西伯利亚白刺离体培养体系建立及试管苗耐盐性研究
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摘要
西伯利亚白刺隶属于蒺藜科(Zygophyllaceae)白刺属(Nitraria L),是典型荒漠植物,在稳定沙漠、保护绿洲中起着重要的作用。同时,它又是沙生植物中少有的药食两用的浆果类植物,具有较高的经济利用价值。本研究以西伯利亚白刺茎段为试材,从探讨最佳外植体取材时间、基本培养基、以及最佳增殖和生根培养基配方入手,建立起无菌离体培养体系。进而研究NaCl处理下西伯利亚白刺试管苗耐盐机理,为西伯利亚白刺种质资源保存和人工繁殖及盐生植物耐盐机理提供理论依据。研究结果如下:
1.以茎段为外植体,首次建立西伯利亚白刺的离体培养体系。外植体采集最佳时间为5月底。最佳启动培养基为MS+6-BA2.0 mg·L~(-1)+NAA 0.5 mg·L~(-1)腋芽启动萌发率91.11%;继代增殖培养基为MS+6-BA 1.0 mg·L~(-1)+NAA 0.2 mg·L~(-1)增殖系数高达4.53;生根培养基为1/2 MS+IBA 0.5 mg·L~(-1)生根率为96.67%。
2.以试管苗叶片为外植体,首次通过愈伤组织途径获得了西伯利亚白刺再生植株。黑暗培养一周后转为光周期培养,最佳诱导愈伤组织培养基为MS+2,4-D2.0 mg·L~(-1),诱导率高达100%,培养25d愈伤组织相对生长量为468.31%。
3.以试管苗为材料,最佳生根培养基附加不同浓度NaCl(0,25,50,100,200 mmol·L~(-1))。随着盐浓度的增加,植株的鲜重、干重以及根冠比呈现先上升后下降的变化趋势,在50 mmol·L~(-1)NaCl处理下,植株的鲜重、干重均比对照明显增加且为各处理的最大值,说明50 mmol·L~(-1)NaCl有利于白刺试管苗的生长。
4.随着NaCl浓度的增加,试管苗叶片中的可溶性糖和脯氨酸含量呈增加趋势:叶绿素含量变化呈现先升后降的趋势,在50 mmol·L~(-1)NaCl时达到最大值;而叶片中丙二醛含量则呈现先降后升趋势,在50 mmol·L~(-1)NaCl时达到最小值。这表明50 mmol·L~(-1) NaCl是西伯利亚白刺试管苗生长最适的盐浓度。
5.随着NaCl浓度增加,试管苗地上部和根系Na~+离子含量呈现增加趋势,在低浓度时(≤50 mmol·L~(-1))显著增加,而高浓度时(≥100 mmol·L~(-1))增加幅度不大;地上部K~+含量下降而根系K~+含量却呈现增加趋势;地上部、根系Ca~(2+)含量降低;白刺试管苗地上部和根系的Na~+/K~+、Na~+/Ca~(2+)比值都呈升高趋势。表明在低盐浓度下,试管苗通过地上部积累Na~+离子降低渗透势,适应盐环境,而在高盐浓度时根部积累一定的K~+对Na~+起到拮抗作用,减少高浓度盐的伤害。
总之,本研究建立了西伯利亚白刺离体培养体系,明确了50 mmol·L~(-1)NaCl是其试管苗生长的最适盐浓度。探讨了在NaCl胁迫下,白刺试管苗主要通过Na~+、K~+离子积累参与渗透调节的耐盐机理。
Nitraria sibirica Pall.is a species of Zygophyllaceae in Nitraria genus,which is an important kind of tree species to control sand.Not only its fruits have higher economic value,but also N.sibirica Pall.plays an important role on local environment protection.In this study,a tissue culture system of N.sibirica Pall.was established by its stem.then we discussed the mechanism of plant salt resistance of N.sibirica Pall.' test-tube plantlet,hoping to offer some references for the tissue culture and the mechanism of salt resistance of the salt plant.The main research conclusions were showed as follow:
1.It is the first time that a tissue culture system of N.sibirica Pall.was successfully established by its stem section.May middle is the best time when explants are collected.The optimal medium for inducing adventitious buds from stem explants was MS+6-BA 2.0mg·L~(-1)+NAA 0.5 mg·L~(-1),the rate of germination was 91.11%;the optimal medium for subculture culture was MS+6-BA 1.0 mg·L~(-1)+NAA 0.2 mg·L~(-1),the multiplication coefficients was 4.53;the optimal medium for rooting culture was 1/2MS+IBA 0.5 mg·L~(-1),the rate was 96.67%.
2.The callus formation and plant regeneration system were established by the leaf of test-tube plantlet,and it is also the first time.Cultured one week in the dark then changed in the Photoperiod of 14 h/10 h.The optimal medium for callus formation was MS+2,4-D 2.0 mg·L~(-1),the formation rates was 100%,the relation growth amount was 468.31%after 25 days.
3.NACl(0,25,50,100,200 mmol·L~(-1)) was added in the optimal rooting culture medium,N.sibirica Pall.'s test-tube plantlet were grown in these mediums.With the increase of NaCl concentration,fresh weight,dried weight and root/shoot rates were firstly increased and then decreased.Under 50 mmol·L~(-1) NaCl,test-tube plantlets' fresh weight and dried weight were more higher than others.It showed that 50 mmol·L~(-1) NaCl was the best salt concentration for the growth of test-tube plantlet.
4.With the increase of NaCl concentration,contents of soluble sugar,proline were increased.Meanwhile the contents of chlorophyll were firstly increase and then decreased,and have the maximum value under 50 mmol·L~(-1) NaCl.The content of MDA in the leaf were firstly decrease and then increased,and have the minimum value under 50 mmol·L~(-1) NaCl.These showed that 50 mmol·L~(-1) NaCl is the best salt concentration for test-tube plantlet growing.
5.With the increase of NaCl concentration,the concentration of Na~+ in shoot and root were increased,under low concentration(≤50 mmol·L~(-1)) NaCl,rapidly increased,but under high concentration(≥100mmol·L~(-1)) NaCl,increased slowly. Meanwhile concentration of K~+ in shoot was decrease,but concentration of K~+ in root was increased and concentration of Ca~(2+) in shoot and root were decreased and Na~+/K~+、Na~+/Ca~(2+) in shoot and root were increased.These showed that low concentration NaCl,Na~+ rapidly increased in shoot in order to adapting to salt stress, but high concentration NaCl,K~+ increased in root to antagonize Na~+,in order to avoid salt damage.
In conclusion,in the experiment,a tissue culture system of N.sibirica Pall.was established.It indicated that 50 mmol·L~(-1) NaCl was the best salt concentration for the growth of test-tube plantlet growing,and discussed that under NaCl stress,the mechanism of salt tolerance the test-tube plantlet of N.sibirica Pall.used by accumulation of Na~+、K~+.
1.以茎段为外植体,首次建立西伯利亚白刺的离体培养体系。外植体采集最佳时间为5月底。最佳启动培养基为MS+6-BA2.0 mg·L~(-1)+NAA 0.5 mg·L~(-1)腋芽启动萌发率91.11%;继代增殖培养基为MS+6-BA 1.0 mg·L~(-1)+NAA 0.2 mg·L~(-1)增殖系数高达4.53;生根培养基为1/2 MS+IBA 0.5 mg·L~(-1)生根率为96.67%。
2.以试管苗叶片为外植体,首次通过愈伤组织途径获得了西伯利亚白刺再生植株。黑暗培养一周后转为光周期培养,最佳诱导愈伤组织培养基为MS+2,4-D2.0 mg·L~(-1),诱导率高达100%,培养25d愈伤组织相对生长量为468.31%。
3.以试管苗为材料,最佳生根培养基附加不同浓度NaCl(0,25,50,100,200 mmol·L~(-1))。随着盐浓度的增加,植株的鲜重、干重以及根冠比呈现先上升后下降的变化趋势,在50 mmol·L~(-1)NaCl处理下,植株的鲜重、干重均比对照明显增加且为各处理的最大值,说明50 mmol·L~(-1)NaCl有利于白刺试管苗的生长。
4.随着NaCl浓度的增加,试管苗叶片中的可溶性糖和脯氨酸含量呈增加趋势:叶绿素含量变化呈现先升后降的趋势,在50 mmol·L~(-1)NaCl时达到最大值;而叶片中丙二醛含量则呈现先降后升趋势,在50 mmol·L~(-1)NaCl时达到最小值。这表明50 mmol·L~(-1) NaCl是西伯利亚白刺试管苗生长最适的盐浓度。
5.随着NaCl浓度增加,试管苗地上部和根系Na~+离子含量呈现增加趋势,在低浓度时(≤50 mmol·L~(-1))显著增加,而高浓度时(≥100 mmol·L~(-1))增加幅度不大;地上部K~+含量下降而根系K~+含量却呈现增加趋势;地上部、根系Ca~(2+)含量降低;白刺试管苗地上部和根系的Na~+/K~+、Na~+/Ca~(2+)比值都呈升高趋势。表明在低盐浓度下,试管苗通过地上部积累Na~+离子降低渗透势,适应盐环境,而在高盐浓度时根部积累一定的K~+对Na~+起到拮抗作用,减少高浓度盐的伤害。
总之,本研究建立了西伯利亚白刺离体培养体系,明确了50 mmol·L~(-1)NaCl是其试管苗生长的最适盐浓度。探讨了在NaCl胁迫下,白刺试管苗主要通过Na~+、K~+离子积累参与渗透调节的耐盐机理。
Nitraria sibirica Pall.is a species of Zygophyllaceae in Nitraria genus,which is an important kind of tree species to control sand.Not only its fruits have higher economic value,but also N.sibirica Pall.plays an important role on local environment protection.In this study,a tissue culture system of N.sibirica Pall.was established by its stem.then we discussed the mechanism of plant salt resistance of N.sibirica Pall.' test-tube plantlet,hoping to offer some references for the tissue culture and the mechanism of salt resistance of the salt plant.The main research conclusions were showed as follow:
1.It is the first time that a tissue culture system of N.sibirica Pall.was successfully established by its stem section.May middle is the best time when explants are collected.The optimal medium for inducing adventitious buds from stem explants was MS+6-BA 2.0mg·L~(-1)+NAA 0.5 mg·L~(-1),the rate of germination was 91.11%;the optimal medium for subculture culture was MS+6-BA 1.0 mg·L~(-1)+NAA 0.2 mg·L~(-1),the multiplication coefficients was 4.53;the optimal medium for rooting culture was 1/2MS+IBA 0.5 mg·L~(-1),the rate was 96.67%.
2.The callus formation and plant regeneration system were established by the leaf of test-tube plantlet,and it is also the first time.Cultured one week in the dark then changed in the Photoperiod of 14 h/10 h.The optimal medium for callus formation was MS+2,4-D 2.0 mg·L~(-1),the formation rates was 100%,the relation growth amount was 468.31%after 25 days.
3.NACl(0,25,50,100,200 mmol·L~(-1)) was added in the optimal rooting culture medium,N.sibirica Pall.'s test-tube plantlet were grown in these mediums.With the increase of NaCl concentration,fresh weight,dried weight and root/shoot rates were firstly increased and then decreased.Under 50 mmol·L~(-1) NaCl,test-tube plantlets' fresh weight and dried weight were more higher than others.It showed that 50 mmol·L~(-1) NaCl was the best salt concentration for the growth of test-tube plantlet.
4.With the increase of NaCl concentration,contents of soluble sugar,proline were increased.Meanwhile the contents of chlorophyll were firstly increase and then decreased,and have the maximum value under 50 mmol·L~(-1) NaCl.The content of MDA in the leaf were firstly decrease and then increased,and have the minimum value under 50 mmol·L~(-1) NaCl.These showed that 50 mmol·L~(-1) NaCl is the best salt concentration for test-tube plantlet growing.
5.With the increase of NaCl concentration,the concentration of Na~+ in shoot and root were increased,under low concentration(≤50 mmol·L~(-1)) NaCl,rapidly increased,but under high concentration(≥100mmol·L~(-1)) NaCl,increased slowly. Meanwhile concentration of K~+ in shoot was decrease,but concentration of K~+ in root was increased and concentration of Ca~(2+) in shoot and root were decreased and Na~+/K~+、Na~+/Ca~(2+) in shoot and root were increased.These showed that low concentration NaCl,Na~+ rapidly increased in shoot in order to adapting to salt stress, but high concentration NaCl,K~+ increased in root to antagonize Na~+,in order to avoid salt damage.
In conclusion,in the experiment,a tissue culture system of N.sibirica Pall.was established.It indicated that 50 mmol·L~(-1) NaCl was the best salt concentration for the growth of test-tube plantlet growing,and discussed that under NaCl stress,the mechanism of salt tolerance the test-tube plantlet of N.sibirica Pall.used by accumulation of Na~+、K~+.
引文
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