Kadzimin, Saleh

Universiti Putra Malaysia Institutional Repository

FOR Category F10900: Agricultural SciencesDEVELOPMENT OF ORCHID VARIETIES BY NqN-CONVENTIONAL TECHNIQUESaleh'KadziminFaculty of AgricultureUniversiti Putra Malaysia, 43400 UPM, Ser£iang, Selangor,MalaysiaKeywords: mutation breeding, gamma irradiation, invitro culture, protoconn-like bodies, orchidaceae.IntroductionOrchid cut flowers has become an important export com-modity in Malaysia, contributing to about 40% of total 'valueof export of cut-flowers since 1995. Orchid gains its popu-larity for its wide range of varieties producing an array ofshapes, colours and sizes. However, many of the existinghybrids have been in the market for more than 15 years, withsome more than 20. With ever changing tastes and prefer-ences of consumers, there is an urgent need for new or im-proved varieties with new colours or size. In orchids, con-ventional breeding and selection has been difficult or impos-sible, costly and time-consuming and often hampered by ge-netic sterility. Induction of mutation by gamma-irradiationhas provided an avenue for creating variability in many plantspecies. --The present study was undertaken to elucidate theeffects of gamma-irradiation with the objective of determin-ing the appropriate dose feasible for inducing desired muta-tion in already established varieties of orchids. The purposeof the study was to develop protocol for the creation of vari-ability in orchids.Materials and MethodsProtocorm-like bodies of Mokara'Chark Kuan and Dendro-bium Jacky, the plant materials used, were initiated throughthe standard culture of shoot tips in Vacin and Went medium.The culture of protocorm-like bodies was maintained inVacin and Went liquid medium with 1.2% coconut water.The culture was irradiated in a series of different doses of 0to 150 Grays in sterile environment (Briggs and Constantin,1977). Reculture was accomplished immediately after irra-diation in semi-solid medium. Survival and plantlet regen-eration as welJ as dry weight accumulation were observed.Random Amplified Polymorphic Deoxyribonucleic Acidanalysis was carried out according to Doyle and Doyle(1990) with some modifications to observe polymorphism inthe genomic deoxyribonucleic acid of the plants after irra-diation. Detected polymorphism was reconfirmed by South-ern analysis.Results and DiscussionGamma-irradiation had resulted in various degrees of lethal-ity to cultures of both varieties. Decrease in survival with in-creasing doses was significantly observed. Optimum dose ofgamma-irradiation for Mokara Chark Kuan was in the rangeof 20-40 Grays. Dendrobium Jacky recorded an optimum at60-70 Grays. Regeneration of protocorm-like bodies ~eclinedwith increase in doses in both varieties. Fresh weight.dryweight and dry weight accumulation generally decreasedwith increase in doses.Random Amplified, Polymorphic - Deoxyribonucleic' -Acidanalysis carried out on 7 Mokara Chark Kuan samples using17 primers from Operon Kit AB resulted in two primers(OPAB-OS and OPAB-ll) producing 2 novel bands for eachprimer indicating polymorphism in the samples. The primersdetected somaclonal variations in 2 non-irradiated samplesprobably initiated through repeated subculture of protocorm-like bodies. They also detected variations caused by gamma-irradiation. When Southern analysis was performed usingthe novel bands as probes, the results supported the findingsobtained from Random Amplified. Polymorphic Deoxyribo-nucleic acid analysis , _ , .. ~ ,Amplification using OPAB-ll as primers were cloned andsequenced. The sequences could be used for SequencedCharacterised Amplified Regions for the specific amplifica-tions of a single band. Homology searches using pALI0Sand pAL202 as query sequences revealed 56.6% and63.4% similarity to Lycopersicon esculuntum copia-like retrotransposon and L. esculuntum gypsy-like ret-rotransposon respectively (Aung and Tan, 1996). There isa possibility that both pALlOS and pAL202 are related totransposable elements. Both repeated subculture and irradia-tion could have activated these transposable elements whichcontributed to the variations. Southern analysis of genomicdeoxyribonucleic acid showed that probe B and pALlOSwere parts of the genomic deoxyribonucleic acid of MokaraChark Kuan.· It further confirmed the polymorphisms de-tected by Random Amplified Polymorphic deoxyribonucleicacid analysis.' There might be some similarities betweenpALlOS and pAL202'thatallowed pAL202 to hybridise topAL lOS: . When Random Amplified Polymorphic deoxyri-bonucleic acid analysis was performed on S DendrobiumJacky samples using 20 primers from Operon Kit AB, nopolymorphism was detected. It is'possible that these primerswere- unable to detect any possible polymorphism that hadoccurred. - Primers containing different combinations of oli-gonucleotides should be considered in future study.ConclusionsGamma-irradiation induced changes in the culture. It causedsignificant effects on fresh weight, dry weight and dry weightaccumulation. Variations caused by repeated subculture ofprotocorm-like bodies and irradiation were detected by Ran-dom Amplified Polymorphic Deoxyribonucleic Acid analy-sis. Phenotypic expression of these variations could not beconfirmed in this study as plantlets take at least another 2-3years to flower.ReferencesAung, A.M. and Tan S.T. 1996. RAPD Analysis of DNA polymor-phisms among three slipper orchids species, Paphiopedilum bar-batum, P. callosum and P. niveum. As Pac J Mol Bioi Biotech-nol.4(3): 196-202.Briggs, R.W. and Constantin M.1. 1977. Radiation types and radia-tion sources. In: Manual on mutation _breeding (Technical Re-ports Series No. 119) IAEA, Vienna. p. 33-40.Doyle, 1.1 and Doyle,I.L 1990. Isolation of plant DNA from freshtissue. Focus. 12(1): 13-15.Supported by JRPA Grant OJ-02-04-004663

Development of orchid varieties by non-conventional technique

http://psasir.upm.edu.my/18170/1/Development%20of%20orchid%20varieties%20by%20non-conventional%20technique.pdf

Development of orchid varieties by non-conventional technique

Abstract

Similar works

Full text

Available Versions

Universiti Putra Malaysia Institutional Repository