Propagation of Rubber Tree Resistant to White Root Disease through Somatic Embryogenesis from Thin Cell Layer and Floral Explants and Assessment of Somaclonal Variation by RAPD and SSR Markers

Abstract

Rubber tree is economically important rubber producing plant of Thailand. At present, a rubber tree plantation is susceptible to white root disease. Therefore, the use of rootstock from early introduce clone that proved to be resistant to white root disease could help sustain growing of rubber tree. Thus, the objectives of this research were to study the effects of plant growth regulators and different explants on callus and somatic embryo (SE) induction of this rubber clone. Both longitudinal thin cell layer (lTCL) from young branch and two different types of explants from young inflorescence gave 100% of sterilization and callus formation on MS medium supplemented with 2.0 mg/l 6-benzyladenine (BA) and 1.5mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) after culture for 4 weeks. The characteristics of callus from flower explant was friable and compact. Whereas the callus from lTCL was compact only. The color of callus from all sources of explants was yellowish green. For proliferation of callus, callus from mix flower gave the highest proliferation rate in terms of fresh weight at 392.05 mg after culture for 4 weeks on MS medium supplemented with the above concentrations of BA and 2,4-D. Upon transferring the callus to the same culture medium and culturing for further 12 weeks somatic embryo (SE) formation at the highest frequency of 39.84% and number of cotyledonary embryos (CEs) at 3.25 embryos /callus were obtained. CEs conversed into embryo axis at 50% and shoot at 25% after transfer to 0.25 mg/l GA3 containing MS medium with the best concentrations of BA and 2,4-D for 4 weeks. The assessment of genetic stability of in vitro derived clones is considered to be a very useful and essential step in this study. For SEs derived from different explants, 2 primers (OPAD-01 and OPAD-10) of RAPD and 3 primers (hmac4 hmct1 and hmct5) of SSR marker gave the same profile of DNA pattern. It was clear that somaclones obtained from our protocol were uniform and successfully used to assess genetic stability in micropropagated plants.