The Study Of Tissue Culture And Protoplast Preparation Of Gardenia Jasminoides Ellis

ObjectiveGardenia(Gardenia jasminoidesEllis) is a small perennial evergreen shrub which belongs to Gardenia genus in Rubiaceae family. Particularly in recent years,the wild resources of gardenia has been severely damaged, resulting in the germplasm resources with good characters is reducing.The planting area and yield of gardenia have been increased substantially because there is a big market demand. But the gardenia germplasm is degradating and the ability of resistance to diseases and pests is decreasing obviously because of the directional cultivation, single variety, lacking of management and other factors.This article aims to improve the genetical characterization decline of gardenia through the method of tissue culture and protoplast fusion.In this paper,gardenia fruit was used as explants to investigate the tissue culture which laid a technical foundation for protecting elite germplasm of gardenia,and protoplast preparation of gardenia was studied that laid a technical foundation for protoplast fusion,hoping for providing technology platform for the improvement of gardenia germplasm in the future.Methods1 Tissue culture of Gardenia1.1 Establishing the sterile system of the gardenia fruit(1)Time filtering of the sterilization that gardenia fruit was used as explants.Set the best sterilizing time with the pericarp and seeds of N-maturity Gardenia as the explant and 0.1% HgCl2 as the sterilizing agent(2)Screening the cutting way of the explant from gardenia fruitScreening the best cutting way of Gardenia fruit before sterilization with the N-maturity Gardenia as the explant when the sterilizing agent was 0.1% HgCl2 and the sterilization time is 9 min 1.2 Induction of callus(1) The screening of maturity of gardenia fruitScreening the best maturity of Gardenia fruit with the fruit at different ripening stages as the explant when the sterilizing agent was 0.1% HgCl2 and the sterilization time is 9 min.(2) Screening the appropriate illumination intensity for the induction of callusScreening the appropriate illumination intensity for the induction of callus from explants of pericarps, placentas with seeds and seeds of gardenia which three all at N-maturity.(3) Screening the combination of plant growth regulators in the course of calli inductionUsing the MS as the based culture medium, the optimum induction medium for callus from explants of pericarps, placentas with seeds, and seeds of gardenia which three all at N-maturity was obtained by screening combinations of 2,4-D and 6-BA at different concentrations.1.3 The differentiation of the callusUsing the MS as the based culture medium, on solid and suspension medium, the optimum induction medium for differentiation of the callus was obtained by screening combinations of 6-BAx 2,4-D and TDZ at different concentrations.1.4 The shoots proliferationUsing the MS as the based culture medium, the optimum induction medium for gardenia regeneration plant was obtained by screening combinations of 6-BA> NAA at different concentrations.2 The separation of gardenia protoplastThe protoplasts from gardenia tender leaves were isolated by the method of orthogonal test with four factors and four levels with Cellulase R-10, Macerozyme R-10, enzymolysis time and concentration of mannitol as the indexes.2.1 The separation of protoplast from G. jasminoides Ellis, f. logicarpa Z.W. Xie et OkadaThe protoplasts from G. jasminoides Ellis, f. logicarpa Z. W. Xie et Okada tender leaves were isolated by the method of orthogonal test with four factors and four levels with Cellulase R-10, Macerozyme R-10, enzymolysis time and concentration of mannitol as the indexes.Results1 Tissue culture of Gardenia1.1 Establishing a sterile system of the gardenia fruit(1)Time filtering of the sterilization that gardenia fruit was used as explantsThe best sterilizing time of the pericarp and seeds of N-maturity gardenia in 0.1% HgCl2 is 9 min.(2)Screening the cutting way with the fruit as the explantThe sterilization effect was better by separating the pericarps and placentas with seeds before sterilisation.1.2 Induction of callus(1) Screening the maturity of explant from the fruitThe gardenia fruit at N-maturity that the placentas with seeds’color was creamy white or yellow-tinged after after separated from the pericarps was better for the induction of callus.(2) Screening the appropriate illumination intensity for the induction of callusIn darkness, the callus induction rate of the 2 explants of pericarps and placentas with seeds was higher than that under the light conditions.,(3) Screening the combination of plant growth regulators in the course of calli induction.The optimal medium for callus induction from pericarp or seed was MS+ 0.5 mg·L-1 2,4-D+0.25 mg· L-1 6-BA. The induction rates were 83.3% and 88.5%, respectively. The optimal medium for callus induction from placentas with seeds was MS+1.0 mg·L-1 2,4-D+1.0 mg·L-16-BA and the induction rate was 78.1%.1.3 The differentiation of the callusOnly the calli induced from seeds could be differentiated to form buds. TDZ stimulated bud differentiation. The optimal medium for bud differentiation was MS+0.05 mg·L-1 NAA+0.10 mg·L-1 TDZ with a differentiation rate of 8.75%.2 The experimental results about protoplast isolation from gardenia2.1 The experimental results about protoplast isolation from gardeniaOptimizing the best separation conditions of the protoplasts from gardenia tender leaves. The optimal conditions for protoplast isolation were that the enzyme solution is 15 g·L-1 cellulase R-10+15 g·L-1 macerozyme R-10 +0.7 mol ·L-1 mannitol+100 mg·L-1 MES, the enzymolysis time is 8 h, and the pH value of enzyme solution is 5.6.2.2 The experimental results about protoplast isolation from G. jasminoides Ellis.f.logicarpa Z. W. Xie et OkadaOptimizing the best separation conditions of the protoplasts from tender leaves of G. jasminoides Ellis.f. logicarpa Z. W. Xie et Okada. The optimal conditions for protoplast isolation were that the enzyme solution is 3 g· L-1 cellulase R-10+10 g· L-1 macerozyme R-10+0.7 mol·L-1 mannitol+100 mg·L-1 MES, the enzymolysis time is 8 h, and the pH value of enzyme solution is 5.6.ConelusionWe established a plant tissue culture system with gardenia seeds as explants, which can serve as a technical platform for gene transformation. we had successfully isolated the active protoplast from tender leaves of gardenia and G. jasminoides Ellis.f. logicarpa Z. W. Xie et Okada, screened the the optimum conditions of their protoplasts separation.