百合杂交育种、遗传转化及胚性愈伤的初步研究
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摘要
百合是多年生球根花卉,属于单子叶植物,具有较高的经济价值和观赏价值,现已成为世界上最重要的鲜切花之一。但是百合的种球容易退化,花粉污染花瓣,病害、虫害等问题严重制约着百合的生产和市场需求,因此,采用各种有效的手段培育出抗性强、无花粉、不退化的百合新品种已成为当务之急。本研究从百合的杂交育种、遗传转化两个方面对百合新品种的创制进行了初步的探索,结果如下:
(1)杂交育种
杂交组合麝香百合(♀)×线叶百合(♂)与晶体百合(♀)×麝香百合(♂)的杂交子房在自然条件下得不到成熟的种子。采用胚抢救的方法,分别授粉后10天、25天进行胚珠培养(以B5、1/2B5为基本培养基)获得了杂交苗;其中杂交组合麝香百合(♀)×兰州百合(♂)授粉10天后采用胚珠培养的方法得到了1.17%的抢救成功率;而杂交组合晶体百合(♀)×麝香百合(♂)授粉25天后进行胚珠培养得到了1.00%抢救成功率。整个实验过程中观察到了胚珠体外的畸形与正常发育情况,并且对杂交苗进行了ISSR分子鉴定。
(2)遗传转化
再生体系建立起了索邦、西伯利亚、麝香百合、元帅四个百合品种的小鳞片、叶片、叶柄的再生体系,其中再生率小鳞片可以达到95%以上,叶柄达到90%以上,叶片达到80%以上。
转化体系以麝香百合、索邦、西伯利亚三个品种的叶片、叶柄、小鳞片作为材料,从基本培养基、浸染时间,共培养时间、菌株选择、菌液浓度、乙酰丁香酮(AS)、外源激素、外植体表面损伤等方面进行了转化体系的探索。结果表明:以索邦的叶片和叶柄作为浸染材料,以没有硝态氮的MS培养基为基本培养基,以农杆菌C58作为转化菌株,表面刻伤后浸染20分钟,共培养4天后进行选择培养效果比较好,得到了较好的GUS的瞬间表达,并且的得到了一定数量的抗性苗。
(3)胚性愈伤状态的调整与再生
麝香百合假鳞茎的横切薄片诱导出的胚性愈伤组织,从氯化钴、ABA、蔗糖浓度以及光照条件四个方面对胚性愈伤组织的状态进行调整,结果表明:0.05μM/L的氯化钴有利于提高体细胞胚的比例和颗粒性,0.02μM/L和0.01μM/L的氯化钴有利于愈伤体积增大和增殖系数提高;ABA随着其浓度提高,对胚性愈伤的长势、干湿程度、增殖体积和增殖系数的抑制效果越明显,但对胚性愈伤比例和颗粒性的抑制效果不显著;光培养条件下,90g/L和60g/L蔗糖对麝香百合胚性愈伤的比例和颗粒性都有较好的影响,但在暗培养条件下,仅仅60g/L蔗糖的效果较好;光培养和暗培养对胚性愈伤的干湿程度和颗粒性都没有显著的影响。综合结果,麝香百合胚性愈伤的最佳增殖方式为:采用光培养,培养基为:MS+NAA(5.4μM/L)+TDZ(0.4μM/L)+CoC12(0.05μM/L)+蔗糖(60g/L)。
Lily is a kind of perennial monocotyledonous bulbar flower which has high economical and ornamental value,and now has been one of the most important fresh flowers.But the bulbs degenerated severely,the pollens stained the petals,and the plant diseases and insect pests restricted the production and marketing remand of lily.so, cultivaring the innovative breeds which is high fastness,no pollens,no degeneration by some effective technology is very necessary.In present study,primary study was conducted for the lily innovative breeds by the technology of cross breeding and genetic transfomation.The results were as fllows:
(1) Cross breeding
Two combinations of pollination was carried out in lilium longiflorum(♀)×Lilium lophophorum var.linearifolium(♂) and lilium longigflorum 'Gelria'(♀)×lilium longiflorum(♂),but no mature seeds were obtained from these two combinations in vivo culture.Ovules excised from each carpel 10 and 25 days after pollination(DAP) were cultured on B5 medium and half-strength B5 medium containing sucrose at different concentrations in vitro.In lilium longiflorum(♀)×Lilium lophophorum var.linearifolium (♂),1.17%of ovules excised at 10 DAP developed into seedlings.In lilium longiflorum'Gelria'(♀)×lilium longiflorum(♂),1.00%of ovules excised at 25 DAP developed into seedlings.In the present study,we observed normal and abnormal embryo development,and identified hybrids based on inter-simple sequence repeat(ISSR) analyse.
(2) Genetic transfomation
Regeneration system In present study,perfect regeneration system has been established,the regeneration rate of pseudo-bulblet,ledfstalk and leaf which come from Oriental hybrid lily(Lilium L.) cvs.'Sorbonne','Siberia','Acapulco'and lilium longiflorum can be reach to 95%,90%and 80%respectively.
Transmit system Pseudo-bulblet,ledfstalk and leaf come from Oriental hybrid lily (Lilium L.) cvs.'Sorbonne','Siberia' and lilium longiflorum were utilized as explants in present study.The transmit system was studyed from basical culture medium,infaction time,co-culture time,Kan selecting concentration,bacterium selecting,hormone and scaring on surface of explants,then we obtained the optimal transmit system:improved MS culture medium(no NH_4NO_3),bacterium C58,infected 20 min,co-cultured 4 days, scared the explant surface,Kan 120,100,8凹?/L separate to pseudo-bulblet,ledfstalk and leaf.
(3) Adjustment of the status of embryogenic callus and plant regeneration of Lilium longiflorum
Experiments were performed in order to obtain embryogenic callus with better status.The effect of different concentrations of CoCl_2,ABA and sucrose,in addition,the condition of illumination were studied.Enhanced proportion and dispersion of somatic embryos were achieved by the use of CoCl_2 with the concentration of 0.05μM/L,and improved propagation volume and propagation coefficient of somatic embryos were obtained using 0.01μM/L and 0.02μM/L CoCl_2;Some negative effect on growing status, water content,propagation volume and propagation coefficient of somatic embryos was observed when the concentration of ABA was gradually increased,while proportion and dispersion of somatic embryos were not affected by different ABA concentrations;It was beneficial for proportion and dispersion of somatic embryos by adding 6.0g/L or 9.0g/L sucrose and cultured in light,whereas,when somatic embryos were cultured at dark,only 6.0g/L sucrose could improve proportion and dispersion;Furthermore,the water content and dispersion of somatic embryos were not affected whether cultured in light or not.Considering all factors in present study,the results demonstrated that the most appropriate condition for the proliferation of somatic embryos was MS medium supplemented with NAA(5.4μM/L),TDZ(0.4μM/L),CoCl_2(0.05μM/) and sucrose (6.0g/L) under light.
(1)杂交育种
杂交组合麝香百合(♀)×线叶百合(♂)与晶体百合(♀)×麝香百合(♂)的杂交子房在自然条件下得不到成熟的种子。采用胚抢救的方法,分别授粉后10天、25天进行胚珠培养(以B5、1/2B5为基本培养基)获得了杂交苗;其中杂交组合麝香百合(♀)×兰州百合(♂)授粉10天后采用胚珠培养的方法得到了1.17%的抢救成功率;而杂交组合晶体百合(♀)×麝香百合(♂)授粉25天后进行胚珠培养得到了1.00%抢救成功率。整个实验过程中观察到了胚珠体外的畸形与正常发育情况,并且对杂交苗进行了ISSR分子鉴定。
(2)遗传转化
再生体系建立起了索邦、西伯利亚、麝香百合、元帅四个百合品种的小鳞片、叶片、叶柄的再生体系,其中再生率小鳞片可以达到95%以上,叶柄达到90%以上,叶片达到80%以上。
转化体系以麝香百合、索邦、西伯利亚三个品种的叶片、叶柄、小鳞片作为材料,从基本培养基、浸染时间,共培养时间、菌株选择、菌液浓度、乙酰丁香酮(AS)、外源激素、外植体表面损伤等方面进行了转化体系的探索。结果表明:以索邦的叶片和叶柄作为浸染材料,以没有硝态氮的MS培养基为基本培养基,以农杆菌C58作为转化菌株,表面刻伤后浸染20分钟,共培养4天后进行选择培养效果比较好,得到了较好的GUS的瞬间表达,并且的得到了一定数量的抗性苗。
(3)胚性愈伤状态的调整与再生
麝香百合假鳞茎的横切薄片诱导出的胚性愈伤组织,从氯化钴、ABA、蔗糖浓度以及光照条件四个方面对胚性愈伤组织的状态进行调整,结果表明:0.05μM/L的氯化钴有利于提高体细胞胚的比例和颗粒性,0.02μM/L和0.01μM/L的氯化钴有利于愈伤体积增大和增殖系数提高;ABA随着其浓度提高,对胚性愈伤的长势、干湿程度、增殖体积和增殖系数的抑制效果越明显,但对胚性愈伤比例和颗粒性的抑制效果不显著;光培养条件下,90g/L和60g/L蔗糖对麝香百合胚性愈伤的比例和颗粒性都有较好的影响,但在暗培养条件下,仅仅60g/L蔗糖的效果较好;光培养和暗培养对胚性愈伤的干湿程度和颗粒性都没有显著的影响。综合结果,麝香百合胚性愈伤的最佳增殖方式为:采用光培养,培养基为:MS+NAA(5.4μM/L)+TDZ(0.4μM/L)+CoC12(0.05μM/L)+蔗糖(60g/L)。
Lily is a kind of perennial monocotyledonous bulbar flower which has high economical and ornamental value,and now has been one of the most important fresh flowers.But the bulbs degenerated severely,the pollens stained the petals,and the plant diseases and insect pests restricted the production and marketing remand of lily.so, cultivaring the innovative breeds which is high fastness,no pollens,no degeneration by some effective technology is very necessary.In present study,primary study was conducted for the lily innovative breeds by the technology of cross breeding and genetic transfomation.The results were as fllows:
(1) Cross breeding
Two combinations of pollination was carried out in lilium longiflorum(♀)×Lilium lophophorum var.linearifolium(♂) and lilium longigflorum 'Gelria'(♀)×lilium longiflorum(♂),but no mature seeds were obtained from these two combinations in vivo culture.Ovules excised from each carpel 10 and 25 days after pollination(DAP) were cultured on B5 medium and half-strength B5 medium containing sucrose at different concentrations in vitro.In lilium longiflorum(♀)×Lilium lophophorum var.linearifolium (♂),1.17%of ovules excised at 10 DAP developed into seedlings.In lilium longiflorum'Gelria'(♀)×lilium longiflorum(♂),1.00%of ovules excised at 25 DAP developed into seedlings.In the present study,we observed normal and abnormal embryo development,and identified hybrids based on inter-simple sequence repeat(ISSR) analyse.
(2) Genetic transfomation
Regeneration system In present study,perfect regeneration system has been established,the regeneration rate of pseudo-bulblet,ledfstalk and leaf which come from Oriental hybrid lily(Lilium L.) cvs.'Sorbonne','Siberia','Acapulco'and lilium longiflorum can be reach to 95%,90%and 80%respectively.
Transmit system Pseudo-bulblet,ledfstalk and leaf come from Oriental hybrid lily (Lilium L.) cvs.'Sorbonne','Siberia' and lilium longiflorum were utilized as explants in present study.The transmit system was studyed from basical culture medium,infaction time,co-culture time,Kan selecting concentration,bacterium selecting,hormone and scaring on surface of explants,then we obtained the optimal transmit system:improved MS culture medium(no NH_4NO_3),bacterium C58,infected 20 min,co-cultured 4 days, scared the explant surface,Kan 120,100,8凹?/L separate to pseudo-bulblet,ledfstalk and leaf.
(3) Adjustment of the status of embryogenic callus and plant regeneration of Lilium longiflorum
Experiments were performed in order to obtain embryogenic callus with better status.The effect of different concentrations of CoCl_2,ABA and sucrose,in addition,the condition of illumination were studied.Enhanced proportion and dispersion of somatic embryos were achieved by the use of CoCl_2 with the concentration of 0.05μM/L,and improved propagation volume and propagation coefficient of somatic embryos were obtained using 0.01μM/L and 0.02μM/L CoCl_2;Some negative effect on growing status, water content,propagation volume and propagation coefficient of somatic embryos was observed when the concentration of ABA was gradually increased,while proportion and dispersion of somatic embryos were not affected by different ABA concentrations;It was beneficial for proportion and dispersion of somatic embryos by adding 6.0g/L or 9.0g/L sucrose and cultured in light,whereas,when somatic embryos were cultured at dark,only 6.0g/L sucrose could improve proportion and dispersion;Furthermore,the water content and dispersion of somatic embryos were not affected whether cultured in light or not.Considering all factors in present study,the results demonstrated that the most appropriate condition for the proliferation of somatic embryos was MS medium supplemented with NAA(5.4μM/L),TDZ(0.4μM/L),CoCl_2(0.05μM/) and sucrose (6.0g/L) under light.
引文
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