Establishment Of In Vitro Propagation Systems And Study On Photoautotrophic Culture Of Ilex Chinensis Sims

Ilex chinensis Sims (Chinese holly) is the evergreen tree, belongs to the family Aquifolium and the genus Ilex. Ilex chinensis Sims is ornamental and greening tree, also has the medicinal value. Conventional procedures for mass propagation are seeding and cutting, seeding propagation has low germination rate and does not keep the excellence characteristics of parent plant; Rate of rooting is also low by cutting propagation.However, the number of cutting stems is finitary. Tissue culture technique can overcome these problems. In this study, The factors affecting in vitro propagation of Chinese holly systematically were studied,and besides studied how to improve photosynthetic capacity of in vitro plantlet by environmental control, which also called Photoautotrophic culture. The effects of photosynthetic environmental control on growth in vitro plantlet were investigated. The results are as follows.Establishment of aseptic culture was conducted, when sterilized by conventional way, stems of Chinese holly were sterilized by HgCl₂, the optimal sterilized time was 15min, when more than this time, the explants were damaged and browning. However, when sterilized time was 10min, the effect of sterilization was not efficient. The optimal length of stem explant was 1.0 cm. more or fewer than 1.0 cm, the effect of sterilization and utilization of explant was not efficient. The medium of MS supplemented with 0.5 mg·L^-1 6-BA and 1.0 mg·L^-1 GA₃ was suitable to germinate of Ilex chinensis, the germination rate could reach 46.7%. When there were only cytokinin and auxin, the better hormone combination was 1.0 mg·L^-1 6-BA and 0.05 mg·L^-1 NAA.The optimal basal medium for propagation was MS which contains high concentration nutrition. For proliferation of Ilex chinensis, MS was propitious to WPM which contains low concentration nutrition. At the same concentrations, 6-BA was more efficient than KT and 2iP in propagation, the rate of propagation descended when the cytokinin concentration rised. When medium supplemented with 0.5～1.0mg-L^-1 6-BA or 0.5 mg·L^-1 2iP and 0.05 mg·L^-1 NAA, the rate of propagation and quality of in vitro plantlet were the best. The optimal auxin was NAA, the NAA should be used at the concentration of 0.05～0.1 mg·L^-1. The different concentration of NAA and IBA for rooting was studied in rooting culture of Ilex chinensis. At the same concentration, NAA was more efficient than IBA, rate of rooting could reach 83.3%.The basal mediums for rooting were compared, 1/2MS(half of macronutrients) was more efficient than MS and WPM. 1/2MS (half of macronutrients) supplemented with 0.4 mg.L^-1NAA was optimal for rooting. No significant difference was indicated as added activated charcoal (AC).Callus induction by leaf of in vitro plantlet was studied. The effects of different cytokinins on callus induction were indicated that: 6-BA had best effects; KT took second place; TDZ was not efficient. MS supplemented with 6-BA 1.0 mg.L^-1 and 2,4-D 0.5 mg.L^-1, rate of callus induction could reach 93.3%. The optimal auxin was 2,4-D, NAA took second place; IAA was not efficient, the 2,4-D should be used at the concentration of 0.5～1.0 mg.L^-1. The effects of light and dark on callus induction had no significant difference. The quality of callus formed in light was better than dark. However, the callus did not regenerate shoots, callus would become brown on medium after 30 days without new subculture. Adventitious roots regenerated from callus when they subculture on MS medium containning 1.0 mg.L^-16-BA and 0.5mg·L^-12,4-D.Photoautotrophic culture of Ilex chinensis was studied, photosynthetic environment was controled by different concentrations of CO₂ and photosynthetic photon flux density(PPFD). Mediums contained different concentration of sucrose.The physiological index,including chlorophylla,chlorophyllb,carotenoid,soluble saccharide,soluble protein,root vigour,fresh weight and dry weight, was measured. The growth of chinese holly plantlets was promoted by environmental control, photosynthetic capacity of in vitro plantlet could be improved, sucrose in medium also influenced plantlets. Plantlets would grow perfectly by photosynthetic environmental control when sucrose in medium was reduced. Some physiological index would go down when the concentration of CO₂ was too high. We primarily concluded the optimal combination was CO₂ 1000μmol·mol^-1,PPFD 100μmol·m^-2·s^-1 and 20 g·L^-1 sucrose in medium.