somatic embryogenesis in palm species.pptx

SOMATIC EMBRYOGENESIS, HISTORY,STAGES OF SOMATIC EMBRYOS,INDUCTION OF SOMATIC EMBRYOGENESIS,SOMATIC EMBRYOGENESIS IN OIL PALM
,GENERAL PRINCIPLES OF SOMATIC EMBRYOGENESIS
,PROBLEMS ASSOCIATED WITH PALM ,SOMATIC EMBRYOGENESIS
,SOMATIC EMBRYOGENESIS IN TENERA PALM AND MACAW PALM, SOMATIC EMBRYOGENESIS IN PEACH PALM, SOMATIC EMBRYOGENESIS IN COCONUT PALM,
Somatic embryogenesis is the process in which a single cell or a small group of cells follow a developmental pathway that leads to reproducible regeneration of non-zygotic embryos which are capable of producing a complete plant.

1. Somatic Embryogenesis: SOMATIC EMBRYOGENESIS IN PALM SPECIES SUBMITTED BY : Boddu Sangavi Reg : 2010120093 M.sc agriculture 2 nd semester Genetics and plant breeding

2. CONTENTS SOMATIC EMBRYOGENESIS HISTORY STAGES OF SOMATIC EMBRYOS INDUCTION OF SOMATIC EMBRYOGENESIS SOMATIC EMBRYOGENESIS IN OIL PALM GENERAL PRINCIPLES OF SOMATIC EMBRYOGENESIS PROBLEMS ASSOCIATED WITH PALM SOMATIC EMBRYOGENESIS SOMATIC EMBRYOGENESIS IN TENERA PALM AND MACAW PALM SOMATIC EMBRYOGENESIS IN PEACH PALM SOMATIC EMBRYOGENESIS IN COCONUT PALM

3. SOMATIC EMBRYOGENESIS : Somatic embryogenesis is the process in which a single cell or a small group of cells follow a developmental pathway that leads to reproducible regeneration of non-zygotic embryos which are capable of producing a complete plant.

4. SOMATIC EMBRYOGENESIS

5. HISTORY 1958—F.C. Steward (USA) and Reinert (Germany), independently, reported the formation of embryos by the somatic cells of carrot (somatic embryogenesis). Since then somatic embryogenesis has been reported in more than 500 species of dicots and monocots Single Cell Origin of Somatic Embryos 1965—Vasil and Hildebrand achieved regeneration of full plants starting from isolated single cells of tobacco. 1966—Kohlenbach succeeded in inducing divisions in isolated mature mesophyll cells of Macleaya cordata which later differentiated somatic embryos. Backs-Hüsemann and Reinert (1970) achieved embryo formation from an isolated single cell of carrot.

6. SINGLE CELL TO PLANT

7. STAGES OF SOMATIC EMBRYOS Globular Heart Cotyledonary Torpedo LOWER OR IMMATURE STAGE MATURE STAGE

8. Induction Development Maturation Germination PLANT REGENERATION THROUGH SOMATIC EMBRYOGENESIS

9. INDUCTION OF SOMATIC EMBRYOGENESIS Rai et al. (2007) Sci. Hort. 113:129-133 INDUCTION MEDIUM : 0.8% agar-solidified full-strength MS medium + 1 mg l-1 2, 4-D + 5% sucrose DEVELOPMENT MEDIUM: 0.8% agar-solidified growth regulator free full-strength MS medium + 5% sucrose c- cotyledonary stage somatic embryo g- globular stage somatic embryo, h- heart stage somatic embryo

10. DEVELOPMENT OF SOMATIC EMBRYOS The development of somatic embryos was asynchronous and somatic embryos at different stages of development could be seen on the same explant at the same time Earlier formed younger stage somatic embryos proceeded towards mature stage of development in 4-7 week old cultures

11. MATURATION OF SOMATIC EMBRYOS Maturation is a key phase between embryo development and germination. During maturation, several storage proteins synthesized which is necessary for the germination of somatic embryos et- elongated torpedo stage somatic embryo, st- short torpedo stage somatic embryo Rai et al. (2009) Sci. Hort.121: 233-236

12. GERMINATION OF SOMATIC EMBRYOS AND PLANTLET DEVELOPMENT Germination medium : 0.8% agar solidified half strength MS medium + 3%sucrose

13. GENERAL PRINCIPLES OF SOMATIC EMBRYOGENESIS Somatic embryogenesis is the preferred in vitro regenerative route for palms, as this morphogenetic pathway may increase the number of regenerated plantlets in comparison with organogenesis. Among other advantages, somatic embryogenesis permits creation of cycling cultures through the use of cell suspensions (Teixeira et al., 1995; Sané et al., 2006) or through secondary somatic embryogenesis (Perez-Nunez et al., 2006). The production of somatic embryos capitalizes upon the totipotency of plant cells and involves the development of bipolar structures resembling zygotic embryos

14. DIRECT SOMATIC EMBRYOGENESIS AND PLANTLET REGENERATION IN OIL PALM The oil palm ( Elaeis guineensis Jacq.) is typically propagated in vitro by indirect somatic embryogenesis, a process in which somatic cells of an explant of choice are, via an intermediate phase of callus growth, induced to differentiate into somatic embryos. The architecture of the oil palm, lacking axillary shoots, does not allow for vegetative propagation. Therefore, somatic embryogenesis is the only alternative to seed propagation, which is hampered by long germination times and low germination rates, for the production of planting material. The current oil palm somatic embryogenesis procedure is associated with several difficulties

15. GENERAL PRINCIPLES OF SOMATIC EMBRYOGENESIS In direct SE, SEs are induced directly from the explant, without an intervening callus phase (Guan et al., 2016). It is suggested that direct SE proceeds from embryogenically predetermined cells, such as the hypocotyl epidermis of young plantlets, the nucellus, synergids, or embryonic cells. On the other hand, indirect embryogenesis proceeds from differentiated cells, which are induced to dedifferentiate into callus cells, followed by the acquirement of an embryogenically determined state

16. DIRECT SOMATIC EMBRYOGENESIS AND PLANTLET REGENERATION IN OIL PALM Globular somatic embryos directly from the explant Globular embryos with clear suspensor region Heart shaped and torpedo shaped embryos Somatic embryo with shoot and root pole Complete plantlet regeneration from somatic embryo

17. INDIRECT SE, which typically includes the following five steps Initiation Of Embryogenic Callus (EC) Cultures Proliferation And Maintenance Of EC Initiation Of Ses (Also Referred To As Expression Of SE) Maturation Of Ses Plantlet Regenerationse Seldomly Occurs Under Natural Conditions (Garcês Et Al., 2014), It can be induced in vitro as a method for multiplication of selected plants by application of appropriate plant growth regulators (PGRs) and growth conditions.

18. Immature zygotic embryos are cultivated on a callus induction medium containing high auxin concentrations to induce cell dedifferentiation DIFFERENT STEPS OF INDIRECT SOMATIC EMBRYOGENESIS FROM OIL PALM ZYGOTIC EMBRYOS After 8 weeks, the formation of primary, nodular callus can be observed.

19. After 16 weeks, nodular calli have developed into embryogenic callus, that can be proliferated on semi-solid or liquid proliferation media. Embryogenic cells are transferred to a culture medium with reduced auxin concentrations to initiate the formation of somatic embryos

20. After a subsequent maturation phase in which the embryos accumulate storage material and acquire desiccation tolerance, germination starts. (F) Plantlets having a well-developed shoot and root system are being formed.

21. IN VITRO SOMATIC EMBRYOGENESIS AND PLANTLET REGENERATION FROM IMMATURE MALE INFLORESCENCE OF ADULT DURA AND TENERA PALMS OF Elaeis guineensis (JACQ.) Tissue swelling and initiation of callus on explants cultured in auxin supplemented media. Immature inflorescence collected from the leaf axil of dura palm. Male inflorescence after removal of external and internal spathes

22. Embryogenic calli induction note the development of globular calli. Asynchronous somatic embryogenesis note the heart and torpedo stages Germination of somatic embryos .

23. Hardened plant of oil palm Plantlet regeneration from somatic embryos Rooting of regenerated plants

24. PROBLEMS ASSOCIATED WITH PALM SOMATIC EMBRYOGENESIS Limited Availability of Explants There is very limited choice of explants that can be used for initiation of embryogenic cultures. Typically, immature leaf explants are being used for commercial production of tissue culture plantlets (Schwendiman et al., 1988; Constantin et al., 2015; Corrêa et al., 2016), but not without difficulties. Low Efficiency of Somatic Embryogenesis Somatic embryogenesis generally is known as a process with low efficiencies, yielding only a small number of plantlets Regeneration rates are low and many incomplete plantlets lacking a well-developed shoot or root system are formed. The combination of limited explant availability and low SE initiation and regeneration rates make it impossible to propagate oil palm at large scale without implementation of a proliferation phase. Proliferation of embryogenic structures does however drastically increase the risk for somaclonal variation (Rival et al., 2013), which is the third main challenge associated with oil palm SE.

25. Phenotype of mantled fruits.. MANTLED ABNORMALITY CAUSED BY TISSUE CULTURE PROCEDURES Affected palms are characterized by abnormal flower development and a consequent reduction in oil yield. Because of the high frequency of abnormality, which is only rarely observed in seed-derived palms, the role of tissue culture in the induction of the mantled phenotype became clear, scaring plantation holders to buy more tissue culture planting material. Mantled fruits are characterized by the presence of extra carpels, arising from a feminization of the staminodes present in female flowers

26. SOMATIC EMBRYOGENESIS FROM LEAF TISSUES OF MACAW PALM [Acrocomia aculeata (Jacq.) Lodd. Ex Mart.] The development of protocols for palm trees by somatic embryogenesis, including Acrocomia aculeata , involves the addition of growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) and4-amino-3,5,6-tri chloro picolinic acid (Picloram)(Moura et al. 2009, Luis & Scherwinski-Pereira2014, Granja et al. 2018), usually employed at high concentrations in the induction phase . In fact, growth regulators play a central role in mediating signal transduction cascades leading to gene reprogramming (Dudits et al. 1991) and subsequent somatic embryogenesis. Selection of explant Immature and unexpanded leaves (palm heart)of Acrocomia aculeata , derived from adult plants

27. ph:palm heart; lp: leafpetiole: sp: immatureaculeus. Bars: a: 30 cm;b: 1 cm MORPHOLOGICALASPECTS OF THE PALMHEART WHEN EXPLANTSARE EXCISED FOR SOMATIC EMBRYOGENESIS INDUCTION IN MACAWPALM (ACROCOMIAACULEATA) Leaf explants on culture medium

28. Explant after 3 months of cultivation, with inception of callus formation. MORPHOLOGICAL ASPECT OF SOMATIC EMBRYOGENESIS IN MACAW PALM (ACROCOMIA ACULEATA) FROM LEAF TISSUES. Explant. Explant at 4 months, with development of primary calli at the edge s

29. Elongated yellowish callus at 9 months of cultivation, showing calli with a yellow nodular appearance. Explants at 6 months with elongated yellowish calli. Elongated yellowish callus at 9 months of cultivation, showing calli formation with white and yellow nodular appearance

30. Germination of somatic embryos with leaf primordia. Petri dish at 9 months of in vitro culture, showing amount of calli formed after multiplication stage. Somatic embryos observed after 12 months of in vitro culture. pc:primary callus, ex: explant, eyc: elongated yellowish callus; WN: whitish nodular callus; YW: yellowish nodular callus; se: somatic embryos; lp: leaf primordia; pl: young plantlets. Bars: 5 mm

31. Callus formed in the vascular bundle region after 90 days of cultivation, showing epidermal face separation. HISTOLOGICAL EVENTS OF MACAW PALM ( Acrocomia aculeata ) DURING SOMATIC EMBRYOGENESIS Leaf after 60 days, showing inception of primary callus in the vascular bundle region.

32. Proembryos delimited by thickening of the cell wall. Anatomical cut of the yellow elongated callus showing formation of white and yellow nodular calli on its surface.

33. Detail of the procambium in the central region of the embryo Cluster of somatic embryos delimited by the protoderm, with elongated cells in the central region (arrow). Detail of the protoderm in the peripheral region of the embryo.

34. SOMATIC EMBRYOGENESIS IN PEACH PALM USING THE THIN CELL LAYER TECHNIQUE TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0–600 µm Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets.

35. development of the shoot meristem SOMATIC EMBRYOGENESIS AND PLANTLET REGENERATION FROM THIN CELL LAYERS OF PEACH PALM Primary callus Primary callus induction from shoot meristem TCL development of embryogenic callus Scale bars: A = 1 mm B,C = 2·5 mm

36. Embryogenic callus on maturation culture medium. Somatic embryo development from primary callus. Scale bars: D = 2·5 mm; E = 1 cm;

37. Scale bars: F, G = 2·5 cm Plantlets obtained from converted somatic embryos. Acclimatized plantlets.

38. HISTOLOGICAL ANALYSES OF PEACH PALM SOMATIC EMBRYOGENESIS a collateral vascular bundle distributed along the tissue and intercalated with a fibre bundle. Histological section of fresh tissue to illustrate the organization of a mature tissue First cell division events observed in cells adjacent to the vascular tissue and the degenerating parenchyma Primary callus induction: note the formation of a meristematic zone

39. Globular somatic embryo, showing polarization signals and a well-defined protodermis. Details from the meristematic zone

40. Mature somatic embryos showing a well-developed procambium and without vascular connection with the matrix tissue. Globular somatic embryos observed in maturation culture conditions.

41. SOMATIC EMBRYOGENESIS FROM BUD AND LEAF EXPLANTS OF DATE PALM ( Phoenix dactylifera L.) CV. NAJDA Somatic Embryogenesis From Bud And Leaf Explants Of Date Palm avoids: (a) using offshoots excessively in case of limited production; (b) causing damage to the mother tree and (c) affecting fruit production by an excessive use of spathes. In addition, such technique uses a material source free of diseases and available independently of seasons.

42. Bars correspond to 0.5 cm ORIGIN OF THE EXPLANTS USED TO INDUCE SOMATIC EMBRYOGENESIS.

43. EFFECT OF EXPLANT TYPE ON SOMATIC EMBRYOGENESIS After one month of culture in the expression medium, somatic embryogenesis was greatly affected by the explant source. The somatic embryogenesis rate was higher when calli were obtained from bud explants, with a range of 60.0–86.0%. Proximal leaf segments showed embryogenic cultures only when 2,4-D or picloram were used; with a range of 12.0–16.0% Distal leaf and root explants did not show any embryogenic response in culture.

44. SOMATIC EMBRYOGENESIS AND PLANTLET REGENERATION FROM BUD SEGMENTS OF DATE PALM CV. NAJDA compact nodular primary callus obtained from foliar explants after 2 months on 2,4-D (2 mg l−1) containing medium friable secondary callus obtained 1 month after chopping of the primary callus

45. suspension culture somatic embryos of stage I,

46. somatic embryos of stage, II and III somatic embryos of stage, II and III after 2, 4, and 6 weeks of culture on PGR-free medium

47. GERMINATED SOMATIC EMBRYO

48. Plantlets of date palm cv. Najda after 6 months in the glasshouse. Bars correspond to 5 cm Shoot elongation and rooting after 3 months of culture on the elongation-rooting medium. Bars correspond to 1 cm Somatic embryo germination after 2 months on the germination medium

49. COCONUT (COCOS NUCIFERA L.) SOMATIC EMBRYOGENESIS Coconut is very recalcitrant to somatic embryogenesis. And although the many obstacles to this are slowly being reduced, in order to successfully micropropagate coconut on a large scale bottlenecks in the protocol still exist, and those include inconsistency of the embryogenic response by explanted tissues, poor somatic embryo maturation and germination, low regeneration rate of the new plantlets and long time required to produce plants (1.5 years) The immature inflorescence explants were superior in producing embryogenic callus and somatic embryos; therefore they were selected as the preferred explant source to use The use of embryogenic suspension cultures improved the yields of the regeneration processes and allowed large-scale propagation of several date palm cultivars Recently, regeneration through somatic embryogenesis from embryogenic suspensions culture was obtained for the first time for the Sahelian cultivar Amsekhsi

50. Stages in coconut somaticembryogenesis developmental pathway from plumular explants: Initial calli 30 days 60 days embryogenic calli90days

51. 120 days somatic embryos after 150 days 180 days after inoculation of plumular explants

52. COCONUT (COCOS NUCIFERA L.) SOMATIC EMBRYOGENESIS All bars = 1mm, except K = 5 mm. TS: translucid structures; ESt: embryogenic structures Embryogenic Callus Formation During In Vitro Culture Inital explant after 30 days 60 days 75 days 90 days of culture in induction medium Non embryogenic callus Formation of somatic embryos: callus with somatic embryos at different stages piece of callus with Proembryos globular embryo Coleoptilar embryo and Germinating embryo

53. Primary callus formed after 12 weeks in callus induction media Compact embryogenic calli friable embryogenic calli formed after 8 weeks in embryo formation media

54. Embryogenic calli with all stages of embryogenic callus development shows shoot induction from compact embryogenic calli after 8weeks in maturation media containing BAP and GA3 Multiple shoot induction from friable embryogenic calli in media containing 10 µM kinetin, 10 µM BAP, 200 µM NAA and 0.5 µM GA3;

55. Elongated shoots from green embryogenic calli

56. Rooted after 8 weeks in root induction media

57. The rate of coconut somatic embryos formation was as well significantly increased (over 300 %) By the simultaneous application of suspension culture step, spermine (0.01 µ M), SSW (10 %) high auxin concentration (500 µ M). Nevertheless the presence of TDZ and other cytokinins in the medium, as well as the absence of activated charcoal, were found to be unable to positively influence the somatic embryogenesis process.

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