Advances in D. <i>melanoxylon</i> Investigations towards Tissue Culture: Problems and Limitations

Abstract

The first attempt on D. melanoxylon tissue culture was conducted from 2010 to 2013 at a high level of expectations. A total of 500 seeds were sterilized at different concentration of reagents and inoculated at different strength of the Murashige and Skoog medium for germination to obtain disease free explants for callus induction trials. A total of 400 nodal segments obtained from germinated seeds were sterilized at different concentration of reagents and inoculated at different hormonal combinations to induce callus formation for seedling multiplication. Results from this tissue culture attempt set a foundation for tissue culture success in D. melanoxylon on the future research. Only 19.8% of seeds inoculated in half strength of Murashige and Skoog medium germinated within 7 days while only 6.8% of seeds inoculated in full strength of Murashige and Skoog medium germinated within 6 days. This germination was at sterilization of 20 minutes in 35% ethanol and 20 minutes in 2.6% sodium hypochlorite. A total of 1% of inoculated D. melanoxylon seedling fragments in Murashige and Skoog media supplemented with hormone combination at 2.0 mg/l BAP + 0.5 mg/l NAA developed callus after16 days from the inoculation day. The final weight of the callus at the last record was 0.62 g. In this induction ex-plants were surface sterilized in 35% ethanol for 20 minutes and 2.6% sodium hypochlorite solution for 20 minutes. The color of callus was green and friable in nature. Other hormonal combinations in this case did not induce callus production. These results suggested that the problems which affect seed germination in the natural environment are also reflected on germination in the Murashige and Skoog medium and in callus induction. Vulnerability to fungal attack is a limitation for successful callus induction and germination in the culture room. More research under improved sterile conditions is needed to improve callus percentage for seedling multiplication.