Methods towaards establishing oil palm haploid technology
MARIA Madon * , CLYDE, M.M. ** , AHMAD TARMIZI Hashim * , AHMAD RAFDHI, M.H. ** , AHAMAD TAJUDDIN, A **Screening for natural haploids by flow cytometry and chromosome counts on 518 culled tissue culture plantlets and 13 twin germinated seeds yielded no haploids. In anther culture experiments, anthers bearing pollen mother cells (PMC) at the uninucleate and tetrad stages were collected and stored at 4oC for one, two, four, six, eight, 10 and 12 days to bring about stress for callus induction. In these experiments, anthers from dura and tenera palms were cultured on two types of media, namely, the modified N6 and, Murashige and Skoog (MS) media. Observations were made based on the qualitative responses of the anthers, and MS medium was found to be more suitable as N6 took 28 weeks to induce any response from the anthers. For dura anthers, the most positive response was obtained from anthers containing uninucleate PMC after four and 10 days of cold pre-treatment while for anthers containing tetrad PMC, the best responses were obtained after eight days of cold pre-treatment when cultured on N6 media. When cultured on modified MS induction media, the best response percentages were obtained from dura anthers containing uninucleate PMC after eight days of cold pre-treatment while anthers containing tetrad PMC showed the best response after four days of cold pre-treatment. Tenera anthers containing tetrad PMC showed the best response after six days of cold pre-treatment when cultured on MS induction media. An experiment was also conducted to determine suitable sucrose levels (3%, 6%, 9%, 12% and 15%) in modified MS media, and to observe the effect of orientation of the anthers bearing tetrad PMC (abaxial surface facing down or up) on callus induction response. It was observed that 3% sucrose and anthers cultured with the abaxial surface facing up are the most suitable conditions for oil palm anther culture. Microspore culture was carried out next as anther culture is very laborious. Various stress factors were used to induce the sporophytic pathway, these being: (1) cold shock, with male flowers incubated at 4oC for one and six days, (2) heat shock at 35°C, 38°C, 42°C and 46°C, (3) immersion of the flower buds in 0.3 M mannitol for one and six days, (4) combinations of 0.3 M mannitol with cold shock (at 4oC) and 0.3 M mannitol with heat shock (at 35oC) for one and six days, and finally (5) anti-tubulin pre-treatment of isolated microspores with 2.5 mg ml-1 colchicine or 50 ìg ml-1 oryzalin. These treatments were followedby culture in two types of media –modified MS media with 5 or 10 mg litre-1 NAA – at 27oC in the dark with continuous shaking. None of the stress factors was able to induce microspore embryogenesis in the oil palm microspores studied.
Tags: BREEDING & GENETIC, HYBRIDIZATION, CHROMOSOME, MPOB PUBLICATION, oil palm
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