In Vitro Culture Of Dendrobium And Assessment Of Genetic Stability Using Molecular Marker

Dendrobium, a genus from Orchidaceae is a kind of elite herbage plants in Chinese traditional medicine, which characterized in multiple functions, such as in senescence-retarding and cancer-preventing, eye-brightening and body-strenthering, immune-improving etc. To satisfy the increasing demand of germplasm conservation, micropropagation, as well as the further exploitation, in vitro culture and genetic stability evaluation to the in vitro materials of these germplasms were carried out in this present study. The main results are as follows:1 Establishment of aseptic cultures for shoot segmentsAfter rinsing with tap water, shoot segments of Dendrobium nobile were firstly dipped in 70% ethanol for 30 s. Secondly, they were surface-sterilized by immersion in: 1) 0.1% (w/v) HgCl₂ for 12min;2) 0.1% (w/v) HgCl₂ with 3-5 drops of Tween 80 for 10 min. The effect of the two menthods were quite different, and contamination rate were 73.3% and 16.7%, respectively. Then the shoot segments were inoculated in MS+ BA (1.0 mg/L) + NAA (0. 4 mg/L) + sucrose (3.0 % )+ agar (0. 7% )for germination. Consequently, in vitro shoots were transferred to MS+ BA (1.2 mg/L) + NAA (0.4 mg/L) + sucrose (3.0%) + agar (0.7%) for multiplication via proto-corn genesis. 1/2MS+NAA (0.5 mg/L) +IBA (0.5 mg/L) + sucrose (2.0%) + agar (0.7%) gave the best effect for streathening seedling. Following a shoot elongation stage, 1/2MS+NAA (1.0 mg/L) +sucrose (2.0%) +agar (0.7%) showed the best effect on rooting. One or two pinecones were putted in the one—off plastic cup with some holes and then layed considerable lichen or bark on, the in vitro plants showed higher than 90% of survival ex vitro after tranplanting.2 Callus induction and regenerationIn vitro shoot segments of Dendrobium nobile were employed to induce callus. Different basal medium, types and concentration of plant growth regulators (PGRs) diversely showed callus formation ability. The oders of callus formation rate is as follows: 1) modified MS₂(m MS₂) >MS>1/2MS>>modified MS₁(m MS₁ ; 2) NAA > IBA ; 3) BA > KT. Taking the above-mentioned factors into consideration, MS+NAA (0.8mg/L) +BA (0.5mg/L) + sucrose (3.0%) +agar (0.7 %) showed the best effect. With the addition of NAA (0.8 mg/L) , BA (0.5mg/L) ,...