Indian Journal of Experimental Biology Vol. 43, October 2005, pp. 921-925

Carbon source dependent somatic embryogenesis and plant regeneration in cotton, Gossypium hirsutum L. cv. SVPR2 through suspension cultures

M Ganesan & N Jayabalan '

Department of Plant Science, Bharathidasan University, Tiruchirappalli 620 024, India

Received 17 August 2004; revised 9 May 2005

Highly reproducible and simple protocol for cotton somatic embryogenesis is described here by using different concentrations of mal tose, glucose, sucrose and fructose. Maltose (30 gil) is the best carbon source for embryogenic callus induction and glucose (30 g/1) was suitable for induction, maturation of embryoids and plant regeneration. Creamy white embryogenic calli of hypocotyl explants were formed on medium containing MS basal salts, myo-inositol (100 mg/1), thiamine HCI (0.3 mg/1), piclorarn ((J 3 mg/1), Kin (0.1 mgll) and maltose (30 g/1). During embryo induction and maturation , accelerated growth was observed in liquid medium containing NH3N04 (1 g/1), picloram (2.0 mg/1), 2 ip (0.2 mg/1), Kin (0.1 mg/1) and glucose (30 g/1). Before embryoid induction, large clumps of embryogenic tissue were formed . These tissues only produced viable embryoids. Completely matured somatic embryos were germinated successfully on the medium fortified with MS sal ts, myo-inositol (50 mg/1), thiamine HCI (0.2 mg/1), GA3 (0.2 mg/1), BA ( 1.0 mg/1) and glucose (30 g/1). Compared with earlier reports, 65% of somatic embryo germination was observed. The abnormal embryo formation was highly reduced by using glucose (30 gil) compared to other carbon sources. The regenerated plantlets were fertile but smaller in height than the seed derived control plants.

Keywords: Carbon source, Cotton, Embryoids, Regeneration Suspension culture,

[IPC Code: LInt Cl7 A01H]

Cotton (Gossypium hirsutum L.) is one of the most important industrial crops. Cultivated for fibre production and essential seed-oil, it plays an important role in the economy of cotton producing countries. The conventional breeding methods require more than five years to produce a cotton variety with desired traits . Today, through genetic· engineering, cotton has been largely improved in short time. Plants with a better quality and quantity of fibre, resistant to insects, herbicide, fungi, bacteria a:1d nematode were obtained through Agrobacterium-mediated transformation and particle gun bombardment1

' 2

. To apply a successful transformation protocol, it is necessary to obtain a highly efficient and reproducible regeneration and/or somatic embryogenesis protocols. To date, there are limited reports regarding high frequency of successful plant regeneration from cotton via somatic embryos2

-9

. As reported in these works, there are some difficulties in producing somatic embryos and in plant regeneration. One of the main drawbacks is the browning of callus within the short period of culture due to phenolic secretion and

•·correspondent author: Phone : 00-91-0431-2407061 Fax: 00-91-0431-2407045 e­mail : jayabalan54@yahoo.co. in

oxidation and the long time required from somatic embryos to plantlet formation (up to 10 months). The large number of abnormal embryo formation and low embryo maturation and germination frequency are other important constraints in cotton somatic embryo. Due to these problems, a good regenerative and reproducible protocol is needed for the cotton via somatic embryogenesis.

Only limited somatic embryogenesis has been reported in Indian cotton varieties because the cotton somatic embryogenesis is mainly genotype dependent2

• In this paper a regeneration protocol for the cotton somatic embryos via suspension culture by using different types and concentrations of carbon sources is described.

Materials and Methods Collection of plant materials and aseptic culture of

seedlings-The acid delinted cotton seeds (Gossypium hirsutum L. cv. SVPR2) were collected from the Cotton Research Institute, Tamil Nadu Agricultural University, Srivilliputhur, Tamil Nadu, India. Seeds were wrapped in two layers of cheesecloth and placed in running tap water for 30 min to remove surface particles. Seeds were sterilized in 70% ethanol for 10 min and kept in 0.1% HgCb for

922 INDIAN J EXP BIOL, OCTOBER 2005

20 min in aseptic conditions after rinsing 5 times with sterile distilled water. Sterilized seeds were cultured in the seed germination medium consisting of MS salts 10

, 8 5 vitamins 1 1, BAP (0.5 mg/1), GA3 (0.2 mg/1),

30 g/1 sucrose, 0.8 g/1 agar and the pH of the medium was adjusted to 5.7-5.8 by using 0.1 N NaOH before autoclaving at 120 lb for 15 min. Initially the cultures were maintained in dark condition for 48 hr at 25° ± 2°C and then under 16 hr light and 8 hr dark photoperiod at a light intensity of 3000 lux.

Initiation, proliferation and selection of embryogenic callus-In this study 3 to I 0-days-old cotyledonary pieces and horizontally sectioned hypocotyl segments were used as explants and placed on callus initiation medium consists of MS salts, myo­inositol (I 0-200 mg/1), thiamine HCI (0.1-0.5 mg/1), picloram (0.1 to 0.5 mg/1) and Kin (0.05 to 0.2 mg/1), 0.8% agar (pH 5.8). After 3 weeks of culture, calli were initiated from both the type of explants. The proembryogenic portions from the callus were identified, isolated and subcultured in the same medium fortified with MS salts, myo-inositol (10-200 mg/1), thiamine HCI (0.1-0.5 mg/1), picloram (0.1 to 0.5 mg/1 ) and Kin (0.05 to 0.2 mg/1), 0.8% agar. Whitish green, brown, watery brown and black coloured non-embryogenic calli were also generated but they were discarded. The selected proembryogenic calli were weekly subcultured for two weeks for the induction of embryogenic callus. Embryogenic calli were identified by creamy white colour and by the presence of small cells, less vacuolate with a densely filled cytoplasm.

Embryoid induction, maluration and plantlet regeneration--Fresh embryogenic calli (250 mg) were transferred to 500 ml conical flasks containing 50 ml of liquid medium containing MS salts, ammonium nitrate ( 1.0 g/1), myo-inositol (I 0 - 200 mg/1), thiamine HCI (0.1 to 0.5 mg/1), picloram (0.1 to 0.4 mg/1), Kin (0.05 - 0 .2 mg/1) and 2iP (0.1 to 0.4 mg/1) without agar. For embryoid induction, suspension cultures were weekly subcultured. At each subculture, half the liquid medium was removed, and fresh medium was replaced. The liquid cultures were maintained at 120 rpm with a light intensity of 2500 lux.

After 8 weeks of subculture the whitish yellow, embryoids were induced in clusters and at this stage embryogenic callus was subcultured in MSC liquid medium consisting of MS salts, ammonium nitrate (I g/1), myo-inositol (10 to 200 mg/1), thiamine HCI

(0.1 to 0.5 mg/1), picloram (0.1 to 0.4 mg/1), Kin (0.05 to 0.2 mg/1) and 2iP (0.1 to 0.4 mg/1) without agar. Embryoids were further subcultured onto the same medium with agar (0.7%) for complete maturation.

Matured embryoids were transferred to somatic embryo regeneration medium (MSD) consisting of MS basal salts, myo-inositol (I 0 to 200 mg/1), thiamine (0.05 to 0.2 mg/1) , 0.8% agar and the hormones such as GA3 (0.1 to 1.0 mg/1 ) and BA (0.5 to 2.0 mg/1) were also added . The embryo n1aturation was confirmed by the formation of globular, heart and torpedo shaped embryos. Germinated embryos (plumule and radicle formation) were transferred to MSD medium for further development into plantlets. After the complete plantlet development (I 00 to 120 days), they were hardened in plastic pots containing sand, soil and vermiculite in I: I: I ratio covered with plastic bags to maintain the humidity . After 35 days, hardened plants were transferred to earthen pots for further development and agronomical evaluations.

Carbon source--Four different carbon sources (sucrose, glucose, fructose and m(:]ltose) and 8 different concentrations (5, 10, 15, 20, 25, 30, 35 and 40 g/1) were tested. The effect of these concentrations on cotton somatic embryogenesis was measured including fresh weight of the embryogenic callus before embryo induction.

Statistical analysis-Means and st andard errors were used throughout the study and t.he values were assessed using a parametric Moojs medi an test 12

. The data were analyzed for variance by Duncan's multiple range test (DMRT) using the SAS program (SAS Institute, Cary, N.C.).

Results and Discussion Seeds of cotton were germinated aseptically and

cotyledon pieces with midrib region and horizontally sectioned hypocotyls were excised and transferred to callus initiation and proliferation media supplemented with different types and concentrations of carbon source. Maximum frequency of callus formation was observed in explants derived from 6-days-old plantlets. From the explants tested, rhe hypocotyl segments produced maximum percentage of embryogenic calli. Creamy white, yellowish green , watery yellow and brown colored calli were obtained and the creamy white calli produced higher number of embryos. Among different callus induction media tested, MSB supplemented with MS salts, myo­inositol (100 mg/1), thiamine HCI (0.3 mg/1), picloram (0 3 mg/1), Kin (0.1 mg/1) and maltose (30 g/1) showed

GANESAN & JAYABALAN: SOMATIC EMBRYOGENESIS & PLANT REGENERATION IN COTION 923

Fig.!- ~Different deveiopmental stages of somatic embryogenesis of cotton variety SYPR2 through suspension culture from hypocotyl exp lants, [(a) Embryogenic callus induction from hypocotyl expiants after 1 week of culture. Bar= 5 mm; (b) Embryogenic cell clusters observed in suspension culture after 6 weeks. Bar = I 00 Jlm; (c) Globular and heart shaped embryos after 8 weeks of culture; (d) Matured embryoids after 3 weeks. Bar = 2 mm; (e) Germinated somatic embryo after 4 weeks. Bar= 5 mm; (f) Rooted whole plant after 3 weeks. Bar= 1 em; (g) Fully regenerated plant with tertiary roots in bottles. Bar= 1.5 em.]

924 INDIAN J EXP BlOL, OCTOBER 2005

the best response, producing cream-colour pro­embryogenic calli which were morphologically similar as described earlier13-14 (Fig. 1)_ The selected proembryogenic calli were then transferred to the same medium to multiply the amount of embryogenic calli .

Browning of callus occurred but after two or three subcultures this was reduced by the addition of maltose to the callus induction medium (Table 1 and Fig. 2). Fructose showed very poor response in embryogenic calli induction and development. The embryogenic calli were identified by the presence of dense cytoplasm, large and more starch granules and small cells as was also noticed in other cases 16. The percentage of non-embryogenic calli formation was also reduced by maltose when compared to other carbon sources. The creamy white embryogenic calli produced the embryoids compared with the above said other types of calli in the suspension cultures. The well-developed embryos were induced from in the medium supplemented with MS salts, 100 mg/1 myo-inositol , thiamine HCl (0.3 mg/1), picloram (2 mg/1) , 2iP (0.2 mg/1) , Kin (0.1 mg/1), glucose 30 g/1 and NH3N04 (1 g/1). Usually, 2,4-D in combination with glucose or sucrose was widely used for the induction of embryogenic calli and embryoids 17 - 23

. In some cases zeatin8

, and combination of NAA, Kin with glucose16 were used for the induction and

proliferation of embryoids. In our study , a combination of picloram, 2-iP, thiamine HCl and Kin produced embryogenic calli and viable embryoids.

After 6-weeks of subculture on the same medium, embryoids were produced from the large clumps of embryogenic tissues. The present study proved that the embryoid induction rate increased when the induction medium was supplemented with NH4N03 (1 g/1 ) and such results were reported in several plants including cotton2

. The number of embryoids increased by the addition of glucose (30 g/1) as carbon source to the embryo induction medium. About 195 embryos I

Q) 0> .!!! c

~ Q)

0.

30

Concentrations (giL) 40

Fig. 2-Effect of different concentrations of carbons sources on embryogenic callus induction.

Table 1-Effect of different carbon sources on somatic embryogenesis of cotton from hypocotyl explar.ts [Values are mean±SE of 3 repeated experiments]

Response

Callus formation (%) Browning of callus (%)

Embryogenic callus Formation (%)

Fresh weight of embryogenic callus After 6 weeks (mg)

Number of Embryos formed per 500 mg of embryogenic callus

Number of plants germinated form embryoids

Number of rooted embryoids without shooting

Name and concentration of carbon source (gil)

Fructose (30) Glucose (30) Sucrose (20)

75 ± 0.8 d 90 ± 0.7 b 50 d 75 b

55± 0.6 d 85 ±0.4 b

75 ± 3.25 d 265 ± 2.4 b

5 ± 1.78 d 195 ± 1.20 a

138 ± 2.0 a

4 ± 0.35 b

88 ± 0.4 c 64c

77 ± 0.3 c

180±3.2c

70 ± L7 c

25 ± 1.2 c

9 ± 0.27 a

Number of shooted embryoids without rooting 4 ± 0.10c 6 ± 0.1b Number of ex plants tested= 60. Each treatment consisted of 6 replicates. Means within a row followed by the same letters are not significant at P=0.05 according to DMRT

Maltose (30)

92 ± 1.05 a 95 a

97 ± 0.9 a

310 ± 1.95 a

158±2.1 b

95 ± 1.35 b

8 ± 0.09 ab

7 ± 0.17a

GANESAN & JA YABALAN: SOMATIC EMBRYOGENESIS & PLANT REGENERATION IN COTTON 925

500 mg of embryogenic calli were obtained. Germination percentage of somatic embryos and formation of whole plants also increased due to the addition of glucose (30 g/1). During germination of embryoids to plants, small cotyledons were observed compared to in vivo seed germinated cotyledons. These cotyledons easily fell within couple of weeks, after the treatment with varying concentrations of carbon source, and morphologically no significant change in cotyledon was observed during this period. Completely matured embryoids, when transferred to the medium supplemented with MS salts, inositol (50 mg/1), thiamine HCI (0.2 mg/1), GA3 (0.2 g/1), BA (1.0 mg/1) and glucose (30 g/1) exhibited 65% regeneration. Lower percentage of regeneration was reported earlier6

· 24

. During the germination of somatic embryos, high percentage of abnormal growth was noticed on media containing maltose (30 g/1) and sucrose (20 g/1). Abnormal embryoid formation and germination was strikingly reduced when the cultures were grown on glucose (30 g/1). After regeneration of embryos, the plants were transferred to plastic cups for hardening. The hardened plants were grown in pots till flowering and fiber yielding. The plants showed the same parental behaviours and were morphologically similar but smaller (70%) than the in vivo seed derived plants.

Untill now, there were difficulties in achieving cotton somatic embryogenesis and mass production of embryos. The conversion rate of embryoids to plantlets was also very low. The protocol described here can be used for the mass propagation or in gene transformation experiments that can lead to improvement of cotton.

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