EFFECTIVE CACAO SOMATIC EMBRYO REGENERATION ON KINETIN SUPPLEMENTED DKW MEDIUM AND SOMACLONAL VARIATION ASSESSMENT USING SSRs MARKERS

Nur Ajijah, Rr. Sri Hartati, Rubiyo Rubiyo, Dewi Sukma, Sudarsono Sudarsono

Abstract


This study aimed to develop the cacao (Theobroma cacao L.) in vitro regeneration system through somatic embryogenesis on kinetin supplemented DKW medium and somaclonal variation assessment using SSR markers.  Callus were initiated from basal petal and staminoid explants cultured on callus induction (CI) medium contained DKW basalt salts and kinetin:2,4-D ratios of  1:15.5, 1:7.8 or 1:3.9 and then transferred onto secondary callus growth (SCG) medium contained WPM basalt salts and kinetin:2,4-D ratios of 1:7.8 or 1:3.9. The calli were then subsequently transferred onto embryo development medium contained DKW basal salts with or without the addition of amino acids, adenine or activated charcoal for the formation of somatic embryos. Nine cacao genotypes were tested for their  ability to develop somatic embryos. Results of this study indicated DKW medium supplemented with Kinetin in combination with 2,4-D effectively induced cacao somatic embryogenesis. The highest somatic embryos formation was abtained from kinetin:2,4-D ratio of 1:3.9 and 1:7.8 in CI and SCG medium respectively. Cacao genotype responses were highly explant type dependent. The developed method resulted in a high percentage of somatic embryo formation (5.6-66.7%), germination (50%)  and plantlet conversion (65%) and a medium percentage of somaclonal variations based on SSRs marker analysis.


Keywords


explants; genotypes; kinetin:2,4-D; SSRs; Theobroma cacao L.

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References


Abdin, M.Z. and A. Ilah. 2007. Plant regeneration through somatic embryogenesis from stem and petiole explants of Indian chicory (Cichorium intybus L.). Indian Journal of Biotechnology, 6(2), 250-255. Retrieved fromhttp://nopr.niscair.res.in/handle/12345678 9/5120

Ajijah, N., I. Darwati, Yudiwanti and Roostika. 2010. Effect of temperature incubation on growth and development of Purwoceng (Pimpinella pruatjan Molk.) somatic embryos (in Indonesian). Jurnal Penelitian Pertanian Tanaman Industri, 16(2), 56-63. Retrieved from http://www.ejurnal.litbang. pertanian.go.id/index.php/jptip/article/view/2046/1776

Ajijah, N., Sudarsono, Rubiyo, and Dewi Sukma (Eds.). 2012. The utilization of somaclo-nal variation for the improvement of cacao (Theobroma cacao L.) resistance against black pod due to Phytophthora palmivora Butl infection. Proceedings from the Meeting of Experts in Biotechnology. Bogor.

Ajijah, N., Rubiyo and Sudarsono. 2014. Callo-genesis and Somatic Embryogenesis of Cacao using Thidiazuron through One Step of Callus Induction (in Indonesian). Jurnal Penelitian Pertanian Tanaman Industri. 20(4): 179-186. Retrieved from http://www.ejurnal.litbang.pertanian.go.id/index.php/jptip/article/view/2580/2220

Al-Khayri, J.M. 2011. Basal salt requirements differ according to culture stage and cultivar in date palm somatic embryogenesis. American Journal of Biochemistry and Biotechnology. 7(1): 32-42. doi: 10.3844/ ajbbsp.2011.32.42

Bidabadi, S.S., S. Meon, Z. Wahab and M. Mahmood. 2010. Study of genetic and phenotypic variability among somaclones induced by BAP and TDZ in micro-propagated shoot tips of banana (Musa spp.) using RAPD markers. Journal of Agricultural Science. 2(3): 49-60. doi: http://dx.doi.org/10.5539/jas.v2n3p49

Brown, J.S., W. Phillips-Mora, E.J. Power, C. Krol C, C. Cervantes-Martinez, J.C. Motama-yor and R.J. Schnell. 2007. Mapping QTLs for resistance to frosty pod and black pod diseases and horticultural traits in Theobroma cacao L. Crop Science. 47(5): 1851-1858. doi: 10.2135/cropsc i2006.11.0753

da Costa Pinto, G.C.C. 2007. Eucalyptus globulus plant regeneration by somatic embryo-genesis (Unpublished doctoral disser-tation). University of Aveiro, Portugal.

Dalila, Z.D., H. Jaafar and A.A. Manaf. 2013. Effects of 2,4-D and kinetin on callus induction of Barringtonia racemosa leaf and endosperm explants in different types of basal media. Asian Journal of Plant Sciences. 12(1): 21-27. doi: 10.3923/ ajps.2013.21.27

Emile, M., N. Nicolas, I.E. Auguste, S. Abdourahamane and D.N. Omoko-lo.2010. Sulphur depletion altered somatic embryogenesis in Theobroma cacao L. biochemical difference related to sulphur metabolism between embryogenic and non embryogenic calli. African Journal of Biotechnology. 9(35): 5665-5675. doi: 10. 5897/AJB10.565

Feher, A., T.P. Pasternak and D. Dudits. 2003. Transition of somatic plant cells to an embryogenic state. Plant Cell, Tissue and Organ Culture. 74(3): 201-228. doi: 10.10 23/A:1024033216561

Ibrahim, M.S.D., Sudarsono, Rubiyo and Syafaruddin. 2012. Effect of medium com-position on embryogenic calli formation of arabica coffee (Coffea arabica) (in Indo-nesian). Jurnal Tanaman Industri dan Penyegar. 3(1): 13-22. Retrived from http://balittri.litbang.pertanian.go.id/index.php/publikasi/category/51-buletin-vol-3-no-1-tahun-2012?download=67%3Apeng aruh-komposisi-media-terhadap-pemben-tukan-kalus-embriogenesis-somatik-kopi-arabika-coffea-arabica

Ibrahim, M.S.D., Rr.S. Hartati, Rubiyo, A. Purwito and Sudarsono. 2013a. Embryogenic callus induction and regeneration potential of arabica coffee by 2,4-dichlorophe-noxyacetic acid and 6-benzyladenine (in Indonesian). Jurnal Tanaman Industri dan Penyegar. 4(2): 91-98. Retrieved from http://balittri.litbang.pertanian.go.id/index.php/publikasi/category/53-buletin-vol-4-no-2-tahun-2013?download=78%3A01.-induksi-kalus-embriogenik-dan-daya-re-generasi-kopi-arabika-menggunakan-2-4-dichlorophenoxyacetic-acid-dan-6-benzyladenine

Ibrahim, M.S.D., Rr.S. Hartati, Rubiyo, A. Purwito and Sudarsono.2013b . Direct and indirect somatic embryogenesis on arabica coffee (Coffea arabica). Indonesian Journal of Agricultural Science. 14(2): 79-86. Retrieved from http://www.ejurnal.litbang. pertanian.go.id/index.php/ijas/article/view/1413/1194

Indonesian Ministry of Agriculture. 2014. Cocoa Outlook (in Indonesian). Ragunan, Jakarta: Central of Agricultural Data and Information System Printing Office.

International Cocoa Organization. 2014. ICCO quartely bulletin of cocoa statistic XI (1) 2013/2014. Retrieved from http://www.i cco.org/about-us/icco-news/266-august-2014-quarterly-bulletin-of-cocoa-statis-tics.html

Jimenez, V.M. 2005. Involvement of plant hormones and plant growth regulators on In Vitro somatic embryogenesis. Plant Growth Regulation, 47(2-3), 91-110. doi: 10.1007/s10725-005-3478-x

Jose, J., K. Nimisha, M.A. Anu and P. Nambisan. 2012. Evaluation of somaclonal variation in callus cultures of Jatropha curcas maintained on different hormonal combi-nations using RAPD markers. World Jour-nal of Agricultural Sciences. 8(6): 616-623. doi: 10.5829/idosi.wjas.2012. 8.6. 1644

Kurniasih, S., Rubiyo, A. Setiawan, A. Purwantara and Sudarsono. 2011. Genetic diversity analysis of cacao (Theobroma cacao L.) germplasm based on SSR markers (in Indonesian). Jurnal Penelitian Pertanian Tanaman Industri. 17(4): 156-162. Re-trieved from http://sitp.rpn.co.id/ up-loads/riset/kakao/Jurnal%20Littri%2017%284%29,%202011%20156-162.pdf

Larkin, P.J. and W.R. Scowcroft. 1981. Soma-clonal variation - a novel source of variability from cell cultures for plant improvement. Theoretical and Applied Genetics. 60(4): 197-214. doi: 10.1007/BF 02342540

Li, Z., A. Traore, S. Maximova and M.J. Guiltinan. 1998. Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron. In Vitro Cellular & Developmental Biology – Plant. 34(4): 293-299. doi: 10.1007/BF0 2822737

Obando, A. and E. Manuel. 2009. Genetic characterization of Theobroma cacao L. in Nicaragua (Master Thesis). Retrieved from http://urn.fi/URN:NBN:fi:hulib-2015 072 11645

Pasternak, T.P., E. Prinsen, F. Ayaydin, P. Miskolczi, G. Potters, H.A. Van Onckelen, D. Dudits and A. Feher. 2002. The role of auxin, pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa. Plant Physiology, 129(4), 1807-1819. doi: http://dx.doi.org/10.1104/pp.000810

Pugh, T., O. Fouet, A.M. Risterucci, P. Brottier, M. Abouladze, C. Deletrez, B. Courtois, D. Clement, P. Larmande, J.A.K. N’Goran and C. Lanaud. 2004. A new cacao linkage map based on codominant markers: development and integration of 201 new microsatellite markers. Theo-retical and Applied Genetics. 108(6): 1151-1161. doi: 10.1007/s00122-003-1533-4

Purwati, R.D., S. Harran and Sudarsono. 2007. In Vitro selection of abaca for resistance to Fusarium oxysporum f.sp. cubense. Hayati Journal of Biosciences. 14(2): 65-70. Retrieved from http://journal.ipb.ac.id/ index.php/hayati/article/view/241/107

Rahayu, E.S., E. Guhardja, S. Ilyas and Sudarsono. 2005. Polyethylene glycol (PEG) induced stress conditions in in vitro culture of peanuts (Arachis hypogaea L.) (in Indonesian). Berkala Penelitian Hayati. 11(1), 39-48. Retrieved from http://www. berkalahayati.org/#/journal/89bc7bb1

Rahayu, E.S. and Sudarsono.2015. In-vitro selection of drought tolerant peanut embryogenic calli on medium containing polyethylene glycol and regeneration of drought tolerant plants. Emirates Journal of Food and Agriculture. 27(6): 475-487. doi: 10.9755/ejfa.2015.04.062

Rodriguez Lopez, C.M., A.C. Wetten and M.J. Wilkinson. 2010. Progressive erosion of genetic and epigenetic variation in callus-derived cocoa (Theobroma cacao) plants. New Phytologist. 186(4): 856-868. doi: 10.1111/j.1469-8137.2010.03242.x

Rubiyo and Siswanto. 2012. Improvement and development production of cocoa in Indonesia (in Indonesian). Jurnal Tanaman Industri dan Penyegar. 3(1), 33-48. Retrieved from http://balittri.litbang. pertanian.go.id/index.php/publikasi/category/51-buletin-vol-3-no-1-tahun-2012? download=69%3Apeningkatan-produksi-dan-pengembangan-kakao-theobroma-cacao-l.-di-indonesia

Subhashini, R., U.S.M. Rao, P. Sumathi and G. Gunalan. 2010. A comparative phyto-chemical analysis of cocoa and green tea. Indian Journal of Science and Tech-nology. 3(2): 188-192. doi: 10.17485/ ijst/2010/v3i2/29676

Tang, H., Z. Ren and G. Krczal. 2000. Somatic embryogenesis and organogenesis from immature embryo cotyledons of three sour cherry cultivars (Prunus cerasus L.). Sci-entia Horticulturae. 83(2): 109-126. doi:10.1016/S0304-4238(99)00073-4

Widoretno, W., E. Guhardja, S. Ilyas and Sudar-sono. 2002. Effectiveness of polyethylene glycol for evaluating response of soybean at germination stage against drought stress (in Indonesian). Hayati. 9(2): 33-36. Retrieved from http://repository.ipb.ac.id/ bitstream/handle/123456789/24928/Wahyu%20W%20%28SS%29.pdf?sequence=1&isAllowed=y

Widoretno, W., E.L. Arumningtyas and Sudarsono. 2003a. In Vitro methods for inducing somatic embryos of soybean and plantlet regeneration (in Indonesian). Hayati. 10(1): 19-24. Retrieved from http://repo-sitory.ipb.ac.id/bitstream/handle/123456789/24941/Wahyu%20Widoretno%20%28SS%29.pdf?sequence=1&isAllowed=y

Widoretno, W., S. Harran and Sudarsono. 2003b. Variation in qualitative and quantitative characters among somaclones of soybean derived from In Vitro selected somatic embryo (in Indonesian). Hayati. 10(3), 110-117. Retrieved from http://re-pository.ipb.ac.id/bitstream/handle/123456789/24822/Sahid%20Harran.pdf?sequence=1&isAllowed=y

Widoretno, W., R. Megia and Sudarsono. 2003c. Responses of soybean somatic embryos to polyethylene glycol and its uses for In Vitro selection of soybean against drought stress (in Indonesian). Hayati.10(4): 134-139. Retrieved from http://reposi-tory.ipb.ac.id/bitstream/handle/123456789/29668/HAY031004wwi2003-4-134139.pdf?sequence=1&isAllowed=y

Yuliasti and Sudarsono. 2011. Responses of soybean mutant lines to aluminium under In Vitro and In Vivo condition. Atom Indonesia. 37(3): 126-132. doi: http://dx. doi.org /10.17146/aij.2011.80

Yusnita, Widodo and Sudarsono. 2005. In vitro selection of peanut somatic embryos on medium containing culture filtrate of sclerotium rolfsii and plantlet regeneration. Hayati Journal of Biosciences. 12(2): 50-56. Retrieved from http://journal.ipb. ac.id/index.php/hayati/article/view/172/39

Yusnita, H. Aswidinnoor, R. Megia, R. Suseno and Sudarsono. 2010. Peanut somaclonal variants resistant to Sclerotium rolfsii derived from in vitro selection with fungal culture filtrates (in Indonesian). Jurnal Hama dan Penyakit Tumbuhan Tropika. 10(1): 35-46. Retrieved from http://journal. unila.ac.id/index.php/jhtrop/article/viewFile/348/561




DOI: http://doi.org/10.17503/agrivita.v38i1.619

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