【作者】 梁茂厂；

【导师】 刘友全；

【作者基本信息】 中南林业科技大学 ， 林木遗传育种， 2008， 硕士

【摘要】 本研究旨在对桂花(Osmanthus fragrans Lour.)组织培养不同发生途径进行研究。实验包括以当年生幼嫩枝条、无菌苗茎段和胚为外植体的无菌短枝扦插再生途径的组织培养技术研究和以叶片、胚为外植体的愈伤组织发生途径的组织培养技术研究。研究结果如下：1桂花无菌短枝扦插再生途径组织培养技术的研究(1)无菌体系建立种子消毒易控制,接种污染率低。当年生幼嫩枝条以0.1%升汞处理3-6 min为宜,其中茎尖污染率低。(2)初代培养与B5、WPM培养基相比,MS培养基更有利于胚的初代萌发；对种胚进行N+注入时,剂量小于1×1014N+·cm-2时利于种胚的萌发；4℃低温冷藏处理210天能有效打破种胚的自然休眠,促进种子提前萌发。与B5、MS培养基相比,WPM培养基更有利于幼嫩茎段的萌发；在控制茎段褐化时,活性炭用量应控制在0.1%以内。(3)幼胚苗增殖培养B5+0.50 mg·L-1TDZ+0.05 mg·L-1NAA利于丛生芽增殖诱导；B5+5.00 mg·L-16-BA+0.10 mg·L-1NAA、B5+1.00 mg·L-1-BA+1.00mg·L-1 GA、WPM+0.50 mg·L-1TDZ+0.05 mg·L-1NAA+0.50 mg·L-1BR利于胚苗伸长增殖培养；离子注入的种胚在胚苗伸长培养过程中平均高2.04 cm；未处理的种胚在胚苗伸长培养过程中平均高2.77 cm,两者差异显著。(4)茎段苗增殖培养B5+7.00 mg·L-16-BA+O.10 mg·L-1NAA、B5+0.10mg·L-1 TDZ+0.05 mg·L-1NAA利于丛生芽诱导；B5+8.00 mg·L-1KT+0.05 mg·L-1NAA、B5+2.00 mg·L-16-BA+1.00 mg·L-1GA、B5+2.00 mg·L-16-BA+0.10 mg·L-1BR、WPM+ 0.50 mg·L-1TDZ+0.05 mg·L-1NAA+0.50 mg·L-1BR、B5+2.00mg·L-16-BA+3.00 mg·L-1生物素利于茎段伸长培养诱导。(5)生根培养1/2MS+0.60mg·L-16-BA+4.00mg·L-1NAA利于生根培养的诱导。2桂花愈伤组织发生途径组织培养技术的研究(1)愈伤组织初代诱导培养WPM+0.12mg·L-12,4-D利于胚愈伤组织诱导；B5+0.12 mg·L-12,4-D+1.50 mg·L-16-BA利于幼嫩叶片愈伤组织诱导。B5+5.00mg·L-16-BA+0.10 mg·L-1NAA利于无菌苗叶片愈伤组织诱导。(2)愈伤组织增殖诱导培养WPM+0.50 mg·L-1 TDZ利于胚愈伤组织的增殖;WPM+0.10 mg·L-1 BR+0.10 mg·L-1 NAA利于叶片愈伤组织和胚愈伤组织的增殖。(3)愈伤组织分化诱导培养B5+0.40 mg·L-1TDZ+0.10 mg·L-1ZT+0.50 mg·L-1BR利于胚愈伤组织的分化；WPM+1.00 mg·L-1TDZ+0.05 mg·L-1NAA、B5+5.00 mg·L-16-BA+0.10 mg·L-1NAA利于叶片愈伤组织的分化。(4)生根培养1/2MS+0.60 mg·L-16-BA+4.00 mg·L-1NAA利于生根诱导培养。更多还原

【Abstract】 The experiment purpose is to do the research of Osmanthus fragrans Lour. plant tissue culture which happened in variety of ways. The experiment contained two facets of tissue culture research.One facet is that taking the young tress,germfree seedling stem and embryo as explants to regenerate in the form of germfree short tress cutting.The other is that taking young leaf,embryo as explants to occur in the form of callus.The results are as follows:1 Tissue culture research of regenerating in the form of germfree short tress cutting(1)Germfree system established The seed which inoculated at low contaminat-ion rate is easy to disinfect. Using Hgcl20.1% to disinfect young new tress for 3-6 minutes is available.The top bud is at low contamination rate.(2)Initial culture comparing with medium B5 and WPM, medium MS is more available for embryo initial germinating;Less than the dosage of 1×1014 N+·cm-2 is available for embryo germinating when N+ immitting in the embryo body.Dealing with the seed in the environment of low temperature 4℃and cold storage for 210 days can break embryo dormancy in effect,accelerating the embryo to bourgeon in advance. Comparing with medium B5 and MS, medium WPM is more available for young stem germinating;While controlling the stem not to be browned,the AC concentration should be controled within 0.1%.(3) Multiplication culture for embryo plants B5+0.50 mg·L-1TDZ+0.05 mg·L-1 NAA is available for tuft buds inducement. B5+5.00 mg·L-1VBA+0.10 mg·L-1NAA, B5+1.00 mg·L-16-BA+1.00 mg·L-1GA and WPM+0.50 mg·L-1 TDZ+0.05 mg·L-1 NAA+0.50 mg·L-1BR are available for elongation and multiplication culture of embryo plants.The plant with ion beam implantation in embryo is at about 2.04 cm high in elongating culture process; The plant with no ion beam implantation in embryo is at about 2.77 cm high in elongating culture process; The diversity between each other is notable(4) Multiplication culture for stem plants B5+7.00 mg·-16-BA+0.10 mg·L-1 NAA,B5+0.10 mg·L-1TDZ+0.05 mg·L-1NAA are available for tuft buds inducement. B5+8.00 mg·L-1KT+0.05 mg·L-1NAA,B5+2.00 mg·L-16-BA+1.00 mg·L-1GA,B5+2.00 mg·L-16-BA+0.10 mg·L-1BR,WPM+0.50 mg·L-1TDZ+0.05 mg·L-1NAA+0.50 mg·L-1 BR and B5+2.00 mg·L-6-BA+3.00 mg·L-1Biotin are available for stem elongation inducement.(5)Rooting culture 1/2MS+0.60 mg·L-16-BA+4.00 mg·L-1 NAA is available for rooting culture inducement.2 Tissue culture research of occuring in the form of callus(1)Initial callus inducement culture WPM+0.12 mg·L-12,4-D is available for embryo callus inducement. B5+0.12 mg·L-12,4-D+1.50 mg·L-16-BA is available for young leaf callus inducement. B5+5.00 mg·L-16-BA+0.10 mg·L-1NAA is available for young sterile plant leaf callus inducement.(2)Multiplication callus inducement culture WPM+0.50 mg·L-1TDZ is available for embryo callus multiplication. WPM+0.10 mg·L-1BR+0.10 mg·L-1 NAA is available for leaf and embryo callus multiplication.(3) Polarization callus inducement culture B5+0.40 mg·L-1TDZ+0.10 mg·L-1 +0.50mg·L-1BR is available for embryo callus polarization. WPM+1.00 mg·L-1TDZ +0.05 mg·L-1NAA and B5+5.00 mg·L-16-BA+0.10 mg·L-1NAA are available for leaf callus polarization.(4)Rooting culture 1/2MS+0.60 mg·L-16-BA+4.00 mg·L-1NAA is available for rooting culture inducement.更多还原