33.. OOrrggaannooggeenneessiiss

3.1 INTRODUCTION

Plant cell cultures are initiated through the formation of a mass of

undifferentiated cells called “callus.” Plant regeneration through callus cultures

(indirect organogenesis) is an effective strategy for successful exploitation of

in vitro techniques for somaclonal variation induction, genetic transformation

and protoplast culture (Sarasan et al., 1994; Lusia and Rojas, 1996; Ahroni et al.,

1997). Recent reports proved that the organogenesis protocols provided useful

systems for the study of regulating mechanisms of plant growth and

development (Castillo and Jordan, 1997). There are several plant species

adopted for the successful plant regeneration through indirect shoot

organogenesis and some of the plant species are recalcitrant to in vitro indirect

organogenesis. Castor is one among the plant species showing extremely low

percentage of callus induction and successive plant regeneration. There are

notable problems remained to be overcome for the callus induction and

subsequent plant regeneration in castor. The extrinsic and intrinsic factors like

type of explants, age of explants, type of media and exogenous and

endogenous plant growth regulators, the amount of carbon sources and

additives present in the media are the main factors to be analyzed.

Despite research efforts over the last three decades, whole plants still

could not be regenerated with reproducible frequencies from friable callus

cultures of castor. The sporadic appearance of shoots from callus cultures of

castor implies that the calli contain at least a few morphogenic cells

interspersed in several non-morphogenic tissues. Failure to isolate a competent

cell line might result in its suppression by the overgrowth of non-competent

cells. Alternatively, the occasional appearance of shoots could be owing to the

activation of recalcitrant calli to undergo caulogenesis caused by a rare

inductive stimulus resulting from the interaction between exogenous and

endogenous conditions. In castor, callus initiation and plantlet regeneration

Organogenesis

31

from vegetative explants are reported by (Athma and Reddy 1983; Sarvesh

et al., 1992). However, regeneration of plants from callus cultures has been

problematic. There are only few reports on plantlet regeneration in castor and

in most of the cases regenerated plantlets were obtained from apical meristems

and shoot tip callus (Reddy et al., 1986; Reddy et al., 1987a, Sangduen et al.,

1987, Genyu et al., 1988; Sujatha and Sailaja, 2005; Malathi et al., 2006). As

genetic transformation involves several manipulations for gene introduction

followed by selection for 2 – 3 subculture cycles, the efficiency of these

regeneration systems for genetic transformation of castor need to be

established.

In this present investigation, several challenging difficulties were

highlighted during the regeneration of castor from callus cultures. The

excretion of secondary metabolites from the explants into the medium,

browning of callus after a short period of culture, a low frequency of green

compact callus formation, formation of loosely arranged organogenic callus,

yellowing of organogenic callus within a short period of culture and very slow

response for shoot proliferation from the selected organogenic callus cultures

were recorded as main regeneration difficulties to be overcome. Hence, this

experiment was undertaken for the production of a good regenerative and

reproducible protocol for castor organogenesis through callus induction by

using a different type of explants.

3.2 MATERIALS AND METHODS

3.2.1 Explant Obtention

Castor in vivo seed germination was achieved by ten days. After seedling

growth hypocotyl (HL) and cotyledonary leaf (CL) explants were taken from

the 10 days old seedling for the organogenic callus studies.

Organogenesis

32

3.2.2 Initiation, proliferation and selection of organogenic callus

Cotyledonary leaf and Hypocotyl were used as explants and placed

horizontally in callus initiation medium which contained mMS medium salts

and varying concentrations of BA (0.5 – 4.0 mg/l), KN (1.0 to 3.0 mg/l) for

organogenic callus induction. After the identification of suitable concentration

of cytokinin for callus induction, combinations of different concentrations of

NAA (0.2 – 2.0 mg/l) were tested for enhanced callus production. After 6

weeks of culture, callus formation was observed from the cut end of the

explants. From the obtained mass, the organogenic nature of the callus was

identified by the presence of green colour with compact nodular texture

(GCN). The organogenic portions were isolated and subcultured in the same

medium. Greenish friable (GF), brown compact (BC), brown friable (BF) and

yellowish green friable (YGF) colored non-organogenic callus were also

observed and they were not selected and discarded for further studies due to

nil response. The selected organogenic callus was weekly subcultured for

another two weeks for the induction of well developed green compact

organogenic callus. For callus induction, maximum of 50 explants were tested

and these experiments were repeated for three times with five replicates.

3.2.3 Adventitious shoot proliferation

8-week-old organogenic callus (250 mg) was transferred to 200 cm3

narrow bottles containing 50 ml of shoot initiation medium. Then the cultures

were subcultured for 2 months with weekly subculture for the initiation of

shoots. During each subculture removal of dead, dark brown cells was done.

Otherwise, the whole callus tissues become necrotic and dead. The plant

growth regulators like, BA, KN and TDZ (0.5 – 2.5 mg/l) with different

concentrations and combination of IBA and IAA (0.05 – 0.8 mg/l) were tested

for proliferation of shoots. During multiple shoot proliferation, maximum of

30 callus cultures were tested and these experiments were repeated for three

times with five replicates.

Organogenesis

33

3.2.4 Effect of carbon source, additives and amino acids on multiple shoot

proliferation

The effect of sucrose, glucose, fructose and maltose (10 – 50 g/l) were

tested for organogenic callus induction and multiple shoot proliferation. In that

same way, the influence of different amino acids like alanine, glutamine,

proline and serine (5 – 25 mg/l) were also tested. During whole plant

regeneration of castor-bean, browning of explants as well as the medium was

noticed. To control phenolic exudation process, different concentrations of

additives like, activated charcoal (50 – 250 mg/l), citric acid, ascorbic acid and

PVP (5 – 25 mg/l) were tested.

3.2.5 Elongation, Root induction and hardening

The multiple shoots were transferred to shoot elongation medium

containing different concentration of GA3 (0.1 – 0.5 mg/l) with PF - 68

(1.0 mg/l) and root induction from the elongated shoots were obtained from

the mMS medium fortified with IBA (0.1 – 0.5 mg/l) and AgNO3 (0.2 –

1.0 mg/l), sucrose 30 g/l and agar (0.8%). After complete regeneration of

shoots with tertiary roots (35 - 40 days after root induction) the regenerated

plants were transferred to plastic pots containing sand, soil and vermiculate in

1:1:1 ratio for hardening The hardened plants were maintained in

environmental plant growth chamber (SANYO, JAPAN) for proper

acclimatization and then the in vitro regenerated plants were transferred to

green house condition for 15 days and successfully transferred to the field.

The survival percentage of all the hardened plants was recorded regularly. For

root induction studies, 30 elongated shoots were tested for each concentration.

The experiments were repeated for three times with five replicates.

3.2.6 Statistical analysis

Means and standard errors were used throughout the study and the

values were assessed using a parametric Moods median test (Snedecor and

Cochran, 1989). The data were analysed for variance by Duncan’s multiple

Organogenesis

34

range test (DMRT) using the SAS programme (SAS Institute, Cary, N.C.). For

organogenesis from the cotyledonary leaf and hypocotyl explants, 50

explants were tested with 5 replicates and each experiment was repeated

three times. During multiple shoot induction 30 callus were tested for each

treatment and the each experiment was repeated 3 times with 5 replications.

3.3 RESULTS AND DISCUSSION

3.3.1 Organogenic callus induction

The HL and CL explants were taken from the 10 days old well

established seedlings. After one week of inoculation, callusing was observed

from both the explants. Individual treatment of KN and BA showed low

response for callus induction. Among the two cytokinins tested media

comprising BA (2.0 mg/l) showed superior response (28.7%) with CL and

25.3% with HL explants. The callus from both the explants was green colour

with compact nature. However, nodular nature is absent in both the explants.

Hence, along with BA (2.0 mg/l) different concentration of NAA (0.2 –

2.0 mg/l) was tested for enhanced callus production. So, to enhance the nature

of the callus combinations of BA with NAA was tested. High proficiency callus

induction of CL explants was noticed on the mMS medium comprising BA

(2.0 mg/l) and NAA (0.8 mg/l) with the maximum of 69.5 % response whereas,

HL explants showed 54.2% of response in the same concentration (Table 3.1). In

this concentration, maximum amount of organogenic callus was observed

when compared with other concentrations in the mMS medium fortified with

30 g/l sucrose and 8 g/l agar. The induced calli from the explants were green,

compact and nodular in nature (Plate 3, 4).

Similar to our results, Sarvesh et al. (1992) reported that when epicotyls

and cotyledonary explants supplemented with BA (2.5 mg/l) + NAA

(0.1 mg/l) produces 96.5% of callus formation. Genyu (1988) by his experiment

proved that BA (1.0 mg/l) + NAA or IAA (1.0 mg/l) developed callus from

young stem explants of castor. Athma and Reddy (1983) speculated that

Organogenesis

35

cotyledonary leaf explants of castor treated with BA (0.5 – 2.0 mg/l) with NAA

(0.5 mg/l) produced 90 – 98% of callusing. Suresh and Rao (1994) achieved

callus from the MS medium supplemented with BAP (4.0 mg/l) + NAA

(4.0 mg/l) from axillary bud and terminal buds of castor.

Similar to our results, in Melia azedarach, organogenic callus cultures

were initiated by using a combination of BA (4.4 µM) and NAA (0.46 µM) and

successful regeneration of plantlets was obtained by using the callus cultures

(Vila et al., 2003). Like our result, in all dicot plants, combination of the high

amount auxin (2, 4 - D or NAA) with a low amount cytokinin (BA or Kin) was

widely used for the initiation of organogenic callus (Caboni et al., 2000; Rugini

and Muganu, 1998; Rani et al., 2006; Haliloglu, 2006) and sometimes cytokinins

alone (BA or KN) was also used for the induction of organogenic callus (Yam

et al., 1990). Our experiments proved that combined effect of BA and NAA

showed best response for organogenic callus induction.

During organogenic callus formation, calli with variation in texture were

noticed and only the hard and compact green calli responded well for the

induction of shoots. Hence, shoot induction and multiplication was achieved

by using the above said calli. Individual effect of different concentrations of

BA, KN and combined effect of KN + NAA were also tested for the induction

of organogenic callus, unfortunately, in these concentrations, abnormalities

like, induction of roots, bulging of explants and necrosis of tissues were clearly

observed. It is also observed that BA or KN when applied alone induced only

waste brownish friable callus from cotyledon explants (Sudha et al., 1998; Tang

and Guo, 2001; Martin, 2002). In our studies also combination of cytokinin with

auxins showed best response because, the nature of the callus varied according

to the exogenous application of the cytokinin and also for the auxin: cytokinin

ratio (Marks and Simpson, 1994; Soniya and Sujitha, 2006). In Tylophora indica,

organogenic callus induction was achieved by the supplementation of

cytokinin alone (Manjula et al., 2000; Handro and Floh, 2001). In these cases,

Organogenesis

36

endogenous hormone level (auxin level) plays a vital role in callus induction

(Fracaro and Echeverrigary, 2001).

In Iris ensata, I. setosa and I. sanguinea supplementation of BA with NAA

showed best response for callus induction and they also proved that

supplementation of auxin and cytokinin alone showed poor response for callus

induction (Boltenkov and Zarembo, 2003). Callus induction from the different

explants of apple (Malus domestica) for organogenesis was also obtained by the

combined treatment of BA and NAA (Caboni et al., 2000; Martin, 2002). In

Dianthus caryophyllus also organogenic callus induction was successfully

induced by the supplementation of auxin and cytokinin synergistically (Kallak

et al. 1997). Previous experiments in our laboratory proved that phenolic

excretion and oxidation is a severe problem during callus induction and callus-

mediated regeneration and this problem was recovered by the addition of

additives along with plant growth regulators (Ganesan and Jayabalan, 2005).

Unexpectedly, in this present investigation, excretion of phenolic compounds

from explants to the medium was strictly avoided by regular sub-culturing of

callus and without addition of additives.

3.3.2 Shoot proliferation from the callus

During shoot proliferation, all the treatments of BA + NAA (Graph 1a),

TDZ + NAA (Graph 1b) and KN + NAA (Graph 1c) showed shoots

proliferation from the obtained cotyledonary leaf and hypocotyl derived callus

cultures. NAA (0.05 – 0.8) was tested in combination with the cytokinins After

one month, cotyledon callus and hypocotyl callus were successfully

regenerated and produced maximum of 17.8 shoots (CL) and 15.4 (HL) shoots

from the callus in the mMS medium fortified with TDZ (1.0 mg/l) and NAA

(0.4 mg/l) (Graph 1b, e; Plate 3). In the case of BA and NAA combination, BA

(2.0 mg/l) and NAA (0.4 mg/l) produced 16 shoots (CL) and 14.1 (HL) shoots

from the callus cultures (Graph 1a, d). At the same time, KIN - NAA

combination showed poor response for multiple shoot induction. Only 7.0 (CL)

Organogenesis

37

and 6.4 (HL) shoots were induced from the callus culture (Graph 1c, f) after 45

days of culture (Plate 3, 4).

From the callus supplemented with IAA, 12.5 and 11.6 shoots were

successfully regenerated from CL and HL callus in the mMS media fortified

with TDZ (1.0 mg/l) and IAA (0.2 mg/l) (Graph 1b, e). While from BA and

IAA combination, BA (1.5 mg/l) and IAA (0.3 mg/l) produced 12.1 and 10.3

shoots from the CL and HL callus cultures (Graph 1a, d). At the same time, KN

and IAA combination showed poor response for multiple shoot induction.

Only 7.6 and 6.4 shoots were induced from CL and HL callus (Graph 1c, f)

culture after 45 days of culture. In this present investigation, we confirmed that

along with any type of cytokinin, addition of NAA favors shoot organogenesis

from the callus cultures of castor-bean.

Callus mediated regeneration is reported from hypocotyl sections

(Reddy et al., 1987a), young stem segments (Genyu, 1988), young leaves

(Reddy and Bahadur, 1989a) and epicotyl / cotyledons (Sarvesh et al., 1992).

However, differentiation of callus into shoots and shoot buds was reported to

be either occasional of low. Similar to our results Reddy and Bahadur (1989b)

found shoot regeneration from leaf callus but produced only 3 – 4 shoots in the

medium fortified with KN 2.0 mg/l + IAA 1.0 mg/l, which is contrast to our

result where 17.8 shoots were produced from cotyledon leaf derived callus

treated with TDZ (1.0 mg/l) with NAA (0.4 mg/l). Suresh and Rao (1994)

achieved shoots from the axillary bud callus when MS medium supplemented

with BAP (0.5 mg/l) with NAA (0.5 mg/l) whereas terminal bud callus was

also obtained when the MS medium supplemented with KIN (0.5 mg/l) with

NAA (0.5 mg/l) in Castor TMV5. Studies by Sarvesh et al. (1992) proved that in

castor 20% of the cultures with shoot buds induced on B5 medium (Gamborg,

1968) supplemented with 2.5 mg/l BA with 0.1 mg/l NAA on transfer to the

same medium produces 6 – 8 shoots per callus which is also very low

comparatively to our result.

Organogenesis

38

Similar to our result, TDZ mediated-shoot proliferation was obtained in

several plants (Fiola et al., 1990; Malik and Saxena, 1992). Usually TDZ used for

somatic embryogenesis and it was reported in many plant species, either alone

or in combination with other growth regulators (Murthy et al., 1998; Faisal and

Anis, 2006). But in our experiments, TDZ along with NAA were proved as best

cytokinin for organogenesis from cotyledon callus cultures. In some cases,

shoot multiplication and regeneration was efficiently achieved by 2iP (0.17 μM)

from the callus derived rhizomes of Cymbidium ensifolium (Chang and Chang,

2000). In Fraxinus angustifolia and Carica papaya also 2iP showed a vital role in

the shoot organogenesis from different explants derived callus (Tonon et al.,

2001; Khatoon and Sultana, 1994). Usually, BA or KN was widely used for

multiple shoot initiation from the callus cultures (Martin, 2002). In our study,

combination of TDZ with NAA yielded notable results in shoots proliferation

form the obtained callus mass.

3.3.3 Effect of carbon sources, additives and amino acids on shoot

proliferation studies from callus cultures

During multiple shoot proliferation, effects of different concentrations of

carbon sources were tested. Among them, sucrose 30 g/l showed best response

for both the explants (Graph 2a, b). In the other concentrations and forms of

carbon sources tested reduced percentage of multiple shoot induction

frequency was observed. At the same time, severe browning of callus was

noticed in other concentration of glucose, fructose and maltose tested. Similar

to our report, in Coyylus avellana sucrose-mediated shoot multiplication was

effectively achieved and maximum of 3 – 4 shoots were regenerated (Yu et al.,

1993). As per previous tissue culture reports, the most commonly used

carbohydrate for plant tissue culture is sucrose. In nature, carbohydrate is

transported within the plant as sucrose and the tissue may have the inherent

capacity for uptake, transport and utilization of sucrose (Sul and Korban, 2004).

Organogenesis

39

During multiple shoot proliferation from callus cultures of CL and HL

explants, severe browning of callus was noticed. The addition of additives

controlled the browning of callus and tissues and simultaneously increased the

percentage of multiple shoot induction (Amin and Jaiswal, 1988). Hence, in this

present investigation, we have evaluated different type of additives to control

the phenolic oxidation. All the four additives tested showed best response for

the suppression of browning of callus tissues. Among the four different

additives tested, supplementation of PVP (15 mg/l) showed superior activity to

control the browning process. At the same time, addition of PVP along with

multiple shoot induction medium enhanced the multiple shoot induction

percentage (72.2%) with CL and 57.8% with HL callus. Maximum of 20 shoots

/callus were initiated by the addition of PVP and in the case of controls only

17.8 shoots were obtained from cotyledonary leaf explants whereas, in

hypocotyl explants 12.2 shoots were obtained which is also higher than the

shoots obtained from control (10.4 shoots/explant) (Table 3.2). Like our results,

control of phenolic compounds by the addition of additives showed best

response in several crops (Amin and Jaiswal, 1988; Quraishi and Mishra, 1998).

The influence of various amino acids like alanine, glutamine, proline

and serine were also evaluated for the multiple shoot initiation from the

obtained callus. All the four amino acids showed enhanced activity on multiple

shoot induction. From the four, 15 mg/l glutamine showed best response for

multiple shoot proliferation and maximum of 22.1 shoots and 17.9 shoots /

callus clump was regenerated from cotyledonary leaf and hypocotyl explants

(Table 3.3; Plate 3,4). Similar effect of glutamine-mediated plant regeneration

was obtained in barley by using microspore explants (Ritala et al., 2001).

Generally, glutamine has been used for the induction of embryogenic callus

and direct and indirect induction of somatic embryos (Kim et al., 1997; Ipekci

and Gozukirmizi, 2002). But in our work, multiple shoot initiation was

effectively supported by the addition of glutamine (15 mg /l) as one of the

media component.

Organogenesis

40

3.3.4 Effect of Pluronic F68 on shoot multiplication and elongation

When the calli were transferred to the regeneration medium containing

TDZ (0.3 mg/l), NAA (0.4 mg/l), PVP (15 mg/l), Glutamine (15 mg/l) and

Sucrose (30 g/l), they showed signs of shoot regeneration by producing tiny

green meristems on the surface of the calli within 6 weeks of culture which

later formed shoot buds. To increase the frequency of shoot regeneration from

the callus PF - 68 (0.5 – 2.0 mg/l) was supplemented along with the above

mentioned PGRs. The percentage of shoot regeneration increased to 94.5% and

90.4% and produced 25.8 shoots and 19.4 shoots /callus on 1.0 mg/l of PF - 68

with CL and HL derived callus (Table 3.4; Plate 3, 4). This suggested that PF -

68 had the potential to promote organogenesis of castor.

There are several standardized protocols are existing for callus-mediated

somatic organogenesis and embryogenesis of economically valuable crops

(Wilkins et al., 2000, 2004). Organogenic callus induction and plant

regeneration from the callus cultures of castor-bean was reported by Ganesh

kumari et al. (2008) from CL explants. The influence of PF – 68 on organogenic

was high compared with the other PGRs. Our results proved that organogenic

callus induction and direct shoot regeneration was possible in castor-bean.

Usually, addition of GA3 with shoot proliferation medium was used for the

elongation of shoots (Caboni et al., 2000). In our studies also shoot elongation

was achieved by combined treatments of PF - 68 (1.0 mg/l) with GA3 (0.3 mg/l)

(Table 3.5). Regular weekly sub-culturing of induced shoots was done on the

same medium for complete elongation and maturation and complete

maturation of shoots required total of 2 – 4 weeks (Plate 3, 4). Wilting of leaves

was totally avoided by this weekly interval subculture (Pretto and Santarem

1997; Reddy et al., 2002).

3.3.5 Root induction and hardening

Induction of rooting is an essential step in plant propagation in vitro. The

classical root induction method uses a shock of high auxin concentration.

Organogenesis

41

However, the roots are stunted and malformed (Moncousin, 1991; Rao and

Purohit, 2006). In the present investigation, root induction was achieved after

7 days of culture. During root induction we have tested different

concentrations of auxins and AgNO3. Our results proved that root induction in

castor-bean was difficult compared to other crop plants. Hence, along with

different auxins we have tested AgNO3 for root induction. In the present study,

compared with individual treatment of auxins and AgNO3, combination of IBA

(1.5 mg/l) with AgNO3 (0.6 mg/l) showed best response (72.5%) for root

induction from elongated shoots and in this concentration, maximum of

5.9 roots with 5.6 cm in root length was induced (Table 3.6; Plate 3, 4). These

results demonstrated that AgNO3 can influence root emergence and growth

and can improve rooting efficiency (Bais et al., 2000). Similar to our results, it

has been accepted that interaction of thiol compounds stimulate rooting in vitro

(Biddington, 1992). He demonstrated that the use of ethylene inhibitors such as

AgNO3 might promote root formation in shoot cultures of apple.

The use of AgNO3, a potent ethylene action inhibitor, for promoting

in vitro rooting (Bais, 2000). In the case of IBA and IAA also root induction was

noticed but in all the concentrations of IBA tested showed low response for

root induction (data not shown) when compared with combined treatment of

IBA and AgNO3. Usually, IBA mediated root induction has been reported for

several plants including Hemidesmus indicus (Sreekumar et al., 2000) and Cunila

galiodes (Fracaro and Echeverrigary, 2001). Our results proved that combination

of IBA with AgNO3 was needed for the high percentage of root induction.

To our knowledge, this is the first report for indirect organogenesis of

castor-bean through cotyledonary leaf and hypocotyl explants. Root induction

was also highly increased when the medium was supplemented with IBA and

AgNO3. The increased root length leads to increase in the survival percentage

of hardened and field grown plants. For hardening process sand, soil and

Organogenesis

42

vermiculated soil were used in 1:1:1 ratio (Plate 3, 4). After proper

acclimatization the hardened plants were transferred to field.

3.4 CONCLUSION

In conclusion, an efficient and simple protocol for in vitro adventitious

shoot multiplication from callus cultures, and whole plant regeneration has

been described. The protocol was optimized by manipulations of different

PGRs, amino acids, carbohydrates and additives for enhanced multiplication.

In conclusion, protocol explained in this research paper provides a rapid plant

regeneration system from callus for castor-bean which could be used for the

somaclonal variation induction, and producing transgenic plants in castor-bean

through Agrobacterium and biolistic methods.

Table 3.1

Effect of cytokinins and auxins on organogenic callus induction from Cotyledonary leaf and Hypocotyl explants on mMS medium supplemented with B5 vitamins.

Cotyledonary leaf explant Hypocotyl explant Concentrations of growth regulators

(mg/l)

Percentage of organogenic

callus formation

Type and nature of callus

Percentage of organogenic

callus formation

Type and nature of callus

KN 1.0 1.5 2.0 2.5 3.0

12.3 ± 0.4p 14.1 ± 1.2op 17.6 ± 1.5mn 15.2 ± 0.5o 10.9 ± 1.1po

GF GF

GLC BF BC

11.5 ± 0.7o 12.5 ± 0.6mn 15.5 ± 1.2l 13.5 ± 1.6m 11.4 ± 1.9op

YGF YGF YWF

BF BC

BA 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

8.5 ± 0.5q 24.7 ± 0.5l 25.3 ± 0.3j 28.7 ± 1.3jk 20.5 ± 0.6lm 18.1 ± 0.4m 13.2 ± 1.4p 7.6 ± 0.5qr

GF GC GC GC GC GC

GLC BF

16.7 ± 0.6k 18.5 ± 0.5j 20.5 ± 0.6i 25.3 ± 1.6h 17.3 ± 1.5jk 14.7 ± 1.7lm 12.4 ± 1.5mn 11.7 ± 1.1no

GF GC GC GC GF GF

GLC BF

BA + NAA 2.0 + 0.2 2.0 + 0.4 2.0 + 0.6 2.0 + 0.8 2.0 + 1.0 2.0 + 1.2 2.0 + 1.4 2.0 + 1.6 2.0 + 1.8 2.0 + 2.0

46.5 ± 1.0f 52.6 ± 1.5e 55.5 ± 0.5d 69.5 ± 1.5a 61.5 ± 1.7b 59.3 ± 1.4bc 53.5 ± 0.7de 44.8 ± 0.5fg 41.5 ± 0.7h 38.5 ± 0.4i

GC GC

GCN GCN GCN GC GC GC GC GC

40.4 ± 1.6g 51.2 ± 1.5c 52.3 ± 1.1b 54.2 ± 1.7a 53.1 ± 0.7ab 52.3 ± 0.2b 51.7 ± 1.4bc 50.4 ± 1.4cd 48.9 ± 1.6e 44.5 ± 1.5f

GC GC

GCN GCN GCN GCN GCN GCN GC GC

GF – Green Friable; GC – Green Compact; GCN – Green Compact Nodular; YWF – Yellowish White Friable; YGF – Yellowish Green friable;

GLC – Greenish Less Compact; BC – Brown Compact; BF – Brown Friable

Number of explants tested = 50. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly

different according to Duncan's Multiple Range Test (DMRT) at 5 % level.

Table 3.2

Effect of different concentrations of additives on multiple shoot proliferation from callus cultures of castor (Ricinus communis L.) on the mMS medium fortified with

TDZ (0.3 mg/l) and NAA (0.4 mg/l)

Cotyledonary leaf explant Hypocotyl explant Additives (mg/l ) Percentage of

response Mean number

of shoots / callus Percentage of

response Mean number

of shoots / callus

Charcoal 50

100 150 200 250

58.4 0.2mn 62.1 0.3k 65.6 0.5hi 63.2 0.2j 60.4 1.0l

16.8 0.4j 17.2 0.8i 17.6 0.5h 17.0 0.2ij 16.5 0.6k

34.0 ± 0.6op 44.5 ± 0.4k 50.4 ± 1.2ef 55.1 ± 0.6c 50.3 ± 1.3fg

7.6 ± 1.2j 8.0 ± 0.5i 8.7 ± 0.2f 8.5 ± 0.5fg 7.4 ± 1.3jk

PVP 5

10 15 20 25

69.3 0.6cd 71.8 0.5ab 72.2 0.2a 70.2 0.1b 67.3 0.3de

18.4 0.2ef 19.2 1.0c 20.0 0.3a 19.0 0.1cd 18.6 0.9e

52.3 ± 1.6d 55.3 ± 1.4b 57.8 ± 1.5a 55.0 ± 0.5cd 50.1 ± 0.4g

9.3 ± 1.6de 10.6 ± 1.4b 12.2 ± 1.8a 9.7 ± 0.1d 8.3 ± 0.4gh

Ascorbic acid 5

10 15 20 25

67.5 1.0f 68.7 0.6e 69.1 0.3d 66.2 0.7g 65.0 0.1i

17.1 0.5ij 17.9 0.1g 19.5 0.2b 18.9 0.1d 17.6 0.2h

51.2 ± 0.3e 52.3 ± 0.2e 54.3 ± 1.4d 50.0 ± 1.6h 48.1 ± 0.7i

8.4 ± 0.6g 9.5 ± 1.5de 10.5 ± 1.7bc 9.7 ± 0.3d 8.4 ± 0.5g

Citric acid 5

10 15 20 25

56.5 1.0p 59.6 0.5m 60.1 0.2lm 58.2 0.1n 57.4 0.3op

15.4 0.5m 16.0 0.2l 16.5 0.1k 15.0 0.4n 14.4 0.3o

41.6 ± 0.4n 43.8 ± 0.5l 45.8 ± 0.6j 42.7 ± 1.5m 39.7 ± 0.6o

6.2 ± 0.3m 7.0 ± 0.5l 7.6 ± 1.2j 7.0 ± 1.7l 6.1 ± 1.5mn

Number of explants tested = 30. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly

different according to Duncan's Multiple Range Test (DMRT) at 5 % level.

Table 3.3

Effect of different concentrations of amino acids on the multiple shoot proliferation from the callus on the medium supplemented with on the mMS medium fortified

with TDZ (0.3 mg/l), NAA (0.4 mg/l) and PVP (15 mg/l)

Cotyledonary leaf explant Hypocotyl explant Amino acids

(mg/l) Percentage

of callus formation

Mean number of shoots / callus

Percentage of callus

formation

Mean number of shoots / callus

Alanine 5

10 15 20 25

56.3 0.2m 57.6 0.1k 57.2 0.5l 52.2 0.1n 49.3 0.6o

18.7 0.3o 19.3 0.1kl 19.0 0.2n 18.3 0.5p 17.5 0.1q

52.7 ± 0.5lm 56.8 ± 0.2j 54.7 ± 0.6kl 55.4 ± 0.1k 52.4 ± 0.3m

8.4 ± 0.3o 10.8 ± 0.6hi 9.4 ± 1.3m 8.0 ± 0.5p 7.4 ± 0.1q

Proline 5

10 15 20 25

64.6 0.7i 65.7 0.4gh 66.5 0.2g 65.4 0.1h 63.2 0.2j

19.2 0.1l 19.9 0.4i 20.5 0.2fg 19.6 0.3jk 19.0 0.7n

58.4 ± 1.4hi 70.3 ± 0.4b 62.7 ± 0.6ef 56.4 ± 0.6jk 53.8 ± 0.5l

8.9 ± 0.7n 11.0 ± 0.6h 12.5 ± 0.2f 9.7 ± 0.5l 8.1 ± 0.5op

Serine 5

10 15 20 25

73.5 0.1e 74.0 0.5de 73.5 0.3e 74.3 0.6d 72.7 0.8f

20.3 0.4h 21.5 0.3c 20.6 0.1fg 21.7 0.2b 19.7 0.1j

59.4 ± 0.3h 61.2 ± 0.1fg 62.1 ± 0.3f 63.7 ± 0.6e 50.3 ± 0.7n

9.0 ± 0.1mn 10.3 ± 0.6j 11.5 ± 0.1g 13.0 ± 0.3de 9.5 ± 0.5lm

Glutamine 5

10 15 20 25

84.3 0.2b 84.9 0.5ab 85.2 0.1a 83.6 0.3c 83.1 0.4cd

21.1 0.3e 21.4 0.1cd 22.1 0.2a 21.0 0.4de 20.7 0.2f

65.4 ± 0.5d 67.6 ± 0.3bc 72.3 ± 0.2a 68.4 ± 0.6b 63.2 ± 0.1ef

10.2 ± 0.1jk 13.4 ± 0.6d 17.8 ± 0.1a 15.4 ± 0.4c 16.5 ± 0.5b

Number of explants tested = 30. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly

different according to Duncan's Multiple Range Test (DMRT) at 5 % level.

Table 3.4

Effect of different concentrations of pluronic F68 on regeneration of shoot buds from organogenic callus cultured on mMS medium fortified with TDZ (0.3 mg/l),

NAA (0.4 mg/l), PVP (15 mg/l ) and glutamine (15 mg/l)

Cotyledonary leaf derived callus cultures

Hypocotyl derived callus cultures Concentrations

of PF – 68 (mg/l) Percentage of

response Mean no. of

Shoots / callus Percentage of

Response Mean no. of

Shoots / callus

Pluronic F - 68 0.5 0.6 0.7 0.8 0.9 1.0 1.2 1.4 1.6 1.8 2.0

90.5 ± 0.4f 91.5 ± 0.2d 92.4 ± 0.1c 93.6 ± 0.2b 94.0 ± 0.5ab 94.5 ± 0.6a 93.2 ± 0.8b 91.4 ± 0.2de 89.5 ± 0.4g 89.0 ± 0.6gh 87.5 ± 0.8h

19.6 ± 0.4g 21.5 ± 0.2f 22.5 ± 0.5e 23.6 ± 0.2c 25.0 ± 1.0ab 25.8 ± 0.6a 24.8 ± 0.3b 23.5 ± 0.5cd 23.3 ± 0.6d

22.5 ± 0.2e

21.5 ± 1.7f

77.4 ± 0.8fg 80.3 ± 0.4f 82.5 ± 0.5de 83.5 ± 0.3d 89.5 ± 0.6ab 90.4 ± 0.1a 89.4 ± 0.5b 85.6 ± 0.1c 82.1 ± 0.6e 78.5 ± 0.5f 77.4 ± 0.7fg

12.4 ± 0.5i 13.6 ± 0.4h 15.7 ± 0.1fg 17.4 ± 1.0de 18.4 ± 0.6bc 19.4 ± 0.3a 18.5 ± 0.6b 18.0 ± 0.3c 17.4 ± 0.5de 16.2 ± 0.5f 15.4 ± 0.5g

Number of explants tested = 30. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly

different according to Duncan's Multiple Range Test (DMRT) at 5 % level.

Table 3.5

Effect of GA3 in combination with PF - 68 on shoot elongation of castor (Ricinus communis L. cv TMV 5)

Growth regulators (mg /l)

Percentage of response

Shoot length (cm)

PF - 68 + GA3 1.0 + 0.1 1.0 + 0.2 1.0 + 0.3 1.0 + 0.4 1.0 + 0.5

51.8 0.5e 56.7 0.6ab 62.3 0.5a 57.4 0.8b 50.5 0.4ef

4.5 0.5cd 5.1 0.8c 5.6 0.6a 5.4 0.7e 4.9 0.3ef

Number of explants tested = 30. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly

different according to Duncan's Multiple Range Test (DMRT) at 5 % level.

Table 3.6

Effect of IBA and AgNO3 on root induction from elongated shoots of Castor (Ricinus communis L. cv TMV 5)

Growth regulators (mg/l )

Percentage of Response

Mean No. of roots / explant

Average root length (cm)

IBA + AgNO3 1.5 + 0.2 1.5 + 0.4 1.5 + 0.6 1.5 + 0.8 1.5 + 1.0

65.0 1.2c 69.5 1.9b 72.5 2.2a 70.5 3.2b 68.5 2.5b

5.2 0.25c 5.4 0.32bc 5.9 0.51a 5.5 0.16b 5.1 0.25d

5.2 0.5bc 5.5 0.9ab 5.6 0.8a 5.3 0.4b 5.0 0.5c

Number of explants tested = 30. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly

different according to Duncan's Multiple Range Test (DMRT) at 5 % level.

Adventitious shoot proliferation from cotyledonary leaf and hypocotyl explants of Castor (Ricinus communis)

Effect of carbohydrates on multiple shoot proliferation from cotyledonary leaf and hypocotyl explants of Castor (Ricinus communis)

Plate 3 Organogenesis from cotyledonary leaf explants of Ricinus communis L.

a. Callus initiation (1.5 x) b. Callus proliferation (1.5 x) c & d. Shoot bud initiation (1.5 x & 1.0 x) e. Multiple shoot initiation (0.5 x) f & g. Shoot elongation (2.0 x & 1.0 x) h. Root initiation (0.5 x) i & j. Hardening of in vitro derived plants (0.1 x & 0.2 x)

Plate 4 Organogenesis from hypocotyl explant of Ricinus communis L.

a. Callus initiaion (1.5 x) b. Callus proliferation (1.0 x) c, d & e. Shoot bud initiation and proliferation (2.0 x, 1.5 x & 1.5 x) f, g & h. Shoot elongation (0.5 x, 0.5 x & 0.3 x) i. Root initiation (0.5 x) j. Hardened plants (0.2 x) k. Well grown plants (0.1 x)