculia urens were transferred to

plastic cups containing autoclaved vermiculite (Hussain et al., 2008). Rooted shoots

of Aegle marmelos were transferred to glass bottles containing different carrier substrates viz.

autoclaved soil, soil, sand and FYM (1:1:1) and coconut husk and were supplemented with ½

MS plant salt mixture (Pati et al., 2008). Regenerated plantlets of Oroxylum indicum

were initially kept in distilled water in flasks covered with beaker for

approximately 8 days and finally transferred to soil: sand (1:1) in cups (Gokhle and

Bansal, 2009). The regenerated plantlets of Morus alba with well developed roots were

transferred to pots containing soilrite (Balakrishnan et al., 2009). The regenerated shoots

of Taxus baccata were transferred to sterilized soil mixture consisting of peat

moss, sand and soil at the rate of 1:1:1 (Abbasin et al., 2010). Well rooted plantlets

of Commiphora mukul were transferred to glass jars filled with quarter vermiculite

and wetted with Hoagland’s solution (Kant et al., 2010). Regenerated plants of

Gomortega keule were transferred to compost and covered with transparent plastic

bags for acclimatization (Muñoz-Concha and Davey, 2011). Use of coco peat as

hardening medium resulted in maximum survival during hardening phase of Acacia

auriculiformis (Girijashankar, 2011). Regenerated plantlets of Warburgia ugandensis were

watered with half strength WPM on alternate days to harden those (Kuria et al.,

2012).

2.2 Assessment of genetic fidelity using molecular markers

In vitro regeneration of plants involves the application of plant growth regulator, such as

auxins and cytokinins. Nevertheless, these plant growth regulators are known to be associated

with genetic instability in plants (Karp, 1989; Cullis, 1992). Changes to these growth

regulator habituations are known to be associated with genetic instability in plants. It is

believed that high concentrations of plant growth regulators can modify the frequency of

ploidy changes and point mutations. Factors such as explants source, time of culture, number

38

of subcultures, plant growth regulator, genotype, media composition, the level of ploidy are

capable of inducing in vitro variability (Silvarolla, 2000; Yu et al., 2008). Several

mechanisms governing somaclonal variation induced during subculturing includes gene

amplification, single nucleotide base change, transposon migration, altered methylation

states, chromosome instability, chromosome inversion, single gene mutations, translocations,

cytoplasmic genetic changes, ploidy changes, rearrangements and partial chromosome

deletions (Duncan et al., 1986, Yu and Buckler, 2006).

Variation also occurs as responses to the stress imposed on the plant in culture conditions and

are manifested in the form of DNA methylations, chromosome rearrangements and point

mutations (Phillips et al., 1994). Traditionally, morphological description, physiological

supervision, karyological analysis, biochemical estimations and field assessment were used to

detect any types of genetic variations, but presently molecular markers have complemented

over traditional methods to detect and monitor the genetic fidelity of tissue culture derived

plantlets and variety identification. Recently, molecular markers have been used in the

detection of variation or confirmation of genetic fidelity during micropropagation (Gupta et

al., 1998; Tyagi et al., 2007). Molecular markers have been used successfully to determine

the degree of relatedness among individuals or group of accessions to clarify the genetic

structure or variation among accessions, population, varieties and species.

Molecular markers are now routinely used for characterization of genetic diversity, DNA

fingerprinting, genome mapping, genome evolution, ecology, taxonomy, and plant breeding.

DNA-based markers are abundant, highly polymorphic and independent of environment or

tissue type. Most DNA-based markers can be classified into three categories depending on

the technique used (Karp and Edwards, 1997): Hybridization-based DNA markers, arbitrarily

primed polymerase chain reaction (PCR)-based markers, sequence targeted and single locus

DNA markers. Restriction fragment length polymorphism (RFLP) is an hybridization-based

markers in which DNA polymorphism is detected by digesting DNA with restriction enzymes

followed by DNA blotting and hybridizations with probes. Arbitrarily primed PCR-based

markers are employed in organisms for which no genome sequence is available. These

markers are RAPD and AFLP. Sequence tagged sites (STS), SSR, Single nucleotide

polymorphisms (SNP) markers belong to sequence targeted and single locus PCR-based

DNA markers. Among the polymerase chain reaction (PCR) based markers frequently used,

RAPD (random amplified polymorphic DNA) is considered to be efficient and cost effective.

The technique only needs a few nanograms of DNA for a fast polymorphism analysis, does

not require prior knowledge of DNA sequence, and does not involve radioactivity (Williams

39

et al., 1990). Isozyme markers provide a convenient method for detecting genetic changes,

but are subject to ontogenic variations. They are also limited in number, and only DNA

regions coding for soluble proteins can be sampled.

A large number of RFLPs were recorded in some tree species like Populus, eucalyptus etc.,

for studying the variation and genetic fidelity of micropropagated plants. There are a number

of reports in literature which demonstrated the detection of genetic fidelity using RAPD/ISSR

markers in various plant species such as Picea mariana (Isabel, 1993), Festuca pratensis

(Valles et al., 1993), Picea abeies (Heinze and Schmidt, 1995), Popolus deltoides (Rani et

al., 1995), Pinus thunburghii (Goto et al., (1998), Eucalypyus ( De Laia et al., 2000),

Actinidia deliciosa (Palombi and Damiano, 2002), Vitex negundo (Ahmad et al,. 2008)

Dendrocalamus hamiltonii (Agnihotri et al., 2009), Capparis decidua (Tyagi et al., 2010).

Bouman et al., (1992) and Bouman and Kuijpers (1994) also found RAPD polymorphism

amongst micropropagated plants of Begonia. Similarly, Rani et al., (1995) reported that the

plants originating from the same clone of Populus deltodeis showed all the amplification

products were monomorphic across all the micropropagated plants. Absence of genetic

variation using the RAPD marker system has been reported in several cases such as

micropropagated shoots of Pinus thunbergii (Goto et al., 1998), somatic embryogenesis-

derived regenerants of oil palm (Rival et al., 1998), micropropagated teak somatic

embryogenesis-derived regenerants (Gangopadhyay et al., 2003). Similarly, RAPD markers

have been applied for characterization of micropropagated Populus tremuloides (Rahman and

Rajora, 2001). About 97% homology between the mother plants and micropropagated plants

has been reported in Syzygium travancorium (Anand, 2003). Random amplified polymorphic

DNA analysis was resulted 30% polymorphism in Robinia pseudoacacia (Bindia and

Kanwar, 2003). Scocchi et al., (2004) also used the RAPD markers to test the genetic

stability of in vitro raised plants of Melia azedarach. Out of six RAPD and four ISSR primer

combinations used for PCR amplification of in vitro raised plants of Azadirachta indica, a

total of 60 bands were scored of which 48.3% were polymorphic (Kota et al., 2006).

Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic

stability of 80 micropropagated Hagenia abyssinica plants (Feyissa et al. 2007). Similarly, a

total number of 925 bands were obtained from the PCR profile of micropropagated shoots of

Mucuna pruriens (Sathyanarayana et al., 2008). RAPD analysis in 90 micropropagated plants

of Eucalyptus globulus was resulted with a total of 115 amplified reproducible bands per

plant produced from 14 random primers (Liu et al., 2009). Reproducible monomorphic

RAPD banding patterns have been obtained using all the tested primers in Phoenix

40

dactylifera (Othmani et al., 2010). RAPD analysis confirmed that all the in vitro derived

plants of Populus alba and Populus tremula were genetically identical to their donor plants

(Khattab, 2011). Nadha et al., (2011) utilized RAPD and ISSR markers to assure the genetic

fidelity of in vitro raised Guadua angustifolia clones. Analysis of RAPD banding patterns

generated by PCR amplification using 20 random primers gave no evidences for somaclonal

variation in Saccharum officinarum (Pandey et al., 2012).

ISSR technique has successfully been used for the assessment of genetic fidelity in Populus

trimuloides (Rahman and Rajora, 2001), Robina ambigua (Guo et al., 2006b). However, Guo

et al., (2006a) reported 15.7% of polymorphic bands in the ISSR analysis for the 63

regenerants of Codonopsis lanceolata. Amplification of the ISSR markers reported

polymorphism in Actinidia deliciosa (Palombi and Damiano, 2002). By using ISSR, a low

genetic variation (3.92%) among the 21 in vitro grown plants of Dictyospermum ovalifolium

was reported by Chandrika et al., (2008). All ISSR profiles of micropropagated plants were

monomorphic, and similar to those of field-grown plants of Vitex nigundo (Ahmad et al.,

2008), Ochreinauclea missionis (Chandrika and Rai, 2009), Crataeva magna (Bopana and

Saxena, 2009) and Nothapodytes foetida (Chandrika et al., 2010), Phoenix dactylifera

(Kumar et al., 2010), Gentiana straminea (He et al., 2011). Genetic stability of the

regenerated plants of Anisodus tanguticus was assessed by 25 ISSR markers (He et al., 2011).

Liu and Yang (2012) reported that out of 21 ISSR primers screened, 16 primers were found

to produce clear, reproducible bands with an average of 6.5 bands per primer in Psidium

guajava. ISSR banding pattern analysis generated 15 primers (112 amplicons) and gave no

evidences for somaclonal variation in Saccharum officinarum (Pandey et al., 2012).

Vendrame et al., (1999) evaluated the applicability of AFLP analysis for the assessment of

somatic embryos of Carya illinoinensis. Singh et al., (2002) observed that the two hundred

and thirty-nine amplified AFLP fragments were monomorphic across the mother tree and its

tissue culture raised progenies of Azadarachta indica. Devarumath (2002) performed the

RFLP fingerprinting of Camellia chinensis using six restriction endonuclease. Amplified

fragment length polymorphism (AFLP) markers were employed to detect genetic fidelity

between in vitro raised plantlets of Papaver bracteatum and mature (Carolan et al., 2002).

Bhatia et al., (2005) observed that the DNA samples obtained from the regenerated shoots

and cotyledonary explants were subjected to amplified fragment length polymorphism

(AFLP) analysis to examine genetic uniformity of tissue-cultured tomato. Tripathi et al.,

(2006) reported the identification of Eucalyptus clones regenerated through tissue culture by

using genetic markers viz., RAPDs/AFLPs. Analysis of the AFLP banding patterns exhibited

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no variation about the number and the size of AFLP bands either among the somatic embryos

derived plants and the mother plant of Phoenix dactylifera (Othmani et al., 2010). Keivani et

al., (2010) observed polymorphic bands in regenerated plants of Medicago sativa.

In isozyme analyses, the particular enzyme is extracted from the plant tissue, and the different

forms separated by gel electrophoresis, on the basis of molecular size, shape and electrical

charge. Gangopadhyay et al., (2004) observed identical isozymic profiles for mother plant

and tissue cultured plants of Pandanus amaryllifolius using acid phosphatase isozymes.

Garcia et al., (2004) reported the genetic fidelity testing using isozyme markers like as,

shikimate dehydrogenase (SDH), isocitric dehydrogenase (IDH), acid phosphatase (ACP),

malate dehydrogenase (MDH) and glutamate oxaloacetate transaminase (GOT). Scocchi et

al., (2004) also used the isozyme markers to access the genetic stability of in vitro raised

plants of Melia azedarach. Similar reports have been observed in tissue culture derived

plantlets of Celastrus paniculatus and date palm (Maruthi et al., 2004; Saker et al., 2000).

Picoli et al., (2008) has also observed the isozyme patterns of different isozyme like esterase,

peroxidase, acid phosphatase, malate dehydrogenase to access the genetic fidelity of

Eucalyptus. Cheniany et al., (2010) used peroxidase and polyphenol oxidase isozymes as

markers for studying the physiological processes of rooting in Persian walnut. The isozymic

profile indicated the genetic conformity of Naringi crenulata and Aegle marmelos among

plantlets obtained through in vitro propagation and mother plant were all true to the type

(Smila et al., 2011).