2. Direct organogenesis

Transcription

1 2. Direct organogenesis 2.1. Introduction Soybean [Glycine max (L.) Merrill] is one of the most important protein and oil rich crop in the world. Till now, many laboratories show a great deal of interest to improve this crop by introducing value added agronomic traits. However, the tissue culture and genetic transformation techniques that are well established for other agriculturally important dicotyledonous species are not very efficient for soybean (Ma and Wu, 2008). Successful application of biotechnology in soybean improvement depends on availability of efficient plant regeneration protocol (Uranbey et al., 2005; Haliloglu, 2006). Regenerating soybean plants using the explants with pre-existing meristem and transforming them by Agrobacterium tumefaciens-mediated DNA transfer has resulted some success (Hinchee et al., 1988; Olhoft et al., 2003; Paz et al., 2004). Recently, researchers reported efficient improvements of T-DNA delivery to soybean using various explants (Zhang et al., 1999; Ke et al., 2001; Olhoft and Somers, 2001; Olhoft et al., 2003; Paz et al., 2006). Yet, the transformation efficiency remains low (Ma and Wu, 2008). Hence, much more improvement is needed to develop an efficient regeneration system, which leads to the higher level of recovery of transgenic plants in desirable genotypes. The limitation in many protocols is mainly due to low frequency of shoot regeneration, long regeneration period, and explant growth difficulties, which prevent the plant from being regeneration-competent. These problems could be overcome, if number of shoots/explant is increased or if the number of meristematic cells in the explants is increased. Soybean is among the most recalcitrant crops for in vitro manipulation (Ma and Wu, 2008). Cheng et al. (1980) first reported successful soybean regeneration with seedling cotyledonary nodes as explants on modified B 5 M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 14

2 media. Many researchers have used different parts of the soybean plant as explants for successful regeneration. These include cotyledonary nodes (Wright et al., 1986; Shan et al., 2005; Zia et al., 2010a), seedling shoot tips (Kartha et al., 1981), immature cotyledon embryos (Barwale et al., 1986), epicotyl and primary leaves (Wright et al., 1987a; Wright et al., 1987b), young embryo axes (McCabe et al., 1988), primary leaf nodes (Kim et al., 1990), and hypocotyls (Dan and Reichert, 1998; Yoshida, 2002). Numbers of plant growth hormones were used in the organogenesis studies of soybean. Mostly cytokinins evoked better shoot bud development (Cheng et al., 1980; Saka et al., 1980). Among cytokinins, BA is widely used for shoot regeneration in soybean (Barwale et al., 1986; Wright et al., 1986; Hinchee et al., 1988; Mante et al., 1989; Kaneda et al., 1997; Dan and Reichert, 1998). Other than BA, TDZ (Kaneda et al., 1997; Yoshida, 2002; Shan et al., 2005), Kn (Ma and Wu, 2008), and Zea (Zia et al., 2010a) were also used for soybean shoot regeneration. Even though a wide range of PGRs have been studied for its effect in shoot induction and plant regeneration, till date, to our knowledge, there are no reports demonstrating the role of polyamines in soybean tissue culture. Polyamines (PAs) are important and interesting group of naturally occurring low molecular weight, polycationic, aliphatic nitrogenous compounds present in all cells (Galston, 1983). Polyamines occur in all higher Eukaryotes (Smith, 1985). They have been implicated in several important cellular processes like cell division, protein synthesis, DNA replication, plant response to abiotic stress (Tabor and Tabor, 1984; Smith, 1985; Van den Broeck et al., 1994; Bais et al., 2000), and have been shown to interact with phytohormones (Altman, 1982; Alabadí et al., 1996; Tonon et al., 2001). M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 15

3 The use of polyamines in plant cell culture system dates back to the classical report of Bagni et al. (1978) who produced callus from tuber explants of Jerusalem artichoke. After that, several in vitro culture systems involving plant organ development has been studied in conjugation with polyamines. Positive influence in exogenous supplementation of polyamines alone or in combination with PGRs to the medium for high regeneration has been reported in various plant species like celery and carrot (Robie and Minocha, 1989; Danin et al., 1993), rice (Bajaj and Rajam, 1995; Bajaj and Rajam, 1996), Panax ginseng (Kevers et al., 2000), Elaeis guineensis (Rajesh et al., 2003), radish (Curtis et al., 2004), apricot (Petri et al., 2005), upland cotton (Sakhanokho et al., 2005), Lagenaria siceraria (Shyamali and Hattori, 2007), Cucumis sativus (Zhu and Chen, 2005; Vasudevan et al., 2008), Capsicum frutescens (Kumar et al., 2007), Araucaria angustifolia (Steiner et al., 2007), banana (Venkatachalam and Bhagyalakshmi, 2008), Phalaenopsis amabilis (Gow et al., 2008), Crocus sativus (Chen et al., 2008), Withania somnifera (Sivanandhan et al., 2011), but still now not its role in shoot regeneration has not been studied in soybean. Hence, in the present investigation, using cotyledonary node and half-seed explants, a study was conducted (1) to determine the effect of cytokinins like N 6 -benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ) in multiple shoot induction, (2) to assess the role of PGRs like gibberellic acid (GA 3 ), zeatin (Zea), and indole-3-acetic acid (IAA) in shoot elongation, (3) to identify the suitable concentration of indole-3-butryic acid (IBA) for rooting. In addition, the synergistic role of polyamines (spermidine, spermine, and putrescine) with PGRs on high frequency regeneration has been studied for both types of explants for the first time. M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 16

4 2.2. Materials and Methods Seed source Five cultivars (PK 416, JS 90-41, Hara soya, Co 1 and Co 2) were selected based on their area of cultivation, extent of resistance to diseases, and agro-climatic conditions (NRCS, Updated February 2012). The seeds of cv. PK 416, JS 90-41, and Hara soya were procured from National Research Center for Soybean (NRCS), Indore, Madhya Pradesh, India, whereas the seeds of cv. Co 1 and Co 2 were obtained from Tamil Nadu Agriculture University (TNAU), Coimbatore, Tamil Nadu, India. The seeds obtained from the above sources were multiplied by adopting the agronomic practices as recommended by the respective Institutes in the experimental garden, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India Seed surface sterilization Among the five cultivars, the cv. PK 416 was randomly selected for standardization of direct organogenesis. The surface sterilization of seeds was performed by following the method of Di et al. (1996). The mature seeds of cv. PK 416 were surface sterilized for 16 hr using chlorine gas produced by mixing 3.5 ml of 12N HCl (Qualigens, Mumbai, India) and 100 ml of chlorine bleach (5.25% sodium hypochlorite) [Qualigens, Mumbai, India] in a tightly sealed vacuum desiccator (Tarsons Products Pvt. Ltd, Kolkata, India) In vitro seed germination and cotyledonary node preparation The sterilized-seeds were inoculated with the hilum proximal to the seed germination medium (SGM) comprising MS salts and vitamins (Murashige and Skoog, 1962) [Sigma, St. Louis, USA], sucrose (87.65 mm) [Sisco Research Laboratories, Mumbai, India], and 0.2% (w/v) phytagel (Sigma, St. Louis, USA) or 0.8% (w/v) agar (Himedia, Mumbai, India). The ph of the medium was adjusted to prior to autoclaving at 1.06 kgcm 2 for 15 min using 1N NaOH (Sigma, St. Louis, USA)/0.1N HCl. About 30 ml of the medium was M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 17

5 dispensed into 150 ml Erlenmeyer flasks (Borosil, Mumbai, India) and 6 seeds were inoculated per flask. The inoculated seeds were then incubated for 3 days under total darkness at 25±2 C and later transferred to 16/8 hr light/dark conditions at a light intensity of 50 µmol m 2 s 1 (Cool white fluorescent lamps; Philips, Kolkata, India) for next 4 days. The cotyledonary node explants (~8 mm) were prepared from 7-day-old seedlings by removing cotyledons, primary shoot, and hypocotyl Seed imbibition and half-seed preparation Disinfected-seeds were soaked in 150 ml Erlenmeyer flask containing 30 ml of sterile distilled water (25 seeds per flask). The flasks containing seeds were incubated in an orbital shaker (Orbitek-Scigenics Biotech Pvt. Ltd, Chennai, India) at 120 rpm under total darkness for 1 day at 25±2 C. The imbibed-seeds were transferred to sterile petri plate ( mm; Borosil, Mumbai, India) for dissection. A longitudinal cut was made through the hilum proximal to separate the cotyledons and seed coat. A half-seed with the plumule, radicle, cotyledonary node, and one cotyledon attached was used for the experiment. The plumule and the edge of radicle were removed to obtain the half-seed explants Effect of cytokinins on multiple shoot induction Cotyledonary node and half-seed explants were cultured on shoot induction medium (SIM) [10 ml/tube] in the culture tubes ( mm; Borosil, Mumbai, India) containing MS salts and vitamins, sucrose (87.65 mm) along with various concentrations of plant growth regulators namely, BA ( μm), Kn ( µm), and TDZ ( µm) [Sigma, St. Louis, USA] as individual component to compare their effect on the regeneration ability. Cotyledonary node explants were inoculated vertically with the shoot apical region facing up whereas half-seed explants were inoculated in such a way that radicle were embedded in the medium. The cultures were maintained at M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 18

6 25±2 C in 16/8 hr light/dark photoperiod conditions at a light intensity of 50 µmol m 2 s 1. TDZ was filter-sterilized (0.22 µm; Pall Gelman Sciences, Mumbai, India), and added to warm autoclaved medium. Separate controls were maintained by culturing both the types of explants on PGR free SIM Effect of polyamines on multiple shoot induction In order to test the effect of polyamines with respect to their shoot induction potential, separate experiments were carried out by culturing both types of explants on SIM supplemented with the best concentration of BA (2.22 μm) in combination with different concentrations of polyamines such as spermidine ( μm), spermine ( μm), and putrescine ( μm) [Sisco Research Laboratories, Mumbai, India] (The medium is designated as SIPAM). All tested-polyamines were filter-sterilized (0.22 µm) before adding to warm autoclaved medium. The cultures were maintained in same conditions as described in section Effect of subculture on shoot production After the initial culture duration (15 days), subculture of explants (cotyledonary node and half-seed) along with emerging shoot buds/ shoots was carried out in SIM and SIPAM with same hormonal concentrations for two times at 15 days interval to determine the effect of subculture on shoot production. In the case of half-seed explants, cotyledons were excised from the explants after 15 days of culture (end of initial culture) on SIM and SIPAM. The cultures were maintained in same conditions as described in section Shoot elongation After 45 days of culture on SIPAM, the cotyledonary node and half-seed explants with multiple shoots were transferred to shoot elongation medium (SEM) comprising MS salts and vitamins, sucrose (87.65 mm) supplemented with various concentrations of GA 3 ( µm), Zea ( µm), and M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 19

7 IAA ( µm) [Sigma, St. Louis, USA] to determine their effect in shoot elongation. The cultures were maintained in same conditions as described in section After 15 days of culture on SEM, a subculture was done for both the types of explants with fresh SEM containing same hormonal concentrations and kept for another 15 days. Separate controls were maintained by culturing both the types of explants on PGR free SEM Effect of polyamines on shoot elongation To study the role of polyamines in shoot elongation, both the types of explants with regenerated shoots were cultured on SEM supplemented with different concentrations of polyamines such as spermidine ( μm), spermine ( μm), and putrescine ( μm) along with GA 3 (1.45 μm) [The medium is designated as SEPAM]. The cultures were maintained in same conditions as described in section After 15 days of culture on SEPAM, a subculture was done for both the types of explants in fresh SEPAM containing same hormonal concentrations for another 15 days. After 30 days, shoots longer than 4 cm were excised and transferred to root induction medium (RIM) Rooting Individual elongated shoots from cotyledonary node and half-seed explants were cultured on root induction medium (RIM) containing MS salts and vitamins, sucrose (87.65 mm) along with various concentrations of IBA ( µm) [Sigma, St. Louis, USA] to optimize the ideal concentration of IBA for root induction. For in vitro rooting, the cultures were maintained as described in section Separate controls were maintained by culturing shoots in hormone free RIM. M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 20

8 Effect of polyamines on rooting Elongated shoots from cotyledonary node and half-seed explants were transferred to RIM supplemented with optimal concentration of IBA (4.93 µm) in combination with different concentrations of polyamines such as spermidine ( μm), spermine ( μm), and putrescine ( μm) [The medium is designated as RIPAM] to study their effect on root induction. The cultures were maintained in same conditions as described in section Acclimatization After 30 days of culture in RIPAM, the rooted-plantlets were gently removed from the culture tubes and were washed with running tap water to remove gelling agent from root surface, and then transferred to plastic cups (8 7cm) containing sterile sand, soil, and vermiculate (1:1:1 v/v/v). All the plantlets were covered with polyethylene bags with minute puncture and grown in growth chamber (Sanyo, Osaka, Japan) at 25±2 C with 85% relative humidity (RH) for 2 3 weeks. The plantlets were irrigated once in two days. Upon growth, the plantlets were transferred to earthen pots (25 25 cm) containing sterile sand, soil, and vermiculate (1:1:1 v/v/v) and grown in the greenhouse under controlled conditions Genotypic variations in cultivars After standardization of optimal concentration of plant growth regulators for cotyledonary node and half-seed explants for multiple shoot regeneration using the cv. PK 416, the same hormonal concentration was applied to test their effect on regeneration frequency of other soybean cultivars (JS 90-41, Hara soya, Co 1, and Co 2). M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 21

9 Statistical analysis For multiple shoot induction, 50 explants of both types were cultured per treatment and each growth regulator treatment was repeated thrice. Percentage of explants responding, number of shoots/explant obtained during initial culture, and subsequent subcultures were tabulated. For shoot elongation, 50 explants with multiple shoots of both types were cultured per treatment and each growth regulator treatment was repeated thrice. Percentage of culture showing response, number of elongated shoots/explant, and shoot length in cm were tabulated after 30 days of culture. For rooting, 50 elongated shoots above 4 cm were cultured per treatment and each growth regulator treatment was repeated thrice. Rooting response, number of roots/shoot, and root length in cm was tabulated after 30 days of culture. Data were statistically analyzed using analysis of variance (ANOVA). Data are presented as means±standard error. The mean separations were carried out using Duncan s multiple range test and significance was determined at 5% level (SPSS 11.5) Results Explants Cotyledonary node explants (Fig. 2.3b) prepared from 7-day-old in vitro seedlings (Fig. 2.3a) and half-seed explants (Fig. 2.4b) prepared from 1-day-old imbibed seeds (Fig. 2.4a) were used Effect of cytokinins on multiple shoot induction The percentage of shoot induction response varied with the type of explant and the concentrations of plant growth regulators used. Among the different concentrations of BA, Kn and TDZ tested, BA at (2.22 µm) was most effective for shoot bud induction in both the types of explants (Table 2.1 cotyledonary node; Table 2.2 half-seed). The culture of both cotyledonary node and half-seed explants on BA containing SIM led to initiation of shoot buds after 7 days and 10 days respectively. In both types of explants, M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 22

10 regeneration of shoot buds occurred as small, nodule-like protrusions from the pre-existing axillary meristematic cells. In cotyledonary node and half-seed explants, the percentage of responses using BA (2.22 µm) in SIM individually were 84.33% and 79.05% with the production of and 8.63 shoots/explant respectively after 15 days of initial culture (Table 2.1 cotyledonary node; Table 2.2 half-seed). Increase or decrease in concentration of BA (2.22 µm) led to reduction in number of shoots for both the types of explants (Table 2.1 cotyledonary node; Table 2.2 half-seed). Next to BA, Kn showed a better response in shoot induction for both the types of explants followed by TDZ. The percentage of response using Kn (4.65 µm) and TDZ (0.46 µm) in the SIM were 73.35% and 61.94% for cotyledonary node explants and 68.32% and 57.62% for half-seed explants with the production of 6.62 and 5.31 shoots/explant (cotyledonary node) and 5.63 and 4.92 shoots/explant (half-seed) respectively after 15 days of initial culture (Table 2.1 cotyledonary node; Table 2.2 halfseed). Increase or decrease in concentration of Kn (4.65 µm) and TDZ (0.46 µm) led to reduction in number of shoots in both the types of explants (Table 2.1 cotyledonary node; Table 2.2 half-seed). Both types of explants cultured in SIM without PGR responded very poorly (18.62% for cotyledonary node explants and 13.61% for half-seed explants) and produced an average of only 1.61 shoots/explant (cotyledonary node) and 1.31 shoots/explant (half-seed) during the same culture period (Table 2.1 cotyledonary node; Table 2.2 halfseed) Effect of polyamines on multiple shoot induction The optimal concentration of BA (2.22 μm) was combined with different concentrations of spermidine, spermine, and putrescine (SIPAM) to study their synergistic role in multiple shoot induction. When compared to individual treatments of BA, combination of optimal concentration of BA with polyamines was effective in increasing multiple shoots in both types of explants (Table 2.3 cotyledonary node; Table 2.4 half-seed). Spermidine exhibited higher M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 23

11 percentage of response for shoot induction when compared to spermine and putrescine (Table 2.3 cotyledonary node; Table 2.4 half-seed). Among the different combinations of BA and spermidine tested, BA with spermidine ( μm) was found effective and showed maximum percentage of response with increased production of multiple shoots in both types of explants [96.94% and shoots/explant for cotyledonary node explants (Fig. 2.3c) and 92.04% and shoots/explant for half-seed explants (Fig. 2.4c)] at the end of initial culture (15 days) [Table 2.3 cotyledonary node; Table 2.4 half-seed]. Next to BA and spermidine, BA and spermine evoked better response followed by BA and putrescine combination. Among the several combinations of BA and spermine tested, BA and spermine (98.84 μm) generated better results at the end of initial culture (93.36% and shoots/explant for cotyledonary node explants and 88.35% and shoots/explant for half-seed explants) [Table 2.3 cotyledonary node; Table 2.4 half-seed]. In BA and putrescine combination, putrescine at μm was found better in shoot induction (89.32% for cotyledonary node explants and 83.91% for half-seed explants) with the production of and shoots/explant from cotyledonary node and halfseed explants respectively at the end of initial culture (Table 2.3 cotyledonary node; Table 2.4 half-seed) Effect of subculture on shoot production Explants cultured continuously (up to 20 days) in the SIM containing the same growth regulator neither enhanced shoot number nor resulted in initiation of additional shoots. Hence, subculture of explants along with the shoots was performed in fresh SIM containing the same growth regulator concentrations for two times at 15 days interval. During the subcultures in SIM supplemented with BA, Kn, and TDZ individually, new shoots started to emerge from the base of already regenerated shoots and the axillary meristematic region retained the regeneration potential. At the end of initial culture in SIM containing BA (2.22 µm), cotyledonary node explants produced shoots/explant and half-seed M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 24

12 produced 8.63 shoots/explant. At the end of 2 nd subculture, the shoot number was increased up to shoots/explant in cotyledonary node explants and shoots/explant in half-seed explants (Table 2.1 cotyledonary node; Table 2.2 half-seed). The average length of shoots was around 0.5 to 1.0 cm for both types of explants at the end of 2 nd subculture (data not shown). In SIM containing Kn (4.65 µm), the shoot number was increased from 6.62 to and 5.63 to shoots/explant in cotyledonary node and half-seed explants respectively at the end of 2 nd subculture (Table 2.1 cotyledonary node; Table 2.2 half-seed). For both the types of explants, shoots initiated in presence of Kn were observed to be weak with an average length of 1 to 1.5 cm (data not shown). In SIM containing TDZ, shoots produced from cotyledonary node and half-seed explants showed abnormality in morphology irrespective of the concentrations tested. The shoots produced were in rosettes, fasciated and exhibited stunted growth with dark green leaves (Fig. 2.3l cotyledonary node; Fig. 2.4l half-seed). At the end of 2 nd subculture, the number of shoots that regenerated from a single explant in SIM containing TDZ (0.46 µm) was increased from 5.31 to for cotyledonary node explants and 4.92 to 9.36 for half-seed explants (Table 2.1 cotyledonary node; Table 2.2 half-seed). Prolonged culture (after 2 nd subculture) in SIM containing individual concentrations BA, Kn and TDZ did not produce new shoots, instead yellowing of leaves was observed (data not shown). Subculture of explants into the SIPAM containing same PGRs concentrations as in initial culture also resulted in increase of shoot number for all the BA and polyamines combinations tested. A maximum number of (Fig. 2.3f) and shoots/explant (Fig. 2.4f) were produced from cotyledonary node and half-seed explants respectively in SIPAM containing BA (2.22 μm) along with spermidine ( μm) at the end of 2 nd subculture (Table 2.3 cotyledonary node; Table 2.4 half-seed). In SIPAM containing BA (2.22 µm) and spermine (98.84 µm), and shoots/explant were produced in M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 25

13 cotyledonary node and half-seed explants respectively at the end of 2 nd subculture (Table 2.3 cotyledonary node; Table 2.4 half-seed). A maximum number of and shoots/explant were achieved from cotyledonary node and half-seed explants respectively in SIPAM fortified with BA (2.22 μm) and putrescine (93.12 μm) at the end of 2 nd subculture (Table 2.3 cotyledonary node; Table 2.4 half-seed). At the end of 2 nd subculture the length of shoots regenerated from both the types of explants in medium supplemented with spermidine and putrescine were about 0.5 to 1.0 cm. In same culture duration, increase in shoot length (1.2 to 1.6 cm) was observed in shoots produced from both the types of explants in spermine containing medium (data not shown). The shoots regenerated in all tested BA and polyamines combination were healthy and exhibited normal morphology. The culmination point for shoot production was attained after two subcultures from initial culture for both the types of explants. Subcultures made after (2 nd subculture) neither increased shoot number nor shoot length Shoot elongation The shoots developed from cotyledonary node and half-seed explants on SIPAM containing BA (2.22 μm) with spermidine ( μm) at the end of the 2 nd subculture were short (<1 cm) and hence transferred to SEM with various concentrations of GA 3, Zea, and IAA individually to induce shoot elongation. SEM containing GA 3 showed better response when compared to Zea and IAA (Table 2.5 cotyledonary node; Table 2.6 half-seed). GA 3 at 1.45 μm evoked 71.34% (24.05 elongated shoots/explant) of response in cotyledonary node explants and 65.06% (19.92 elongated shoots/explant) in half-seed explants with an average shoot length of 5.43 and 5.23 cm, respectively after 30 days of culture (Table 2.5 cotyledonary node; Table 2.6 half-seed). The elongated shoots regenerated from GA 3 amended medium were thick and healthy. In SEM containing Zea (4.57 μm), the percentage of response was 62.32% (19.93 elongated shoots/explant) in the case of cotyledonary node explants and 56.34% M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 26

14 (14.33 elongated shoots/explant) in half-seed explants with an average shoot length of 4.40 and 4.10 cm respectively after 30 days of culture (Table 2.5 cotyledonary node; Table 2.6 half-seed). The shoots elongated in medium containing various concentrations of Zea were thin and weak. The response of IAA for shoot elongation in both the types of explants was very low when compared to GA 3 and Zea. In SEM containing IAA (0.58 μm), the percentage of response was 54.31% (14.62 elongated shoots/explant) in the case of cotyledonary node explants and 48.61% (11.36 elongated shoots/explant) in half-seed explants with an average shoot length of 3.33 and 2.90 cm, respectively after 30 days of culture (Table 2.5 cotyledonary node; Table 2.6 half-seed). Both types of explants cultured in SEM without PGR responded very poorly (18.31% for cotyledonary node explants and 15.01% for half-seed explants) and they produced an average of only 6.62 elongated shoots/explant (cotyledonary node) and 5.61 elongated shoots/explant (half-seed) during the same culture period. The average shoot length was 1.86 and 1.60 cm for cotyledonary node and half-seed explants, respectively in same culture duration (Table 2.5 cotyledonary node; Table 2.6 half-seed). After 30 days of culture on SEM, the percentage of explants responding was moderate while number of elongated shoots/explant obtained, remained comparatively low for all the concentrations of PGRs tested Effect of polyamines on shoot elongation In order to further optimize the medium for shoot elongation, both the types of explants with shoots were transferred to SEM containing GA 3 (1.45 µm) in combination with polyamines such as spermidine, spermine and putrescine [SEPAM]. The data were scored after 30 days of culture on SEPAM. Spermine exhibited greater response for shoot elongation when compared to spermidine and putrescine (Table 2.7 cotyledonary node; Table 2.8 halfseed). SEPAM containing GA 3 and spermine (74.13 μm) combination was most effective and showed maximum percentage of response with higher number of M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 27

15 elongated shoots/explant in both the types of explants. The percentage of response was 87.32% [34.62 elongated shoots/explant (Fig. 2.3g)] for cotyledonary node explants and 82.02% [29.31 elongated shoots/explant (Fig. 2.4g)] for half-seed explants (Table 2.7 cotyledonary node; Table 2.8 halfseed). The average shoot length was 7.60 cm in shoots regenerated from cotyledonary node explants and 7.23 cm in half-seed explants respectively (Table 2.7 cotyledonary node; Table 2.8 half-seed). Spermidine which showed maximum response for multiple shoot induction in both the types of explants exhibited less response in shoot elongation when compared to spermine. In SEPAM containing GA 3 and spermidine ( µm), the percentage of response was 81.62% (30.64 elongated shoots/explant) for cotyledonary node explants and 75.31% (26.34 shoots/explant) for half-seed explants (Table 2.7 cotyledonary node; Table 2.8 half-seed). The average shoot length was 6.60 cm in cotyledonary node explants and 6.30 cm for halfseed explants respectively (Table 2.7 cotyledonary node; Table 2.8 halfseed). In SEPAM containing GA 3 and putrescine (62.08 µm), the percentage of response was 76.05% (27.01 elongated shoots/explant) for cotyledonary node explants and 69.92% (22.93 shoots/explant) for half-seed explants (Table 2.7 cotyledonary node; Table 2.8 half-seed). The average shoot length was 6.03 cm in cotyledonary node explants and 5.83 cm for half-seed explants respectively (Table 2.7 cotyledonary node; Table 2.8 half-seed) Rooting The rooting response as well as the nature of roots varied with the concentrations of IBA used. Maximum frequency of rooting (87.33%) as well as production of normal roots (5.34 roots/shoot) was observed when IBA at 4.93 μm was used in RIM (Table 2.9). The average root length was cm at the aforesaid concentration of IBA (Table 2.9). Higher level of IBA (>4.93 μm) showed decrease in response of root induction and also induced callus development from the base of the shoots. Culture of elongated shoots on RIM M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 28

16 without any PGR resulted in poor response (30.32%) and formed less number of roots/shoot (1.31) [Table 2.9] with an average length of 2.33 cm (Table 2.9) Effect of polyamines on rooting To increase the rooting response of in vitro-raised elongated shoots, RIM containing IBA (4.93 µm) was supplemented with different concentrations of polyamines (spermidine, spermine and putrescine) [RIPAM]. Spermidine and spermine which are found to show best response in shoot production and shoot elongation in both the types of explants showed a negative effect for in vitro rooting. In all the combinations of IBA and spermidine tested, elongated shoots started to produce callus at the base and in the case of IBA and spermine combination, shoots started to elongate instead of producing roots. Most of the combination showed nil response for rooting except few combinations which developed very less number of roots after prolonged culture in the same medium (data not shown). In contrast, putrescine which showed less response in shoot induction and elongation showed promising effect in increasing the rooting response. In RIPAM containing IBA (4.93 µm) and putrescine (62.08 µm), maximum frequency of rooting (94.04%) as well as production of normal roots (7.34 roots/shoot) were recorded [Table 2.10; (Fig. 2.3h cotyledonary node; Fig. 2.4h half-seed)]. The average root length was cm on the same concentration of IBA and putrescine (Table 2.10) Acclimatization The well-rooted plantlets were transferred to plastic cups containing sterile sand, soil, and vermiculate (1:1:1 v/v/v) and covered with polythene bags to ensure high relative humidity (85% RH). The plantlets were maintained under controlled environmental conditions for two weeks and were irrigated with water once in two days during this period to avoid desiccation. When signs of new shoot growth were evident (2 3 weeks) [Fig. 2.3i &j cotyledonary node; Fig. 2.4i &j half-seed], polythene bags were gradually removed and the survived M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 29

17 plantlets (90%) were subsequently transferred to earthen pots containing composition mixture as mentioned above and grown in greenhouse (Fig. 2.3k cotyledonary node; Fig. 2.4k half-seed) Genotypic variations in cultivars Genotype influenced the shoot regeneration response as well as the average number of shoots produced/explant. Among the five genotypes tested to study, cv. PK 416 responded most favorably with the highest percentage of response (96.94% for cotyledonary node and 92.04% for half-seed) with the highest number of shoots, i.e. an average of and shoots/explant for cotyledonary node and half-seed explants respectively in SIPAM containing BA (2.22 μm) and spermidine ( μm) [Fig. 2.1 cotyledonary node; Fig. 2.2 half-seed]. This was followed by Co 1 (CN 81.32%, H-S 76.33%), Hara soy (CN 70.35%, H-S 63.99%), Co 2 (CN 64.67%, H-S 59.95%), and JS (CN 49.93%, H-S 43.38%) [Fig. 2.1 cotyledonary node; Fig. 2.2 halfseed] Discussion Explants In the present study, cotyledonary node (7-day-old) and half-seed (1-dayold) explants were successfully used for multiple shoot production in cv. PK 416, JS 97-41, Hara soy, Co 1, and Co 2. Cotyledonary node and half-seed as explant source for multiple shoot induction has been studied earlier for many soybean genotypes (Wright et al., 1986; Shan et al., 2005; Ma and Wu, 2008; Zia et al., 2010a). In the present investigation, preparation of explants and response of explants towards various plant growth regulators are described. For the first time, a study to determine the effect of polyamines such as spermidine, spermine, and putrescine on multiple shoot induction, elongation and rooting for both types of explants has also been carried out in soybean. M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 30

18 Cotyledonary node and half-seed explants when cultured on SIM without PGR showed a very poor response towards shoot induction [1.61 shoots/explant (cotyledonary node) and 1.31 shoots/explant (half-seed)]. The above obtained results strongly emphasis that cytokinin supplementation to SIM is very essential for multiple shoot induction in both the types of explants. Wright et al. (1986) and Carmen et al. (2001) showed histologically that exogenously applied cytokinins alters the development of axillary meristems, promotes proliferation of the meristematic cells in the axillary buds, increases the number of bud primordia which originated from the pre-existing axillary meristems. In this study, multiple shoots were developed from pre-existing axillary meristems found in both the types of explants by supplying appropriate cytokinins to the SIM. Further multiple shoot production was increased by the addition of exogenously supplied polyamines to the SIM making this protocol efficient to be used in transformation of soybean cultivars Effect of cytokinins on multiple shoot induction In the present study, the effects of three cytokinins (BA, Kn and TDZ) were investigated on multiple shoot induction from cotyledonary node and halfseed explants. The results confirmed that BA produced greatest effect (84.33% for cotyledonary node explants and 79.05% for half-seed explants) on shoot induction among three plant growth regulators tested. Successful regeneration protocols in soybean on medium containing only BA has been reported (Barwale et al., 1986; Wright et al., 1986; Reichert et al., 2003; Shan et al., 2005). The superiority of BA on shoot induction over other PGRs such as IBA and Kn (Ma and Wu, 2008) and Zea and Kn (Zia et al., 2010a) has also been reported in soybean tissue culture. In contrast to the present investigation, Kaneda et al. (1997) reported that TDZ showed better response on shoot induction from cotyledonary node and hypocotyl explants of soybean when compared to BA. M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 31

19 Our observation revealed that low concentration of BA (2.22 µm) showed maximum response for shoot induction. In SIM containing BA (2.22 µm), one explant of cotyledonary node and half-seed explant generated and 8.63 shoots respectively after initial culture of 15 days, which was in agreement with the previous report of using low concentration of BA (2.22 µm) to induce efficient shoot regeneration in soybean (Shan et al., 2005). In the present study, Kn was not much efficient on induction of multiple shoots as compared to BA. However, Kn at 4.65 µm showed reliable response for multiple shoot induction (73.35% for cotyledonary node explants and 68.32% for half-seed explants). Ma and Wu (2008) and Zia et al. (2010a) reported shoot induction in soybean from whole cotyledonary node and cotyledonary node explants using Kn as a cytokinin supplement in shoot induction medium. In the present study, shoot abnormality was observed in both the types of explants for all the concentrations of TDZ tested ( µm). This type of abnormalities appeared in peanut when cotyledon explants were cultured in presence of TDZ (Akasaka et al., 2000; Kathiravan et al., 2006). Abnormalities such as fasciations and shoots in rosettes are mainly due to the phenyl group of TDZ and are reported as heritable traits commonly associated with long term use of TDZ (Preece et al., 1987; Huetteman and Preece, 1993; Sahoo and Chand, 1998). Shoot formation in rosettes and fascination were also observed in regeneration systems of faba beans (Mohamed et al., 1992) and pigeon pea (Singh et al., 2003). Our observation revealed that usage of TDZ even at very low concentration (0.46 µm) resulted in the production of shoots in rosettes from both the types of explants. In contrast to our study, Kaneda et al. (1997) reported multiple shoots regeneration with normal shoot morphology from cotyledonary nodes and hypocotyl segments of soybean on media supplemented with high M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 32

20 concentration of TDZ (9 µm). This may be due to the difference in explants used or might be a genotypic effect of cultivars Effect of polyamines on multiple shoot induction The next aim in the present study was to assess the effect of polyamines on multiple shoot induction with the optimal concentration of BA (2.22 μm). Polyamines have been regarded as a new class of plant growth regulators or hormonal secondary messengers and as one of the reserves of carbon and nitrogen at least in cultured tissues (Flores and Filner, 1985; Altman and Levin, 1993). In the present study, exogenous application of polyamines in combination with BA (2.22 µm) in SIPAM exhibited a synergistic effect and resulted in highest shoot induction frequency. Polyamines are known to promote shoot multiplication in various plant systems as reported by Chi et al. (1994) and Bais and Ravishankar (2002). Scholten (1998) suggested that regeneration and differentiation in a series of plant species could be drastically improved by the application of polyamines. Desai and Mehta (1985), Kaur-Sawhney et al. (1986), Galston and Sawhney (1990), Chi et al. (1994), and Walden et al. (1997) suggested that polyamines are important for cell growth, somatic embryogenesis, and shoot morphogenesis. In the present study, exogenous administration of polyamines resulted in the restoration of morphogenetic potential and increased the percentage of explant response. There are many reports available in other plant species describing the positive role of polyamines in shoot induction as evidenced in the present study. Changen et al. (1994) had shown that spermidine, spermine and putrescine were all involved in adventitious shoot formation from cotyledons of melon. Zhu and Chen (2005) reported that adventitious shoot formation could be enhanced in cotyledon explants of cucumber by supplementation of 5 mm putrescine, 1 mm spermidine or 0.1 mm spermine in MS medium. In addition, they reported that, explants grew well within all the concentrations of putrescine ( mm), M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 33

21 while at elevated levels, spermidine and spermine inhibited explant response at which cotyledons senesced and died. Similar results were observed in Brassica rapa (Chi et al., 1994). Shyamali and Hattori (2007) reported that putrescine at 15 mm showed 56% regeneration and spermidine at 1 µm showed 34.67% regeneration in the presence of BA using cotyledon explants of bottle gourd. Kumar et al. (2007) reported that exogenously supplied putrescine, spermidine, and spermine enhanced adventitious shoot formation from decapitated seedling explants of Capsicum frutescens and they concluded that 50 mm putrescine along with 10 µm BA in shoot bud induction medium was indispensable for adventitious shoot formation (83%) followed by 50 mm spermine (75%) and 50 mm spermidine (70%). However, in the present study, spermidine rather than spermine or putrescine was most effective in multiple shoot induction from the cotyledonary node and half-seed explants. This evidence showed that spermidine, spermine and putrescine may play dissimilar roles in different species or in different explants, as reported by Zhu and Chen (2005) in cucumber. Our results revealed that in SIPAM containing BA (2.22 µm) and optimal level of spermidine ( μm), 96.94% of the cotyledonary node explants produced an average of shoots/explant and 92.04% of the half-seed explants produced an average of shoots/explant at the end of initial culture. The results obtained were in agreement with the report of Vasudevan et al. (2008) in Cucumis sativus using shoot tip explants, in which a combination of BA (4.44 µm) and spermidine (68 μm) evoked maximum response of shoot induction (92%) compared to other polyamine combinations (spermine and putrescine) tested. Sivanandhan et al. (2011) achieved maximum number of multiple shoots (46.4 shoots/nodal explant) with 94% of nodal explants in Withania somnifera on medium containing BA (6.66 µm), IAA (1.72 µm) and spermidine ( µm). They reported that spermidine was superior over spermine and putrescine on shoot production from nodal explants. A similar observation was made by M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 34

22 Tanimoto et al. (1994) for adventitious shoot formation from stem segments of Torenia. Another support for the results obtained in the present investigation was received from Petri et al. (2005) in apricot using leaf as explant. In their report, the application of putrescine at several concentrations did not make any significant difference with the control while spermidine significantly improved regeneration at 2 mm. Although the addition of spermine (98.84 μm) and putrescine (93.12 µm) in SIPAM containing BA (2.22 µm) produced greater percentage of response as well as number of shoots/explant when compared to BA alone, the results obtained were not as significant as spermidine. In our present study, it is assumed that spermidine provided nitrogen source, in addition showed synergistic effect along with BA and enhanced shoot differentiation from both the types of explants Effect of subculture on shoot production Prolonged culture of both the types of explants in same medium with growth regulators up to 20 days did not increase shoot number. Hence, after initial culture (after 15 days), subculture of explants in SIM and SIPAM with respective plant growth regulators was carried out which resulted in a drastic increase in shoot number. The shoot number was increased from to shoots/cotyledonary node explant and 8.63 to shoots/half-seed at the end of 2 nd subculture in SIM containing BA (2.22 µm). In the case of SIPAM containing BA (2.22 µm) and spermidine ( μm), the shoot number was increased from to shoots/cotyledonary node explant and to shoots/half-seed explant at the end of 2 nd subculture. These results are in agreement with the results of Shan et al. (2005) in soybean. They reported that the multiplication of shoot buds and increased production of shoots were possible with repeated subculture of cotyledonary node explants in MS medium M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 35

23 containing TDZ (0.46 µm). Murch et al. (2000) optimized regeneration conditions from etiolated hypocotyl explants in Hypericium perforatum and concluded that an initial culture for 9 days and subsequent subculture into fresh growth regulator free medium increased the frequency of regeneration. On the other hand, in Phaseolus vulgaris, Malik and Saxena (1992) showed that the regeneration commenced after 2 weeks and that the shoot number increased after another 2 weeks of culture in the presence of TDZ though the subcultures were not performed as reported in the present study Shoot elongation Elongation of shoot buds into shoots is a critical step in legume regeneration. Shoots obtained in the presence of BA (2.22 µm) and spermidine ( μm) were short and failed to elongate on repeated subcultures and needed a separate medium for shoot elongation. Hence, in the present study, the explants of both types were cultured in SEM containing different concentrations of GA 3, Zea, and IAA for shoot elongation. Efficient shoot elongation of explants was achieved in SEM supplemented with GA 3. The SEM containing GA 3 (1.45 µm) resulted with 71.34% of response for cotyledonary node and 65.06% of response for half-seed after 30 days of culture. Our results were in agreement with the report of Kumar et al. (2007) in Capsicum frutescens using decapitated seedling explants. In their report, multiple shoots induced in MS medium containing BA (26.63 µm), IAA (2.28 µm), and AgNO 3 (10 µm) along with polyamines (spermidine, spermine and putrescine) failed to elongate even upon prolonged culture (2 months on shoot bud induction medium) and successful elongation (68%) within 45 days was achieved only upon the transfer of explants to MS medium containing GA 3 (2.8 µm) and AgNO 3 (10 µm). In contrary to the present investigation, Vasudevan et al. (2008) reported shoot induction and elongation in the medium containing same hormonal concentration (MS + BA (4.44 μm), leucine (88 μm), and polyamines (spermidine, spermine and putrescine) using shoot tip explants of cucumber. M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 36

24 In soybean, Franklin et al. (2004) achieved efficient shoot elongation in MS medium containing only GA 3 (0.29 µm) using mature and immature cotyledon explants. In the present study, shoots elongated in presence of GA 3 were normal and healthy. In contrast to our findings, Shan et al. (2005) reported that shoots developed from cotyledonary node explants of soybean at the same concentration of GA 3 as in the present study (1.45 µm) were thin and long and about 50% of the shoots became vitrified. Next to GA 3, Zea showed good results in shoot elongation. In our results, Zea at 4.57 µm evoked maximum response (62.32% for cotyledonary node explants and 56.34% for half-seed explants) for shoot elongation which is in agreement with the results of Zia et al. (2010a) where they used same concentration of Zea (4.57 µm) for maximum shoot elongation. Previous reports also suggest the use of Zea for shoot elongation during Agrobacterium-mediated transformation of soybean (Zhang et al., 1999; Liu et al., 2004; Paz et al., 2006; Olhoft et al., 2007). IAA showed less response for shoot elongation (54.31% for cotyledonary node explants and 48.61% for half-seed explants). Chakraborti et al. (2006) reported 80% of shoot elongation using half-seed explant of Cicer arietinum in MS medium containing IAA (1.15 µm). In SEM without any PGR, both the types of explants exhibited a very poor response towards shoot elongation (18.31% for cotyledonary node explants and 15.01% for half-seed explants). In contrast to the present findings, Kaneda et al. (1997) achieved shoot elongation from adventitious shoots of hypocotyl segments in half strength L 2 medium without phytohormone in soybean Effect of polyamines on shoot elongation Nas (2004) reported that addition of polyamines to the culture medium for Corylus avellana showed a strong effect on shoot elongation and stimulated elongation up to 83%. In addition, the author reported that shoot elongation continued up to 4.0 cm while in the absence of polyamines shoot elongation only reached 2.0 cm. M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 37

25 In the present investigation, addition of polyamines to SEM containing optimum concentration of GA 3 (1.45 µm) showed a positive correlation and further increased percentage of shoot elongation, mean number of elongated shoots/explant as well as shoot length for both the types of explants with the maximum response shown by GA 3 and spermine combination. In SEPAM containing GA 3 (1.45 µm) and spermine (74.13 µm), the percentage of response was increased up to 87.32% for cotyledonary node explants and 82.02% for halfseed explants. Further, the elongated shoots/explant also increased in same medium (34.62 elongated shoots/explant for cotyledonary node and elongated shoots/explant for half-seed explants). The average shoot length was 7.60 cm for cotyledonary node explants and 7.23 cm in half-seed explants. In the present study, spermidine and putrescine combination with GA 3 also generated better response when compared to individual treatment of GA 3. In a similar study, Bais et al. (2000) reported the promotive role of GA 3 and polyamine (putrescine) combination with respect to shoot elongation. In their study, GA 3 (1.45 µm) along with putrescine (40 mm) and 2-iP (2.0 mg/l) resulted in higher response in terms of shoot numbers (34.6 shoots/explant) and shoot elongation (7.6 cm) Rooting Although the promotive effect of auxins in eliciting rooting response was well established (D'Silva and D'souza, 1992), their type and level in the nutrient medium were found to vary from tissue to tissue and species to species (Rao and Padmaja, 1996). In the present study, elongated shoots derived from both the types of explants produced well developed roots (5.34 roots/shoot) in RIM containing IBA (4.93 µm). The mean root length was cm at the same concentration of IBA. IBA induced in vitro rooting in many soybean genotypes (Kaneda et al., 1997; Reichert et al., 2003; Ma and Wu, 2008; Zia et al., 2010a). Wright et al. (1987a), and Shan et al. (2005) achieved rooting in medium M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 38

26 without PGRs which was found contrast to our results, in which shoots derived from both explants exhibited relatively a low response without IBA supplementation to the medium Effect of polyamines on rooting In the present study, rooting response is further increased by the addition of polyamines to RIM containing IBA. IBA and putrescine combination showed better response while spermidine and spermine showed negative impact towards rooting. The rooting percentage was increased up to 94.04% with the production of 7.34 roots/shoot in RIPAM containing IBA (4.93 µm) and putrescine (62.08 µm). The results obtained were in agreement with Sivanandhan et al. (2011) in Withania somnifera, in which putrescine at 124 µm showed 100% of rooting in the elongated shoots. Similar results were also obtained by Vasudevan et al. (2008) in Cucumis sativus. In their report, 98% of shoots produced welldeveloped roots with an average of 9.2 roots/shoot on MS medium containing a combination of putrescine (62 μm) along with BA (4.44 μm) and leucine (88 μm). In addition, they reported that treatments with the other two polyamines, spermidine and spermine, caused no response except at the highest tested concentration [spermidine (136 µm) and spermine (98 µm)] and produced a lower number of roots, which are in agreement with the present findings. Geneve and Hackett (1990) recorded root development and elongation in Hedera helix by addition of putrescine Acclimatization The rooted plantlets were acclimatized by transferring them into plastic cups. After 2 3 weeks of acclimatization in environmental growth chamber, the plants were transferred to earthen pots and grown in greenhouse. 90% of plantlets transferred in pots could be successfully established in greenhouse. Similarly, Ma and Wu (2008) achieved 94.5% survival rate by acclimatizing soybean plantlets in mixture of sand, soil and vermiculite in the ratio of 1:1:1 M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 39

27 (v/v/v) in greenhouse. In another study, Franklin et al. (2004) acclimatized soybean plantlets in pots containing Redi-earth (Scotts, OH, USA) and achieved 87% of survival rate of the regenerated plants in the greenhouse Genotypic variations in cultivars Genotype is an important determinant in development of soybean regeneration system (Barwale et al., 1986; Delzer et al., 1990; Komatsuda and Ko, 1990). Selection of appropriate genotype is a vital part in soybean (Ma and Wu, 2008). In our study, however, genotype differences were observed with regard to shoot regeneration, but the outcome was acceptable (Fig. 2.1 cotyledonary node; Fig. 2.2 half-seed). It appears that optimizing the medium with suitable plant growth regulator combinations can overcome genotypeassociated problems with regeneration in the soybean regeneration system using cotyledonary node and half-seed as explants. Similar to the present investigation, genotypic difference in shoot regeneration was reported in soybean by Barwale et al. (1986), Graybosch et al. (1987), Delzer et al. (1990), Franklin et al. (2004) and Ma and Wu (2008) Conclusion The present work demonstrates multiple shoot production from cotyledonary node and half-seed explants of five Indian soybean cultivars. Cotyledonary node explants responded most favorably when compared to halfseed explants for all PGRs treatments tested. The synergistic effect of polyamines with different plant growth regulators resulted in increased percentage of shoot production, elongation and rooting. The present observation is the first report in soybean. The procedure enables the production of a large number of regenerated plantlets in a relatively short period. The protocol described herein will be useful for soybean genetic transformation experiments to transfer variety of novel or useful agronomic traits for effective crop improvement. M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 40

28 Table 2.1. Effect of cytokinins on multiple shoot induction from cotyledonary node explants derived from 7-day-old in vitro seedlings of soybean cv. PK 416 on shoot induction medium (SIM). Plant growth regulators (μm) Control BA Kn TDZ Percentage of explants responding (%) 18.62±0.30 l 73.61±0.37 d 84.33±0.21 a 81.65±0.33 b 76.02±0.29 c 71.31±0.21 e 67.03±0.21 f 73.35±0.36 da 61.36±0.36 ga 54.91±0.31 h 48.31±0.30 i 61.94±0.37 g 54.32±0.33 ha 43.91±0.23 j 28.6±0.30 k Mean number of shoots/explant Initial culture (After 15 days) 1.61±0.16 ha 7.03±0.36 c 10.34±0.55 a 9.61±0.61 aa 8.35±0.30 b 6.91±0.31 ca 5.36±0.39 d 6.62±0.30 cb 4.62±0.42 dbe 3.96±0.31 eaf 2.95±0.23 fcga 5.31±0.36 da 3.91±0.23 ebfa 3.06±0.33 fbg 2.32±0.26 gbh 1 st subculture (After 15 days) 1.61±0.16 k 12.32±0.51 bac 16.32±0.33 a 15.35±0.53 aa 13.03±0.47 b 10.96±0.37 da 9.31±0.47 e 11.62±0.33 cad 7.94±0.37 fag 6.06±0.33 hai 5.31±0.30 ia 8.32±0.39 eaf 7.01±0.33 gah 4.92±0.27 ibj 3.90±0.27 ja 2 nd subculture (After 15 days) 1.61±0.16 m 15.94±0.64 d 22.33±0.44 a 20.92±0.27 b 17.31±0.49 c 13.64±0.22 eaf 12.64±0.52 fag 14.02±0.25 e 10.93±0.43 ha 8.04±0.25 j 6.32±0.42 kal 11.61±0.33 gah 9.63±0.47 i 7.34±0.36 jak 5.32±0.30 la Control: Treatment without Plant growth regulators. For each treatment, 50 explants were used and repeated three times. Values represent the means±standard error. Mean values followed by the same letters within a column are not significantly different according to Duncan s multiple range test at 5% level.

29 Table 2.2. Effect of cytokinins on multiple shoot induction from half-seed explants derived from 1-day-old imbibed seeds of soybean cv. PK 416 on shoot induction medium (SIM). Plant growth regulators (μm) Control BA Kn TDZ Percentage of explant responding (%) 13.61±0.33 l 68.62±0.54 d 79.05±0.36 a 75.32±0.42 b 70.35±0.55 c 65.02±0.36 e 63.31±0.36 f 68.32±0.39 da 57.01±0.29 ga 49.32±0.30 ha 44.61±0.30 i 57.62±0.30 g 49.92±0.37 h 37.63±0.37 j 23.35±0.30 k Mean number of shoots/explants Initial culture (After 15 days) 1.31±0.15 i 5.92±0.31 cad 8.63±0.37 a 7.32±0.26 b 6.63±0.37 bac 5.02±0.25 eaf 4.61±0.30 fbg 5.63±0.16 dae 3.96±0.23 ga 3.06±0.21 h 2.33±0.21 hb 4.92±0.27 ebfa 3.91±0.23 gb 3.01±0.29 ha 2.31±0.15 hc 1 st subculture (After 15 days) 1.31±0.15 k 9.32±0.30 bac 13.34±0.30 a 12.65±0.40 aa 10.02±0.29 b 8.66±0.26 cad 7.02±0.21 e 8.61±0.40 cbda 5.93±0.37 fag 5.05±0.21 hai 4.32±0.30 iaj 7.91±0.37 db 6.62±0.30 eaf 5.31±0.30 gah 4.03±0.21 ja 2 nd subculture (After 15 days) 1.31±0.15 k 12.31±0.36 d 18.62±0.26 a 16.35±0.26 b 14.01±0.33 c 10.61±0.26 e 9.31±0.30 fa 11.63±0.40 da 8.37±0.26 g 7.01±0.25 h 5.32±0.21 iaj 9.36±0.33 f 8.32±0.44 ga 6.01±0.25 i 5.02±0.25 ja Control: Treatment without Plant growth regulators For each treatment, 50 explants were used and repeated three times. Values represent the means±standard error. Mean values followed by the same letters within a column are not significantly different according to Duncan s multiple range test at 5% level.

30 Table 2.3. Effect of polyamines on multiple shoot induction from cotyledonary node explants derived from 7-day-old in vitro seedlings of soybean cv. PK 416 on shoot induction polyamine medium (SIPAM) containing BA (2.22 µm). Polyamines (μm) Control Spermidine Spermine Putrescine Percentage of explants responding (%) 84.33±0.21 ja 88.31±0.44 fbga 91.93±0.37 d 94.62±0.30 b 96.94±0.31 a 89.65±0.33 e 86.31±0.33 ib 88.63±0.30 ebfag 91.34±0.42 da 93.36±0.30 c 87.02±0.29 hai 86.34±0.30 ia 87.95±0.31 gbh 89.32±0.47 eaf 85.01±0.25 j 80.63±0.45 k Mean number of shoots/explant Initial culture (After 15 days) 10.34±0.55 jb 15.61±0.43 cbda 17.33±0.22 ba 18.02±0.30 b 19.34±0.42 a 16.36±0.29 c 11.91±0.37 hai 13.63±0.22 fag 14.92±0.37 dbe 15.95±0.22 cad 12.62±0.37 gbh 11.33±0.44 iaj 13.02±0.21 ga 14.31±0.36 eaf 10.94±0.34 ibja 8.65±0.30 k 1 st subculture (After 15 days) 16.32±0.33 ia 23.92±0.31 e 26.63±0.26 c 28.34±0.42 b 29.66±0.33 a 25.31±0.44 d 18.67±0.45 h 21.62±0.40 f 23.36±0.26 ea 24.91±0.27 da 20.01±0.25 ga 18.61±0.40 ha 20.32±0.33 g 22.95±0.31 eb 17.03±0.29 i 14.62±0.33 j 2 nd subculture (After 15 days) 22.33±0.44 ka 31.02±0.25 e 34.33±0.26 c 36.64±0.26 b 39.02±0.33 a 32.94±0.31 d 24.02±0.25 ja 27.63±0.30 h 30.04±0.25 f 32.37±0.42 da 25.35±0.33 i 24.31±0.36 j 26.92±0.31 ha 28.91±0.34 g 23.03±0.21 k 19.91±0.45 l Control: Treatment with SIM containing BA (2.22 µm) For each treatment, 50 explants were used and repeated three times. Values represent the means±standard error. Mean values followed by the same letters within a column are not significantly different according to Duncan s multiple range test at 5% level.

31 Table 2.4. Effect of polyamines on multiple shoot induction from half-seed explants derived from 1-day-old imbibed seeds of soybean cv. PK 416 on shoot induction polyamine medium (SIPAM) containing BA (2.22 µm). Polyamines (μm) Control Spermidine Spermine Putrescine Percentage of explants responding (%) 79.05±0.36 ja 83.32±0.49 fbg 87.01±0.53 d 89.64±0.33 b 92.04±0.55 a 85.36±0.42 ea 81.92±0.31 hb 83.97±0.45 f 86.34±0.44 dae 88.35±0.49 c 82.61±0.26 fcgah 80.63±0.47 i 82.34±0.51 gbha 83.91±0.37 fa 79.91±0.45 iaj 75.61±0.40 k Mean number of shoots/explants Initial culture (After 15 days) 8.63±0.37 la 13.92±0.27 cbd 15.62±0.22 ba 16.32±0.33 b 17.61±0.30 a 14.63±0.22 c 10.31±0.26 iaj 12.03±0.33 fag 13.34±0.59 dae 14.32±0.21 ca 11.01±0.36 hai 9.62±0.16 jak 11.31±0.42 gah 12.62±0.26 eaf 9.33±0.26 kal 7.04±0.25 m 1 st subculture (After 15 days) 13.34±0.30 ia 20.62±0.33 e 23.38±0.26 c 25.03±0.36 b 26.61±0.30 a 22.31±0.42 cad 15.61±0.45 ha 18.63±0.33 f 20.34±0.36 ea 21.92±0.40 da 17.06±0.33 ga 15.65±0.42 h 17.32±0.47 g 19.93±0.31 eb 14.01±0.33 i 11.62±0.37 j 2 nd subculture (After 15 days) 18.62±0.26 ja 26.36±0.36 e 29.62±0.22 c 31.97±0.45 b 34.31±0.33 a 27.62±0.30 da 19.91±0.37 hai 23.63±0.26 f 25.91±0.37 ea 28.63±0.37 cad 21.94±0.43 g 20.63±0.45 h 23.32±0.42 fa 25.33±0.42 eb 19.36±0.33 iaj 16.32±0.36 k Control: Treatment with SIM containing BA (2.22 µm) For each treatment, 50 explants were used and repeated three times. Values represent the means±standard error. Mean values followed by the same letters within a column are not significantly different according to Duncan s multiple range test at 5% level.

32 Table 2.5. Effect of GA 3, Zea and IAA on shoot elongation of regenerated shoots from cotyledonary node explants derived from 7-day-old in vitro seedlings of soybean cv. PK 416 on shoot elongation medium (SEM) after 30 days of culture. Plant growth regulators (µm) Control Percentage of explants responding (%) 18.31±0.51 o Mean number of elongated shoots/explant 6.62±0.30 j Mean shoot length (cm) 1.86±0.04 ma GA ±0.25 e 19.62±0.22 cb 4.53±0.06 cad ±0.33 a 24.05±0.33 a 5.43±0.07 a ±0.40 b 21.93±0.43 b 5.09±0.06 b ±0.42 d 20.37±0.36 c 4.63±0.06 c ±0.30 ga 17.62±0.40 da 4.13±0.06 e Zea ±0.45 i 15.62±0.30 e 3.50±0.07 gah ±0.51 c 19.93±0.23 ca 4.40±0.07 da ±0.30 f 17.95±0.37 d 3.93±0.05 f ±0.27 h 17.01±0.25 db 3.66±0.06 g ±0.33 k 13.32±0.30 g 3.20±0.07 ia IAA ±0.39 g 14.62±0.30 f 3.33±0.06 hai ±0.33 j 12.91±0.27 ga 2.96±0.06 j ±0.30 l 11.32±0.44 h 2.53±0.07 k ±0.60 m 9.95±0.40 i 2.23±0.05 l ±0.42 n 9.06±0.36 ia 2.03±0.06 m Control: Treatment without Plant Growth Regulators. For each treatment, 50 explants were used and repeated three times. Values represent the means±standard error. Mean values followed by the same letters within a column are not significantly different according to Duncan s multiple range test at 5% level.

33 Table 2.6. Effect of GA 3, Zea and IAA on shoot elongation of regenerated shoots from half-seed explants derived from 1-day-old imbibed seeds of soybean cv. PK 416 on shoot elongation medium (SEM) after 30 days of culture. Plant growth regulators (µm) Control Percentage of explants responding (%) 15.01±0.49 m Mean number of elongated shoots/explant 5.61±0.33 ka Mean shoot length (cm) 1.60±0.02 la GA ±0.63 d 16.01±0.36 ca 4.30±0.07 ca ±0.42 a 19.92±0.45 a 5.23±0.06 a ±0.52 b 18.63±0.40 b 4.93±0.07 b ±0.44 ca 16.96±0.50 c 4.46±0.06 c ±0.39 g 14.05±0.44 da 3.83±0.05 e Zea ±0.33 i 10.61±0.40 fbg 3.13±0.06 h ±0.42 c 14.33±0.57 d 4.10±0.07 d ±0.49 e 12.62±0.40 e 3.63±0.07 f ±0.52 h 11.30±0.49 fa 3.33±0.06 g ±0.36 ja 8.91±0.27 hai 2.86±0.06 ia IAA ±0.47 f 11.36±0.47 f 2.90±0.07 i ±0.51 ia 9.63±0.56 gah 2.53±0.07 j ±0.49 j 8.31±0.39 ia 2.23±0.07 k ±0.31 k 7.02±0.29 j 2.09±0.06 ka ±0.45 l 6.31±0.36 jak 1.73±0.07 l Control: Treatment without Plant Growth Regulators. For each treatment, 50 explants were used and repeated three times. Values represent the means±standard error. Mean values followed by the same letters within a column are not significantly different according to Duncan s multiple range test at 5% level.

34 Table 2.7. Effect of polyamines on shoot elongation of regenerated shoots from cotyledonary node explants derived from 7-day-old in vitro seedlings of soybean cv. PK 416 on shoot elongation polyamine medium (SEPAM) containing GA 3 (1.45 µm) after 30 days of culture. Polyamines (µm) Control Percentage of explants responding (%) 71.34±0.33 g Mean number of elongated shoots/explant 24.05±0.33 jbk Mean shoot length (cm) 5.43±0.07 hb Spermidine ±0.40 eb 25.01±0.53 iaj 5.70±0.07 g ±0.36 db 27.64±0.47 eaf 6.03±0.06 f ±0.42 ca 30.64±0.49 ca 6.60±0.06 d ±0.39 d 29.32±0.47 d 6.43±0.07 dbea ±0.31 fb 22.90±0.37 kb 5.53±0.06 gbh Spermine ±0.36 da 28.61±0.37 dae 6.53±0.06 dae ±0.40 c 31.34±0.47 c 6.93±0.05 c ±0.33 a 34.62±0.33 a 7.60±0.06 a ±0.55 b 33.04±0.36 b 7.23±0.06 b ±0.43 ea 26.32±0.36 gah 6.36±0.06 eb Putrescine ±0.27 f 25.33±0.42 hai 5.69±0.05 ga ±0.36 e 27.01±0.44 fag 6.03±0.06 fa ±0.30 fa 24.64±0.30 ibja 5.49±0.05 ha ±0.36 ga 22.96±0.45 ka 4.96±0.05 i ±0.36 h 20.32±0.42 l 4.33±0.06 j Control: Treatment with SEM containing GA 3 (1.45 µm) For each treatment, 50 explants were used and repeated three times. Values represent the means±standard error. Mean values followed by the same letters within a column are not significantly different according to Duncan s multiple range test at 5% level.

35 Table 2.8. Effect of polyamines on shoot elongation of regenerated shoots from half-seed explants derived from 1-day-old imbibed seeds of soybean cv. PK 416 on shoot elongation polyamine medium (SEPAM) containing GA 3 (1.45 µm) after 30 days of culture. Polyamines (µm) Control Percentage of explants responding (%) 65.06±0.42 i Mean number of elongated shoots/explant 19.92±0.45 edf Mean shoot length (cm) 5.23±0.06 hb Spermidine ±0.54 gb 20.62±0.42 eb 5.26±0.06 ha ±0.47 fa 23.31±0.49 da 5.53±0.07 f ±0.44 d 26.34±0.39 bac 6.30±0.07 d ±0.44 eaf 25.02±0.33 cb 5.90±0.06 e ±0.33 hb 18.31±0.49 ga 5.13±0.05 hc Spermine ±0.50 e 23.36±0.47 d 6.19±0.07 da ±0.49 c 26.05±0.49 ca 6.56±0.06 c ±0.55 a 29.31±0.55 a 7.23±0.07 a ±0.43 b 27.62±0.40 b 6.93±0.05 b ±0.40 g 21.03±0.66 ea 5.86±0.06 ea Putrescine ±0.42 h 21.32±0.42 e 5.46±0.06 fag ±0.58 ga 22.93±0.54 db 5.83±0.04 eb ±0.39 ha 20.34±0.47 ec 5.29±0.04 gah ±0.42 ia 18.61±0.42 fag 4.76±0.04 i ±0.51 j 16.02±0.39 h 4.19±0.04 j Control: Treatment with SEM containing GA 3 (1.45 µm) For each treatment, 50 explants were used and repeated three times. Values represent the means±standard error. Mean values followed by the same letters within a column are not significantly different according to Duncan s multiple range test at 5% level.

36 Table 2.9. Effect of IBA on rooting of elongated shoots of soybean cv. PK 416 on root induction medium (RIM). IBA (µm) Rooting response (%) Mean number of roots/shoot Mean root length (cm) Control 30.32±0.63 e 1.31±0.15 da 2.33±0.05 f IBA ±0.42 ba 3.92±0.23 bac 6.93±0.04 c ±0.47 a 5.34±0.42 a 10.03±0.05 a ±0.45 b 4.36±0.44 b 7.33±0.03 b ±0.40 c 3.01±0.33 ca 5.16±0.06 d ±0.49 d 1.92±0.27 d 2.66±0.03 e Control: Treatment without Plant growth regulator For each treatment, 50 elongated shoots (>4 cm) were used and repeated three times. Values represent the means±standard error. Mean values followed by the same letters within a column are not significantly different according to Duncan s multiple range test at 5% level.

37 Table Effect of putrescine on rooting of elongated shoots of soybean cv. PK 416 on root induction polyamine medium (RIPAM) containing IBA (4.93 µm). Putrescine (µm) Rooting response (%) Mean number of roots/shoot Mean root length (cm) Control 87.33±0.47 d 5.34±0.42 cad 10.03±0.05 d Putrescine ±0.42 b 6.63±0.33 aab 11.33±0.03 b ±0.33 a 7.34±0.33 a 13.06±0.06 a ±0.50 c 6.02±0.29 bac 10.30±0.04 c ±0.31 e 4.91±0.27 da 8.69±0.04 e ±0.37 f 3.62±0.22 e 6.46±0.02 f Control: Treatment with RIM containing IBA (4.93 µm) For each treatment, 50 elongated shoots (>4 cm) were used and repeated three times. Values represent the means±standard error. Mean values followed by the same letters within a column are not significantly different according to Duncan s multiple range test at 5% level.

38 Figure 2.1. Genotypic effect on multiple shoot induction from cotyledonary node explants (7-day-old) from various cultivars of soybean in SIPAM containing BA (2.22 µm) and spermidine ( µm) at the end of 2 nd subculture. For each treatment, 50 explants were used and repeated three times. The bars represent mean±standard error.

39 Figure 2.2. Genotypic effect on multiple shoot induction from half-seed explants (1-day-old) from various cultivars of soybean in SIPAM containing BA (2.22 µm) and spermidine ( µm) at the end of 2 nd subculture. For each treatment, 50 explants were used and repeated three times. The bars represent mean±standard error.

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