Embryogenesis 27 mar 15

somatic embryogenesis in cereals crops

1. WELCOME 3/27/2015 Deptt of Plant Biotechnology 1

2. In vitro Regeneration System for Indirect Somatic Embryogenesis of Cereals Crops. 3/27/2015 Deptt of Plant Biotechnology 2 AVINASH SHARMA ID. No:- PALB 3235 Sr. M.Sc. (Plant Biotech)

3. EMBRYOGENESIS:- Plant embryogenesis refers to the process of development of plant embryos, being either a sexual or asexual reproductive process that forms new plants. Embryogenesis may occur naturally in the plant as a result of sexual fertilization, and those embryo are called zygotic embryos and develop into seeds, which can germinate and give rise to seedlings. Plant cells can also be induced to form embryos in plant tissue culture; these embryo are called somatic embryos. 3/27/2015 Deptt of Plant Biotechnology 3

4. Stages of Embryogenesis:- 3/27/2015 Deptt of Plant Biotechnology 4

5. Types of Embryogenesis:- Two types of embryogenesis:- A) Zygotic embryogenesis B) Somatic embryogenesis 3/27/2015 Deptt of Plant Biotechnology 5

6. Zygotic Embryogenesis:- The zygotic embryo is formed following double fertilization of the ovule, forming the plant embryo and the endosperm which together go into the seed, this process is known as zygotic embryogenesis. Seeds may also develop without fertilization through pathways referred to as apomixis. 3/27/2015 Deptt of Plant Biotechnology 6

7. Somatic Embryogenesis:- Somatic embryogenesis is a process by which somatic cells or tissues develops into differentiated embryos. Embryos regenerate from somatic cells or tissues ( haploid or diploid etc) it is termed as Somatic Embryogenesis. 3/27/2015 Deptt of Plant Biotechnology 7

8. 3/27/2015 Deptt of Plant Biotechnology 8 Somatic embryogenesis was first induced in suspension culture (Stewart et al, 1958) and in callus culture (Reinert, 1959) of carrot, Umbelliferae and Solanaceae dicotyledonous families have produced somatic embryos. SE occur most frequently in tissue culture as an alternative organogenesis for regeneration of whole plant. In literature, somatic embryos are referred to by many names such as embryo like structures, adventitious or vegetative embryos, Embryoids; and the process is termed as adventitious , asexual or somatic embryogenesis.

9. Stages of Somatic Embryogenesis:- 3/27/2015 Deptt of Plant Biotechnology 9

10. Stages of Somatic Embryogenesis:- 3/27/2015 Deptt of Plant Biotechnology 10

11. Contd:- INDUCTION Development and Maturation Globular Heart stage Torpedo Germination and Conversion Globular stage: Embryo is small and round (multicellular). Heart stage (Bilateral symmetry): Shape changes to heart shape with more cotyledon development. Torpedo shaped stage: Consists of initial cells for the shoot/root meristem. Mature stage: Embryo becomes cylindrical. 3/27/2015 Deptt of Plant Biotechnology 11

12. Induction Auxin required for induction Pro embryonic masses are formed. 2,4-D are mostly used. NAA, DICAMBA are also used. 3/27/2015 Deptt of Plant Biotechnology 12

13. Development Auxin must be removed for embryo development. Continuous use of Auxin inhibits embryogenesis. Stages are similar to those of Somatic embryogenesis:- Globular Heart Torpedo Cotyledonary Germination (Conversion) 3/27/2015 Deptt of Plant Biotechnology 13

14. Maturation Require complete maturation with apical meristem, radicle and cotyledons. Often obtained repetitive embryony. Storage protein production necessary. Often require ABA for complete maturation. ABA often required for normal morphology. 3/27/2015 Deptt of Plant Biotechnology 14

15. Morphological Stages of Maize cv. Gaurav 3/27/2015 Deptt of Plant Biotechnology 15

16. Routes of Somatic Embryogenesis:- Two routes to somatic embryogenesis Direct somatic embryogenesis The embryos initiate directly from explants in the absence of callus formation. Embryos are formed due to PEDCs cell. Indirect somatic embryogenesis Callus from explants takes place from which embryos are developed. Embryos are formed due to IEDCs cells. 3/27/2015 Deptt of Plant Biotechnology 16

17. Examples of Direct Somatic Embryogenesis:- 3/27/2015 Deptt of Plant Biotechnology 17 Figure :- Isolation of mature embryo from imbibed cereal grain. (a) A curved-tip scalpel blade is inserted beneath the Coleoptilar region of the Mature embryo; (b) With a swift and smooth scooping motion the mature embryo is dislodged from its attachment to the scutellum ; (c) Isolated mature embryo which will be inoculated with abaxial surface in contact with culture medium. Ganeshan et al ., 2006.

18. Contd:- Mature embryos culture in the Murashige and Skoog, 1962 medium with supplements 1gm/l enzymatic casein hydrolysate, 0.7 gm/l L-proline. 4.5 µM of TDZ and 4.4 µM of BAP are best combination of growth regulators in which Durum Wheat produces 35 number of shoots per explant and Mature embryos of CDC Dancer oat produces 16 shoots per explant. Explants for direct embryogenesis include microspores, ovules, scutellum, endosperm, embryos and seedlings. 3/27/2015 Deptt of Plant Biotechnology 18

19. Indirect Somatic Embryogenesis:- In Indirect SE, callus is produced from explants. Embryoids(suspensory cell to cotyledon) are produced from callus tissue. Explants are roots, shoots, leaf cells, anthers, seeds etc. Steps involved in Plant regeneration of Rice variety through Indirect SE:- 3/27/2015 Deptt of Plant Biotechnology 19

20. Formation of callus b) Greening of callus c) Embryo at globular stage d) Torpedo stage of embryo e) Cotyledonary stage and regeneration of embryo f-g) Multiple shoot regeneration h) Complete plantlets i ) Hardening of plantlets. (Rice Variety:- Swarna) Mondal et al ., 2011 (a) (b) (c) (d) (e) (f) (g) (h) ( i ) 3/27/2015 Deptt of Plant Biotechnology 20

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23. Factors affecting Somatic Embryogenesis:- Genotype:- Genetically engineered / transgenic plant does not regenerate through SE because due to variation. Methylation occurs in the DNA during mitosis then SE occurs. If Methylation occur in the cytosine bases or H3 protein then SE get stop. 3/27/2015 Deptt of Plant Biotechnology 23

24. 3/27/2015 Deptt of Plant Biotechnology 24 2) Explant:- Totipotent somatic cell are used. Immature inflorescence and Scutellar tissue of immature seeds are used for the research. Ex:- Triticum aestivum . Epidermis, Procambial tissue are also produced somatic embryo.

25. 3/27/2015 Deptt of Plant Biotechnology 25 During Proembryonic phase, 2,4-D generates DNA Hyper methylation so that cells in a highly active mitotic stage. High concentration of auxin produces root in somatic embryo. 2,4-D is one of the growth regulator that produces callus from cereals and conc. of 2,4-D 0.1-10µM 3) Auxin:- Polar transport of auxin produces somatic embryo. Auxin concentration will be more then somatic embryogenesis get stop. Ex:- Maize. Auxin induces indirect somatic embryogenesis in monocots.

26. 3/27/2015 Deptt of Plant Biotechnology 26 4) Cytokinins:- Cytokinin promote axial growth. Cytokinin produces globular embryo from initial embryo. Cytokinin combination with auxin, induces somatic embryogenesis and produce callus in cereals. Cytokinin ratio more than auxin then it produces Shoots.

27. 3/27/2015 Deptt of Plant Biotechnology 27 5) Gibberellic acid:- GA promote elongations of embryo axis, cell division. It synthesized of photosynthetic pigments in developing somatic embryo. It improve photosynthetic activity, Extra storage reserves in vitro germination. Hypo cotyledon are used as explant then GA inhibit somatic embryogenesis. Addition of Uniconazole, Paclobutrazol inhibit somatic embryogenesis. GA higher in suspensory embryo than the proper embryo. So GA requires early embryo development.

28. 3/27/2015 Deptt of Plant Biotechnology 28 6) Abscisic acid (ABA):- ABA control tolerance and seed dormancy during later stage of embryogenesis. ABA induced somatic embryogenesis in high osmotic stress and high temperature in auxin free medium. Primary embryo contain more conc. of ABA than secondary embryo. Treatment of Fluridone inhibit ABA synthesis and primary embryo does not produce secondary embryo.

29. 7) Polyamines:- Spermidine, Spermine and Putrescine are added as growth regulators and secondary messenger. Polyamines serve as nitrogen source for plants. It act as a free radical scavengers by protecting senescing membranes against lipid per oxidation. In Maize, Putrescine are most effective with varying concentration of GA3. Spermine act as a antioxidant in a medium. It help in vegetative growth, pollen development, regulation of DNA duplication, transcription of genes, cell division, development of organs. 3/27/2015 Deptt of Plant Biotechnology 29

30. 8) Phytosulfokine It modulate the culture media. It promote somatic embryogenesis by activating cell division of embryogenic cells, in presence auxin. Phytosulfokine increases the cell through differentiation process. 9) Phenolic compounds:- Phenolic compounds are inhibit somatic embryogenesis. 4hydroxy benzyl alcohol inhibits the globular stages. Vanillyl benzyl ether are inhibit the suspensor development. Recently identification of 4 [(phenyl methoxy) methyl] phenol involves in seed development stills unknown. 3/27/2015 Deptt of Plant Biotechnology 30

31. Differences between Zygotic and Somatic embryo:- Zygotic embryo Fertilized egg or zygote. Contain seed coat. Produce seed. Plantlets are healthy. Not like to mother plant. Propagation is low. Somatic embryo Sporophytic cells. Did not contain seed coat. Only form embryo. Plantlets are weaker Like to mother plant. Propagation is high. 3/27/2015 Deptt of Plant Biotechnology 31

32. Advantages and Disadvantages of Somatic Embryogenesis:- Higher propagation rate. Suitable for Suspension culture. Artificial seed production. Somaclonal variation. Germplasm conservation. Labour savings. 3/27/2015 Deptt of Plant Biotechnology 32

33. Disadvantages Response tissue specific (explants). Low frequency embryo production. Incomplete embryo production. May create unwanted genetic variation (Somaclonal variation). Inability to generate large numbers of normal, free living plantlets. Plantlets are weaker. 3/27/2015 Deptt of Plant Biotechnology 33

34. CASE STUDY 3/27/2015 Deptt of Plant Biotechnology 34

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36. Introduction Rice is the staple diet for two billion people world wide . It is the major food for over half of those living Asia. It is feared that world population would be around 10 billion by 2050. Diminishing of cultivated land. Attack of pests and insects are responsible for decrease in production. There is a constant need to improve crops to overcome all these hazards. Somatic embryogenesis in rice has been reported culture of leaf tissue, root tissue, inflorescence and protoplast. 3/27/2015 Deptt of Plant Biotechnology 36

37. Materials and method:- Explant collection:- Explant material for this research were rice seeds. Variety APMS-6B obtained from DRR (Hyderabad). Rice caryopses containing Scutellar region of embryo, were isolated by removing lemma and palea from the seeds . 3/27/2015 Deptt of Plant Biotechnology 37

38. Surface sterilization of Seeds:- Sterilization of rice caryopses using 70% alcohol for 3min. Followed by shaking in 30% Chlorox containing 2-3 drops of Tween-20 on an orbital shaker at 120 rpm for 20min. Explants were rinsed with sterile with sterile double sterilization water for 6 times. Cultured onto the medium with different treatment. 3/27/2015 Deptt of Plant Biotechnology 38

39. Preparation of Media:- Two basic media used in this study:- First one was:- half MS (Murashige & Skoog, 1962) supplements with 500mg/l glutamine, 100 mg/l proline. Second one was:- N6 media supplemented with 500mg/l L -Glutamine. Both media were solidified with 0.2% agar. pH adjusted with 5.8. 3/27/2015 Deptt of Plant Biotechnology 39

40. Callus Induction Media:- Different concentrations of 2, 4-D [0.1, 1.5, 2.5,3.5 and 5 mgL-1 (w/v)] were used as the treatments for embryogenic callus induction. Media were kept in dark condition for 1 week, 25±2°C at room temperature. After 1 week transferred the cultures under 16 hrs lighting , provided by fluorescent bulbs with 15.75 µmolm-²s-¹ for eight weeks. 3/27/2015 Deptt of Plant Biotechnology 40

41. Somatic Embryo Germination Media:- MS medium containing different concentrations of BAP (0, 1, 2, 3, 4and 5 mg/l), in combination with different concentrations of NAA (0, 0.5, 1.0, 1.5, 2.5 and 4.0 mgL-1) were used as treatments for the germination of somatic embryos. Media were kept in the incubation room 25±2°C with 16 hrs of light provided by fluorescent bulbs and a light intensity of 16.75 µmolm-²s-¹ for eight weeks. Calculate the Callus induction frequency(%) and Regeneration frequency(%). 3/27/2015 Deptt of Plant Biotechnology 41

42. Results:- After 3 days of culture callus started to grow from Scutellar embryo. Embryo derived callus subsequently started to enlarge and some yellowish to greenish nodules grew around explants after ten days. After 2 months of culture calli almost covered the explants surface. For callus induction MS medium supplemented with different concentration of 2,4-D(0, 1.0, 1.5, 2.5, 3.5 and 5 mg/l) was used in which 3.5 , 5 mg/l 2,4-D showed high callus induction percentage. It can be observed from Table 1 3/27/2015 Deptt of Plant Biotechnology 42

43. Table 1. Callus induction percent of rice in Somatic Embryogenesis S. No Conc. Of 2,4-D (mgL-¹) Callus Induction Frequency % from rice 1. No callus 2. 1.0 76±35 3. 1.5 80±40 4. 2.5 88±45 5. 3.5 95±30 6. 5.0 86±45 3/27/2015 Deptt of Plant Biotechnology 43 The result showed that the increased concentration of 2,4 –D more than 3.5 mgL-¹ decreased the callus formation percentage.

44. Contd:- MS media supplemented with 0.8% agar, 70gm/l sucrose, 4gm/l Casein, 3mg/l BAP and 4 mg/l NAA was used for derived calli. 3 mg/l BAP concentration showed good results in Shoot induction, it can be observed from Table 3. 4 mg/l NAA concentration showed good results in Shoot induction, it can be observed from Table 2. 3/27/2015 Deptt of Plant Biotechnology 44

45. S.No. Conc. Of NAA (mg/l) Shoot Induction % No. of Shoots 1. 31.33 2.6±0.48 2. 0.5 25.65 2.5± 0.64 3. 1 33.45 3.0± 0.54 4. 1.5 41.60 3.5± 0.64 5. 2.5 45.60 4.0± 0.59 6. 4.0 48.55 4.5± 0.60 Table 2. Effect of Transplantation PGRs in rice Table 3. Effect of Transplantation PGRs in rice S.No. Conc. Of BAP (mg/l) Shoot Induction % No. of Shoots 1. 30.33 2.0±0.87 2. 1 23.45 1.8±0.48 3. 2 31.85 2.2±0.16 4. 3 40.68 3.0±0.18 5. 4 38.67 2.5±0.64 6. 5 35.45 2.4±0.35 3/27/2015 Deptt of Plant Biotechnology 45

46. Contd:- MS medium supplements with different concentrations of NAA (0, 0.5, 1.0, 1.5, 2.0 mg/l) in combination with different concentrations of BAP (0, 1, 2, 3, 4, and 5 mg/l). Result showed that combination of 3mg/l BAP + 1.5 mg/l NAA showed highest result. Further combination increases cause the decrement of percent of Shoot induction. It can be observed from Table 4. 3/27/2015 Deptt of Plant Biotechnology 46

47. Table 4. Effect of BAP + NAA 3/27/2015 Deptt of Plant Biotechnology 47 S.No. BAP + NAA (mg/l) Shoot Induction % No. Of Shoots 1. 1 + 0.5 26.85 2.1± 0.63 2. 2 + 1.0 29.65 2.5 ±0.83 3. 3 + 1.5 39.60 3.5± 0.54 4. 4 + 2.0 35.45 3.2± 0.45 5. 5 + 4.0 30.40 3.0± 0.54

48. APMS -6B Variety Seeds Regenerate through Indirect Somatic Embryogenesis 3/27/2015 Deptt of Plant Biotechnology 48 Fig 1. Seed inoculation in MS medium Fig 2. Callus formation by 2, 4-D Fig 3. Shoot induction by differ. Conc. Of BAP and NAA Fig -4 Transplantation

49. Conclusion Somatic embryogenesis is an efficient plant regeneration system. It is potentially useful tool for genetic transformation. Cross linking between hormone and transcription factors is likely to play an important part in SE. But mechanism of plant embryogenesis is unclear and comphrensive work in future it by studying the interaction of various factors thereby entire picture of regulatory mechanism of embryogenesis would be transparent. 3/27/2015 Deptt of Plant Biotechnology 49

50. Conclusion Indirect Somatic embryogenesis reduces the breeding cycle. Indirect somatic embryogenesis are used in the crop improvement. Indirect somatic embryogenesis are produce virus free plants. Indirect somatic embryogenesis are better than the Direct somatic embryogenesis. 3/27/2015 Deptt of Plant Biotechnology 50

51. References:- Joshi, R., KUMAR, P., 2013, Regulation of Somatic Embryogenesis in Crops: A Review, Agri. Reviews, 34 (1): 1-21, 2013. DHLLION, N. K., GOSAL, S. S., 2013, Analysis of Maize Inbred Lines for their response to Somatic Embryogenesis, J. Cell and Tiss. Res, 13 (1): 3557-3563. SAH, SK., KAUR, A., SANDHU, JS., 2014, High Frequency Embryogenic Callus Induction and Whole Plant Regeneration in Japonica Rice Cv . Kitaake, J. Rice Res ., 2 : 125. ANAND, P., TIWARI, A., SAXENA, A., ARNOLD, R., TIWARI, S., 2014 , Studies on Optimization OF Protocol for Somatic Embryogenesis and Regeneration of Rice (APMS – 6B), Euro. J. Mol. Biol. Biochem ., 1 (1):13-17. 3/27/2015 Deptt of Plant Biotechnology 51

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