EMBRYOGENESIS ; 27 MARCH 15. 3.00 pm

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somatic embryogenesis in cereals crops

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WELCOME 3/25/2015 Deptt of Plant Biotechnology 1

In vitro Regeneration System for Indirect Somatic Embryogenesis in Cereal Crops.:

In vitro Regeneration System for Indirect Somatic Embryogenesis in Cereal Crops. 3/25/2015 Deptt of Plant Biotechnology 2 AVINASH SHARMA ID. No:- PALB 3235 Sr. M.Sc. (Plant Biotech)

CONTENTS:

CONTENTS Definition of Somatic Embryogenesis. Stages of Somatic Embryogenesis. Process involved in formation of Somatic Embryos. Types of Somatic Embryogenesis. Factors affecting Somatic Embryogenesis. Case Study. 3/25/2015 Deptt of Plant Biotechnology 3

Somatic Embryogenesis:-:

Somatic Embryogenesis:- Somatic embryogenesis is a process by which somatic cells or tissues develops into differentiated embryos. Embryos regenerate plants from somatic cells or tissue including haploid cells it is termed as Somatic Embryogenesis. 3/25/2015 Deptt of Plant Biotechnology 4

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3/25/2015 Deptt of Plant Biotechnology 5 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. 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.

Stages of Somatic Embryogenesis:-:

Stages of Somatic Embryogenesis:- 3/25/2015 Deptt of Plant Biotechnology 6

Contd:-:

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. 3/25/2015 Deptt of Plant Biotechnology 7

Induction:

Induction Auxin required for induction Pro embryogenic masses are formed. 2,4-D are mostly used. NAA, DICAMBA are also used . 3/25/2015 Deptt of Plant Biotechnology 8

Development :

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) 3/25/2015 Deptt of Plant Biotechnology 9

Maturation:

Maturation Embryo are mature 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. 3/25/2015 Deptt of Plant Biotechnology 10

Morphological Stages of Maize cv. Gaurav:

Morphological Stages of Maize cv. Gaurav 3/25/2015 Deptt of Plant Biotechnology 11

Types of Somatic Embryogenesis:-:

Types of Somatic Embryogenesis:- Two types of 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. 3/25/2015 Deptt of Plant Biotechnology 12

Examples of Direct Somatic Embryogenesis:-:

Examples of Direct Somatic Embryogenesis:- 3/25/2015 Deptt of Plant Biotechnology 13 Figure :- Isolation of mature embryo from imbibed durum grain. Isolated mature embryo which will be inoculated with abaxial surface in contact with culture medium. Ganeshan et al ., 2006.

Contd:-:

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 produced 35 number of shoots per explant and Mature embryos of CDC Dancer oat produced 16 shoots per explant. Explants for direct embryogenesis include microspores, ovules, scutellum, endosperm, embryos and seedlings. 3/25/2015 Deptt of Plant Biotechnology 14

Indirect Somatic Embryogenesis:-:

Indirect Somatic Embryogenesis:- In Indirect SE, callus is produced from explants. Embryoids are produced from callus tissue. Explants are roots, shoots, leaf cells, anthers, seeds etc. 3/25/2015 Deptt of Plant Biotechnology 15

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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 ) 3/25/2015 Deptt of Plant Biotechnology 16 Steps involved in Plant regeneration of Rice variety through Indirect SE:-

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Factors affecting Somatic Embryogenesis:-:

Factors affecting Somatic Embryogenesis:- Genotype:- Genetically engineered / transgenic plant does not regenerate through Somatic embryogenesis. Methylation occurs in the DNA during mitosis then SE occurs. If Methylation occur in the cytosine bases or H3 protein then Somatic embryogenesis get stop. 3/25/2015 Deptt of Plant Biotechnology 19

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3/25/2015 Deptt of Plant Biotechnology 20 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.

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3/25/2015 Deptt of Plant Biotechnology 21 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. During Pro-embryogenic phase, 2,4-D generates DNA Hyper methylation so that cells in a highly active mitotic stage. High concentration of auxin produces root from 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

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3/25/2015 Deptt of Plant Biotechnology 22 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.

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3/25/2015 Deptt of Plant Biotechnology 23 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. GA higher in suspensory embryo than the proper embryo. Addition of Uniconazole, Paclobutrazol inhibit somatic embryogenesis.

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3/25/2015 Deptt of Plant Biotechnology 24 6) Abscisic acid (ABA):- ABA control tolerance 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.

7) Polyamines:-:

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. 3/25/2015 Deptt of Plant Biotechnology 25

8) Phytosulfokine :

8) Phytosulfokine It modulate the culture media. It promote cell division of embryogenic cells, in presence auxin. Phytosulfokine increases the cell through differentiation process. 3/25/2015 Deptt of Plant Biotechnology 26

9) Phenolic compounds:- :

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. 3/25/2015 Deptt of Plant Biotechnology 27

Advantages and Disadvantages of Somatic Embryogenesis:-:

Advantages and Disadvantages of Somatic Embryogenesis:- Advantages: Higher propagation rate. Suitable in Suspension culture. Artificial seed production. Germplasm conservation. Labour savings. 3/25/2015 Deptt of Plant Biotechnology 28

Disadvantages:

Disadvantages Response tissue specific (explants). Low frequency embryo production. Incomplete embryo production. Inability to generate large numbers of normal, free living plantlets. Plantlets are weaker. 3/25/2015 Deptt of Plant Biotechnology 29

CASE STUDY:

CASE STUDY 3/25/2015 Deptt of Plant Biotechnology 30

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Introduction:

Introduction Rice is the staple diet for two billion people world wide . 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. 3/25/2015 Deptt of Plant Biotechnology 32

Materials and method:-:

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 . 3/25/2015 Deptt of Plant Biotechnology 33

Surface sterilization of Seeds:-:

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. 3/25/2015 Deptt of Plant Biotechnology 34

Preparation of Media:-:

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. 3/25/2015 Deptt of Plant Biotechnology 35

Callus Induction Media:-:

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. 3/25/2015 Deptt of Plant Biotechnology 36

Somatic Embryo Germination Media:-:

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(%). 3/25/2015 Deptt of Plant Biotechnology 37

Results:-:

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 3/25/2015 Deptt of Plant Biotechnology 38

Table 1. Callus induction percent of rice in Somatic Embryogenesis:

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. 0 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 3/25/2015 Deptt of Plant Biotechnology 39 The result showed that the increased concentration of 2,4 –D more than 3.5 mgL-¹ decreased the callus formation percentage.

Contd:-:

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. 3/25/2015 Deptt of Plant Biotechnology 40

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S.No. Conc. Of NAA (mg/l) Shoot Induction % No. of Shoots 1. 0 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. 0 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 3/25/2015 Deptt of Plant Biotechnology 41

Contd:-:

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. 3/25/2015 Deptt of Plant Biotechnology 42

Table 4. Effect of BAP + NAA:

Table 4. Effect of BAP + NAA 3/25/2015 Deptt of Plant Biotechnology 43 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

APMS -6B Variety Seeds Regenerate through Indirect Somatic Embryogenesis:

APMS -6B Variety Seeds Regenerate through Indirect Somatic Embryogenesis 3/25/2015 Deptt of Plant Biotechnology 44 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

Conclusion :

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 is necessary to be studied with the interaction of various factors for entire picture of regulatory mechanism of embryogenesis to be transparent. 3/25/2015 Deptt of Plant Biotechnology 45

Conclusion:-:

Conclusion:- Indirect Somatic embryogenesis reduces the breeding cycle. Indirect somatic embryogenesis are used in the crop improvement. Frequency of somaclonal variation is also very high in Indirect somatic embryogenesis Indirect somatic embryogenesis are better than the Direct somatic embryogenesis. 3/25/2015 Deptt of Plant Biotechnology 46

References:-:

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, J,S., 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. 3/25/2015 Deptt of Plant Biotechnology 47

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