Study On Rapid Micropropagation And Cell Culture Of Dendrobium Candidum Wall Ex Lindl

Dendrobium candidum Wall ex Lindl is a perennial herbaceous epiphytic Orchid,which is also the second class national protected and valuable medicinal material. Ingeneral, it plays physiological functions in antitumorigenesis and enhancing immunity. Atpresent, the market supply and demand of Dendrobium candidum is severely imbalanced.Although, there are reports about its in vitro regeneration system, the proliferationcoefficient is still very low. Thus, it is significant to expanse Dendrobium candidummedicine sources.In this paper, the rapid micropropagation and cell culture system are established withthe wild Dendrobium candidum from Yunan, the main results are as follows:(1) The rapid micropropagation system of Dendrobium candidum via axillary budwas established. The result shows that types of explants, including doses and ratio of NAAand6-BA play important roles on induction rate. The stem with the first internodecontributes to the highest induction rate of66.7%, and the stem with the second or thirdinternode is less efficient with an induction rate of53.3%. The ratio of NAA and6-BA ispositively correlated with induction and proliferation rate of clustered buds. The optimizedclustered buds induction medium is1/2MS+0.5mg/L NAA+4mg/L6-BA.The inductionrate is73.3%,and the proliferation coefficient reaches19.87.(2) The rapid propagation system of Dendrobium candidum was established via PLB(direct somatic embryogenesis).The factors influencing PLB induction, differentiation,and rooting were studied. The most suitable mediums are as follows:PLB Induction medium: MS+1mg/L TDZ+0.5mg/L NAA+0.5mg/L2，4-DPLB differentiation medium: MS+1mg/LNAA+10mg/L6-BARooting and strengthening medium:1/2MS++1mg/L6-BA+2mg/LNAA+6mg/LIBA+200g/L potato juiceThe proliferation coefficient reaches52.3according to this culture system.Plantlets of5cm height or above were transplanted to the substrate (bottomedsphagna1~2cm+vermiculite and pine bark mixture (the volume ratio is1:2to1:4)+topsphagna3~5cm), and the survival rate is as high as98.3%. (3) The technical system of Dendrobium candidum callus induction and cellacclimation was established. The result shows that the best induction medium is:MS+0.5mg/L2,4-D+0.25mg/L or0.5mg/L KT and the callus can be obtained in7to10days. The callus clumps enrichment method was used to realize directional screening andacclimation of cells. The subculture cycle is15days in1to2months and later20days.After the cells became granular, they were transplanted to the media of MS+5mg/L NAA+0.5mg/L6-BA or KT,30days per cycle. After6to9months, Dendrobium candidum celllines could be obtained.(4) Comparing the content of polysaccharide of different kinds of tissue cultures forquality analysis of their medicinal potentials，the result shows that the wet-dry ration ofDendrobium candidum callus is7.7%, the water-soluble polysaccharide and the totalpolysaccharide content are18.18%and24.67%; the wet-dry ration of PLB is4.4%, twokinds of polysaccharides content are10.4%and15.7%;wet-dry ration of seedling is6.7%,two kinds of polysaccharides content are7.8%and13.8%. To sum up, the polysaccharideproductivity of callus is the highest among the four tissue cultures, and the three additionaltissue cultures are equivalent. Compared with the wild plantlets from Yunnan (25%~30%),the productivity of callus is still competitive. The widely accepted evaluation criteria forDendrobium candidum is the content of polysaccharide which is positively correlated withphysiological function. So, Dendrobium candidum callus is the most potential alternativemedicine source.