Somatic embryogenesis in cotton

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1 Somatic embryogenesis in cotton

CONTENTS:

2 CONTENTS Introduction Establishment of Axenic culture Pathways of Somatic embryogenesis Callus initiation Callus proliferation Selection of embryogenic calli Somatic embryo induction Somatic embryo development Maturation of somatic embryos Germination of somatic embryos Hardening and transfer to soil Conclusion Future thrust

INTRODUCTION:

3 INTRODUCTION Cotton is the most important fiber crop economically and the world’s second most important oilseed crop after soybean, beside producing fibers, its seeds are also used for hulls, linters and meal (Nobre et al., 2001) . Botanically , cotton belongs to malvaceae family , the tribe gossypieae and the genus gossypium . The cotton genus consists of 45 diploid and 5 tetraploid species ,only four species of cotton were domesticated and grown as annual crop mainly under sub tropical and tropical environment . Among them two species G ossypium arboreum (2n=26) and G. herbaceum ( 2n = 26) are diploid and Gossypium hirsutum (2n=52) and G. barbadence (2n = 52) are tetraploid. At present India ranks third in cotton production after USA and China. Gujarat stands first in position for cotton production in India (Patel, 2005) Since cotton is highly susceptible to biotic and abiotic stresses, it requires intensive crop management. Although conventional breeding programs have made steady improvement in agronomic traits, not much genetic diversity exist for further improvement. However transformation techniques have provided ample scope for introduction of foreign genes in cotton, which involves the efficient regeneration system.

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4 Regeneration through somatic embryogenesis over organogenesis is preferred because of single cell origin of the somatic embryos, thus reducing the chimeric transformation event. In vitro cultured cotton cells have been induce to undergo somatic embryogenesis in numerous laboratories using varied strategies, after first one was reported by Price and Smith in 1979 ( Shoemaker et al .,1986; Chen et al., 1987; Trolinder and Goodin, 1987; Gawel and Robacker ,1990; Zhang et al ., 2001; Sun et al .,2004) Although regeneration efficiency via somatic embryogenesis has been improved, problems remained are :- High frequency of abnormal embryo development The excretion of secondary metabolites from the explant to the medium Low maturation and conversion rate of somatic embryos into plantlet

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5 A generalized procedure of axenic culture establishment Collection of plant material Washing with sterile deionized water Surfactant application Application of chemical sterilizing agent Triple wash with water Inoculation of plant material on nutrient medium Incubation Germination Explant When seed were taken

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6 Table:1 Summary of different procedures to establish the aseptic culture as a source of explant Contd…. Authors Chemicals used in Sterilization Time span Media used pH range Light condition Temp. range Haq et al., (2005) 30% bleach (5.25% v/v NaOCl) 30 min MS salts+ B 5 vitamins 5.7-5.8 previously in dark 28 ±2 C Aydin et al., (2004) Ethanol+ 20% bleach 20 min -do- 5.5 16 hrs light / 8 hrs dark 25 C Ganesan and Jayabalan (2004) 70 % ethanol + 0.1% HgCl 2 30 min -do- 5.7-5.8 Dark period 16/8 hrs 25 ± 2 C Wu Jiawe et al., (2004) 0.1% HgCl 2 (w/v) 8 min ½ MS salts 5.7 7 day dark 28 ± 2 C Sakhanakho et al., (2004) 100 % ethanol + 23 % bleach +Tween-20 20 min MS salts 5.7 16/8 hrs photo period 28 ± 2 C Kumaria et al., (2003) 20% ethanol + 0.1% HgCl 2 20 min -do- 5.8-6.0 16/8 hrs photo period 28 ± 2 C

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7 Authers chemicals used in Sterilization Time span Media used pH range light condition Temp. range Mishra et al., ( 2003) 20 % bleach 20 min Stewort’s germination media 5.8 16/8 hrs 28 ±1 C Sun et al., ( 2003) 0.1% HgCl 2 5-8 min ½ MS salts 5.8 Dark 28 ±1 C Rakitin et al , (2001) 96 % ethanol - ½ MS salts + 10 g/lit G 5.8 16/8 hrs 26 ±1 C Zhang et al., (2001a) 0.1% HgCl 2 - ½ MS salts + B 5 vitamins 5.8 16/8 hrs 28 ±1 C Sakhanakho et al., (2001) 100 % ethanol + 23 % bleach 20 min MS medium 5.7 16/8 hrs 28 ±1 C Zhang et al., ( 2001b) 0.1% HgCl 2 20 min ½ MS salts + B 5 vit 5.8 16/8 hrs 28 ±1 C Nobre et al ., (2001) 15 % bleach (v/v) 15 min MS salts + vitamins 5.7 16/8 hrs 28 ±1 C Kumar and Pental, (1999) 70 % ethanol + Bleach 0.1% HgCl 2 15 min ½ MS salts 5.8-5.9 16/8 hrs 28 ±1 C Trolinder and Goodin, (1987) Tween-20 + 70% ethanol + 10 % Clorex 20 min MS medium 5.8 16/8 hrs 28 ±1 C Contd…

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8 General pathways of cotton somatic embryogenesis

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9 (Cells of explant directly undergoes embryogenesis in absence of callus proliferation ) (Cells of explant first undergo callus proliferation and embryoids develop within the callus tissue ) Somatic embryogenesis PEDC IEDC Explant Pathways Direct somatic embryogenesis Indirect somatic embryogenesis

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10 Fig-2 : Diagram showing the stages of plant regeneration via indirect somatic embryogenesis.

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11 Fig- 1:Direct cotton somatic embryogenesis and plant regeneration. (a) Embryo formation directly from leaf explant b) Direct somatic embryo development (c)Somatic embryos at various developmental stages (d) Regenerated plant China Zhang et al., (2001d)

CALLUS INITIATION :

12 CALLUS INITIATION The most important factors affecting the process of callus initiation are - Source of explant and its physiological condition Type of explant Media used Plant Growth regulators Nitrogen source T he first step of indirect somatic embryogenesis is “initiation of callus” in which the explant starts to convert into unorganized mass of cells. This process decides the fate of material and its further growth , differentiation and development.

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13 Fig- 3: Callus initiated on cotton ( Gossypium hirsutum L .) hypocotyl explants (A,D) Yellowish-green callus produced by Coker 312 hypocotyl explants cultured. (B, E) Hard, dark green, compact, callus produced on Maxxa hypocotyl (C, F) Parrot-green, callus produced on Maxxa stem. New Delhi Mishra et al., (2003)

Table :2. Effect of plant age, bract region and light intensity on callus initiation and embryogenic response :

14 Table :2. Effect of plant age, bract region and light intensity on callus initiation and embryogenic response Parameter tested Epidermal strip cultured(n) Callus initiation(%) Embryogenic callus / total callus Plant age and bract region Base (9-10 months) 337 36.3 + 15.8 a 4/61 b Top -do- 279 13.2 + 7.1 a 0/26 Base (4-5 months) 159 17.8 + 10.5 4/28 b Top -do- 55 0.0 0/26 Light condition ( μ mol /m / s ) 15.8 75 16.0 5/12 b 21.0 101 29.0 0/25 b 26.3 94 31.3 0/22 b a: Mean callus initiation frequency + SE; means followed by c Epidermal strips excised from basal bract regions of younger different letters are significantly different at the P = 0.05 level plants. b: Sum of embryogenic calluses regenerated following three consecutive passages in callus initiation medium UK Nobre et al., (2001)

Table :3 . Frequency of callus induction, callus mass with frequency of somatic embryo producing calli in cotton cv. Nazilli M-503 and Nazilli 143. :

15 Table :3 . Frequency of callus induction, callus mass with frequency of somatic embryo producing calli in cotton cv. Nazilli M-503 and Nazilli 143. Media used BA + kinetin (mg l -1 ) Explant Nazili (503) callus induction (%) callus mass (g/e x.) Somatic embryo (%) Nazili (143) callus induction (%) Callus mass (g/ex) Somatic embryos (%) MS1 0.5 + 0.5 Hypocotyl Node Apex 33.3 ±3.0 65.0 + 3.1 58.3 + 2.9 2.4 2.5 1.9 40.0 + 1.2 46.0 + 1.4 71.4 + 2.0 56.6 + 2.6 60.0 + 2.9 41.6 + 4.8 2.1 3.4 2.1 100.0 + 0.0 100.0 + 0.0 35.7 + 1.1 MS2 0.5+1.0 Hypocotyl Node Apex 40.0 + 4.7 70.0 + 0.9 50.0 + 2.4 4.0 2.4 3.4 41.6 + 1.3 42.8 + 1.3 83.3 + 0.3 53.3 + 2.4 70.0 + 0.9 66.6 + 3.2 3.7 1.5 2.8 100.0 + 0.0 100.0 + 0.0 66.6 + 1.8 MS3 0.5+2.0 Hypocotyl Node Apex 53.3 + 2.8 20.0 + 2.3 41.6 + 4.8 1.9 2.6 2.3 31.2 + 1.2 60.0 + 1.6 66.6 + 1.8 40.0 + 4.7 70.0 + 0.9 50.0 + 2.4 2.2 2.9 3.5 83.3 + 1.8 42.0 + 1.3 100.0 + 0.0 MS4 1.0+0.5 Hypocotyl Node Apex 100.0 + 0.0 100.0 + 0.10 0.0 + 0. 2.9 3.5 3.1 66.6 + 1.8 100.0 + 0. 100.0 + 0. 86.6 + 3.2 60.0 + 2.9 91.6 + 3.2 2.6 1.9 0.9 76.9 + 2.1 88.8 + 1.9 73.3 + 2.0 Turkey Aydin et al., (2004) Nazilli M-503 Nazilli -143 Contd…

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16 Means + SD. For each treatment, 30, 20 and 12 hypocotyl, node and apex explants were recorded, respectively in 3 replicates Turkey Aydin et al ., (2004) Medium BA + kinetin (mgl -1 ) Explant Nazilli 503 Nazilli-143 callus induction (%) Callus mass (g/ex) Somatic embryo (%) callus induction (%) Callus mass (g/ex) Somatic embryo (%) MS5 1.0+1.0 Hypocotyl Node Apex 46.6+5.5 70.0+0.9 41.6+4.8 4.2 2.8 2.5 85.7+1.8 71.4+2.0 20.0+0.7 60.0+2.9 55.0+2.5 66.6+3.2 2.2 3.9 1.0 55.5+1.4 54.5+1.4 75.0+2.0 MS6 1.0+2.0 Hypocotyl Node Apex 100.0+0.0 100.0+0.0 91.6+3.2 1.9 2.5 3.9 43.3+0.3 100.0+0.0 100.0+0.0 83.3+3.0 95.0+3.3 91.6+3.2 2.5 2.8 3.0 100.0+0.0 100.0+0.0 48.8+1.4 MS7 2.0+0.5 Hypocotyl Node Apex 86.6+3.2 80.0+2.9 75.0+0.4 2.5 2.2 3.2 46.1+1.4 50.0+1.3 88.8+1.9 100.0+ 0 100.0+ 0 91.6+3.2 2.3 2.3 1.5 100.0+0.0 100.0+0.0 55.5+1.4 MS8 2.0+1.0 Hypocotyl Node Apex 33.3+1.6 30.0+1.4 33.3+1.6 1.8 2.6 2.4 20.0+ 0.7 16.0+ 0.7 25.0+ 0.6 20.0+ 2.3 20.0+ 2.3 16.6+ 1.9 1.1 3.1 4.8 50.0+1.3 25.0+0.0 50.0+1.3 Contd…

Table : 4 Comparision between the effect of different PGRs on induction and embryogenesis from various explants :

17 Table : 4 Comparision between the effect of different PGRs on induction and embryogenesis from various explants Hormones used Number of explants Explants Ratio of callus initiation (%) Embryogenic ratio (%) Callus Weight (g) 0.1 mg l -1 ZT 33 18 25 Cotyledon Hypocotyl Root 21.2 100.0 64.0 9.09 5.56 8.00 0.129 0.356 0.135 3.0 mgl -1 ZT 40 42 21 Cotyledon Hypocotyl Root 27.5 100.0 100.0 0.00 0.00 0.00 0.402 1.291 0.107 0.1 mgl -1 2,4-D + 0.1 mgl -1 ZT 48 48 36 Cotyledon Hypocotyl Root 100.0 100.0 100.0 0.00 0.00 0.00 4.872 5.687 2.999 0.1 mgl -1 2,4 – D + 0.1 mgl -1 ZT + 0.1 mgl -1 IAA 60 60 60 Cotyledon Hypocotyl Root 100.0 100.0 100.0 0.00 0.00 0.00 5.299 6.017 3.680 0.1 mgl -1 2.4-D 50 50 50 Cotyledon Hypocotyl Root 100.0 100.0 100.0 0.00 0.00 0.00 1.000 1.532 0.788 China Zhang et. al.., (2001)

Table : 5 Callus Induction response of different explants on induction media with different combinations of PGRs:

18 Table : 5 Callus Induction response of different explants on induction media with different combinations of PGRs Explant used PGR Composition ( M) Callus Description No. of explants per plate No. of explants with callus (mean + S.E.) % of explants with callus (mean + S.E) Colour Texture Hypocotyl* 2,4-D (0.90) + Kinetin( 2.32) Light Green Loose 7 6.7 + 0.48 a 95.7 + 6.9a IBA( 2.46) +Kinetin (2.32) Green Compact 7 6.5 ± 45 b 85.7 ± 10.4 a Cotyledon* 2,4-D( 0.90) + Kinetin (2.32) gray Green Loose 7 5.6 + 0.95 ab 80 + 19.2 ab IBA( 2.46) +Kinetin (2.32) Green Hard 7 2.75 + 0.96 b 39.3 + 13.6 b Explants were scored for culture induction 30 days after culture (*n=10, **n=8) Different letters in one column represent significant differences by the Duncan’s Multiple range test (p<0.05) China Wu et.al., (2004)

Table : 6 Callus induction and proliferation in cotton (G. hirsutum L.) cv. Coker – 312 various hormonal concentrations:

19 Table : 6 Callus induction and proliferation in cotton ( G. hirsutum L.) cv. Coker – 312 various hormonal concentrations Medium hormones (mg/L) Callus induction and its proliferation, 11 weeks culture 2,4-d Kinetin No. of explants No. of calli induced Callus induction (%) *Callus (mg) Callus Wt.(g) Callus growth ratio (%) MS 2a 0.1 0.1 30 28 93.33 99.00 3.10 31.31 MS 2b 0.1 0.2 30 28 93.33 99.00 3.10 31.31 MS 2c 0.1 0.3 28 27 96.71 92.42 6.18 66.68 MS 2d 0.1 0.4 30 16 53.33 96.90 3.65 37.67 MS 2e 0.1 0.5 35 35 100.00 98.70 5.80 58.76 MS 2f MS 2e + KNO 3 (1.90 g/L) 32 29 90.62 100.30 7.26 72.35 MS 2g MS 2e + NH 4 NO 3 (1.90 g/L) 32 29 90.62 100.30 7.26 72.35 Call were cultured as 7 replicates (100 + 10) per medium, “Callus WT (mg): original weight of call in milligrams; callus wt(g): weight of the callus in grams after 11 weeks; growth ratio% (original weight of callus) / (final weight of callus after 11 weeks of culture)x 100 Pakistan Haq and Zaffer, (2004)

Selection of Embryogenic Callus:

20 Selection of Embryogenic Callus

Deferences between embryogenic calli and non- embryogenic calli:

21 Deferences between embryogenic calli and non- embryogenic calli Embryogenic tissue Small in size Cells having dance cytoplasm and cells forming clumps Small vacuoles Presence of normal nucleus Isodiamatric in shape Higher no. of Multivasicular bodies Surface of cell aggregates is smooth Absence of well defined epidermal layer Shows development of treachery elements initials High enzymatic activity High amount of RNA and DNA High rate and unequal division of cells Non embryogenic tissue Large in size Cells having poor cytoplasmic content not forming clumps Large vacuoles Presence of abnormal nucleus Shape balloon type Less no. of Multivasicular bodies Surface of cell aggregates is rough Absence of well defined epidermal layer Shows development of treachery elements initials Low enzymatic activity Low amount of RNA and DNA Low rate and equal division of cells

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22 A, non embryogenic calli B, embryogenic calli C, Embryos after selection Fig- 4: Morphology of embryogenic and non embryogenic callus China Zhang et al. , (2001c)

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23 Fig-Transverse section of callus from non etiolated explant of variety ISA 205 N at one month Cnem = non embryogenic cells without nucleus Fig- Transverse section of callus from non etiolated explant of variety ISA GI 7 at one month Paren = parenchyma cells Vaiss = vascular bundals Belgium Kouadio et al., (2004)

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24 Fig- Transverse section of callus from etiolated explant of variety ISA 205 N at one month Cnam = non embryogenic cells Cem = embryogenic cells with nucleus (intensive division ) Fig- Transverse section of callus from etiolated explant of variety ISA GI 7 at one month Vaiss = vascular bundle Cem = embryogenic cells with nucleus (which start mitotic division ) Proem = proembryogenic cells Belgium Kouadio et al., (2004)

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25 Fig-9 : Higher magnification of friable callus illustrating the grainy, nodular texture of high quality callus . Fig-10 : The biosynthesis of anthocynin (red pigmentation) is a good indicator of regeneration China Wu et al., (2003)

Fig- 5 : GROWTH INDEX OF NON EMRYOGENIC CALLUS ON MEDIUM WITH KANAMYCIN:

26 Fig - 5 : GROWTH INDEX OF NON EMRYOGENIC CALLUS ON MEDIUM WITH KANAMYCIN Growth index USA Zhang et al .,(2001b) (O,10,25,50,75,100,150 mg/L KANAMYCIN CONCENTRATION)

Fig-6 :GROWTH INDEX OF EMBRYOGENIC CALLUS ON MEDIUM WITH KANAMYCIN:

27 Fig-6 : GROWTH INDEX OF EMBRYOGENIC CALLUS ON MEDIUM WITH KANAMYCIN (O,10,25.50,75,100,125,150mg/L of kanamycin concentration) index Growth USA Zhang et al .,(2001b)

Fig-7 : INHIBITION RATIO OF KANAMYCIN ON GROWTH OF COTTON NON - EMBRYOGENIC CALLUS:

28 Fig-7 : INHIBITION RATIO OF KANAMYCIN ON GROWTH OF COTTON NON - EMBRYOGENIC CALLUS {10, 15, 50,75.100,125, 150, STANDS FOR KANAMYCIN CONCENTRATION (mg/L) } Inhibition (%) Days after culture USA Zhang et al .,(2001b)

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29 Table : 7 Effect of haemoglobin on the somatic embryogenesis Response Concentration of haemoglobin (mg/l) Control 100 200 300 400 500 600 Callus formation (%) 89 + 0,2d 96 + 0.8b 96 + 0.7b 98 + 4a,b 99 + 1.05a 95 + 0.8b,c 90 + 0.28c Embryogenic callus formation (%) 84 + 0.45d 95 + 0.6b 95 + 0.4b 97 + 0.3a 97 + 0.9a 90 + 1.2c 90 + 0.1c Fresh weight of embryogenic callus after 4 weeks (mg) 260 + 0.45f 285 + 3.2e 290 + 2.4d 328 + .2b 342 + 1.9a 300 + 2.4c 284 + 1.8e,f Number of embryos formed/ 500mg of embryogenic calli 128 + 4.0e 135 + 1.7a,c 158 + 2.1c,d 170 + .7b 195 + 1.2a 160 + 2.1c 138 + 0.2d Number of plants germinated from embryoids 68 + 2.22f 84 + 2.34d 95 + 1.35c 105 + .2b 138 + 2.0a 95 + 0.95c 80 + 0.15e Percentage of plants germinated from embryoids 53.1 + 1.5g 62.2 + 2.3d 66.4 + 1.3e 73.5 + 1.4b 75.8 + .9a 59 + 1.9e 57.9 + 0.1e,f Number of embryoids with roots but without shoots 21 + 0.50a 12 + 0.2b 11 + 0.09b 9 + 0.27e 11 + .3b,e 11 + 0.2b,c 12 + 0.08b Number of embryoids with shoots but without rooting 24 + 0.48a 7 + 0.6b,c 7 + 0.17b 6 + 0.1c 8 + 0.10b 7 + 0.09b 4 + 0.02c Tiruchirappalli (India) Ganesan and Jayabalan.(2004) a. no. of explant tested: 60.Values are means ±standard error of three repeated experiments. each treatment as six replicates. Means within a row followed by the same letter are not significant at 0.005 according to DNMRT

Induction of somatic embryos:

30 Induction of somatic embryos During somatic embryo induction several embryos formed asynchronously due to highly organized division of cells which occurs through unorganized callus cycles, therefore influenced by several factors, some of them are worth discussing like – Source of explant and its physiological condition Explant type Media composition Nitrogen source Plant growth regulators Genotype

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31 Table :8 Effect of source of explant on overall embryogenic response of Gossypium hirsutum L. cv Coker 312. Explant Source 3 NAA 4 ≥5 Total* 3 2,4 – D 4 ≥ 5 Total* Immature tissue seed 21.0 8.5 0 29.5 6.2 2.2 3.7 13.1 b cotyledon 10.0 6.0 4.1 20.1 8.1 2.6 0 10.7 ab upper hypocotyl 15.2 4.1 5.6 24.9 12.2 3.7 4.4 20.3 a mid hypocotyl 10.0 9.6 4.8 25.4 10.1 4.8 8.9 24.8 a lower hypocotyl 10.7 7.4 5.2 23.3 11.9 10.8 5.0 27.7 a Mature tissue leaf tip 11.9 4.1 1.0 17.0 1.1 1.9 0 3.0 b Leaf base 18.9 3.7 0.0 22.7 0.04 0 0 0.04 b Stem 15.9 4.4 0.4 20.7 0.07 0 0 0.07 b U.S.A. Trolinder and Goodin, (1987a) ( Data were aggregated over all NAA + cytokinin and 2,4-D + cytokinin treatment ) Where 3= a minimum of 5 embryos,4=minimum of 25 embryos and 5 = 50% of callus mass contain embryos . totals with the same letter within a column are not significantly different at α = 0.05 according to DNMRT of rank means .

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32 Fig-11: Response of explant taken from hypocotyl of 3 days old seedling USA Trolinder and Goodin (1987a)

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33 Fig- 12: The effect of PGRs on induction of somatic embryo when explant derived from cotyledon of 3 days old seedling USA Trolinder and Goodin (1987a )

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34 Fig- 13 : The effects of plant growth regulators on induction of somatic embryogenesis when explant derived from leaf tip tissue of mature plants. USA Trolinder and Goodin (1987a )

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35 Fig-14 : The effects of plant growth regulators on induction of somatic embryogenesis when explant derived from leaf base tissue of mature plants. USA Trolinder and Goodin (1987a)

Table: 9 The effect of media on somatic embryo induction in cotton (Gossypium hirsulum L) cv Cocker 312 callus:

36 Table: 9 The effect of media on somatic embryo induction in cotton ( Gossypium hirsulum L) cv Cocker 312 callus Medium Treatments Number of embryo types after 4 weeks culture Embryo maturation (%) Anthocyanin ( µg/g) Globular cotyledonary MS 0 MS basal 100.0 4.62 4.02 10.4.04 + 3.49 MS 3a MSo+0.5mg/L ZT 78 0.82 1.05 - Ms 3b MSo+KNO 3 (1.90 g/l) 74.0 308 4.12 - MS 3c MSo+KNO 3 (1.90 g/l) 70.2 13.30 27.49 350.54 + 4.51 MS 3d ½ MSo+KNO 3 .(1.90g/l) 79.1 8.08 10.15 302.83 + 7.35 MS 3e ½ MSo+1/2 KNO 3 + ½NH 4 NO 3 88.5 18.77 21.22 252.25 + 5.34 MS 3f ½ MSo+NH 4 NO 3 (0.90 g/l) 44.1 3.93 8.92 420.41 + 3.65 MS 3g ½ MSo+NH 4 NO 3 (1.90 g/l) 20.1 0.25 1.28 540.93 + 4.58 MS 3h ½ MSo+ NH 4 NO 3 (1.90 g/l) 34.2 0.04 0.20 620.62 + 7.48 MS 3i MSo+NH 4 NO 3 (1.90 g/l) 0.0 0.00 0.0 658.70 + 552 Embryo maturation (%) = cotyledenary embryos/globular embryos x 100 Pakistan Haq and Zafar, (2004)

Table :12 Average number of somatic embryos induced from calli of several cotton genotypes cultured on various embryo initiation / germination media.:

37 Table : 12 Average number of somatic embryos induced from calli of several cotton genotypes cultured on various embryo initiation / germination media. S15G0.5NAA = 0.05 NAA + 15 gl -1 sucrose 0 Putrescine 25 Putrescine 5 Putrescine 99.2 + 2.2 91.2 + 2.4 187.9 + 3.8 61.4 + 6.5 55.2 + 3.4 68.9 + 2.0 25.2 + 6.8 14.6 + 5.2 39.7 + 2.6 2.5 + 1.0 3.9 + 3.6 9.6 + 3.5 93.4 + 9.7 51.6 + 1.9 97.0 + 3.7 EMMS 2 = 0.5 NAA + 0.05 Kn + 30 gl -1 glucose 0 Putrescine 25 Putrescine 5 Putrescine 112.2 + 18.4 98.7 + 3.0 111.5 + 4.1 10.8 + 3.5 16.2 + 3.3 86.4 + 3.7 0.3 + 0.2 4.9 + 3.9 16.0 + 0.6 0 + 0 0 + 0 0 + 0 47.2 + 3.0 32.0 + 1.9 103.5 + 5.9 EMMS 4 = 0.1 NAA + 0.5 Kn + 30 gl -1 glucose 0 Putrescine 25 Putrescine 5 Putrescine 37.3 + 5.2 70.0 + 2.0 71.8 + 2.0 59.0 + 6.9 7.5 + 2.9 64.0 + 2.0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 0 + 0 43.4 + 1.9 4.4 + 2.3 35.9 + 9.4 USA Vickers et. al., ( 2001) Coker 312 PD 97019 PD 97021 PD 97100 GA 98033

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38 Table : 14 . Varietal differences on plantlet producing capacity of SEs on various media Genotype Medium Coker 312 PD 97019 PD 97021 PD 97100 GA 98033 S15g.0.5NAA 95 7 5 3 12 S15g.0.5NAA-0.25Put 92 8 8 5 10 S15g.0.5NAA-0.5Put 97 12 5 7 13 EMMS 2 100 15 5 0 8 EMMS 2 -0.25Put 93 17 7 0 12 EMMS 2 -0.5Put 93 17 3 0 15 EMMS 4 92 10 0 0 10 EMMS 4 0.25Put 97 12 0 0 15 EMMS 4 0.5Put 92 8 0 0 17 USA Vickers et al., (2001) Put = putrescine

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39 Table : 15 Mean number of embryos per culture for each treatment combination Genotype Initiation medium Proliferation medium Number of cultures Mean embryos /culture Coker 312 Medium 1 Liquid 29 388 a Coker 312 Medium 1 Semi-solid 29 184bc Coker 312 Medium 2 Liquid 30 62cd Coker 312 Medium 2 Semi-solid 29 18d T25 Medium 1 Liquid 31 228 b T25 Medium 1 Semi-solid 29 125bcd T25 Medium 2 Liquid 31 226 b T25 Medium 2 Semi-solid 25 142 bcd U.S.A Gawel and robackar, (1990) Means followed by different letters are significantly different at 0.05 probabilty level,Duncan’s multiple range test Note : Medium 1 = NAA + Kn Medium 2 = 2,4– D + Kn

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40 The somatic embryos shows somewhat similar growth patterns to the zygotic one , in case of cotton , it typically pass through globular ,heart, torpedo and cotyledonary stages. However, not all embryos go through normal sequence and normal morphology, notably elongation of cotyledons of cotton somatic embryos observed as rudimentary only. This obvious differences arises simply due to the physical pressure exerted by the seed coat on zygotic embryo contributes to one type of regulatory development whereas the development of somatic embryos is affected by in vitro environment. Somatic embryo development

Fig- 15: Developmental stages of an cotyledonary embryo :

41 Fig- 15: Developmental stages of an cotyledonary embryo USA Trolinder and Goodin (1987b) 0 1 A B C D

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42 Figure : . (E) globular embryo ,bar =1 mm . (H) cotyledonary embryo , bar = 2mm. (F) heart shaped embryo , bar = 0.2 mm . (K) late cotyledonary embryo , bar = 0.2 mm (G) torpedo shaped embryo ,bar = 1 mm. bar = 2 mm.. G China Sun et al., (2003)

Table:16 Effects of various concentrations nutrient concentrations along with different treatments on the development of somatic embryos :

43 Table:16 Effects of various concentrations nutrient concentrations along with different treatments on the development of somatic embryos Concentration of MS nutrients Treatment Number of embryos Percentage maturation embroys Globular Torpedo Cotyledonary Full Strength P Fp+P M Fp+P 52.68 + 6.25c 67.32 + 9.93b 135.6 + 8.34a 135.6 + 8.34a 0.00 + 0.00 5.67 + 1.89a 0.51 + 0.17b 0.00 + 0.00 0.66 + 0 . 15c 22.68 + 4.49a 18.00 + 1.36b 23.82 + 3.08a 1.25 33.7 13.3 20.0 ½ Strength P Fp + P M Fp+M 74.00 + 7.30 121.5 + 5.00b 144.0 + 5.47 142.5 + 5.52a 0.00 + 0.00 0.00 + 0.00 0.00 + 0.00 0.18 + 0.06a 1.50 + 0.05d 1.22 + 0.34c 5.50 + 1.12a 4.17 + 0.49b 2.0 1.0 3.8 2.9 1/5 Strength P Fp+P M Fp+M 104.3 + 12.5d 171.6 + 6.78a 152.1 + 6.53b 142.8 + 4.36c 0.00 + 0.000 0.00 + 0.00 0.18 + 0.06a 0.18 + 0.06a 2.67 + 0.59c 5.01 + 1.31a 3.66 + 0.33b 2.67 + 0.29c 2.6 2.9 2.4 1.9 P Parafilm for sealing, Fp+p filter paper along with Parafilm. M porous tape for sealing, Fp + M filter paper along with porous tape Mean within each column followed by the same letter are not significantly different at the 0.05 probability level according to Duncan’s Multiple Range Test. Delhi Kumaria et.al ,. (2003) ( + standard error) per 800 + 100 mg embryogenic callus after 30 days of culture

Table :17. Summary of effects of basal salt, hormones and KNO3 on embryo development. :

44 Table : 17. Summary of effects of basal salt, hormones and KNO 3 on embryo development. Treat. No. Mean and Significant differences Hormone Salt KNO 3 S S 1 S 2 % Germination % Plant Recovery 1 2 + MS + - 4.2 11.0 34.6 20.0 5.6 83.4 42.7 36.8 4.6 2.4 3 2/3 MS + 10.1 40.4 10.8 58.9 6.5 4 5 6 - MS - + - 2.7 24.0 28.7 16.7 34.2 110.3 2.3 40.3 53.6 49.9 47.6 41.9 3.2 4.3 1.7 7 2/3 MS + 6.8 81.2 31.6 63.6 1.3 8 - 38.8 71.4 60.4 45.4 4.0 Mean within each column followed by the same letter are not significantly different at the 0.05 probability level according to Duncan’s Multiple Range Test. S Initial plating S 1 Subculture 1 S 2 Subculture 2 U.S.A. Tolinder & Goodin (1988b)

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45 Oligosaccharide concentration, M No. cells per 1 ml suspension, % of control No. embryos per 1 ml suspension, % of control 10 -9 107 + 12 23 + 3 10 -8 335 + 29 166 + 14 10 -7 169 + 15 377 + 25 Table: 18 Effect of various concentrations of oligosaccharide on cell density and no. of embryos in suspension culture. Callus weight (mg) 210 + 24 (210) 290 + 18 (155) 550 + 48 (305) Russia Rakitin et al ,.( 2001)

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46 China Sun et al (2003) Fig: Growth of somatic embryos on solid and suspension cultures

Embryo maturation :

47 Embryo maturation Difficulties associated with the maturation of somatic embryos, is a critical limiting step in genetic transformation for the production of large number of transgenic plants (Kumaria et al ,.2003) Maturation of embryos refers to their physiological capacity to follow proper germination. It is the terminal event of embryogenesis and characterized by the attainment of mature embryo morphology and sometimes rapid growth continues to occur leading to precocious germination . As such, factors that influence and / or enhance somatic embryo maturation have been unfortunately less explored

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48 3. Matured somatic embryos Fig- 16 : Morphology of matured embryos 2. Maturation of somatic embryos 1. Dehydrated somatic embryos USA Sakhanokho et al .,(2001)

Table :19 Effect of plant growth regulators on cotton somatic embryogenesis :

49 Table :19 Effect of plant growth regulators on cotton somatic embryogenesis Medium (mg1 -1 ) Explant Somatic Embryogenic rate (%) a Number of mature embryos / explant a Resulting growth MS+0.1 2,4-D+0.1 zeatin Leaf Stem 90.0 + 3.2 100.0 + 0.0 0 d 0 d Heart embryo Heart embryo MS+0.1 2,4-D+0.1 zeatin Leaf Stem 87.9 + 5.3 96.4 + 0.0 0 d 0 d Torpedo embryo Torpedo embryo MS+0.1 2,4-D+0.1 zeatin Leaf Stem 56.7 + 2.6 63.3 + 2.9 0 d 0 d Globular embryo Globular embryo MS + 0.1 zeatin Leaf Stem 76.7 + 0.5 86.2 + 3.2 14.3 b 13.4 b Plantlet Plantlet MS + 0.5 zeatin Leaf Stem 77.1 + 1.1 87.1 + 5.3 9.0 b 7.3 bc Plantlet Plantlet MS+0.1 zeatin + 2000 activated carbon Leaf Stem 55.3 + 2.8 60.0 + 0.0 28.0 a 28.1 a Plantlet Plantlet MS + 0.1 2,4-D + 0.1 zeatin + 2000 activated carbon Leaf Stem 53.5 + 8.7 63.5 + 3.1 28.7 a 27.2 a Plantlet Plantlet Pakistan Haq and Zaffer, (2004) a. Values represents Mean ± SD of the percentage of explant s with embryos in a treatment , b. Means followed by different letters are significantly different at 5% level by LSD test. for each treatment ,30 30 explants were recorded in 3 replicates of 10 explant.data represent average no. of somatic embryos/explant in a given treatment after 40 days of colture

Table :20 The effect of nitrogen source on embryo maturation:

50 Table :20 The effect of nitrogen source on embryo maturation Medium Treatments Embryo Maturation (%) Plantlets Development(%) MS 2a MSo+NH 4 NO 3 20.94 30.32 MS 2b MSo + KNO 3 27.49 46.24 Callus taken from MS 2a after 2 weeks 56.51 82.05 Pakistan Haq et al., (2005)

Embryo germination and plant recovery :

51 Embryo germination and plant recovery Obtaining cotton plants from somatic embryos is often more difficult than would be expected due to low and abnormal germination.Early literature concerning reports of somatic embryogenesis in cotton did not include detail data on plant recovery except of Trolinder and Goodin (1987). When plants were obtained , the recovery rate was very low or not reported, suggesting that the majority of somatic embryo germination and plant recovery ranged from 0 to 50%. Only, recently research has been conducted by Zhang et al .,(2000) and some suggestion have been made by Hussain et al., (2004) favors use inert substrate to enhance germination .

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52 Fig- 17: Germination of cotton somatic embryo. Delhi Mishra et al. (2003)

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53 Fig- 18: Effect of Different inert supportive materials on germination and plantlet formation from somatic embryos. Pakistan Hussain et al., (2004)

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54 Fig-19: Growth of root and shoot on supportive medium after germination of embryo USA ` Trolinder and goodin (1988b)

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55 Fig-20 : Percentage of germination of somatic embryos in respect to days of culture China Zhang et al., (2000a)

Table : 21. Germinability ability of various developmental stage common somatic embryos :

56 Table : 21. Germinability ability of various developmental stage common somatic embryos Developmental stage Rooting Shooting Hypocotyl elongation Callus growth Forming secondary embryos Globular shape embryo - - - + + Heart shaped embryo - - - + + Early torpedo embryo + - - + + Late torpedo embryo + + + - - Cotyledon stage embryo + + + - - Note: + promote - inhibit China Zhang et al., (2000)

Table: 22 Effect of embryo size and cotyledons on percentage of germination (% G) and plantlet recovery (% P) from embryos:

57 Table: 22 Effect of embryo size and cotyledons on percentage of germination (% G) and plantlet recovery (% P) from embryos Treatment Domed 3 mm 5 mm %G %P %G %P %G %P 1 10 0 40 0 70 20 2 -- -- -- - - -- 3 40 0 10 0 50 20 4 -- -- -- -- -- -- 5 50 20 50 0 90 30 6 30 0 30 0 70 20 7 10 0 50 0 70 10 8 0 0 20 0 60 30 Domed embryos (embryos without cotyledons) of Treatment 5 were larger in size than other treatments > 5 mm. Treatments which did not have embryos > 3 mm size were not compared and are represented by dashes USA Trolinder and Goodin, (1987 b) 1 = MS + hormones + KNO3 2 = MS + hormones – KNO3 3 = 2/3 MS + hormones + KNO3 4 = 2/3 MS + hormones – KNO3 5 = MS – hormones + KNO3 6 = MS – hormones – KNO3 7 = 2/3 MS – hormones + KNO3 8 = 2/3 MS – hormones - -KNO3

Table: 23 Germination reponse of cotton somatic embryos to basal medium:

58 Table: 23 Germination reponse of cotton somatic embryos to basal medium Basal Medium No. of Embroys % Rooted % Shooted % Germination % Plantlet Recovery BT 53 50.9 18.7 52.8 3.8 SH 50 36.0 18.9 40.0 10.0 MS 49 22.4 30.6 38.7 9.8 China Zhang et al ., (2000) BT = Basley and Ting (1979). SH = Stewerts and Hsu(1980) MS = Murashige and Skoog(1962)

Table : 24 Germination response of cotton somatic embryos to sucrosea:

59 Table : 24 Germination response of cotton somatic embryos to sucrose a Sucrose (gl -1 ) 0 5 10 20 40 No. of embryos tested 21 30 30 30 30 Rooted % 0.0 43.3 53.3 56.7 50.0 Shooted % 4.8 36.7 66.7 83.3 63.3 Germination % 4.8 60.0 76.7 90.0 73.3 Plant recovery % 0.0 26.7 43.3 50.0 40.0 a MS medium supplemented with 0.1 mg l -1 NAA China Zhang et al ., (2000)

Table : 25 Germination response of somatic embryos to NAA:

60 Table : 25 Germination response of somatic embryos to NAA NAA (mgl -1 ) 0 0.01 0.1 0.2 1.0 2.0 5.0 10.0 No. of embryos tested 30 30 30 30 30 21 17 26 Callus growth % 100.0 100.0 100.0 93.3 73.3 68.6 5.9 0.0 Rooted % 43.3 50.0 56.7 56.7 53.3 66.7 82.4 38.5 Shooted % 50.0 60.0 83.3 60.0 33.3 23.8 5.9 0.0 Germination % 56.7 70.0 90.0 70.0 46.7 66.7 88.2 38.5 Plant recovery % 33.3 40.0 50.0 46.7 40.0 23.8 0.0 0.0 China Zhang et al., (2000 )

Table:26 Response of cotton somatic embryos to Kinetin :

61 Table: 26 Response of cotton somatic embryos to Kinetin Kn (mgl -1 ) 0 0.1 0.5 1.0 2.0 5.0 7.0 10.0 No. of embroys tested 30 26 27 25 16 16 13 15 Callus growth % 100.0 61.5 37.0 16.0 0.0 0.0 0.0 0.0 Rooted % 43.3 38.5 33.3 24.0 18.8 12.5 0.0 0.0 Shooted % 50.0 46.2 37.0 36.0 37.5 18.8 23.1 6.7 Germination % 56.7 53.8 48.1 48.0 50.0 31.3 23.1 6.7 Plant recovery % 33.3 30.8 2.2 12.0 6.3 0.0 0.0 0.0 China Zhang et al.. (2000)

Table: 27 Effect of hormone and media on embryo germination and and plantlet regeneration from somatic embryos of Gossypium hirsutum L. (cv Coker 312) :

62 Table : 27 Effect of hormone and media on embryo germination and and plantlet regeneration from somatic embryos of Gossypium hirsutum L. (cv Coker 312) Hormones Stewart & Hsu Beasley & Ting Murashige & Skoog % G %P % G % P % G % P Basal medium 48 2.7 41 8.5 31 3.4 IAA 47 10.6 49 6.5 58 5.9 GA 3 39 1.3 49 3.9 28 1.1 Kinetin 47 3.7 37 7.1 46 7.3 IAA + Kinetin 52 1.2 59 5.3 54 5.8 Kinetin + GA 3 49 1.6 46 4.8 40 0 IAA + Kinetin+ GA 3 45 2.7 64 2.7 50 0 Mean 47.5 + 4.3 3.3 + 3.1 50.4 + 9.3 5.9 + 2.1 44.7 + 10.8 3.5 + 2.8 * 0.1 mg / l of each hormone was utilised G = germination P = Plants recovered USA Trolinder and Goodin, (1988b)

Hardening and Transfer to Soil :

63 Hardening and Transfer to Soil

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64 Primary hardening processes using jars Fig-21: Regenerated cotton plantlet growing in jar USA Sakhanokho et al .,(2001)

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65 Fig-22 : Primary and Secondary hardening

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66

Table:28 Different substrates used in cotton hardening procedure:

67 Table:28 Different substrates used in cotton hardening procedure Substrate M S Media + 1:1:1 Mixture of sand, silt & peat moss 1:1:1 Mixture of Sand + Soil and Vermiculite Vermiculite + Sphagnum moss M S Media Steworts germination media – soil Sterile soil + ½ strength MS medium MS basal – Promix Standard potting compost + perlite (1:1) Vermiculite + Soil (1:1) Sterile Soil + Sand (1:1) Vermiculite + Solrite + Soil Vermiculite Referances Hussain et al., (2004) Ganesan and Jayabalan (2004) Sakhanokho et al., (2004) Kumaria et al., (2003) Mishra et al., (2002) Sun et al., (2003) Sakhanokho et al., ( 2001) Zhang et al., (2001a) Zhang et al .,(2001b) Zhang et al., (2000) Kumar et al .,(1998) Trolinder and Goodin (1987)

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68 Synthetic Seed Production Fig-23 : Synthetic seeds

Conclusion :

69 Conclusion Somatic embryogenesis in cotton has been readily induced from callus obtained from cotyledons, stems, roots, petioles and hypocotyls. However, the best response has been obtained from hypocotyls Regeneration in Gossypium remains largely cultivar/genotype dependent Somatic embryogenesis can be induced in two ways viz. Direct somatic embryogenesis and Indirect embryogenesis Murashige and Skoog (1962) with Gamborg (B 5) vitamins gave better callus initiation Embryogenic calli can possibly be identified on the basis of callus morphology and cytology Among various auxins, NAA recorded greater embryogenic response In general, explant derived from immature tissues produce embryos more easily Nitrate as a source of nitrogen in the medium is found beneficial for somatic embryogenesis Liquid culture step is helpful to different embryogenic developmental processes Activated charcoal is beneficial for embryo maturation Vermiculite is helpful for germination of embryos and plantlet recovery

Future thrust :

70 Future thrust The embryo maturation and germination are very critical and limiting steps in cotton regeneration. Therefore, further extensive studies are required Development of suitable selection system for identification and isolation of embryogenic calli is still required for maximum plantlet recovery The standardization for genotype independent cotton regeneration is required.

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71 THANK YOU

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