Chapter 1

In Vitro Plant Regeneration through Somatic Embryogenesis and Direct Shoot

Organogenesis in Cenchrus ciliaris

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In Vitro Plant Regeneration through Somatic Embryogenesis and Direct Shoot Organogenesis in Cenchrus ciliaris

1.1 REVIEW OF LITERATURE 1.1.1 Tissue culture studies in cereals and grasses An elementary requirement for all applications of biotechnology is the regeneration of whole plants from cells or tissues or organ cultured in vitro. The first protocol describing successful regeneration from callus culture in a Poaceae species was established by Japanese scientists working in Rice (Nishi et al., 1968; Kawata & Ishihara, 1968; Tamura, 1968). Subsequently, regeneration of important crop plants was reported like in wheat (Shimada et al., 1969); minor millet (Rangan, 1974; 1976) and maize (Green & Phillips, 1975). The first successful regeneration of green plants in a forage grass species was calli derived from triploid rye grass embryos (Ahloowalia, 1975). Since then regeneration has been reported for all major cereals, many forage and turf grasses, sugarcane, and from species of various bamboo genera. Reports of regeneration in previously untested graminaceous species continue to appear in the literature. Regeneration procedures for grasses using tissue culture techniques have been explained in many reviews [Potrykus, (1980); Vasil & Vasil, (1980); Conger, (1981); Conger & Gray, (1984); Vasil & Vasil, (1986); Bhaskaran & Smith, (1990)]. From 1980 onwards, there has been an incredible improvement in the ability to achieve long term and high frequency regeneration from tissue culture of Graminaceous species.

An important factor in successful callus induction and plant regeneration in grasses is the use of meristematic tissues as explants. These include immature and mature embryos, flag-boot like inflorescence, and basal leaf tissue. Another major constituent is the inclusion of a strong auxin, usually 2,4-dichlorophenoxy acetic acid in the

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medium. The requirement for a cytokinin is less clear and results have been variable. (Wenck et al., 1988; Denchev & Conger, 1994). The culture response is also influenced by many physiological (growth condition of donor plants) and biological factors (genotype, explants, carbon source, and additives in the medium; (Schulze, 2007). A brief account of various factors determining the success of plant tissue culture and their optimization in gramineae is described below. 1.1.2 Plant regeneration through tissue culture The rate of plant regeneration in tissue culture is dependent on various factors like genotype and explants (type and age). Usually, explants from healthy and vigorous plant is preferred to establish a successful regeneration system. Immature tissues like axillary buds, shoot apex, immature embryos, leaf and stem segments have been reported as best responsive explants (Rao, 1987). Given the optimum combination of growth regulators, any explant has the potentiality to form whole plant. This maintenance of genetic potential is known as ‘totipotency’. According to which all cells are capable of developing into a whole plant if optimum environmental conditions are provided. 1.1.3 Modes of plant regeneration in vitro Plant regeneration in tissue cultures can follow two different pathways. These are organogenesis, involving the development of axillary buds following inhibition of apical dominance and somatic embryogenesis, where bipolar stractures called somatic embryos are formed which eventually develop into individual plants (Vasil, 1987). The formation of callus from an explant generally contains 3 stages: induction, cell division and differentiation. During induction phase, the metabolism is prepared for cell division. At the actively cell division phase, the cells of the explant are reverted to meristematic or dedifferentiated state. Third phase is the appearance of cellular differentiation and expression of certain metabolic pathways (Dodds & Roberts, 1985).

1.1.4 In vitro somatic embryogenesis of monocotyledonous plants Somatic (asexual) embryogenesis is a process in which any somatic tissue is potent to regenerate into whole plant through formation of embryo-like structures (Rao, 1996;

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Jiménez, 2005). These embryo-like structures are analogous to sexually produced zygotic embryos and like zygotic embryos, originate from single cell (Rao, 1996).

Somatic embryo production can be either direct, where somatic cells develop directly into embryos or indirect, where cells form an intervening callus phase. Indirect somatic embryogenesis occurs more often, however direct embryogenesis can be found commonly in reproductive tissues like nucellus, styles or pollen (Slater et al., 2003). Germination of somatic embryos is usually induced on hormone-free culture medium or medium that contains low levels of an auxin or low levels of both auxin and cytokinin. Under these conditions, the somatic embryos which are developmentally capable of germinating do so without maturing. Unlike mature zygotic embryos, which germinate vigorously into sizeable plants within several days, germinating somatic embryos generally lack vigor and may require many days to develop into healthy, rapidly growing plants (Carman, 1995).

The process of somatic embryogenesis comprises of two phases; induction phase and expression phase (Jiménez, 2005). Induction of somatic embryos depends on the availability of exogenous and endogenous plant growth regulators (PGRs). Auxin plays an important role during the induction phase of somatic embryos and other PGRs are required for balancing the hormonal constitution in the somatic embryos. (Dodeman et al., 1997; Jiménez, 2005; Jiménez & Thomas, 2006; Rao, 1996; Feher, 2006).

For most species, the commonly used auxin is 2,4-Dichlorophenoxyacetic acid (2,4-D) for the induction of somatic embryo (Jiménez, 2005). Most of the plant regeneration observed from callus cultures of graminaceous species prior to 1980 was probably by organogenesis. The clear documentation of somatic embryogenesis in the early 1980’s (Morrish et al., 1987; Vasil, 1987; Vasil & Vasil, 1986) represented a major break through in cell and tissue culture of the gramineae and provided the potential for genetic manipulation at the cell level. In 1982, somatic embryogenesis in orchard grass from mature embryos and basal leaf tissue was reported by McDaniel et al., (1982); Hanning & Conger; (1982). It was shown that somatic embryos in leaf segments arise directly from the mesophyll (Conger et al., 1983) and histological studies provided strong evidence for a single cell origin (Trigiano et al., 1989). However, developing

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regeneration methods that meet the physical and chemical requirements of the plant cells is still an empirical process. Identifying optimum in vitro culture conditions can be challenging due to the wide number of factors that control the induction, development and conversion of the somatic embryo into a plant. 1.1.4.1 In vitro environmental factors affecting plant regeneration A. Type of explants Explant (include immature and meristematic cells) with an ability to express totipotency is the most suitable plant part for tissue culture (Mantell et al., 1985). Generally, plant parts such as immature embryos and inflorescence, young leaves, leaf base, anthers of plants etc. regenerate more easily in vitro than generative ones. Explants should be isolated from healthy plants for successful response to tissue culture (Hess & Carman., 1998). In general, young tissues and organs have a high regeneration capacity than the mature ones. Generally, mature tissues of grasses yield either non-regenerable or only root forming calli. Initially 1970’s era used such easily available explants such as immature and mature embryos for plant regeneration in Hordeum vulgare (Norstog, 1970; Green & Phillips, 1974; Harms et al., 1976; Rangan, 1976) and later in 1975, shoot apex explants were used by Cheng & Smith. In addition, roots as explants from wheat and barley seedlings were tested for morphogenic ability, but only callus were induced (Bhojwani & Hayward, 1977). Mesocotyls were also used by Jelaska et al., (1984) for callus initiation and plant regeneration. Mature seed or embryos were intensively analysed and callus cultures were successfully induced in rice (Nishi et al., 1968; Abe & Futsuhara, 1986), maize (Harms et al., 1976; Huang & Wei, 2004), oat (Carter et al., 1967), wheat (Cure & Mott, 1978; Mackinnon et al., 1987), barley (Bayliss & Dunn, 1979) and finger millet (Eapen & George, 1989).

First successful plant regeneration from immature inflorescence explants was reported for Sorghum (Brettle et al., 1980), Italian ryegrass (Dale et al., 1981), Napier grass (Wang & Vasil, 1982) and Wheat (Maddock et al., 1983). Thus, continuous efforts are being carried out to re-evaluate the morphogenic potential of alternate explants.

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B. Genotypes It is well known that successful in vitro callus formation and plant regeneration is highly dependent on genotype (Sears & Deckard, 1982; Mathies & Simpson, 1986; Fennel et al., 1996; Haliloglu & Baenzinger, 2005). Gonzalez et al., (2001) studied the influence of genotype on callus induction and plant regeneration of Triticum turgidum. They evaluated 12 genotypes of durum wheat and found that 3 genotypes showed higher responses. Strategies were developed to reduce genotype limitiation in pearl millet, as differences between genotypes were minimal when explants such as shoot apices were used instead of immature zygotic embryos (Lambe et al., 1999). A strong genotype specificity was also observed for other explants like anthers and microspores (Powell, 1988; Kuhlmann & Foroughi-Wehr, 1989; Logue et al., 1993). Several researchers have reported that genotype has significant effect on the formation of embryogenic callus and plant regeneration capacity (Ozgen et al., 1996; 1998; Rashid et al., 2002; Sarkar & Biswas, 2002; Li et al., 2003; Zale et al., 2004; Chen et al., 2006; Patnaik et al., 2006; Ahmet & Adak, 2007; Bi et al., 2007). In depth knowledge about the mechanism of such control is still lacking (Bregitzer & Campbell, 2001; Tyankova & Zagorska, 2001).

C. Culture medium The composition of growth medium is an important factor affecting growth and morphogenesis of plant tissues. Culture medium is one of the most important factors to be considered for in vitro plant cell culture and it can be used in either solid or liquid state. Also, it must supply the essential macro-micro minerals, vitamins, amino acids or other nitrogen supplements, carbon source, organic supplements, solidifying agents and growth regulators required for growth and development. The most common medium used for in vitro plant cultures was developed by Murashige & Skoog, (1962) reported to be used in plant regeneration of several species (Hanzel, et al., 1985; Mathias & Simpson, 1986; Luhrs & Lorz, 1987; Elena & Ginzo, 1988; Redway et al., 1990; Bregitzer, 1992; Fennel et al., 1996). B5 medium (Gamborg et al., 1968) has also been reported to produce reproducible results (Dale & Deambrogio, 1979; Kott & Kasha, 1984; Goldstein & Kronstad, 1986; Kachhwaha & Kothari, 1996).

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D. Micronutrients The effect of microelements on the induction of embryogenic callus from immature embryo of wheat was studied. The reduction of microelements from MS, such as iron and manganese caused a marked decrease in embryogenic callus yield. Exclusion of metals like boron copper, cobalt, iodine and molybdenum from the medium had little effect on the induction of embryogenic callus. The original MS medium contained 0.1 mg cupric sulphate. Reports in wheat, triticale and barley have indicated that the optimal copper concentration for these two cereal species was 5 µM (Purnhauser & Gyulai, 1993; Dahleen, 1995; Bregitzer et al., 1998; Nuutila et al., 2000).

Additionally, incorporation of silver nitrate (an inhibitor of ethylene) in the medium, was found to effectively increase shoot formation in wheat and maize tissue cultures (Purnhauser et al., 1987; Songstad et al., 1988). Manipulation of ethylene production by addition of ethylene precursor, l-aminocyclopropane l-carboxylic acid or ethylene antagonist, silver nitrate resulted in improvement of plant regeneration. E. Plant growth regulators (PGRs) PGRs are synthetic compounds with hormone- like activity which are given to the plant under in vitro or ex vitro conditions. In vitro responses of explants depend on the type and amount of PGRs in the culture medium. To analyze the role of auxins and cytokinins during culture initiation, extensive studies were undertaken. Somatic embryogenesis of cereals is influenced by the type of auxin used. Higher concentrations of 2,4-D reduce the frequency of regeneration and causes abnormalities (Deambrogio & Dale, 1980; Nabors et al., 1983; Ziauddin & Kasha, 1990; Baillie et al., 1993).

Other auxins such as IAA, IBA, Dicamba, were also evaluated to replace 2,4-D. In wheat and barley, 3,6-dichloro-O-anisic acid (Dicamba) alone or in combination with 2,4-D and picloram was found to be more suitable for somatic embryogenesis induction and higher frequency of plant regeneration than 2,4-D alone in the medium (Hunsinger & Schauz, 1987; Carman et al., 1987; Castillo et al., 1998; Trifonova et al., 2001; Mendoza & Kaeppler, 2002; Przetakiewicz et al., 2003; Halamkova et al., 2004; Satyavathi et al., 2004). Few studies indicated that 2,4,5-trichlorophenoxyacetic acid

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(2,4,5-T) is a better auxin source for callus proliferation (Lazar et al., 1983) as well as for shoot and root formation (Jelaska et al., 1884).

With respect to the cytokinins, their influence on the establishment and maintenance of regenerable cultures, inhibitory (Dale & Deambrogio, 1979; Bayliss & Dunn, 1979; Thomas & Scott, 1985; Chang et al., 2003) as well as enhancing effects have been described (Lazar et al., 1983; Rengel & Jelaska, 1986; Carman et al., 1987; Kachhwaha & Kothari, 1996; Cho et al., 1998; Jiang et al., 1998; Dahleen & Bregitzer, 2002). F. Gelling agent When plant cells or tissues are to be cultured on the surface of the medium, it must be solidified. Even though agar is the most frequently used gelling agent in culture media, the water potential of a medium solidified with gel is more negative than that of liquid medium, due to their matric potential (Amador & Stewart, 1987; George et al., 2008). Other examples of gelling agents include Phytagel or Gellan gum, Guar gum (Babbar et al., 2005), in Banana (Tyagi et al., 2007; Atici et al., 2008; Fira et al., 2008; Ozel et al., 2008; Agrawal et al., 2010; Saglam & Ciftci, 2010). G. Carbon source Carbon sources have been reported to have a significant effect on in vitro plant regeneration. In general, sucrose is the carbohydrate of choice as carbon source, probably because it is the most common carbohydrate in the plant phloem (Murashige & Skoog, 1962; Thorpe, 1982; Lemos & Baker, 1998). The major importance of sucrose, sorbitol, mannitol and maltose as well as salts and polyethylene glycol were evaluated for increased osmotic value. ABA formation under appropriate osmotic conditions is induced by the sugars (Leon & Sheen, 2003). Eapen & George, (1989) found that glucose or sucrose stimulated development of somatic embryos. More embryogenic calli formed instead of soft calli when sucrose was replaced by maltose as the carbon source in callus induction (Mendoza & Kaeppler, 2002; Wang & Wei, 2004). Maltose has frequently been used to increase androgenic resposes in rye (Flehinghaus et al., 1991), barley (Roberts-Ochschlager et al., 1990), wheat (Moieni et al., 1997) and Triticale (Gonzalez & Jouve, 2000).

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H. Organic supplements Initiation of embryogenic cultures can be improved by inclusion of organic supplements like proline, casein hydrolysate and glutamine in the medium. The addition of 0.25 to 1.0 g/l Casein Hydrolysate (Lührs & Lörz, 1987; Bregitzer, 1992), 500 mg/l Glutamine (Redway et al., 1990) or 0.2 mM L-Tryptophan (Carman et al., 1987) was found to increase the induction of embryogenic cell cultures. Moreover, addition of 10 mg/l Proline was reported to maintain morphogenic competence of callus induced from immature wheat embryos for over two years (Kothari & Varshney, 1998; Yadav & Chavala, 2002). Besides that, an optimization of nitrogen composition of media due to an altered nitrate:ammonia ratio resulted in a significant improvement of regeneration frequency (Nuutila et al., 2000; Chauhan & Kothari, 2004). I. Temperature/Light The growth of tissues under in vitro conditions is strongly influenced by temperature and light. The best growth can be obtained when light-dark cycles and temperature during day and night in natural environment are considered under laboratory conditions (Capite, 1955). A cold pre-treatment doubled the embryogenic response of maize cultures in vitro (Pescitelli et al., 1990). In addition, light has a significant effect in plant development. The importance of light in plant regeneration of wheat cultures has been reported in several studies (Liang et al., 1987). J. A new whole plant For in vitro grown plants to survive in ex vitro environmental conditions, they require a gradual transfer procedure for acclimitozation. Compared to tissue culture conditions, the external environment in greenhouse and fields have relatively low humidity, high light intensity and septic environment, which are usually stressful and intolerable for the in vitro grown plants (Hazarika, 2003). In addition, physiological and anatomical features of micropropagated plantlets necessitate gradual acclimitization to greenhouse or field conditions (Kozai, 1991).

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1.1.5 Direct shoot organogenesis The process of organogenesis is defined as the development of organs directly from explants or through intervening callus phase. Organogenesis or plant regeneration from somatic embryos is similar to germination process of zygotic embryos and can occur by three methods. Development of adventitious organs can be (i) directly from explant, or (ii) from callus cultures or (iii) from axillary buds. (Slater et al., 2003). Various explants sources, such as whole seeds, immature inflorescence, immature and mature embryos, scutellum, immature leaf, monocotyl, apical meristem, coleoptilar node, and root have been used to establish regenerable tissue culture in cereals (Rakszegi et al., 2001).

In vitro differentiation of multiple shoots through adventitious shoot formation without an intervening callus phase is known as direct shoot organogenesis. The increase in the number of basal shoots from culms regenerated in tissue culture is quite common and resembles basal tillering habit of grasses. Shoots regenerated this way are well suited for clonal propagation as they are genetically uniform. Wakizuka & Yamaguchi, (1987) in a detailed study reported formation of an apical dome like structure between the two embryonic leaves in finger millet, the dome when sectored and sub-cultured continued to produce, in high frequency, multiple shoots through several passages. All such examples of shoot multiplication may be interpreted as continued proliferation of shoot buds from pre-existing organized meristem (Kothari & Chandra, 1995). The direct contact of plant tissues with the appropriate growth regulators (or increasing the concentration) from the very beginning would probably enhance their action on retarding main shoot elongation as well as on developing axillary and adventitious shoot meristems (Lakshmanan et al., 2006).

Development of high frequency in vitro shoot organogenesis helps in overcoming genotypic barriers, explants availability throughout the year, achieving faster multiplication rate/unit resource and in shortening the period of in vitro culture. More than that, direct regeneration does not result in somaclonal mutations making it more suitable for micropropagation as well as for the development of transgenics. Recent

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reports have indicated that transgenic plants regenerated through a more-or-less long term callus phase had an increased risk of somaclonal variation, problems in transgene inheritance and stability of transgene expression (Choi et al., 2003; Bregitzer & Tonks, 2003; Sticklen & Oraby, 2005).

Explants used for direct regeneration in monocots are nodal segment (Meliza & Khatamian, 1998), young leaf segments (Gill et al., 2006, Khan et al., 2009), caryopsis (Vikrant & Rashid, 2002), intact seedling (Gupta & Cogner, 1998), although shoot tip or shoot apex containing shoot apical meristem is the most widely used explant in Gramineae (Sharma et al., 2004; Zhong et al., 1992; Bao et al., 2001; Zhang et al., 1996; 2010; Devi et al., 2000; Lakshmanan et al., 2006; Yookongkaew et al., 2007; Jha et al., 2009; Kumar & Chandra 2009; Kumar & Bhat, 2012). The main advantage of using shoot apex is that explant can be obtained within three days of germination of mature seeds and there is no requirement for maintaining a constant supply of donor plants. Direct regeneration from shoot apex is less time consuming and easy. Previously, direct proliferation of shoots has been reported in many instances when shoot meristems were cultured. This was due to axillary bud proliferation as has been observed in Dactylis glomerata (Conger, 1981), Oryza (Oinam & Kothari, 1995) Triticum aestivum (Rao & Kothari, 1992), Hordeum vulgare (Kachhwaha & Kothari, 1994).

Highly regenerative direct shoot regeneration system has been successfully established from several cereals and grasses, by using a combination of 2, 4-D and BA. Studies on cereal morphogenesis (Zhong et al., 1998) showed that shoot apex multiplication occurs by differentiation of the shoot apical meristem or of axillary buds from the leaf bases. Gill et al., (2006) reported that young leaf (segment) explant of Saccharum (from field grown plant) produced multiple shoot clumps within four weeks of culture on MS medium supplemented with NAA + Kinetin and BA. Highest frequency of 83.12% was observed in M1 media with NAA (5mg/l) + Kinetin (0.5 mg/l) in variety COJ83. Lakshmanan et al., (1995) (thin cell layer culture) identified hormonal constraints that affect in vitro morphogenesis of sugarcane and successfully exploited this knowledge to

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develop a rapid and efficient direct organogenesis and somatic embryogenesis system that could be used for both mass production and genetic transformation. 1.1.6 Tissue culture studies in Cenchrus

In vitro plant regeneration was reported in Cenchrus (Sankala & Sankala, 1989) followed by both in C. ciliaris and C. setigerous (Kackar & Shekhawat, 1991; Murty et al., 1992). Young immature inflorescence explants were most preferred for callus induction in initial studies, though the frequency of regenerable calli and plant regeneration were not documented.

Callus induction in Cenchrus species was attempted with varying concentrations of 2,4-D (1.0 to 20.0 mg/l) in combination with other growth regulators such as IAA, Kinetin (Sankala & Sankala, 1989), or with media adjuvants like ascorbic acid (Kackar & Shekhawat, 1991) or coconut milk water (Ross et al., 1995) in MS medium. According to recent workers, 2,4-D at a range of 2.5- 6.0 mg/l for Cenchrus ciliaris and at 4.0 to 14.0 mg/l for Cenchrus setigerus were found optimal for callus induction and maintenance. Earlier, (Sankala & Sankala, 1989) callus production could be initiate using 2,4D (1.0 mg/l) along with IAA (5 mg/l) and kinetin (0.5 mg/l). The role of ascorbic acid as an essential anti-oxidant in callus culture of Cenchrus ciliaris has also been emphasized (Kackar & Shekhawat, 1991). Addition of 5% coconut water was also found beneficial for raising calli (Ross et al 1995). However, Ross et al., (1995) preferred mature seed tissue as a more readily available source of explants since recognizing appropriate developmental stage of immature inflorescence or embryos was difficult in Cenchrus species. The type of calli in C. ciliaris and C. setigerous could be mainly distinguished into a slow growing, hard, nodular and white compact embryogenic callus (Sankala & Sankala, 1989; Kackar & Shekhawat, 1991; Dethier et al., 1993).

Dethier et al., (1993) described culture of other explants like shoot apices or initiation of suspension cultures. In their study, callus induction medium comprised of MS salts (Murashige and Skoog, 1962) and B5 vitamins (Gamborg et al., 1968) supplemented

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with 30 g/l sucrose, 13.6 µM 2,4-D, and 7.5 g/l phytagar. Morphologically distinct callus lines were identified separated from the original explant material.

However, Ross et al., (1995) also found a third type of callus characterized by yellowish and relatively compact texture, often containing shiny mucilaginous cell. Mostly the regeneration of plantlets from calli was through somatic embryogenesis. All the earlier workers found that transfer of calli to hormone free MS medium could induce the formation of proembryoids at varying degrees, while prolonged maintenance of callus in the same induction medium resulted in gradual appearance of proembryoids in 20 to 50% of calli in C. ciliaris (Ross et al., 1995). Rooting of regenerated shoots from the embryoids occurred on hormone free media. Nevertheless, the use of NAA (0.1 mg/l) in the medium was helpful for root development and easier acclimatization of the regenerants (Kackar & Shekhawat, 1991).

A highly efficient and reproducible in vitro plant regeneration system is an absolute prerequisite for producing transgenic plants. Routine application of molecular improvement independent of the chosen method of transformation is still impeded by the lack of readily available highly efficient and long-term regenerable cell and tissue culture systems in Cenchrus. However, because of the reproductive system of this grass, its genetic improvement through conventional breeding methods is difficult and time-consuming and is, therefore, restricted to the selection of elite lines from natural variants (Bhat et al., 2001; Batra & Kumar, 2002; 2003).

Colomba & co-workers (2006) obtained regenerated plants using cultured mature embryos of 14 cultivars of Cenchrus. Mature embryos of Buffel grass apomictic cultivars were used to induce embryogenic callus formation using a basal medium supplemented with 3% sucrose and with of five different 2,4-D and four BA concentrations. They compared the effects of cultivar and culture medium on callus induction and plant regeneration, and clear differences were found between genotypes.

Regeneration in Cenchrus proceeds through somatic embryogenesis, in which the developmental sequence starts from callus and progress sequentially through globular to

scutellar mature somatic embryo (difference between sexual and apomictic plant of derived from seed, shoot apices and immature inflorescences cultured on different concentration of 2,4D and BA with MS media. embryogenic calli: off Gradual hardening of the plants and their survival has also been established by theinvestigations.

Previous tissue culture studies on buffel grass have demonstrated plant regenfrom embryogenic calluswell as immature or (shoot apices) from Cenchrus initiated by Kumar & was obtained yielding with 3.0 mg/l TDZ (Kumar & Bhat, (2012))

Table 1.1 An account of different explantregulators used for in vitro

19

scutellar mature somatic embryo (Yadav et al., 2009). They reported sexual and apomictic plant of Cenchrus in the response of calli

derived from seed, shoot apices and immature inflorescences cultured on different ion of 2,4D and BA with MS media. They also observed two types of

embryogenic calli: off white, compact, and nodular callus and a white compact callusGradual hardening of the plants and their survival has also been established by the

Previous tissue culture studies on buffel grass have demonstrated plant regenfrom embryogenic callus induced from seed, shoot apex and immature inflorescence

mature embryos. Using extremely immature shoot meristems enchrus cultures capable of plant regeneration w Bhat, (2012). Direct shoot production without callus interphase 20 shoots per explant by addition of MS medium

(Kumar & Bhat, (2012)).

of different explant source, media composition and combinations of growth in vitro plant regeneration in Cenchrus ciliaris

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They reported significant in the response of calli

derived from seed, shoot apices and immature inflorescences cultured on different observed two types of

white, compact, and nodular callus and a white compact callus. Gradual hardening of the plants and their survival has also been established by their

Previous tissue culture studies on buffel grass have demonstrated plant regeneration immature inflorescence as immature shoot meristems

capable of plant regeneration was successfully Direct shoot production without callus interphase

MS medium supplemented

source, media composition and combinations of growth

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1.2 MATERIALS AND METHODSPlant material Ten apomictic cultivars of buffel grass (IGDBC15-8/32/10, DBC18-2/41/15, DBC242/65/26, DBC25-6/67/28, DBC26were evaluated for their response to experiments. These cultivars were grown in the field or garden under uniform conditions at the Department of Botany,of Delhi. Seeds were collected from field grown plants (Figure 1.1) and used for culturing 1.2.1 Explants and media preparationFor optimization of callus induction, callus growth, embryogenic calli, somatic embryogenesis and plantlet regeneration foExplants such as an year old mature seeds, shoot apices from 2immature inflorescences (1.5 to 3 cm) about to emerge out of the boot leaf were used for this study. (Figure 1.2) In addition, shoot apices were also used to induce multiple shoots through direct shoot organogenesis.

Figure1.2 Three different explants of experiment.

Mature seeds and immature inflorescences (after removal of boot leaf) were surface sterilized by rinsing in 70% ethanol for 1 minute, followed by treatment with 0.1% aqueous HgCl2 for 5 minutes with occasional stirring. Finally, the explants were washed

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METHODS

Ten apomictic cultivars of buffel grass (IG-3108, IG-718, IG-74, 2/41/15, DBC24-2/60/22, DBC25-

6/67/28, DBC26-3/69/30, and DBC27-6/73/34) were evaluated for their response to in vitro culture in separate

se cultivars were grown in the field or garden at the Department of Botany, University

Seeds were collected from field grown plants and used for culturing.

edia preparation optimization of callus induction, callus growth, embryogenic calli, somatic

embryogenesis and plantlet regeneration four genotypes of buffel grass were used. Explants such as an year old mature seeds, shoot apices from 2-3 days old seedlings and

inflorescences (1.5 to 3 cm) about to emerge out of the boot leaf were used In addition, shoot apices were also used to induce multiple

shoots through direct shoot organogenesis.

Figure1.2 Three different explants of Cenchrus ciliaris used for regeneration and transformation

Mature seeds and immature inflorescences (after removal of boot leaf) were surface sterilized by rinsing in 70% ethanol for 1 minute, followed by treatment with 0.1%

r 5 minutes with occasional stirring. Finally, the explants were washed

Figure 1.1 ciliaris L. (Buffel grass)

optimization of callus induction, callus growth, embryogenic calli, somatic r genotypes of buffel grass were used.

3 days old seedlings and inflorescences (1.5 to 3 cm) about to emerge out of the boot leaf were used

In addition, shoot apices were also used to induce multiple

used for regeneration and transformation

Mature seeds and immature inflorescences (after removal of boot leaf) were surface sterilized by rinsing in 70% ethanol for 1 minute, followed by treatment with 0.1%

r 5 minutes with occasional stirring. Finally, the explants were washed

Figure 1.1 Cenchrus L. (Buffel grass)

3-4 times with sterile distilled water to remove any traces of HgClinflorescences were cut into small pieces of 3To obtain shoot apex explant, surface sterilized seeds were germinated on MS (Murashige & Skoog 1962) medium25°C. After 2-3 days, emerging shoot apicespart of mesocotyl were excised and used as explants for both indirect somatic embryogenesis and direct shoot organogenesis. Table 1.2 Composition of MS mediu

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4 times with sterile distilled water to remove any traces of HgClinflorescences were cut into small pieces of 3-5 mm length using sterile scalpel blade. To obtain shoot apex explant, surface sterilized seeds were germinated on MS (Murashige & Skoog 1962) medium (Table 1.2) on a sterile petriplate placed

, emerging shoot apices consisting of shoot apical meristem and a part of mesocotyl were excised and used as explants for both indirect somatic embryogenesis and direct shoot organogenesis.

Composition of MS medium

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4 times with sterile distilled water to remove any traces of HgCl2. For culturing, 5 mm length using sterile scalpel blade.

To obtain shoot apex explant, surface sterilized seeds were germinated on MS on a sterile petriplate placed in light at

consisting of shoot apical meristem and a part of mesocotyl were excised and used as explants for both indirect somatic

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In all the experiments, MS media used were enriched with sucrose (30 g/l) and solidified with 8 g/l agar (Bactoagar, Himedia). The pH of medium was adjusted to 5.8 before adding agar. The media were sterilized by autoclaving at 121°C for 17 min. and 25 ml of media was dispensed into 100 ml autoclaved conical flask (Borosil) and closed with sterile cotton plugs. Around 8-10 explants were placed on the MS medium in each flask aseptically.

Auxins (2,4-D, IBA) and cytokinins (BA, KINETIN, TDZ) were the two major phytohormones used at different concentrations and combinations in MS media for induction, and growth of callus, shoot and root as well as in direct shoot organogenesis. 1.2.2 Induction of callus, formation of somatic embryos and plant regeneration After surface sterilization, explants (seed, shoot apex and immature inflorescence) of four genotypes (IG-3108, IG-718, IG-74, DBC15-8/32/10) were placed on MS media supplemented with five levels of 2,4-D (2, 3, 5, 6 and 7 mg/l) in combination with BA (0.5 mg/l) for callus induction (Figure 1.3). Additional by different concentrations of growth adjuvants such as L. Proline, L. Glutamine and Casein Hydrolysate (100, 200, 300, 400 and 500 mg/l) along with 3 mg/l 2,4-D and 0.5 mg/l BA were tested to improve embryogenic callus growth and quality that could enhance the rate of somatic embryogenesis in genotype IG-3108. Somatic embryos were transferred to regeneration MS media supplemented with various concentrations of BA (1, 2, 3 and 4 mg/l) in combination with 2,4-D at 0.25 mg/l.

Figure 1.3 Three different explants of Cenchrus ciliaris on MS media containing 3 mg/l 2,4-D+ 0.5 mg/l BA (a) seed, (b) shoot apex, and (c) Immature inflorescence explants.

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23

The cultures were maintained in dark at 25 ± 2°C for inducing callus and somatic embryogenesis whereas for shoot regeneration, were maintained under light/dark cycle (16/8 hours) provided through fluorescent tube with a light intensity of 50 µ mol m-2 s-1 at 25 ± 2°C.

To analyze the influence of genotype and culture medium on somatic embryogenesis induction, the following variables were recorded per cultivar, replicate and culture medium:

1. Total number of calli per total number of cultivated explants x 100. Callus formation was considered independent of its size and the data were recorded after 20 days of culturing of primary explants before the first subculture.

2. Total number of embryogenic calli per fixed amount of callus (~100 mg fresh callus) x 100. Data on percentage embryogenic calli was recorded at different time intervals for different explants, 20 days after second subculture for immature inflorescence and third subculture for seed and shoot apex explants.

3. Total number of somatic embryo per fixed amount of embryogenic calli (~100 mg fresh callus) x 100. Data on percentage of somatic embryo formation was recorded at different concentration of growth adjuvents such as proline, glutamine and casein hydrolysate after 20 days of formation of embryogenic calli.

4. Total number of somatic embryos per fixed amount of embryonic calli (~100 mg embryogenic calli) x 100. Mean number of shoots per callus (~100 mg callus) was also calculated. Data on shoot regeneration frequency and the number of shoots per callus was recorded 25 days after transferring to regeneration medium.

1.2.3 Multiple shoots formation and plant regeneration Eight cultivars (IG-3108, DBC15-8/32/10, DBC18-2/41/15, DBC24-2/60/22, DBC25-2/65/26, DBC25-6/67/28, DBC26-3/69/30, and DBC27-6/73/34) of buffel grass were used for multiplication of shoot. Aseptically grown shoot apices were cultured on MS media containing BA or Kinetin or TDZ at different concentrations (1, 2, 3, 4 and 5

Chapter 1

24

mg/l). Cultures were maintained underneath light/dark cycle (16/8 hours) provided by white fluorescent tube with a light intensity of 50 µ mol m-2 s-1 at 25°C. The explants were subcultured onto fresh media every 15 days. When the explants started to multiply, well grown axillary as well as adventitious shoots were separated with the help of a sterile scalpel under the hood and put in the same media for further multiplication. For shoot elongation, single shoots were subcultured on MS medium. The shootlets derived from each explants were tracked individually to determine the total number of plants produced from single shoot apex and their subsequent genetic identity. Multiple shoot induction frequency was calculated as mean number of shoots formed per shoot apex. Explant responded (%) was calculated based on the number of shoot apices that produced multiple shoots over total number of shoot apices inoculated. Observation was taken after 5 weeks of inoculation. 1.2.4 Root induction from shoots Semi solid MS medium containing 30 g/l sucrose along with 8 g/l agar and supplemented with or without 0.8% charcoal (Qualigens) or ½ strength MS medium with or without 0.8% activated charcoal or MS with 2 mg/l IBA were tested for rooting. Root induction frequency was calculated as the total number of roots formed per total number of shoots cultured in each flask x 100. Number of roots per shoot was calculated as a mean of number of roots formed in single shoots. After 20-21 days of transfer, the plantlets were taken out from the culture tubes and washed thoroughly with sterile distilled water to remove the adhering media. For hardening, the in vitro regenerated plantlets were maintained on liquid medium containing 0.4% sucrose for one week. And then placed in test tubes containing sterile tap water for 1 day and subsequently transferred to the pots for hardening. 1.2.5 Acclimatization of regenerated plants These plantlets were transferred to agro-peat (mixture of solarite and vermiculite) in plastic pots and nutrients were provided through Hoagland’s solution (Table 1.3). These

pots were grown inside the culture room and covered with plastic bags witsmall holes to retain humidity. After 15 days, the bags were removed in order to reduce humidity. Finally, the plants were transferred to sterile garden soils, acclimatized by gradual removal of a surrounding plastic bag over several days, then greenhouse and grown to maturity and finally transferred to grow in sunlight under field conditions until maturity.

Table 1.3 Composition of Hoagland's Media (Plant Nutrient Solution)

1.2.6 Statistical analysisStatistical analysis was ver. 16 software to analyze the influence of explants, genotypes and media combinations on callus, shoot and root induction. Analysis of Variance (ANOVA) was performed for all the data sets. experiments. Each test consisted of 10 explants per each flask (100flasks/ treatment) and the experimentation was repeated twice. Significance of differences among means was compared usingdifference at P = 0.05.

25

pots were grown inside the culture room and covered with plastic bags witsmall holes to retain humidity. After 15 days, the bags were removed in order to reduce humidity. Finally, the plants were transferred to sterile garden soils, acclimatized by gradual removal of a surrounding plastic bag over several days, then greenhouse and grown to maturity and finally transferred to grow in sunlight under field conditions until maturity.

Composition of Hoagland's Media (Plant Nutrient Solution)

Statistical analysis Statistical analysis was carried out using Statistical Package for Social Sciences (ver. 16 software to analyze the influence of explants, genotypes and media combinations on callus, shoot and root induction. Analysis of Variance (ANOVA) was performed for all the data sets. All the ten genotypes were used in differexperiments. Each test consisted of 10 explants per each flask (100 flasks/ treatment) and the experimentation was repeated twice. Significance of differences among means was compared using two way ANOVA and Least significant

Chapter 1

pots were grown inside the culture room and covered with plastic bags with numerous small holes to retain humidity. After 15 days, the bags were removed in order to reduce humidity. Finally, the plants were transferred to sterile garden soils, acclimatized by gradual removal of a surrounding plastic bag over several days, then maintained in a greenhouse and grown to maturity and finally transferred to grow in sunlight under field

carried out using Statistical Package for Social Sciences (SPSS) ver. 16 software to analyze the influence of explants, genotypes and media combinations on callus, shoot and root induction. Analysis of Variance (ANOVA) was

n genotypes were used in different ml) in 3 replicas (3

flasks/ treatment) and the experimentation was repeated twice. Significance of two way ANOVA and Least significant

Chapter 1

26

1.2.7 Scanning electron microscopy studies Embryogenic calli (somatic embryo) of Cenchrus ciliaris were fixed in 2% gluteraldehyde prepared in phosphate buffer (pH 6.8-7.2) for 24 hours at 4°C. Then calli were dehydrated through a graded acetone series and stored in 70% acetone. The calli were dried to their critical point and were coated with gold particles. The specimens were examined and photographed in a LEO 435 VP Scanning Electron Microscope (Cambridage U.K. mode) at AIIMS New Delhi.

1.2.8 Histological studies Fresh calli of various textures (non-regenerating and regenerating) and developing multilple shoots were fixed in Carnoy’ solution of acetic acid and alcohol (1:3) for 24 hours and subsequently stored in 70% ethanol. The samples for study were selected and passed through tertiary butyl alcohol dehydration- infiltration series and embedded in paraffin wax according to Johnsen, (1940). The sections were cut at 10 µm thickness by hand driven microtome and spreaded on thoroughly cleaned micro slides. Cleaning of micro slides was done by soaking them overnight in chromic acid solution and thereafter washing thoroughly with water and finally with 95% alcohol and dried. Mayer’s adhesive (Johnsen, 1940) was used for floating the adhesive smeared slides to facilitate spreading on the sections.

The sections were dewaxed with xylene and brought to water through graded alcohol series. The sections of calli were stained with a series of Safranin and Fast Green FCF

(Johnsen, 1940) stain series was used for staining of sections of calli. The sections were cleared with Xylene and mounted in DPX mount. The photographs of the sections were taken with upright Compound microscope with automatic exposimeter photo micrographic attachment (Nikon, Japan).

1.3 RESULTS The present study was conducted to investigate optimal concentrations and combinations of plant growth regulators in the medium for efficient regeneration of buffel grass via seed, shoot apex and immature inflorescence culture and somatic

embryogenesis (through callus culture).cultured on MS media containing differentas, 2,4-D, IBA, BA, kinetin, TDZ and growth adjuvantsummarized in a flow diagram

Figure 1.4 Flow chart showing pathways of Somatic Embryogenesis Organogenesis (B) in Cenchrus cilliaris 1.3A Plant regeneration through 1.3A.1 Callus inductionExplants from four cultivars (IGCenchrus were used for callus induction. In 20 days, and immature inflorescences) exhibited morphological changes leading to the induction of callus. Maximum callus induction frequency, 87% ± 2.2, was obtained from seed explants of genotype IG2,4-D, whereas the minimum callus induction frequency, 25% ± 3.4, was observed from

27

rough callus culture). The morphogenic pathway of various explants cultured on MS media containing different concentrations of the growth regulators such

D, IBA, BA, kinetin, TDZ and growth adjuvants (L. Pro, L. Glu, CH) have been diagram (Figure 1.4).

1.4 Flow chart showing pathways of Somatic Embryogenesis (A)Cenchrus cilliaris

Plant regeneration through somatic embryogenesis Callus induction

Explants from four cultivars (IG-3108, IG-718, IG-74 and DBC15were used for callus induction. In 20 days, all explants (seeds, shoot apices

and immature inflorescences) exhibited morphological changes leading to the induction of callus. Maximum callus induction frequency, 87% ± 2.2, was obtained from seed explants of genotype IG-3108 that were subcultured on MS medium

D, whereas the minimum callus induction frequency, 25% ± 3.4, was observed from

A B

Chapter 1

The morphogenic pathway of various explants concentrations of the growth regulators such

(L. Pro, L. Glu, CH) have been

(A) and Direct Shoot

74 and DBC15-8/32/10) of all explants (seeds, shoot apices

and immature inflorescences) exhibited morphological changes leading to the induction of callus. Maximum callus induction frequency, 87% ± 2.2, was obtained from seed

S medium containing 3 mg/l D, whereas the minimum callus induction frequency, 25% ± 3.4, was observed from

Chapter 1

immature inflorescence explants of genotype medium containing 2 mg/l of 2,4and 7 mg/l) in MS media tested, highest percentage of callus induction w3 mg/l 2,4-D (87%) followed by 2Among the four genotypes, IG83.3% ± 2.1, IG-74 with 75% ± 4.3 and callus induction (Figure 1.5). Such a trend of genotypic response was similar for all the explants tested, indicating that genotype had significant inffrequency.

Figure 1.5 Frequency of callus induction ± SE from three explants [Seed, Shoot Apex (SA), Immature Inflorescence (Im In)] of four genotypes in MS media supplemented with 0.5with varying levels of 2,4-D.

Along with genotype, significant differences were also observed among various explants and 2,4-D levels tested. explants and 2,4-D levels were significant among the explants. Strong interaction among variables indicated that the combinations of genotype, explant and hormone levels significantly influenced callus induction.

The quality of callus induced was watery and loose and pale yellow shoot apex explants whereas calli of immature inflorescence were mand white (Figure 1.6).

28

immature inflorescence explants of genotype DBC15-8/32/10 that were induced on MS containing 2 mg/l of 2,4-D (Figure 1.5). Of the five levels of 2,4-D

and 7 mg/l) in MS media tested, highest percentage of callus induction was observed in ) followed by 2 mg/l 2,4-D (80%) in all the explants and genotypes.

Among the four genotypes, IG-3108 was found to be the best followed by IG74 with 75% ± 4.3 and DBC15-8/32/10 with 66.6% ± 2 percentage of

. Such a trend of genotypic response was similar for all the explants tested, indicating that genotype had significant influence on callus induction

allus induction ± SE from three explants [Seed, Shoot Apex (SA), Immature Inflorescence (Im In)] of four genotypes in MS media supplemented with 0.5

Along with genotype, significant differences were also observed among various D levels tested. Interactions between explants and genotype

were significant among the explants. Strong interaction among variables indicated that the combinations of genotype, explant and hormone levels significantly influenced callus induction.

The quality of callus induced was watery and loose and pale yellow in case of seed and explants whereas calli of immature inflorescence were mostly hard, compact

that were induced on MS D (2, 3, 5, 6

as observed in explants and genotypes. followed by IG-718 with

with 66.6% ± 2 percentage of . Such a trend of genotypic response was similar for all the

luence on callus induction

allus induction ± SE from three explants [Seed, Shoot Apex (SA),

Immature Inflorescence (Im In)] of four genotypes in MS media supplemented with 0.5 mg/l BA

Along with genotype, significant differences were also observed among various Interactions between explants and genotype, and

were significant among the explants. Strong interaction among variables indicated that the combinations of genotype, explant and hormone

in case of seed and ostly hard, compact

Figure 1.6 Callus induction in three different explants + 0.5 mg/l BA. (a) Calli induced from seed explant,Calli induced from immature inflorescence explant.

1.3A.2 Callus growth and qualityCalli produced from three different explants (seed, shoot apex and immature inflorescence) were multiplied in MS media with varying concentrations of 2,4constant level of BA.embryogenic calli) and immature inflorescence (embryogenic calli) in all genotypes on MS medium containing 3

Table 1.4 Rates of callus growth from three different explants on MS media containing 0.5with varying levels of 2,4-D.

callus growth was recorded after 20 days of first subculture.+ = callus growth rate, − = callus became brown within 15 days.(+ = Very slow callus growth,growth).

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Figure 1.6 Callus induction in three different explants cultured on MS media containing 3 mg/l 2,4Calli induced from seed explant, (b) Calli induced from Shoot apex explant and

Calli induced from immature inflorescence explant.

growth and quality Calli produced from three different explants (seed, shoot apex and immature inflorescence) were multiplied in MS media with varying concentrations of 2,4constant level of BA. Maximum callus growth was recorded embryogenic calli) and immature inflorescence (embryogenic calli) in all genotypes on

aining 3 mg/l 2,4-D and 0.5 mg/l BA (Table 1.4).

Table 1.4 Rates of callus growth from three different explants on MS media containing 0.5D.

callus growth was recorded after 20 days of first subculture. = callus became brown within 15 days.

= Very slow callus growth, + + = Slow callus growth, + + + = Moderate callus growth and

Chapter 1

on MS media containing 3 mg/l 2,4-D

Calli induced from Shoot apex explant and (c)

Calli produced from three different explants (seed, shoot apex and immature inflorescence) were multiplied in MS media with varying concentrations of 2,4-D and

Maximum callus growth was recorded from seed (non-embryogenic calli) and immature inflorescence (embryogenic calli) in all genotypes on

Table 1.4 Rates of callus growth from three different explants on MS media containing 0.5 mg/l BA

= Moderate callus growth and + + + + =Fast callus

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30

In case of seed, maximum non-embryogenic callus growth was observed at a 2,4-D concentration of 3 mg/l. Calli became embryogenic when the level of 2,4-D was increased to 6 mg/l in all the genotypes. The morphogenic potential of the induced callus through seed and shoot apex was assessed in Cenchrus by transferring the callus on same induction medium (3 mg/l 2,4-D). Callus derived from seed explants were initially loose, watery in nature and became embryogenic after three to four subcultures. On the contrary, callus induced from immature inflorescences were nodular, hard, and compact and milky white in color (Figure 1.7), entered the culture process rapidly, and had high regenerability. Shoot apex-derived callus required two to three subcultures before becoming embryogenic.

Figure 1.7 Callus growth and quality in observed from different explants. Seed: (a) non-embryogenic calli (four week old culture), (b) embryogenic calli (ten week old culture); Shoot apex: (c) non-embryogenic calli , (d) embryogenic calli and Immature Inflorescence: (e) non-embryogenic calli (after one month), (f) embryogeneic calli (two month old culture). 1.3A.3 Embryogenic callus induction The maximum embryogenic callus production was recorded from immature inflorescence-derived calli of IG-3108 genotype (89.6% ± 4.0) on MS medium containing 3 mg/l 2,4-D and 0.5 mg/l BA, whereas the minimum amount of

embryogenic callus was obtained from shoot apex explants of genotype on MS medium supplemented with (Figure 1.8).

Figure 1.8 Frequency of eImmature inflorescence (Im In)] of four genotypes in MS media supplemented with 0.5 mg/l BA with varying levels of 2,4-D.

The texture of embryogenic calli was hard, compact, nodular and m(Figure 1.9). There was a significant influence of genotype, varying levelexplant on embryogenic callus obtained.levels were also found to be significant. Strong interaction among genotypesand hormone levels significantly influenced the callus induction.media containing varyinBA level (0.5 mg/l) was tested for maintaining embryogenic calli. It wasall the genotype, could be maintained on MS medium with 6BA.

For improving embryogenic callus quaembryos, few growth adjuvantsHydrolysate (CH) were used. These organic nitrogenous additives marked significant effect on somatic embryo formation when they were used in the embryogenic callus maintenance medium.

31

embryogenic callus was obtained from shoot apex explants of genotype on MS medium supplemented with 5 mg/l 2,4-D and 0.5

Frequency of embryogenic calli ± SE from three explants [Seed, Shoot apex (SA), Immature inflorescence (Im In)] of four genotypes in MS media supplemented with 0.5 mg/l BA

D.

The texture of embryogenic calli was hard, compact, nodular and mThere was a significant influence of genotype, varying level

explant on embryogenic callus obtained. The interactions between explants and media levels were also found to be significant. Strong interaction among genotypesand hormone levels significantly influenced the callus induction. In case of seed,media containing varying levels of 2,4-D (2, 3, 5, 6 and 7 mg/l) along with optimum BA level (0.5 mg/l) was tested for maintaining embryogenic calli. It was

, could be maintained on MS medium with 6 mg/l 2,4

For improving embryogenic callus quality and development of large number of somatic growth adjuvants like L. Proline (Pro), L. Glutamine (Glu) and Casein

Hydrolysate (CH) were used. These organic nitrogenous additives marked significant effect on somatic embryo formation when they were used in the embryogenic callus

Chapter 1

embryogenic callus was obtained from shoot apex explants of DBC15-8/32/10 D and 0.5 mg/l BA

mbryogenic calli ± SE from three explants [Seed, Shoot apex (SA),

Immature inflorescence (Im In)] of four genotypes in MS media supplemented with 0.5 mg/l BA

The texture of embryogenic calli was hard, compact, nodular and milky white in color There was a significant influence of genotype, varying levels of 2,4-D, and

The interactions between explants and media levels were also found to be significant. Strong interaction among genotypes, explants

In case of seed, MS mg/l) along with optimum

BA level (0.5 mg/l) was tested for maintaining embryogenic calli. It was observed that mg/l 2,4-D and 0.5 mg/l

lity and development of large number of somatic like L. Proline (Pro), L. Glutamine (Glu) and Casein

Hydrolysate (CH) were used. These organic nitrogenous additives marked significant effect on somatic embryo formation when they were used in the embryogenic callus

Chapter 1

32

Figure 1.9 Embryogenic callus induction in three different explants cultured on MS media containing 400 mg/l proline, 400 mg/l glutamine and 300 mg/l casein hydrolysate. (a) Calli induced from Seed explant on 6 mg/l 2,4-D + 0.5 mg/l BA; (b) Calli induced from Shoot apex explant and (c) Calli induced from Immature Inflorescence explant on 3 mg/l 2,4-D + 0.5 mg/l BA.

1.3A.4 Effect of growth adjuvants Effect of different growth adjuvants viz. L-Proline, L-Glutamine and Casein Hydrolysate on induction of embryogenic calli from immature inflorescence-derived calli (with 3 mg/l of 2,4-D in MS medium) from IG-3108 was observed. The frequency of somatic embryogenesis increased with the addition of each growth adjuvants such as proline (90% at 400 mg/l), glutamine (88% at 400 mg/l) and casein hydrolysate (81% at 300 mg/l) compared to control (50%). There was a slight further increase (92% at 400, 400, 300 mg/l Pro, Glu and CH respectively) in the frequency of SE when all the three adjuvants are added together (Table 1.5). All three amino acids significantly enhanced rate of somatic embryogenesis. Most of the somatic embryos germinated and developed plantlets after 1-2 weeks of incubation in supplemented medium. 1.3A.5 Formation of somatic embryos The somatic embryos (SEs) developed from embryogenic callus turned green when the auxin levels were gradually reduced and cytokinin levels increased in the presence of light after 2 weeks of culture. The somatic embryos developed into small filamentous

(cup shaped) globular shaped embr(Figure 1.10). The SEs showed different stages of somatic embryogenesis such as globular, scutellar and coleoptile stages. At high cytokinin (BA) levels (3 mg/L), these somatic embryos turned green resulting ishoots in the presence of light.

Table 1.5 Effect of various concentrationembryogenesis through callus culture of cv. IG

1.3A.6 Histological,

studies of development stages of somatic embryo Stereomicroscopic studies showed embryogenic calli composed of compact cell masses, hard and white in colour, in contrastmasses of cells, soft, irregular in shap

33

(cup shaped) globular shaped embryos from the nodular compact embryogenic calli (Figure 1.10). The SEs showed different stages of somatic embryogenesis such as globular, scutellar and coleoptile stages. At high cytokinin (BA) levels (3 mg/L), these somatic embryos turned green resulting into organogenesis with protruding green shoots in the presence of light.

Table 1.5 Effect of various concentrations and combinations of growth adjuvantembryogenesis through callus culture of cv. IG-3108.

Histological, stereomicroscopic and scanning electron microscopic of development stages of somatic embryo

microscopic studies showed embryogenic calli composed of compact cell masses, hard and white in colour, in contrast, non-embryogenic calli had composed of masses of cells, soft, irregular in shape and pale yellow in colour (Figure 1.10a

Chapter 1

yos from the nodular compact embryogenic calli (Figure 1.10). The SEs showed different stages of somatic embryogenesis such as globular, scutellar and coleoptile stages. At high cytokinin (BA) levels (3 mg/L), these

nto organogenesis with protruding green

of growth adjuvants on somatic

scanning electron microscopic

microscopic studies showed embryogenic calli composed of compact cell masses, embryogenic calli had composed of loose

e and pale yellow in colour (Figure 1.10a). In

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34

presence of higher level of cytokinin and light, calli converted into green and somatic embryo started to germinate when further stages of embryo development were visible i.e. globular, scutellar type of embryos, scutellum, coleoptiles, young leaf primordia, were fully developed (Figure 1.10b-e).

Figure 1.10 Development of somatic embryos from embryogenic callus culture in Cenchrus ciliaris: Stereomicroscopic photographs of somatic embryo development from callus (a) Proliferating white nodular embryogenic calli (NEC) containing somatic embryo (SE) formed after subculture on 3 mg/l 2,4-D; (b) A bunch of globular embryos (GE); (c) Yellow green somatic embryo (SE) formed from nodular embryogenic calli; (d) Formation of scutellar embryo (SE); (e) Calli turn green (GC) and forms well developed somatic embryo to start regenerating into shoot ( on 3 mg/l BA after seven days). Scanning Electron Microphotographs showing (f) Development of many proembryos of globular stage, scutellum (SC) with coleoptile (CO) and scutellar notch (SN) along with a embryogenic callus (EC) mass; (g) Well-developed germinating somatic embryos, organized in form of disc-shaped scutellum (SC) before origin of coleoptile; (h) Formation of fused somatic embryo with lateral scutellar notch (SN, which partially separates the future embryonic axis from scutellum) and differentiates into scutellum. Histological sections of callus piece showing: (i) Young somatic embryo (SE) of epidermal origin, small projections on the surface of callus (marked by arrows). Well developed upper epidermis (UE) and parenchyama cell layers (PC) visible; (j) An oval shaped somatic proembyo (PE) with well defined protodermis (PT) formed from meristematic cell (MC); (k) An enlarged view of globular staged somatic embryo with procambial strands (PS), (l) Scutellar notch (SN) at the terminal region of embryo and a globular somatic embryo (GE) without connection to mother tissue and suspensor like; (m) Somatic embryo with developing shoot meristem (SM); (n) Fully developed somatic embryo with dome shaped shoot apical meristem (SAM), coleorhiza (CR), coleoptile (CO) and vascular bundles (VB).

Chapter 1

35

SEM further confirmed different stages of somatic embryogenesis in genotype IG-3108. It showed the presence of lobed, more or less individualized and organized globular structures. Such structures were delimited with isodiametrical cells and have developed a lateral notch, a rapid differentiation of embryos with a well defined scutellum with coleoptile occurred (Figure 1.10f-h).

Histological studies with structural details of somatic embryogenesis and plant regeneration were also observed in this system. Cross sections showed the initiation and development of somatic embryos. At the peripheral region of embryogenic calli, multiplying meristematic cells were observed. These cells were small, compact and cytoplasmically dense (Figure 1.10i). Initiation of embryogenesis with continous divisions of single cells resulted in two form, six and eight celled embryos, which after a week formed globular embryo (Figure 1.10k). These pro-embryo structures continued a series of organized division and gave rise to globular somatic embryos (Figure 1.10l). Initially the embryo was attached to the embryogenic callus with a prominent procambial strands. These somatic embryos showed signs of polarization with apical and radical meristems in opposite poles. The shoot apex is organized and the coleoptile developed as a circular primordium around the shoot apex (Figure 1.10m). The shoot apex showed a lateral position while the root apex was seen opposite the shoot apex. The somatic embryos exhibited a scutellum, coleoptile, shoot apex and coleorhiza with an independent vascular system that is not connected to the maternal tissue (Figure 1.10n). Dome shaped shoot apical meristem with leaf primordia were visualized, (Figure 1.10n) which confirmed the induction of somatic embryogenesis.

1.3A.7 Shoot regeneration The regenerability of the somatic embryos was clearly visible as green tiny shoot primordia within 3-4 days of transfer to regeneration medium and after a week the green shoots emerged out of the callus (Figure1.11a-d). Well-grown shoots with leaves from all four genotypes were successfully formed upon germination of somatic embryos as well as by shoot organogenesis subcultured on MS medium containing varying levels of BA (1, 2, 3 or 4 mg/l) in combination with 0.25 mg/l 2,4-D (Figure 1.11e-g). It was observed that regeneration of callus and also number of shoots formed

Chapter 1

36

was faster from immature inflorescence derived calli in comparison to seed as well as shoot apex derived calli (Figure 1.11h-k). Maximum frequency of shoot induction (85% ± 2.2) and highest number of shoots per callus (12 ± 1.0), was observed on MS medium containing 3 mg/l BA and 0.25 mg/l 2,4-D in IG-3108 whereas lowest number of shoots per calli was observed on MS with BA (3 mg/l) and 2, 4-D (0.25 mg/l) in seed followed by shoot apex explants of genotype DBC15-8/32/10 (5.3 ± 0.4) (Table 1.6). Genotype, explants, and BA levels as well as interaction between the later two factors had significant influence on the shoot induction frequency. The absence of a significant interaction between genotype and concentration of BA level indicated that the exogenous supply of cytokinin (BA) influences the response from different explants but not in type of genotype.

Figure 1.11 Development of shoot primordia and shoot regeneration in Cenchrus cilliaris. (a) Two weeks old cultures showing creamish green callus when transferred to MS medium supplemented with 3 mg/l BA and 0.25 mg/l 2,4-D; (b) Greenish with purplish (because of anthocyanin) callus when transferred to MS medium supplemented with 3 mg/l BA and 0.25 mg/l 2,4-D, (c) Proliferating green calli with somatic embryo start to regenerate after three weeks; (d) Germination of the embryoid with the leaf protruding from the coleoptile after four weeks. (e-k) Five week old culture showing complete shoot regeneration in MS media containing 3 mg/l BA and 0.25 mg/l 2,4-D: (e), 3mg/l BA and 0.25mg/l 2,4-D (f,g), shoot elongation (h,i) and multiplication (j,k)

Table 1.6 Frequency of Shoot regeneration and the number explants of four genotypes in MS

1.3B Plant Regeneration from shoot apices1.3B.1 Direct shoot organogenesisIn vitro direct shoot organogenesis was attempted by inducing multiple shoots from shoot apex explant of Shoot apices consisted of shoot apical meristem, which formed shoot primordia directly resulting into multiple shootshigh cytokinin containing MS medium followed by shoot elongation in decreased cytokinin medium. The wellfor efficient root induction.

Multiple shoot formation without visible intervening callus phase was achieved by using three cytokines, BA, Kinetin and a synthetic ureagenotypes (IG-3108, DBC152/65/26, DBC25-6/67/28, DBC26

Different concentrations of all the three cytokinins (1shoot formation in all genotypes of buffel grass using shoot apex induced in all treatments with varying frequencies. Out of three cytokines tested,supplemented with TDZ showed a significantly superior response in terms of multiple shoot induction (as well as number of shoots induced per shooshoots grown within 2–

37

ency of Shoot regeneration and the number of shoots per callus in three explants of four genotypes in MS media containing 0.25 mg/l 2,4-D with varying levels of BA.

Plant Regeneration from shoot apices 1 Direct shoot organogenesis direct shoot organogenesis was attempted by inducing multiple shoots from

shoot apex explant of Cenchrus using high concentration of cytokinin in the media. Shoot apices consisted of shoot apical meristem, which formed shoot primordia directly ulting into multiple shoots (Figure 1.12). Initially, multiple shoots were induced o

high cytokinin containing MS medium followed by shoot elongation in decreased cytokinin medium. The well-developed shoots were then transferred to rooting medium

cient root induction.

Multiple shoot formation without visible intervening callus phase was achieved by using three cytokines, BA, Kinetin and a synthetic urea-cytokinin, Thidiazuron (TDZ) in eight

3108, DBC15-8/32/10, DBC18-2/41/15, DBC246/67/28, DBC26-3/69/30, and DBC27-6/73/34) of Cenchrus ciliaris

Different concentrations of all the three cytokinins (1-5 mg/l) were tested for multipleshoot formation in all genotypes of buffel grass using shoot apex explants. Shoots were induced in all treatments with varying frequencies. Out of three cytokines tested,supplemented with TDZ showed a significantly superior response in terms of multiple shoot induction (as well as number of shoots induced per shoot apex); with distinct

–3 weeks of culture initiation in all genotypes.

Chapter 1

of shoots per callus in three different D with varying levels of BA.

direct shoot organogenesis was attempted by inducing multiple shoots from using high concentration of cytokinin in the media.

Shoot apices consisted of shoot apical meristem, which formed shoot primordia directly ultiple shoots were induced on

high cytokinin containing MS medium followed by shoot elongation in decreased developed shoots were then transferred to rooting medium

Multiple shoot formation without visible intervening callus phase was achieved by using cytokinin, Thidiazuron (TDZ) in eight

DBC24-2/60/22, DBC25-Cenchrus ciliaris.

5 mg/l) were tested for multiple-explants. Shoots were

induced in all treatments with varying frequencies. Out of three cytokines tested, media supplemented with TDZ showed a significantly superior response in terms of multiple

t apex); with distinct 3 weeks of culture initiation in all genotypes.

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Figure 1.12 Direct shoot organogenesis in Cenchrus cilliaris using shoot apices in different media: (a-b) Ten days old culture on MS media supplemented with 3 mg/l BA, 3 mg/l KIN (c-d); (e-h) 3 mg/l TDZ showing formation of multiple shoot clumps; (i-l) Four week old cultures showing multiplication of in-vitro shoots; (m-p) Multiple shoots/ Adventitious shoots containing 10-15 single shoots; (q-t) Single plantlets; (u) Shoot elongation on MS medium.

Maximum shoot induction frequency varied widely among the eight cultivars of buffel grass with values ranging from 16.6% to 93.3%. Genotype IG-3108 and DBC15-8/32/10 showed the highest frequency of multiple shoot induction with values of 93.3% ± 2.1 and 90% ± 3.6 respectively, when the shoot apices were cultured on MS medium containing TDZ (3 mg/l). Maximum number of shoots produced per shoot apex was 19.5 ± 0.34 and was observed on MS medium containing 3 mg/l TDZ in genotype IG-3108 followed by 18.6 ± 0.33 in genotype DBC15-8/32/10 on same medium and the minimum number of shoots (2.1 ± 0.61) was observed on medium supplemented with 1 mg/l kinetin in DBC25-6/67/28 genotype. The number of shoots produced per shoot apex explant increased as the levels of BA, Kinetin and TDZ increased up to 3 mg/l, whereas at cytokinin concentrations above 4 mg/l, the multiple shoot induction rate decreased (Table 1.7).

On the contrary, the number of shoots produced per explant decreased as the BA, kinetin and TDZ level was increased but in shoots produced per shoot apex and percentage explants responded decreased at higher concentration upto 3 mg/l. In case of

TDZ the shoots formed at this concentration were unhealof nodal part) within weeks. Table 1.7 Number of shoots induced per shoot apex and the explants responded from eight genotypes on MS media containing various levels of three different cytokinins (BA, KIN and TDZ).

There was a significant influencenumber of shoots formed per explant.shoot apex indicated that the genotypes and the various combinations of media had significant effect on the number of multiple shoot induction.media, the growth of primary shoot gets retarded and 2from each shoot apex onfor 3-4 weeks on elongation media before inducing rooting 1.3B.2 Histological observation of multiple shoot formation Incubation of shoot apex explants on induction medium caused a dramatic enlargement and swelling of tissues. side of the first leaf about one week after and soon a few more shoots emerged from the side of the first shoot. Multiple shootthe main stem stopped grchange was the initiation of meristematic activity in the epidermal and sub epidermal cell layers on the basal region of the first leaf

39

TDZ the shoots formed at this concentration were unhealthy and turned brown (on base of nodal part) within weeks. Table 1.7 Number of shoots induced per shoot apex and the explants responded from eight genotypes on MS media containing various levels of three different cytokinins (BA, KIN and TDZ).

a significant influence of genotype and the levels of BA/ kinetin/ TDZ on number of shoots formed per explant. The analysis of variance for number of shoots per shoot apex indicated that the genotypes and the various combinations of media had

effect on the number of multiple shoot induction. After 30 daymedia, the growth of primary shoot gets retarded and 2-3 axillary shoots were induced from each shoot apex on MS medium without any hormone, which were grown

4 weeks on elongation media before inducing rooting.

.2 Histological observation of multiple shoot formation Incubation of shoot apex explants on induction medium caused a dramatic enlargement and swelling of tissues. In the earlier differentiated shoot, the first shoot emerged at the side of the first leaf about one week after and soon a few more shoots emerged from the side of the first shoot. Multiple shoots were formed by four weeks after culture and later the main stem stopped growing and died. After three days of culture the first visible change was the initiation of meristematic activity in the epidermal and sub epidermal cell layers on the basal region of the first leaf (Figure 1.13).

Chapter 1

thy and turned brown (on base

Table 1.7 Number of shoots induced per shoot apex and the explants responded from eight genotypes on MS media containing various levels of three different cytokinins (BA, KIN and TDZ).

of genotype and the levels of BA/ kinetin/ TDZ on The analysis of variance for number of shoots per

shoot apex indicated that the genotypes and the various combinations of media had After 30 days on culture

3 axillary shoots were induced , which were grown in vitro

.2 Histological observation of multiple shoot formation Incubation of shoot apex explants on induction medium caused a dramatic enlargement

differentiated shoot, the first shoot emerged at the side of the first leaf about one week after and soon a few more shoots emerged from the

were formed by four weeks after culture and later owing and died. After three days of culture the first visible

change was the initiation of meristematic activity in the epidermal and sub epidermal

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Figure 1.13 Histological sections of in vitro cultured shoot apex explants of Cenchrus cilliaris. These sections show developing stages of multiple shoots from shoot apex explants (a-b) After two days of culture, shoot apex in proliferation medium (3 mg/l TDZ); (c-d) After one week, adventitious bud induced on the basal region of first leaf; (e) Fully developed adventitious shoot from main stem; (f) Coleoptile ruptured after expansion of main stem; (g) Developing tiller buds/ shoots from main stem; (h) Development of adventitious shoots; (i-j) Development of more tiller shoots from main stem; (k-l) Full shoots with leaf primordia developed from main stem; (m) Apical meristem with leaf primordia, (n) Expansion of main stem on one side and developing adventitious shoots on the other. (o-q) Development of multiple shoots (Total no of shoots = 5). [(VB) vascular bundles, (LP) leaf primordia, (Co) coleoptile, (L) leaf, (AM) apical meristem, (ABP) adventitious bud primordia, (AS) adventitious shoots, (MT) main stem, (MS) meristematic tissue, (TB) tiller bud, (MTB) main tiller bud, (MTS), main tiller shoots]

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In a later stage of development of the shoot from axillary bud, apical meristem was flanked by two leaf primordia. Shoot bud primordia were also observed on the leaf surface in the transverse sections (Figure 1.13). Shoot buds with well developed leaf primordia and shoot apical meristem were visible. These developing buds had the dome-shaped meristems surrounded by 2 leafy primordia which are connected to the vascular system. 1.3C Rhizogenesis, hardening and acclimatization Well-formed shoots (5-6 cm long) of all genotypes were transferred to rooting medium comprising of either ½ MS or MS with auxin (IBA), with or without charcoal. Successful rooting was induced after three weeks of transfer to the tested rooting media. A maximum root induction frequency of 90.0% ± 2.2 and maximum number of roots produced per shoot of 5.3 ± 0.4 was recorded on MS medium containing 2 mg/l IBA (Table 1.8). In vitro flowering was observed in 1% of the plants cultured on MS medium after a long period of 2-3 months (Figure 1.14). Analysis of variance showed significant difference between genotypes and various combinations of media used. Rooted plants were hardened, transferred to pots, and grown in green house (Figure 1.15).

Figure 1.14 In-vitro flowering in different genotypes of Cenchrus cilliaris.

Chapter 1

Table 1.8 Root induction frequency and the number of roots per shoot of four genotypes in five different media combinations.

Figure 1.15 Rooting and hardening of from basal part of shoots; (b) rooting inrooting in MS+ 2 mg/l IBA media; plantlets before hardening, in sterile tap water;(i-j) regenerant plants transferred to vermhardened in soil.

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Table 1.8 Root induction frequency and the number of roots per shoot of four genotypes in five

ardening of in vitro grown plants in different media: (a) Initiation of roots rooting in ½ MS basal medium; (c) rooting in MS basal medium;

(e) single plantlets with roots cultured in liquid MS medium;g, in sterile tap water; (g-h) plantlets with efficient root and shoot growth;

egenerant plants transferred to vermiculite/ soilrite in pots; (k) well estab

Table 1.8 Root induction frequency and the number of roots per shoot of four genotypes in five

Initiation of roots

MS basal medium; (d) tured in liquid MS medium; (f)

t root and shoot growth; ell established plants

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1.4 DISCUSSION The genus Cenchrus includes more than 25 species which are very important warm season forage grasses for livestock and have value as bio-energy feedstock. Cenchrus ciliaris (L.) known as buffel grass is a native species of Southern Asia and East Africa. India is known for their quality forages and apomictic mode of reproduction in this grass. Their genetic improvement requires in vitro manipulation, therefore in the present study, tissue culture and plant regeneration was attempted. Although several reports of in vitro plant regeneration from various Cenchrus explants, such as immature embryos (Sankhla & Sankhla, 1989; Kackar & Shekhawat., 1991) mature embryo (Murty et al., 1992; Ross et al., 1995; Colomba et al., 2006; Bhat et al., 2001) immature inflorescence (Yadav et al., 2009) are available, improvement in culture environment to simplify the method for induction of embryogenic calli and to enhance the frequency of somatic embryogenesis and conversion of embryos to plants is desirable. Moreover, widening of the explant source to include seeds and shoot apices is desirable to overcome seasonal dependency and laborious experimentation. Additionally, a comparison of developmental morphogenesis leading to somatic embryo development could help in improving the embryogenic response. Shoot apex explants were used for developing transgenic plants where shoot apical meristem is targeted. So far only one report on the production of multiple shoots using shoot apices has been published recently (Kumar & Bhat, 2012) which used different concentrations of TDZ (1-5 mg/l) as optimum for the production of adventitious shoots from enlarged shoot apical meristem. But, the effects of other cytokinins such as BA and kinetin were not compared which could have had obvious advantage in improving the rate of multiple shoot production.

In the current study, various explants such as seed, shoot apex and immature inflorescence of four genotypes (IG-3108, IG-718, IG-74, DBC15-8/32/10) were used for inducing somatic embryogenesis while shoot apex explants of eight genotypes (IG-3108, DBC15-8/32/10, DBC18-2/41/15, DBC24-2/60/22, DBC25-2/65/26, DBC25-6/67/28, DBC26-3/69/30, DBC27-6/73/34) were used for direct shoot organogenesis.

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1.4A Plant regeneration through somatic embryogenesis In the current study, the frequency of callus induction was maximum at 3 mg/l 2,4-D from seed explants compared to other two explants, shoot apices and immature inflorescence which differed significantly. Genotype IG-3108 showed better callusing response than other three genotypes for all the explants tested, and the inference that genotype and explants had significant influence on callus induction frequency from our study is consistent with the results of Colomba et al., (2006); Yadav et al., (2009) in C. ciliaris and Kumar et al., (2005) in Dichanthium annulatum. Once the callus has been induced, its growth depends on the inherent callus growth potential of the genotypes, the varying levels of growth hormone such as, auxin and cytokinin concentrations and the composition of culture medium (Gupta et al., 2002). Maximum callus growth was found in seed (in case of non embryogenic calli) and immature infloresence (embryogenic calli) derived callus in all genotypes cultured on same media containing 3 mg/l 2,4-D and 0.5 mg/l BA. After 3-4 subcultures embryogenic and non embryogenic calli were observed. As recorded in our study, two distinct types of calli (one fast growing non-regenerative calli and other slow growing regenerative calli were also reported by Kackar & Shekhawat, (1991); Colomba et al., (2006) & Yadav et al., ( 2009) in Cenchrus, Vikrant & Rashid, (2003) in Paspalum, Kumar et al., (2005) in Dichanthium. Thus, our results are consistent with earlier reports on Cenchrus and other Graminaceous genera.

According to our result, maximum rate of embryogenic callus was induced from immature inflorescence-derived calli of IG-3108 genotype as reported by Yadav et al., (2009). The embryogenic callus multiplied faster in seed explants as the level of 2,4-D increased from 3 mg/l to 6 mg/l and further upto 7 mg/l (Colomba et al., 2006), where as shoot apex and immature inflorescence derived calli were maintained on same medium (3 mg/l 2,4-D). In the present study, seed derived calli required higher levels of 2,4-D after first subculture. These combinations of phytohormones and subculturing periods in our study showed significant improvement in embryogenic callus induction compared to earlier ones.

For increasing the frequency of somatic embryos, growth adjuvants like proline, glutamine and casein hydrolysate were used at different concentrations (100-500 mg/l)

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45

along with MS+ 6 mg/l 2,4-D for seed and 3 mg/l 2,4D+ 0.5 mg/l BA for other two explants. Amino acids such as L-Proline, L-Glutamine, play a key role for induction of somatic embryogenesis in sugarcane. The development of somatic embryos in the presence of amino acids have been reported in maize (Carvalho etal., 1997), wheat (Yadava & Chawla, 2002) and also in sugarcane (Sinha et al., 2000; Desai et al., 2004). In our study, 400 mg/l proline, 400 mg/l glutamine and 300 mg/l CH gave better response for somatic embryo development than control medium (MS + 2,4-D + BA). Callus induction from immature embryos was enhanced by using casein hydrolysate and proline in turf-type tall fescue reported by Bai & Qu, (2001) and in maize (Armstrong & Green, 1985; Gleddie et al., 1983; Dhillon & Gosal et al., 2013). Sun et al., (2004) observed that the addition of proline and casein hydrolysate increased the frequency of callus induction from immature embryos of maize (Sharma et al., 2012; Shohael et al., 2003). Whereas, in sugarcane, addition of adjuvants had no significant improvement on callus induction in a recent work published by Roy & coworkers, (2011).

Globular shaped embryos were induced from all the explants when the auxin levels were decreased during subculture (Colomba et al., 2006; Yadav et al., 2009). Progression of somatic embryogenesis from globular to scutellar embryo was observed in our study. Earlier reports by Yadav et al., (2009) failed to induce embryogenic calli and subsequent induction of somatic embryos from seed and shoot apex in Cenchrus ciliaris. While in the present study, shoots were successfully induced on MS media containing 3 mg/l BA and 0.25 mg/l 2,4-D from all explants derived callus. Histological studies of somatic embryogenesis have been carried out in several genera of the Gramineae. It has been suggested that somatic embryos arise de novo from proliferating undiffierentiated parenchyma cells in cultured mature embryos, or directly from mesophyll cells (Mcdaniel et al., 1982; Conger et al., 1983). Single cell origin of somatic embryos was not unequivocally determined (Lu & Vasil, 1985; Brown and Thorpe 1986). But in some previous studies, it was demonstrated that somatic embryos either developed from single cells in embryogenic calli obtained from cultured mature embryos or inflorescence of Pennisetum americanum (Vasil & Vasil, 1982; Boti & Vasil, 1984; Ho & Vasil 1983), in young leaves or by simple folding of the original

Chapter 1

46

embryo scutellum followed by the de novo formation of an embryogenic axis (Dunstan et al., 1978; Vasil & Vasil, 1985). Maximum shoot induction (85% ± 2.2) and number of shoots (12 ± 1.0) per 100 mg calli was recorded from immature inflorescence derived calli in genotype IG-3108. This represents an improvement in shoot regeneration frequency over earlier studies such as Kackar & Shekhawat, (1991), who reported shoot regeneration from immature inflorescence in an auxin-free medium. Our results corroborate the findings of Colomba et al., (2006); Yadav et al., (2009) who obtained shoot regeneration in 3-4 weeks after embryogenic cultures were transferred to regeneration medium containing NAA and BA. As indicated in our study, significant genotypic differences were also observed in shoot differentiation and maintenance of regeneration capacity in Sorghum (Cai & Butler, 1990), barley (Hanzel et al., 1985; Rao et al., 1992), wheat (Rajyalakshmi et al., 1991) and oat (Cummings et al., 1976). 1.4B Plant regeneration through multiple shoot induction Development of high frequency in-vitro direct shoot organogenesis method will help in overcoming genotypic barriers and ensure availability of explants throughout the year with faster multiplication rate/ unit sources while shortening the time duration of in-vitro plant culture. Additionally, direct plant regeneration does not produce somaclonal mutations which are most desirable for micro-propagation and the development of transgenics plants. Recent reports have shown that transgenic plants regenerated through a more-or-less long term callus phase have an increased risk of somaclonal variation, problems in transgene inheritance and stability of expression (Choi et al., 2000; Bregitzer & Tonks, 2003). The reason behind this investigation was to develop a simple and highly efficient instant in- vitro plant regeneration system for foreign gene transfer in Cenchrus ciliaris which would be less genotype dependent than the existing ones. Our enhanced system of in vitro multiplication using meristematic shoot segments provide an alternate target for genetic transformation of buffel grass. To our knowledge, this is the first report in which combinations of BA/ Kinetin/ TDZ have been tried in various combinations for promoting high rate of shoot organogenesis.

Varying concentrations of BA, Kinetin and TDZ (1 to 5 mg/l) were used in our study to induce multiple shoot formation in eight different genotypes. Although all the

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combination of cytokinins resulted in multiple shoots, maximum number of shoots produced per shoot apex explant was 19 on MS medium containing 3 mg/l TDZ. Although TDZ has been characterized to have powerful cytokinin like activity resulting in a significant improvement in shoot multiplication in diverse recalcitrant species, this compound was introduced into cereal tissue culture for that purpose with a remarkable delay (Schulze et al., 2007). In family Poaceae, Gupta & Conger, (1998) were first to describe application of TDZ for differentiation of multiple shoots in switchgrass. Recent reports have indicated TDZ as more effective cyotokinin compared to BA for shoot organogenesis in rice (Rashid, 2002; Yookongkaew et al., 2007). Ganeshan et al., (2003) cultured leaf base/ apical segments directly on TDZ containing medium to induce multiple shoots, which resulted in the development of 2-8 shoots per explant with a significant genotype and medium interaction. In 2006, they were able to successfully induce multiple shoots in wheat using TDZ. Eight cultivars of barley were tested by Sharma et al., (2005), who observed approximately five fold increased number of shoots and shoot buds per explant than Ganeshan et al., (2003). Several studies have reported the differentiation of multiple shoot clumps from shoot apex (Zhong et al., 1992; Devi et al., 2000; Zhang et al., 1996). Kinetin was also reported to induce multiple shoots in finger millet (Kumar et al., 2001).

Histological observation showed that multiple shoots induced by Cenchrus shoot apex culture on high concentration of cytokinin medium consisted of two types of bud- axillary bud and adventitious bud. In the axillary bud, it was clear that high concentration of cytokinin broke apical dominance and triggered the formation of multiple shoots from the primordium tillers. Adventitious buds of multiple shoots were induced from the basal region of the first leaf by high cytokinin concentration. (Mcclean & Grafton, 1989; Abdulla & Grace, 1987; Kazuhiro & Hattori, 1995; Detrez et al., 1988). The site around the base of leaf may have ability to express the meristematic activity of shoot organogenesis (Zaffari et al., 2000; Barker & Steward, 1962 Okole & Schultz, 1996).