Study On Tissue Culture And Volatile Oil Composition Of Rosa Chinensis 'Double Delight'

This experiment used annual branches?stems,leaves,petioles?and buds?petals,filaments?of Rosa chinensis‘Double Delight'as experimental materials,which studied the main influencing factors in the primary culture,subculture,rooting culture,seedling cultivation and transplantation of rose by using the techniques of plant tissue culture,orthogonal design and response surface design and other experimental methods.The in vitro rapid propagation technology system of the Rosa chinensis‘Double Delight'was established,and the technical system can provide theoretical basis and technical guidance for its large-scale production.The headspace solid phase microextraction?HS-SPME?combined with gas chromatography-mass spectrometry?GC-MS?was used to study the volatile oil components of the Rosa chinensis‘Double Delight'including the petals of flower buds,petals in full bloom,stems and leaves.And the types and contents of volatile substances were determined.It aims to develop the potential value of the plant and lay the foundation and theoretical basis for its research and comprehensive development and utilization in other aspects.The main results were as follows:1.Primary culture stage?1?Disinfection of explants:the best disinfection treatment of stem segments was 75%C₂H₅OH 30s+0.1%HgCl₂ 6min,the survival rate was 97.78%;and the optimal disinfection treatment of leaves and petioles was 75%C₂H₅OH 45 s+0.1%HgCl₂ 8 min,and the survival rate was 84.45%and 85.56%,respectively.The best disinfection treatment of petals and filaments was75%C₂H₅OH 30s+0.1%HgCl₂ 7min,and the survival rates were 97.78%and100%,respectively.?2?Adventitious bud induction:The stem segmemta with buds point was used as the test material,and the optimal induction medium was MS+3.00 mg·L^-1 6-BA+0.30 mg·L^-1 NAA,and the induction rate was 95.56%.?3?Callus induction:callus were induced in the stem section internodes,stock plant leaves,petioles,tissue culture seedling leaves,petals and filaments,and the best explants were induced callus by seedling leaves.The optimal induction medium was MS+1.79 mg·L^-1 2,4-D+2.12mg·L^-1 NAA+0.15 mg·L^-1,and the induction rate was 93.33%.2.Subculture stage?1?adventitious bud proliferation:MS+1.00 mg·L^-1 6-BA+0.10 mg·L^-1 IBA was the best adventitious bud proliferation medium;30 g·L^-1 sucrose was the best carbon source concentration;40 day was the best secondary culture cycle,and the proliferation coefficient could reach 6.89.The tissue culture seedlings grow well and the leaves were emerald green.?2?Callus differentiation:The optimal callus differentiation medium was MS+2.00 mg·L^-16-BA+0.20 mg·L^-1 IBA+0.20 mg·L^-1 GA₃,and the callus differentiation rate was 43.20%.3.Rooting stage?1?Rooting in the bottle:Rooting outside the bottle was the best way to root.The optimum medium for rooting in the optimum bottle was 1/4MS+0.05 mg·L^-1 IBA+2.50 g·L^-1 activated carbon?Ac?,and the rooting rate is 95.56%.The adventitious root is mostly white,and the root length is about 5.3cm.?2?Rooting outside the bottle:The seedlings were cultivated indoors for 3 days,and the cuttings were placed in a matrix?V fine sand:Vermiculite:V perlite=1:1:1?mixed with rooting nutrient solution(1/6MS+0.05 mg·L^-1 IBA+0.15 mg·L^-1 NAA).At the same time,the air humidity was maintained at 90%,and the rooting rate outside the bottle could reach 88.89%.4.Acclimatizating and transplanting stageThe optimal acclimatizating time before transplanting of tissue culture seedlings were 5days.The optimum transplanting substrate was V fine sand:V vermiculite:V perlite:V coconut meal=1:2:3:1,the survival rate of transplanting was 96.67%.5.Analysis of volatile oil components:?1?Headspace solid phase micro-extraction head screening:The headspace solid phase micro-extraction head of the optimal Rosa chinensis‘Double Delight'volatile oil extraction was85?m CAR/PDMS in terms of the amount and relative content of the extracted components.?2?GC-MS Analysis:12 kinds of 126 constituents of volatile components,including terpenes,alcohols,aromatic hydrocarbons,esters and aldehydes and so on,were indentificated in the full bloom petal,flower bud petals,leaves and stem segments.All portion indentificated the relative content of the alcohols compounds were the highest,being 36.78%,36.36%,48.90%,and 39.43%,respectively.There were indentificated 12 kinds of 62 constituents of volatile substances in the petals of full bloom,and phenylethyl alcohol?29.97%?was its main aroma component;there were indentificated 11 kinds of 68 constituents volatile substances in the petals of flower bud,and the main aroma component was 3,5-dimethoxytoluene?22.24%?and phenylethyl alcohol?21.14%?;there were indentificated 11 kinds of 52 components in the leaves,among which linalool?46.08%?was the main volatile substance;8 kinds of 28 components were indentificated in the stem segment,and the main volatile substance was 1-nonanol?37.62%?.