Abdul Aziz, Maheran

Abdul Rashid, Azmi

Kabir, Md. Humayun

Mahmood, Marziah

Universiti Putra Malaysia Institutional Repository

FOR Category F10800: Biological SciencesINDUCTION OF SOMATIC EMBRYOGENESIS FOR fIN VITRO GENETIC MANIPULATION OF LOCALBANANA CULTIVARSM.A. Aziz, H. Kabir, A.A. Rashid and M. MahmoodFaculty of AgricultureUniversiti Putra Malaysia, 43400 UPM, Serdang, Selangor,MalaysiaKeywords: banana, scalps, somatic embryogenesis,callus, embryogenic cell suspension.IntroductionThe production and export potential of our local banana cul-tivars are greatly hampered by diseases and undesirable postharvest qualities. Genetic improvement of the crop throughclassical breeding methods remains difficult due to high ste-rility and polyploidy nature of the crop. Interest is now di-verted towards the genetic improvement of banana throughgene transfer techniques. Prerequisite to the successful ex-ploitation of the gene transfer technology is the establishmentof a reliable in vitro culture system that allows regenerationof mature plants. Regeneration through somatic embryro-genesis have been reported in various cultivated triploids -Grand Naine (AAA Cavendish') (Novak et al. 1989), Blug-goe (ABB Bluggoe') (Dheda et al. 1991) and diploid Musa(AA) ssp. malaccensis (Navarro et al. 1997). An attempt atinducing somatic embryogenesis in our local banana culti-vars, particularly in cv. Mas, is described.Materials and MethodsShoot tips, immature male flowers and thin corm slices wereused as explants. Shoot tips removed from suckers were ster-ilised in 80% ethanol (I min) and in 100% clorox (30 min),and rinsed thrice in sterile distilled water. The shoot tipswere placed on modified Murashige and Skoog (1962) (MS)medium supplemented with lOJ.LMand 100J.lMbenzylarnino-purine (BAP) in combination with 1.0J.LMindole acetic acid(!AA). Subculture was performed at 4-week intervals.Scalps obtained were transferred to half strength liquid MSmedium with 5.0J.LM2,4-Dichlororphenoxyacetic acid (2,4-D) and 1.0J.LMzeatin and agitated at 70 rpm. The culturemedium was refreshed at 3 weeks interval. Young male in-florescence, reduced to approximately 5.0 cm in size by re-moval of the external bracts, were sterilised in 70% ethanol(1 min) and the first 15 rows of flowers below the apicalmeristem were dissected and cultured on modified MS me-dium with 20J.LM2,4-D, 5.0J.lM lAA and 5.0J.LMnap-thaleneacetic acid (NAA). Thin corm slices were obtainedfrom below the shoot tip of in vitro plantlets and cultured onMS medium supplemented with 0.5, 10, 20, 40 and 80J.LM2,4-D. Callus produced were subcultured onto MS mediumwith 4.7J.LMabscisic acid (ABA). Embryogenic callus thatproliferated were transferred to various regeneration media:MS medium with I1J.LM!AA and 2.0J.LMBAP, MS mediumwith 5.0, 10 and 20J.LMBAP, and MS medium withoutgrowth regulators.Results and DiscussionFrom the seven cultivars of banana and plantain tested: Mas(AA), Berangan (AA), Intan (AA), Tandok (AAB), Raja(AAB)' Gros Michel (AAA) and Grand Naine (AAA), cv.Mas responded the earliest to form scalps (after 5 subcul-tures). Scalps were obtained only after 6 to 8 subculturesinthe rest of the cultivars, and the scalps were inferior inquality compared to those of cv. Mas. MS medium withl00J.LMBAP and 1.0J.LMlAA promoted scalp formation,whilst MS medium with 1OJ.LMBAP and 1.0J.LM!AA pro-duced only stunted shoots. Dheda et al. (1991) however, re-ported obtaining scalps on MS medium with 1OJ.LMBAP and1.0J.LMlAA in cv. Bluggoe, thus indicating a cultivar specificresponse to scalp formation in banana. Scalps of cv. Mas'placed in liquid medium responded to form meristematicglobules on their surface and releasing them into the liquidmedium after 5 to 8 weeks in culture. The number of meris-tematic globules released increased rapidly up to the thirdsubculture and gradually stabilised in later subculture. Withsubculture, cells were simultaneously released from the mer-istematic globules. Cells released from meristematic globulesof 5 to 6 month-old culture were embryogenic, whilst cellsreleased earlier were more heterogeneous, elongated andhighly vacuolated. In cv. Bluggoe, embryogenic cell suspen-sion was attained from scalps after 4 to 7 months in liquidculture medium (Schoofs, 1997). Young male flowers fromrows 5 to 10 responded to form callus after 1 month in cul-ture. Three months later, whitish, globular and embryogeniccallus appeared especially around each flower. Subculture onMS medium with 4.7J.1MABA proliferated more of suchcallus. Thin corm slices developed yellowish callus after amonth in culture on 0.5J.1M2,4-D. Higher concentrations of2,4-D caused blackening of the callus. More calluses prolif-erated and became globular and embryogenic after 3 monthsin culture. Embryogenic calluses were placed on regenerationmedia to form somatic embryos and germinate into wholeplants.ConclusionsVarious explants of banana are potential sources for embryo-genic callus formation. Exogenous supplies of growth regu-lators, particularly auxins, are vital in inducing embryogenicstructures. Somatic embryogenesis attained will be an idealsystem for improving the local Musa spp. through non-conventional strategies such as genetic transformation, pro-toplast fusion, mutagenesis and somaclonal variation, and formass propagation and cryopreservation.ReferencesDhed'a D., Dumortier, F., Panis, B., Vulysteke, D. and De Langhe,E. 1991. Plant regeneration in cell suspension cultures of thecooking banana cv. Bluggoe (Musa spp. ABB group). Fruits. 46:125-135.Murashige, T. and Skoog, F. 1962. A revised medium for rapidgrowth and bioassays with tobacco tissue cultures. Physiol. Plant.IS: 473-497.Navarro, C., Escobedo, R.M. and Mayo, A. 1997. In vitro plant re-generation from embryogenic cultures of a dipliod and a triploid,Cavendish banana. Plant Cell Tiss. Org. Cult. 51: 17-25Novak, FJ., Afza, R., Van Duren, M., Perea-Dalles, M., Conger,B.V. and Xiaolong, T. 1989. Somatic embryogenesis and plantregeneration in suspension cultures of dessert (AA and AAA) andcooking (ABB) bananas (Musa spp.). BiolI'echnol. 7: 147-158.Schoofs, H. 1997. The origin of embryogenic cells in Musa. Ph.Dthesis. KU Leuven, Belgium. Dissertatione de Agricultural no.330. p. 1-258.Supported by JRPA Grant OJ-02-04-0033191

Induction of somatic embryogenesis for in vitro genetic manipulation of local banana cultivars

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Induction of somatic embryogenesis for in vitro genetic manipulation of local banana cultivars

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