豆梨离体再生体系建立与rol B基因转化研究
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摘要
豆梨是南方梨树常用砧木之一,适应性强,抗病,与梨的嫁接亲和性强。本研究以豆梨为试验材料,研究了影响豆梨子叶、茎段、叶片再生的一些因素,并将通过农杆菌介导方法将rolB基因导入豆梨子叶内,为豆梨的组织培养和遗传转化提供依据。主要研究结果如下:
1.摸索出了豆梨种子二次消毒法,筛选出了比较适宜豆梨子叶的培养基,以MS+6-BA4.0mg/L+NAA0.2mg/L的子叶不定芽再生率最高,培养条件中的暗培养时间及子叶的接种方式均影响子叶的不定芽产生:不定芽继代时在MS+6-BA1.0mg/L+NAA0.1mg/L培养基上的4代不定芽平均增殖系数最高达9.12,1cm以上的不定梢经MS+IBA3.0mg/L~(-1)培养基诱导20天转入MS+蔗糖5.0g/L上正常生根,生根后大多可以移栽成活。
2.豆梨叶片、叶柄和节间经诱导均能产生愈伤组织,叶片在NN69+TDZ2.5mg/L+0.1NAAmg/L的培养基上有最高不定芽再生率为50%,叶柄和节间在NN69+6-BA2.5mg/L+NAA0.2mg/L的培养基上最高不定芽再生率分别为25%和15%。
3.通过对子叶预培养时间、共培时间、菌液浸染时间、选择压等遗传转化影响因素的研究,初步建立了豆梨遗传转化体系:子叶暗培养4天后,经OD600值为0.6的农杆菌浸染10分钟后,黑暗共培60小时,转入筛选培养基上暗培养至抗性愈伤组织产生,后转入光培养获得抗性不定芽,此芽继代3-4次后扩繁。
4.实验获得了7株具有Km抗性的豆梨转化植株,经PCR检测有4株呈阳性,后续研究有待进行。
P.calleryana Dcne.is the mostly common used rootstock in South China,with high adaptability and resistance,which is compatible with both Chinese pear and European pear Cotyledons,stem pieces and leaves of P.calleryana Dcne.were used as explant to investigate some influencing factors and rolB gene was introduced into cotyledons of it mediated by Agrobacterium tumefaciens.A practical method was adopted for issue culture and gene transformation of P.calleryana Dcne.The main results were as follow:
1.Two-time sterilized method and the medium for Cotyledon culture was optimized and selected.The cotyledons could regenerate the most adventitious buds on MS+6-BA 4 mg/L +NAA 0.2 mg/L.The buds proliferated about 9.12 times in every subculture during one to four subculture on MS+6-BA 1.0 mg/L+NAA 0.1 mg/L.Shoots higher than one centimeter rooted regularly induced on 1/4 MS+IBA 3mg/Ltwenty days then transferred onto MS+sugar5.0 g/L.Most of the rooted shoots survived after planted.
2.Internode and leaves of P.calleryana Dcne.could produced callus after induced.Half of leaves produced buds on NN69+TDZ2.5 mg/L+ NAA 0.1 mg/L while 27.5 percent of the internodes produced buds on NN69+6-BA2.5 mg/L+NAA 0.2 mg/L.
3.Factors which influenced the percentage of gene transformation such as pre-culture time, co-culture time,time of dipping and intension of selection were studied The tentative transformation system of P.calleryana Dcne was developed as follows:first the cotyledons were pre-cultured in darkness four days,then dipped into agrobacterium suspension for ten minutes.After the treated cotyledons were co-cultured in darkness for another sixty hours,they were cultured on the selection medium until the resistance callus formed The callus were sub-cultured in light to obtain resistance buds.Those buds were propagated after three to four times of sub-culture.
4.In the study,seven P.calleryana Dcne plants with resistance to Km were obtained.Four of them were detected positive to PCR.Continued detection were expected to be carried out.
1.摸索出了豆梨种子二次消毒法,筛选出了比较适宜豆梨子叶的培养基,以MS+6-BA4.0mg/L+NAA0.2mg/L的子叶不定芽再生率最高,培养条件中的暗培养时间及子叶的接种方式均影响子叶的不定芽产生:不定芽继代时在MS+6-BA1.0mg/L+NAA0.1mg/L培养基上的4代不定芽平均增殖系数最高达9.12,1cm以上的不定梢经MS+IBA3.0mg/L~(-1)培养基诱导20天转入MS+蔗糖5.0g/L上正常生根,生根后大多可以移栽成活。
2.豆梨叶片、叶柄和节间经诱导均能产生愈伤组织,叶片在NN69+TDZ2.5mg/L+0.1NAAmg/L的培养基上有最高不定芽再生率为50%,叶柄和节间在NN69+6-BA2.5mg/L+NAA0.2mg/L的培养基上最高不定芽再生率分别为25%和15%。
3.通过对子叶预培养时间、共培时间、菌液浸染时间、选择压等遗传转化影响因素的研究,初步建立了豆梨遗传转化体系:子叶暗培养4天后,经OD600值为0.6的农杆菌浸染10分钟后,黑暗共培60小时,转入筛选培养基上暗培养至抗性愈伤组织产生,后转入光培养获得抗性不定芽,此芽继代3-4次后扩繁。
4.实验获得了7株具有Km抗性的豆梨转化植株,经PCR检测有4株呈阳性,后续研究有待进行。
P.calleryana Dcne.is the mostly common used rootstock in South China,with high adaptability and resistance,which is compatible with both Chinese pear and European pear Cotyledons,stem pieces and leaves of P.calleryana Dcne.were used as explant to investigate some influencing factors and rolB gene was introduced into cotyledons of it mediated by Agrobacterium tumefaciens.A practical method was adopted for issue culture and gene transformation of P.calleryana Dcne.The main results were as follow:
1.Two-time sterilized method and the medium for Cotyledon culture was optimized and selected.The cotyledons could regenerate the most adventitious buds on MS+6-BA 4 mg/L +NAA 0.2 mg/L.The buds proliferated about 9.12 times in every subculture during one to four subculture on MS+6-BA 1.0 mg/L+NAA 0.1 mg/L.Shoots higher than one centimeter rooted regularly induced on 1/4 MS+IBA 3mg/Ltwenty days then transferred onto MS+sugar5.0 g/L.Most of the rooted shoots survived after planted.
2.Internode and leaves of P.calleryana Dcne.could produced callus after induced.Half of leaves produced buds on NN69+TDZ2.5 mg/L+ NAA 0.1 mg/L while 27.5 percent of the internodes produced buds on NN69+6-BA2.5 mg/L+NAA 0.2 mg/L.
3.Factors which influenced the percentage of gene transformation such as pre-culture time, co-culture time,time of dipping and intension of selection were studied The tentative transformation system of P.calleryana Dcne was developed as follows:first the cotyledons were pre-cultured in darkness four days,then dipped into agrobacterium suspension for ten minutes.After the treated cotyledons were co-cultured in darkness for another sixty hours,they were cultured on the selection medium until the resistance callus formed The callus were sub-cultured in light to obtain resistance buds.Those buds were propagated after three to four times of sub-culture.
4.In the study,seven P.calleryana Dcne plants with resistance to Km were obtained.Four of them were detected positive to PCR.Continued detection were expected to be carried out.
引文
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