2. Direct organogenesis - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/9478/10/10_chapter...

M. Arun, Ph.D. Thesis, Department of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. 14

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 B5

2. Direct Organogenesis


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).



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 N6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ) in

multiple shoot induction, (2) to assess the role of PGRs like gibberellic acid

(GA3), 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.



2.2. Materials and Methods

2.2.1. 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.

2.2.2. 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).

2.2.3. 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 5.6–5.8 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



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–2s–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.

2.2.4. 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 (100 × 15 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.

2.2.5. 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 (15 × 150 mm;

Borosil, Mumbai, India) containing MS salts and vitamins, sucrose (87.65 mM)

along with various concentrations of plant growth regulators namely, BA (1.11–

8.88 μM), Kn (2.33–18.60 µM), and TDZ (0.46–1.82 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



25±2°C in 16/8 hr light/dark photoperiod conditions at a light intensity of 50

µmol m–2s–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.

2.2.6. 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 (34.42–172.11 μM), spermine (24.71–123.55 μM), and putrescine

(31.04–155.20 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 2.2.5.

2.2.7. 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 2.2.5.

2.2.8. 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 GA3 (0.72–5.78 µM), Zea (2.29–11.41 µM), and



IAA (0.58–2.86 µM) [Sigma, St. Louis, USA] to determine their effect in shoot

elongation. The cultures were maintained in same conditions as described in

section 2.2.5. 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.

2.2.9. 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 (34.42–172.11 μM),

spermine (24.71–123.55 μM), and putrescine (31.04–155.20 μM) along with

GA3 (1.45 μM) [The medium is designated as SEPAM]. The cultures were

maintained in same conditions as described in section 2.2.5. 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).

2.2.10. 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 (2.47–

12.31 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 2.2.5. Separate controls were maintained by culturing shoots in

hormone free RIM.



2.2.11. 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

(34.42–172.11 μM), spermine (24.71–123.55 μM), and putrescine (31.04–155.20

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 2.2.5.

2.2.12. 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.

2.2.13. 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).



2.2.14. 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).

2.3. Results

2.3.1. 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.


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,



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 10.34 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 – half-

seed). 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 – half-

seed).


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



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

(137.69 μM) was found effective and showed maximum percentage of response

with increased production of multiple shoots in both types of explants [96.94%

and 19.34 shoots/explant for cotyledonary node explants (Fig. 2.3c) and 92.04%

and 17.61 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 15.95 shoots/explant for cotyledonary node

explants and 88.35% and 14.32 shoots/explant for half-seed explants) [Table 2.3

– cotyledonary node; Table 2.4 – half-seed]. In BA and putrescine combination,

putrescine at 93.12 μM was found better in shoot induction (89.32% for

cotyledonary node explants and 83.91% for half-seed explants) with the

production of 14.31 and 12.62 shoots/explant from cotyledonary node and half-

seed explants respectively at the end of initial culture (Table 2.3 – cotyledonary

node; Table 2.4 – half-seed).


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 10.34 shoots/explant and half-seed



produced 8.63 shoots/explant. At the end of 2nd subculture, the shoot number

was increased up to 22.33 shoots/explant in cotyledonary node explants and

18.62 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 2nd subculture (data not shown). In SIM

containing Kn (4.65 µM), the shoot number was increased from 6.62 to 14.02

and 5.63 to 11.63 shoots/explant in cotyledonary node and half-seed explants

respectively at the end of 2nd 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 2nd subculture, the number of shoots that

regenerated from a single explant in SIM containing TDZ (0.46 µM) was

increased from 5.31 to 11.61 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 2nd 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 39.02

(Fig. 2.3f) and 34.31 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 (137.69 μM) at the end of 2nd subculture (Table 2.3 –

cotyledonary node; Table 2.4 – half-seed). In SIPAM containing BA (2.22 µM)

and spermine (98.84 µM), 32.37 and 28.63 shoots/explant were produced in



cotyledonary node and half-seed explants respectively at the end of 2nd

subculture (Table 2.3 – cotyledonary node; Table 2.4 – half-seed). A maximum

number of 28.91 and 25.33 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 2nd subculture (Table 2.3 – cotyledonary

node; Table 2.4 – half-seed). At the end of 2nd 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 (2nd subculture) neither increased shoot number

nor shoot length.


The shoots developed from cotyledonary node and half-seed explants on

SIPAM containing BA (2.22 μM) with spermidine (137.69 μM) at the end of the

2nd subculture were short (<1 cm) and hence transferred to SEM with various

concentrations of GA3, Zea, and IAA individually to induce shoot elongation.

SEM containing GA3 showed better response when compared to Zea and IAA

(Table 2.5 – cotyledonary node; Table 2.6 – half-seed). GA3 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 GA3 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%



(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 GA3 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.


In order to further optimize the medium for shoot elongation, both the

types of explants with shoots were transferred to SEM containing GA3 (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 – half-

seed). SEPAM containing GA3 and spermine (74.13 μM) combination was most

effective and showed maximum percentage of response with higher number of



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 – half-

seed). 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 GA3 and spermidine (103.27 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 half-

seed explants respectively (Table 2.7 – cotyledonary node; Table 2.8 – half-

seed). In SEPAM containing GA3 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).

2.3.7. 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 10.03 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



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).


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 13.06 cm on the same

concentration of IBA and putrescine (Table 2.10).


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



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).


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 39.02 and 34.31 shoots/explant for

cotyledonary node and half-seed explants respectively in SIPAM containing BA

(2.22 μM) and spermidine (137.69 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 90-

41 (CN – 49.93%, H-S – 43.38%) [Fig. 2.1 – cotyledonary node; Fig. 2.2 – half-

seed].

2.4. Discussion

2.4.1. Explants

In the present study, cotyledonary node (7-day-old) and half-seed (1-day-

old) 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.



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.


In the present study, the effects of three cytokinins (BA, Kn and TDZ)

were investigated on multiple shoot induction from cotyledonary node and half-

seed 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.



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 10.34 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 (0.46–1.82 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



concentration of TDZ (9 µM). This may be due to the difference in explants used

or might be a genotypic effect of cultivars.


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 (0.05–15 mM),



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

(137.69 μM), 96.94% of the cotyledonary node explants produced an average of

19.34 shoots/explant and 92.04% of the half-seed explants produced an average

of 17.61 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 (137.69 µM). They

reported that spermidine was superior over spermine and putrescine on shoot

production from nodal explants. A similar observation was made by



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.


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 10.34 to 22.33

shoots/cotyledonary node explant and 8.63 to 18.62 shoots/half-seed at the end

of 2nd subculture in SIM containing BA (2.22 µM). In the case of SIPAM

containing BA (2.22 µM) and spermidine (137.69 μM), the shoot number was

increased from 19.34 to 39.02 shoots/cotyledonary node explant and 17.61 to

34.31 shoots/half-seed explant at the end of 2nd 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



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.


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

(137.69 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 GA3, Zea, and IAA for shoot elongation. Efficient shoot elongation of

explants was achieved in SEM supplemented with GA3. The SEM containing

GA3 (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 AgNO3 (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 GA3 (2.8 µM) and AgNO3 (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.



In soybean, Franklin et al. (2004) achieved efficient shoot elongation in

MS medium containing only GA3 (0.29 µM) using mature and immature

cotyledon explants. In the present study, shoots elongated in presence of GA3

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 GA3 as in the present study (1.45 µM) were thin and long and

about 50% of the shoots became vitrified. Next to GA3, 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 L2 medium without phytohormone in soybean.


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.



In the present investigation, addition of polyamines to SEM containing

optimum concentration of GA3 (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 GA3 and spermine combination. In SEPAM

containing GA3 (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 half-

seed explants. Further, the elongated shoots/explant also increased in same

medium (34.62 elongated shoots/explant for cotyledonary node and 29.31

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 GA3 also generated

better response when compared to individual treatment of GA3. In a similar

study, Bais et al. (2000) reported the promotive role of GA3 and polyamine

(putrescine) combination with respect to shoot elongation. In their study, GA3

(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).

2.4.7. 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 10.03 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



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.


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 well-

developed 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.


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



(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.


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 genotype-

associated 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).

2.5. 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 half-

seed 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.

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).

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.

Plant growth regulators

(μM)

Percentage of explants

responding

(%)

Mean number of shoots/explant

Initial culture

(After 15 days)

1st subculture

(After 15 days)

2nd subculture

(After 15 days)

Control

BA

1.11

2.22

4.44

6.66

8.88

Kn

2.33

4.65

9.30

13.95

18.60

TDZ

0.46

0.91

1.37

1.82

18.62±0.30l

73.61±0.37d

84.33±0.21a

81.65±0.33b

76.02±0.29c

71.31±0.21e

67.03±0.21f

73.35±0.36da

61.36±0.36ga

54.91±0.31h

48.31±0.30i

61.94±0.37g

54.32±0.33ha

43.91±0.23j

28.6±0.30k

1.61±0.16ha

7.03±0.36c

10.34±0.55a

9.61±0.61aa

8.35±0.30b

6.91±0.31ca

5.36±0.39d

6.62±0.30cb

4.62±0.42dbe

3.96±0.31eaf

2.95±0.23fcga

5.31±0.36da

3.91±0.23ebfa

3.06±0.33fbg

2.32±0.26gbh

1.61±0.16k

12.32±0.51bac

16.32±0.33a

15.35±0.53aa

13.03±0.47b

10.96±0.37da

9.31±0.47e

11.62±0.33cad

7.94±0.37fag

6.06±0.33hai

5.31±0.30ia

8.32±0.39eaf

7.01±0.33gah

4.92±0.27ibj

3.90±0.27ja

1.61±0.16m

15.94±0.64d

22.33±0.44a

20.92±0.27b

17.31±0.49c

13.64±0.22eaf

12.64±0.52fag

14.02±0.25e

10.93±0.43ha

8.04±0.25j

6.32±0.42kal

11.61±0.33gah

9.63±0.47i

7.34±0.36jak

5.32±0.30la

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).

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.


(μM)

Percentage of explant

responding

(%)

Mean number of shoots/explants

Initial culture

(After 15 days)

1st subculture

(After 15 days)

2nd

subculture

(After 15 days)

Control

BA

1.11

2.22

4.44

6.66

8.88

Kn

2.33

4.65

9.30

13.95

18.60

TDZ

0.46

0.91

1.37

1.82

13.61±0.33l

68.62±0.54d

79.05±0.36a

75.32±0.42b

70.35±0.55c

65.02±0.36e

63.31±0.36f

68.32±0.39da

57.01±0.29ga

49.32±0.30ha

44.61±0.30i

57.62±0.30g

49.92±0.37h

37.63±0.37j

23.35±0.30k

1.31±0.15i

5.92±0.31cad

8.63±0.37a

7.32±0.26b

6.63±0.37bac

5.02±0.25eaf

4.61±0.30fbg

5.63±0.16dae

3.96±0.23ga

3.06±0.21h

2.33±0.21hb

4.92±0.27ebfa

3.91±0.23gb

3.01±0.29ha

2.31±0.15hc

1.31±0.15k

9.32±0.30bac

13.34±0.30a

12.65±0.40aa

10.02±0.29b

8.66±0.26cad

7.02±0.21e

8.61±0.40cbda

5.93±0.37fag

5.05±0.21hai

4.32±0.30iaj

7.91±0.37db

6.62±0.30eaf

5.31±0.30gah

4.03±0.21ja

1.31±0.15k

12.31±0.36d

18.62±0.26a

16.35±0.26b

14.01±0.33c

10.61±0.26e

9.31±0.30fa

11.63±0.40da

8.37±0.26g

7.01±0.25h

5.32±0.21iaj

9.36±0.33f

8.32±0.44ga

6.01±0.25i

5.02±0.25ja

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)


responding

(%)

Mean number of shoots/explant

Initial culture

(After 15 days)

1st subculture

(After 15 days)

2nd subculture

(After 15 days)

Control

Spermidine

34.42

68.84

103.27

137.69

172.11

Spermine

24.71

49.42

74.13

98.84

123.55

Putrescine

31.04

62.08

93.12

124.16

155.20

84.33±0.21ja

88.31±0.44fbga

91.93±0.37d

94.62±0.30b

96.94±0.31a

89.65±0.33e

86.31±0.33ib

88.63±0.30ebfag

91.34±0.42da

93.36±0.30c

87.02±0.29hai

86.34±0.30ia

87.95±0.31gbh

89.32±0.47eaf

85.01±0.25j

80.63±0.45k

10.34±0.55jb

15.61±0.43cbda

17.33±0.22ba

18.02±0.30b

19.34±0.42a

16.36±0.29c

11.91±0.37hai

13.63±0.22fag

14.92±0.37dbe

15.95±0.22cad

12.62±0.37gbh

11.33±0.44iaj

13.02±0.21ga

14.31±0.36eaf

10.94±0.34ibja

8.65±0.30k

16.32±0.33ia

23.92±0.31e

26.63±0.26c

28.34±0.42b

29.66±0.33a

25.31±0.44d

18.67±0.45h

21.62±0.40f

23.36±0.26ea

24.91±0.27da

20.01±0.25ga

18.61±0.40ha

20.32±0.33g

22.95±0.31eb

17.03±0.29i

14.62±0.33j

22.33±0.44ka

31.02±0.25e

34.33±0.26c

36.64±0.26b

39.02±0.33a

32.94±0.31d

24.02±0.25ja

27.63±0.30h

30.04±0.25f

32.37±0.42da

25.35±0.33i

24.31±0.36j

26.92±0.31ha

28.91±0.34g

23.03±0.21k

19.91±0.45l

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.

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)


responding

(%)

Mean number of shoots/explants

Initial culture

(After 15 days)

1st subculture

(After 15 days)

2nd subculture

(After 15 days)

Control

Spermidine

34.42

68.84

103.27

137.69

172.11

Spermine

24.71

49.42

74.13

98.84

123.55

Putrescine

31.04

62.08

93.12

124.16

155.20

79.05±0.36ja

83.32±0.49fbg

87.01±0.53d

89.64±0.33b

92.04±0.55a

85.36±0.42ea

81.92±0.31hb

83.97±0.45f

86.34±0.44dae

88.35±0.49c

82.61±0.26fcgah

80.63±0.47i

82.34±0.51gbha

83.91±0.37fa

79.91±0.45iaj

75.61±0.40k

8.63±0.37la

13.92±0.27cbd

15.62±0.22ba

16.32±0.33b

17.61±0.30a

14.63±0.22c

10.31±0.26iaj

12.03±0.33fag

13.34±0.59dae

14.32±0.21ca

11.01±0.36hai

9.62±0.16jak

11.31±0.42gah

12.62±0.26eaf

9.33±0.26kal

7.04±0.25m

13.34±0.30ia

20.62±0.33e

23.38±0.26c

25.03±0.36b

26.61±0.30a

22.31±0.42cad

15.61±0.45ha

18.63±0.33f

20.34±0.36ea

21.92±0.40da

17.06±0.33ga

15.65±0.42h

17.32±0.47g

19.93±0.31eb

14.01±0.33i

11.62±0.37j

18.62±0.26ja

26.36±0.36e

29.62±0.22c

31.97±0.45b

34.31±0.33a

27.62±0.30da

19.91±0.37hai

23.63±0.26f

25.91±0.37ea

28.63±0.37cad

21.94±0.43g

20.63±0.45h

23.32±0.42fa

25.33±0.42eb

19.36±0.33iaj

16.32±0.36k

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.

Table 2.5. Effect of GA3, 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.

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.


(µM)


responding

(%)

Mean number of elongated

shoots/explant

Mean shoot length (cm)

Control

GA3

0.72

1.45

2.89

4.34

5.78

Zea

2.29

4.57

6.85

9.13

11.41

IAA

0.58

1.15

1.72

2.29

2.86

18.31±0.51o

59.02±0.25e

71.34±0.33a

65.62±0.40b

60.61±0.42d

53.35±0.30ga

50.61±0.45i

62.32±0.51c

56.33±0.30f

51.96±0.27h

44.62±0.33k

54.31±0.39g

48.63±0.33j

43.32±0.30l

38.63±0.60m

36.06±0.42n

6.62±0.30j

19.62±0.22cb

24.05±0.33a

21.93±0.43b

20.37±0.36c

17.62±0.40da

15.62±0.30e

19.93±0.23ca

17.95±0.37d

17.01±0.25db

13.32±0.30g

14.62±0.30f

12.91±0.27ga

11.32±0.44h

9.95±0.40i

9.06±0.36ia

1.86±0.04ma

4.53±0.06cad

5.43±0.07a

5.09±0.06b

4.63±0.06c

4.13±0.06e

3.50±0.07gah

4.40±0.07da

3.93±0.05f

3.66±0.06g

3.20±0.07ia

3.33±0.06hai

2.96±0.06j

2.53±0.07k

2.23±0.05l

2.03±0.06m

Table 2.6. Effect of GA3, 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.

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.


(µM)


responding

(%)


shoots/explant


Control

GA3

0.72

1.45

2.89

4.34

5.78

Zea

2.29

4.57

6.85

9.13

11.41

IAA

0.58

1.15

1.72

2.29

2.86

15.01±0.49m

53.33±0.63d

65.06±0.42a

60.62±0.52b

55.33±0.44ca

47.06±0.39g

43.31±0.33i

56.34±0.42c

50.65±0.49e

45.62±0.52h

37.31±0.36ja

48.61±0.47f

42.32±0.51ia

37.61±0.49j

32.94±0.31k

29.65±0.45l

5.61±0.33ka

16.01±0.36ca

19.92±0.45a

18.63±0.40b

16.96±0.50c

14.05±0.44da

10.61±0.40fbg

14.33±0.57d

12.62±0.40e

11.30±0.49fa

8.91±0.27hai

11.36±0.47f

9.63±0.56gah

8.31±0.39ia

7.02±0.29j

6.31±0.36jak

1.60±0.02la

4.30±0.07ca

5.23±0.06a

4.93±0.07b

4.46±0.06c

3.83±0.05e

3.13±0.06h

4.10±0.07d

3.63±0.07f

3.33±0.06g

2.86±0.06ia

2.90±0.07i

2.53±0.07j

2.23±0.07k

2.09±0.06ka

1.73±0.07l

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 GA3 (1.45 µM) after 30 days of culture.

Control: Treatment with SEM containing GA3 (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.

Polyamines (µM)


responding

(%)


shoots/explant


Control

Spermidine

34.42

68.84

103.27

137.69

172.11

Spermine

24.71

49.42

74.13

98.84

123.55

Putrescine

31.04

62.08

93.12

124.16

155.20

71.34±0.33g

75.66±0.40eb

78.33±0.36db

81.62±0.42ca

79.32±0.39d

72.91±0.31fb

79.02±0.36da

82.64±0.40c

87.32±0.33a

85.31±0.55b

75.92±0.43ea

73.92±0.27f

76.05±0.36e

73.32±0.30fa

70.31±0.36ga

67.02±0.36h

24.05±0.33jbk

25.01±0.53iaj

27.64±0.47eaf

30.64±0.49ca

29.32±0.47d

22.90±0.37kb

28.61±0.37dae

31.34±0.47c

34.62±0.33a

33.04±0.36b

26.32±0.36gah

25.33±0.42hai

27.01±0.44fag

24.64±0.30ibja

22.96±0.45ka

20.32±0.42l

5.43±0.07hb

5.70±0.07g

6.03±0.06f

6.60±0.06d

6.43±0.07dbea

5.53±0.06gbh

6.53±0.06dae

6.93±0.05c

7.60±0.06a

7.23±0.06b

6.36±0.06eb

5.69±0.05ga

6.03±0.06fa

5.49±0.05ha

4.96±0.05i

4.33±0.06j

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 GA3 (1.45 µM) after 30 days of culture.

Control: Treatment with SEM containing GA3 (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.

Polyamines (µM)


responding

(%)


shoots/explant


Control

Spermidine

34.42

68.84

103.27

137.69

172.11

Spermine

24.71

49.42

74.13

98.84

123.55

Putrescine

31.04

62.08

93.12

124.16

155.20

65.06±0.42i

69.61±0.54gb

72.32±0.47fa

75.31±0.44d

73.06±0.44eaf

66.61±0.33hb

73.91±0.50e

77.37±0.49c

82.02±0.55a

79.91±0.43b

70.61±0.40g

67.62±0.42h

69.92±0.58ga

67.03±0.39ha

64.31±0.42ia

61.06±0.51j

19.92±0.45edf

20.62±0.42eb

23.31±0.49da

26.34±0.39bac

25.02±0.33cb

18.31±0.49ga

23.36±0.47d

26.05±0.49ca

29.31±0.55a

27.62±0.40b

21.03±0.66ea

21.32±0.42e

22.93±0.54db

20.34±0.47ec

18.61±0.42fag

16.02±0.39h

5.23±0.06hb

5.26±0.06ha

5.53±0.07f

6.30±0.07d

5.90±0.06e

5.13±0.05hc

6.19±0.07da

6.56±0.06c

7.23±0.07a

6.93±0.05b

5.86±0.06ea

5.46±0.06fag

5.83±0.04eb

5.29±0.04gah

4.76±0.04i

4.19±0.04j

Table 2.9. Effect of IBA on rooting of elongated shoots of soybean cv. PK 416 on root induction medium (RIM).

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.

IBA

(µM)

Rooting response

(%) Mean number of

roots/Shoot Mean root length

(cm)

Control

IBA

2.47

4.93

7.39

9.85

12.31

30.32±0.63e

75.92±0.42ba

87.33±0.47a

76.65±0.45b

64.97±0.40c

43.32±0.49d

1.31±0.15da

3.92±0.23bac

5.34±0.42a

4.36±0.44b

3.01±0.33ca

1.92±0.27d

2.33±0.05f

6.93±0.04c

10.03±0.05a

7.33±0.03b

5.16±0.06d

2.66±0.03e

Table 2.10. Effect of putrescine on rooting of elongated shoots of soybean cv. PK 416 on root induction polyamine medium (RIPAM) containing IBA (4.93 µM).

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.

Putrescine

(µM)

Rooting response

(%)

Mean number of roots/Shoot

Mean root length (cm)

Control

Putrescine

31.04

62.08

93.12

124.16

155.20

87.33±0.47d

90.33±0.42b

94.04±0.33a

88.91±0.50c

81.92±0.31e

73.64±0.37f

5.34±0.42cad

6.63±0.33aab

7.34±0.33a

6.02±0.29bac

4.91±0.27da

3.62±0.22e

10.03±0.05d

11.33±0.03b

13.06±0.06a

10.30±0.04c

8.69±0.04e

6.46±0.02f

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 (137.69 µM) at the end of 2nd subculture.

For each treatment, 50 explants were used and repeated three times. The bars represent

mean±standard error.

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 (137.69 µM) at the end of 2nd subculture.

For each treatment, 50 explants were used and repeated three times. The bars represent

mean±standard error.

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