Dendrobium ‘Earsakul’ is an important commercial orchid in Thailand. Breeding new Dendrobium varieties for improved quality and yield is crucial. The objectives of this research were to perform in vitro mutagenesis of Dendrobium ‘Earsakul’ protocorm-like bodies (PLBs) using sodium azide (NaN3) and to select and evaluate the putative mutants using morphological characters, molecular markers, and the cytological method. The percentages of mortality of PLBs increased as concentrations of NaN3 increased. At 2 weeks, the lethal dose 30 (LD30) and LD50 were obtained with 0.1 and 0.5 mm NaN3, respectively. These two NaN3 concentrations were used for in vitro mutagenesis with reverse osmosis water (ROW; control 1) and 0 mm NaN3 (control 2) as controls. After the plants were cultured for 6 months, morphological differentiation was observed in some putative mutants: reduced height, higher numbers of nodes, reduced node length, shorter and thicker leaves, and shorter and fewer roots, compared with controls. When genetic profiles of 24 putative mutants were compared with controls, altered DNA profiles were found in 20 of 24 putative mutants (83.33%). Sixty-three polymorphic bands were produced from a total of 181 bands (34.81%) amplified by 10 polymorphic intersimple sequence repeat (ISSR) primers. When genetic diversity and relatedness, which were evaluated by ISSR analysis, and morphological characters were compared, the two markers were found to be uncorrelated. ISSR had a higher mutant differentiation capability than the morphological characters, indicating its higher efficiency. The chromosome numbers were similar in putative mutants and controls (2n = 2x = 24), suggesting that neither of the concentrations of NaN3 had any effect on the chromosome numbers in this experiment. These results indicate that NaN3 can be used effectively to mutagenize Dendrobium ‘Earsakul’ PLBs, and ISSR is a powerful tool for the identification of mutants. Chemical name: sodium azide (NaN3); reverse osmosis water (ROW).
Anakkaorn Wannajindaporn, Chitpan Kativat, and Piyada Alisha Tantasawat
Piyada A. Tantasawat, Apinya Khairum, Kitiya Arsakit, Oythip Poolsawat, Paniti Pornbungkerd, and Chitpan Kativat
The effects of culture media on growth and proliferation of ‘Earsakul’ dendrobium (Dendrobium) protocorm-like bodies (PLBs) were evaluated in a two-step culture. After culturing on each of the four first step media for 4 months and on each of the four second step media for 4 months, the greatest total PLB fresh weight, increase in number of PLBs and growth rates were obtained when using Vacin and Went medium 1 (VW1) in both culture steps compared with those in 15 other medium combinations. Starting from 0.5 g of PLBs, culturing on VW1 for 8 months achieved a total of 415.25 g of PLBs, a multiplication rate of 830-fold. The supplementation of tomato (Solanum lycopersicum) and substitution of ‘Hom Thong’ banana [Musa acuminata (AAA group)] with ‘Khai’ banana [M. acuminata (AA group)] in this new medium promoted growth and proliferation of dendrobium PLBs 2.4-fold over the control medium, suggesting its usefulness in commercial micropropagation.