柑橘试管苗种质保存及其内源激素变化研究
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摘要
本研究收集了42份柑桔种质资源。以柑桔成熟胚为外植体,建立柑桔离体再生体系;以雪柑珠心胚试管苗为材料,探讨柑桔试管苗限制生长保存方法并采用ISSR技术进行遗传稳定性的检测;采用限制保存方法对42份柑桔种质资源的成熟胚进行离体试管苗保存;进一步测定可溶性蛋白含量,POD、CAT等细胞保护酶活性以及游离脯氨酸含量等相关生理指标及采用HPLC技术测定内源激素的含量变化,探讨柑橘试管苗限制生长保存机理,为完善柑桔种质离体保存方法提供科学依据。主要研究结果如下:
1.柑桔试管苗再生体系的建立。以雪柑珠心胚试管苗为材料,分别研究其增殖、生根、移栽的最佳条件。试验结果表明:最佳增殖培养基为MS+1.0mg·L~(-1)BA+0.01mg·L~(-1)NAA,增殖系数达到9.70;通过对NAA、IBA不同浓度试验表明,最佳生根培养基为MS+0.4mg·L~(-1)IBA,IBA适合于柑橘生根诱导;最佳移栽基质为锯末:糠灰(1∶1)的混合基质。
2.柑桔试管苗离体保存方法的探讨。以雪柑珠心胚试管苗生长3个月后的茎切段为材料,比较了不同浓度的蔗糖、琼脂、甘露醇和蔗糖、多效唑等因素对柑桔试管苗限制生长保存的影响。结果表明:附加50～80 g·L~(-1)蔗糖的MS培养基,保存12个月时,存活率为85.6%;附加0.5 mg·L~(-1)多效唑,保存12个月时,存活率为86.8%。两者的保存效果相当。从保存成本和保存效果综合考虑,我们认为柑橘试管苗限制生产保存的适宜培养基为附加0.5 mg·L~(-1)多效唑。保存18个月后,进行柑桔生根试管苗移栽,植株生长健壮。
3.试管苗限制生长保存遗传稳定性的ISSR分析。以限制生长法保存了18个月与保存前的雪柑试管苗为材料,用ISSR-PCR标记技术对雪柑试管苗保存前后的遗传变异进行检测。结果表明:相同引物不同材料的ISSR图谱完全一致,说明在检测范围内没有发生DNA水平的变异,离体保存试管苗的遗传稳定性好,也说明这种限制生长保存方法的可行性。
4.42份柑桔种质资源离体保存材料的建立。收集了柑桔类主要品种的成熟胚42份,进行离体保存。试验结果表明,MS基本培养基附加0.5 mg·L~(-1)多效唑,适用大部分柑桔类品种成熟胚离体保存,其中雪柑成熟胚培养,保存15个月时存活率高达89.1%。对保存后成活的的植株进行移栽,能正常生长,且生长健壮。
5.柑桔试管苗限制生长保存中生理指标的测定。以保存在附加多效唑与未附加多效唑的培养基上的永春芦柑珠心胚试管苗为材料,进行试管苗限制生长保存过程中可溶蛋白含量、POD、CAT等细胞膜保护酶活性以及游离脯氨酸含量等生理指标的测定。结果表明:在附加多效唑与未附加多效唑的培养基上保存的永春芦柑试管苗,在保存过程中,可溶蛋白含量、POD、CAT活性都是呈先上升后下降的趋势;游离脯氨酸含量的变化则呈上升趋势。但是,在附加多效唑的培养基上保存的试管苗,其可溶蛋白含量、POD、CAT活性呈抛物线状变化,且比为添加多效唑的下降时间向后推迟了1～2个月;而游离脯氨酸含量,在附加多效唑的培养基上保存的试管苗,呈上升趋势,但增长幅度低于对照。
6.柑桔试管苗离体种质保存过程中内源激素含量的HPLC测定。以保存在附加多效唑与未附加多效唑的培养基上的永春芦柑珠心胚试管苗为材料,采用HPLC法进行GA_3、IAA、ABA和ZT等4种内源激素含量的测定。结果表明:
(1)在保存前期,其内源GA_3、IAA和ZT的含量呈上升趋势,且试管苗增长速度快。在保存后期,GA_3、IAA和ZT的含量开始呈下降趋势,试管苗增长速度也开始减慢。即GA_3、IAA和ZT的含量变化趋势一致,都是呈先上升后下降的趋势。而ABA则一直呈现上升趋势。
(2)在保存前期,附加有多效唑的试管苗,其GA_3、IAA和ZT的含量都低于对照,在保存后期,附加有多效唑的试管苗的含量开始上升,且高于对照。而ABA含量则在保存前6个月时,高于对照,之后低于对照。
(3)IAA/ABA的比值,在保存的前4个月时上升,且到保存第4个月时,达到最大。之后,比值开始下降。而附加有多效唑的试管苗的比值,保存前期,低于对照,后期,则下降平缓,且高于对照。
(4)IAA/ZT的比值,从整个保存阶段来看,呈上升趋势。而附加有多效唑的试管苗的比值,保存前期,变化平缓,后期,增长速度快。对照的比值,保存前期,增长速度稍快些,之后趋于平缓。
In this experiment,42 accessions of citrus germplasm were collected for in vitro conservation.The main research contents were as follows:citrus mature embryos were used as the explants for establishing the in vitro regeneration system;the tube plantlets from the nucellar embryos of Citrus sinensis cv.Xuegan were used as the conservation materials for obtaining the effective parameters of restricting growth conservation and the genetic stability of conserved plantlets were detected by ISSR technique;42 accessions of citrus germplasm were conserved under the optimized conditions;some physiological indexes such as soluble protein contents,activities of the cell protective enzymes(POD and CAT),as well as free proline contents were measured,and the changes of endogenous hormones contents were also measured by HPLC for discussing the in vitro preservation mechanism,which would provide a scientific basis for the better in vitro preservation of citrus germplasm.The main results were as follows:
1.Establishment of the in vitro regeneration system of citrus plants.The plantlets from the nucellar embryos of Citrus sinensis cv.Xuegan were used as materials to study the best conditions of the proliferation,rooting and transplanting.The results showed that the best proliferation medium was the MS medium supplemented with 1.0 mg/L 6-BA and 0.01 mg/L NAA,and the proliferation coefficient was 9.70;the plantlets cultured on the MS medium supplemented with 1.0 mg/L IBA rooted best;and the best transplanting medium was the mixture of sawdust and husk(1:1).
2.Study on germplasm conservation in vitro of plantlets in citrus plants.The stem segments which from the nucellar embryos of Citrus sinensis cv.Xuegan growing for 3 months,were used as materials,and the effective parameters of restricting growth conservation was optimized by adjusting different concentrations of sucrose,different concentrations of agar,different concentrations of sucrose and mannitol,and PP_(333).The results showed that the survival rate after conservation for 12 months was 85.6%on the MS medium supplemented with 50～80 g·L~(-1)of sucrose;the survival rate after conservation for 12 months was 86.8%on the MS medium supplemented with 0.5mg·L~(-1)PP_(333). Both of the preservation effects were the same.Considering from the effects of conservation and preservation costs,the MS medium supplemented with 0.5mg·L~(-1)PP_(333)was the best for conservation in vitro of plantlets in citrus plants.After preservation for 18 months,citrus plantlets rooted and transplanted well,and the plants grew robustly.
3.Establishment of the examining system of genetic stability of the conserved plantlets by ISSR-PCR.The tube plantlets of citrus conserved before and after preservation for 18 months were used as materials to analyze the stability of the genetic variation by ISSR-PCR.The results showed that the patterns of ISSR were completely identical,which suggested that there be no variation in the DNA level.,the plantlets from the preservation with the genetic stability,and which also showed the feasibility of the restricting conservation.
4.Establishment of the conservation materials of tube plantlets from the mature embryos in 42 accessions of main citrus plants.42 accessions of citrus germplasm were collected for in vitro preservation.The results showed that the MS medium supplemented with 0.5mg·L~(-1)PP_(333)applied for the mature embryos in vitro preservation of most citrus cultivars,and the survival rate of Citrus sinensis cv.Xuegan after conservation for 15 months was as high as 89.1%.The transplanting seedlings of the preservation in vitro grew normally and robustly.
5.Measurement of the physiology indexes during the restricting growth conservation of the citrus tube plantlets.The plantlets from the nucellar embryos of ponkan in Yongchun County conserved both in the medium adding 0.5mg·L~(-1)PP_(333)and in the medium without PP_(333), were used to measure those physiological indexes such as soluble protein contents,activities of the cell protective enzymes(POD,CAT),as well as free praline contents.The results showed that in the preservation process,the soluble protein content,activity of POD and activity of CAT increased after the first downward trend,and the free proline content of the upward trend.However,the plantlets in the medium supplemented with 0.5mg·L~(-1)PP_(333),the soluble protein content,activity of POD and activity of CAT showed as a parabola-shaped curve,and the fall time of the plantlets in the medium adding PP_(333)postponed by 1 to 2 months.And the trend of free proline content in the medium adding PP333 was upward,while the growth rate was lower than that of the control.
6 Measurement of endogenous hormones by HPLC during restricting growth conservation of tube plantlets in citrus plants.The plantlets from the nucellar embryos of ponkan in Yongchun County conserved both in the medium adding 0.5mg·L~(-1)PP_(333)and in the medium without PP_(333),were used to determinate the changes of endogenous hormones of GA_3,IAA,ABA and ZT by HPLC method.The results showed:
(1)At the preservation of the early stage,the contents of GA_3,IAA and ZT were rising,and the plantlets in vitro grew rapidly.AT the preservation of the late stage,the contents of GA_3,IAA and ZT were slowing down,and the pIantlets in vitro grew fast;and the ABA showed an upward trend.
(2)At the preservation of the early stage,the contents of GA_3,IAA and ZT of the plantlets in the medium adding PP333 were lower than that of control,and at the preservation of the late stage,which started to rise,and higher than that of the control.The content of ABA in the preservation of the first six months was higher than that of the control,then lower than that of the control.
(3)The ratio of IAA/ABA in the preservation of the first four months was up,and that the preservation of the first four months,the maximum,and then the ratio began to drop.At the preservation of the early stage,the ratio of IAA/ABA of the plantlets in the medium adding PP_(333) was lower than that of control,and then declined slowly,and higher than the control.
(4)At the whole preservation stage,the ratio of IAA/ZT was the upward trend.The ratio of pre-preservation of the plantlets in the medium adding PP333 changed gently,late growth rapidly;and the ratio of the control,with a growth rate slightly faster,then more gently.
1.柑桔试管苗再生体系的建立。以雪柑珠心胚试管苗为材料,分别研究其增殖、生根、移栽的最佳条件。试验结果表明:最佳增殖培养基为MS+1.0mg·L~(-1)BA+0.01mg·L~(-1)NAA,增殖系数达到9.70;通过对NAA、IBA不同浓度试验表明,最佳生根培养基为MS+0.4mg·L~(-1)IBA,IBA适合于柑橘生根诱导;最佳移栽基质为锯末:糠灰(1∶1)的混合基质。
2.柑桔试管苗离体保存方法的探讨。以雪柑珠心胚试管苗生长3个月后的茎切段为材料,比较了不同浓度的蔗糖、琼脂、甘露醇和蔗糖、多效唑等因素对柑桔试管苗限制生长保存的影响。结果表明:附加50～80 g·L~(-1)蔗糖的MS培养基,保存12个月时,存活率为85.6%;附加0.5 mg·L~(-1)多效唑,保存12个月时,存活率为86.8%。两者的保存效果相当。从保存成本和保存效果综合考虑,我们认为柑橘试管苗限制生产保存的适宜培养基为附加0.5 mg·L~(-1)多效唑。保存18个月后,进行柑桔生根试管苗移栽,植株生长健壮。
3.试管苗限制生长保存遗传稳定性的ISSR分析。以限制生长法保存了18个月与保存前的雪柑试管苗为材料,用ISSR-PCR标记技术对雪柑试管苗保存前后的遗传变异进行检测。结果表明:相同引物不同材料的ISSR图谱完全一致,说明在检测范围内没有发生DNA水平的变异,离体保存试管苗的遗传稳定性好,也说明这种限制生长保存方法的可行性。
4.42份柑桔种质资源离体保存材料的建立。收集了柑桔类主要品种的成熟胚42份,进行离体保存。试验结果表明,MS基本培养基附加0.5 mg·L~(-1)多效唑,适用大部分柑桔类品种成熟胚离体保存,其中雪柑成熟胚培养,保存15个月时存活率高达89.1%。对保存后成活的的植株进行移栽,能正常生长,且生长健壮。
5.柑桔试管苗限制生长保存中生理指标的测定。以保存在附加多效唑与未附加多效唑的培养基上的永春芦柑珠心胚试管苗为材料,进行试管苗限制生长保存过程中可溶蛋白含量、POD、CAT等细胞膜保护酶活性以及游离脯氨酸含量等生理指标的测定。结果表明:在附加多效唑与未附加多效唑的培养基上保存的永春芦柑试管苗,在保存过程中,可溶蛋白含量、POD、CAT活性都是呈先上升后下降的趋势;游离脯氨酸含量的变化则呈上升趋势。但是,在附加多效唑的培养基上保存的试管苗,其可溶蛋白含量、POD、CAT活性呈抛物线状变化,且比为添加多效唑的下降时间向后推迟了1～2个月;而游离脯氨酸含量,在附加多效唑的培养基上保存的试管苗,呈上升趋势,但增长幅度低于对照。
6.柑桔试管苗离体种质保存过程中内源激素含量的HPLC测定。以保存在附加多效唑与未附加多效唑的培养基上的永春芦柑珠心胚试管苗为材料,采用HPLC法进行GA_3、IAA、ABA和ZT等4种内源激素含量的测定。结果表明:
(1)在保存前期,其内源GA_3、IAA和ZT的含量呈上升趋势,且试管苗增长速度快。在保存后期,GA_3、IAA和ZT的含量开始呈下降趋势,试管苗增长速度也开始减慢。即GA_3、IAA和ZT的含量变化趋势一致,都是呈先上升后下降的趋势。而ABA则一直呈现上升趋势。
(2)在保存前期,附加有多效唑的试管苗,其GA_3、IAA和ZT的含量都低于对照,在保存后期,附加有多效唑的试管苗的含量开始上升,且高于对照。而ABA含量则在保存前6个月时,高于对照,之后低于对照。
(3)IAA/ABA的比值,在保存的前4个月时上升,且到保存第4个月时,达到最大。之后,比值开始下降。而附加有多效唑的试管苗的比值,保存前期,低于对照,后期,则下降平缓,且高于对照。
(4)IAA/ZT的比值,从整个保存阶段来看,呈上升趋势。而附加有多效唑的试管苗的比值,保存前期,变化平缓,后期,增长速度快。对照的比值,保存前期,增长速度稍快些,之后趋于平缓。
In this experiment,42 accessions of citrus germplasm were collected for in vitro conservation.The main research contents were as follows:citrus mature embryos were used as the explants for establishing the in vitro regeneration system;the tube plantlets from the nucellar embryos of Citrus sinensis cv.Xuegan were used as the conservation materials for obtaining the effective parameters of restricting growth conservation and the genetic stability of conserved plantlets were detected by ISSR technique;42 accessions of citrus germplasm were conserved under the optimized conditions;some physiological indexes such as soluble protein contents,activities of the cell protective enzymes(POD and CAT),as well as free proline contents were measured,and the changes of endogenous hormones contents were also measured by HPLC for discussing the in vitro preservation mechanism,which would provide a scientific basis for the better in vitro preservation of citrus germplasm.The main results were as follows:
1.Establishment of the in vitro regeneration system of citrus plants.The plantlets from the nucellar embryos of Citrus sinensis cv.Xuegan were used as materials to study the best conditions of the proliferation,rooting and transplanting.The results showed that the best proliferation medium was the MS medium supplemented with 1.0 mg/L 6-BA and 0.01 mg/L NAA,and the proliferation coefficient was 9.70;the plantlets cultured on the MS medium supplemented with 1.0 mg/L IBA rooted best;and the best transplanting medium was the mixture of sawdust and husk(1:1).
2.Study on germplasm conservation in vitro of plantlets in citrus plants.The stem segments which from the nucellar embryos of Citrus sinensis cv.Xuegan growing for 3 months,were used as materials,and the effective parameters of restricting growth conservation was optimized by adjusting different concentrations of sucrose,different concentrations of agar,different concentrations of sucrose and mannitol,and PP_(333).The results showed that the survival rate after conservation for 12 months was 85.6%on the MS medium supplemented with 50～80 g·L~(-1)of sucrose;the survival rate after conservation for 12 months was 86.8%on the MS medium supplemented with 0.5mg·L~(-1)PP_(333). Both of the preservation effects were the same.Considering from the effects of conservation and preservation costs,the MS medium supplemented with 0.5mg·L~(-1)PP_(333)was the best for conservation in vitro of plantlets in citrus plants.After preservation for 18 months,citrus plantlets rooted and transplanted well,and the plants grew robustly.
3.Establishment of the examining system of genetic stability of the conserved plantlets by ISSR-PCR.The tube plantlets of citrus conserved before and after preservation for 18 months were used as materials to analyze the stability of the genetic variation by ISSR-PCR.The results showed that the patterns of ISSR were completely identical,which suggested that there be no variation in the DNA level.,the plantlets from the preservation with the genetic stability,and which also showed the feasibility of the restricting conservation.
4.Establishment of the conservation materials of tube plantlets from the mature embryos in 42 accessions of main citrus plants.42 accessions of citrus germplasm were collected for in vitro preservation.The results showed that the MS medium supplemented with 0.5mg·L~(-1)PP_(333)applied for the mature embryos in vitro preservation of most citrus cultivars,and the survival rate of Citrus sinensis cv.Xuegan after conservation for 15 months was as high as 89.1%.The transplanting seedlings of the preservation in vitro grew normally and robustly.
5.Measurement of the physiology indexes during the restricting growth conservation of the citrus tube plantlets.The plantlets from the nucellar embryos of ponkan in Yongchun County conserved both in the medium adding 0.5mg·L~(-1)PP_(333)and in the medium without PP_(333), were used to measure those physiological indexes such as soluble protein contents,activities of the cell protective enzymes(POD,CAT),as well as free praline contents.The results showed that in the preservation process,the soluble protein content,activity of POD and activity of CAT increased after the first downward trend,and the free proline content of the upward trend.However,the plantlets in the medium supplemented with 0.5mg·L~(-1)PP_(333),the soluble protein content,activity of POD and activity of CAT showed as a parabola-shaped curve,and the fall time of the plantlets in the medium adding PP_(333)postponed by 1 to 2 months.And the trend of free proline content in the medium adding PP333 was upward,while the growth rate was lower than that of the control.
6 Measurement of endogenous hormones by HPLC during restricting growth conservation of tube plantlets in citrus plants.The plantlets from the nucellar embryos of ponkan in Yongchun County conserved both in the medium adding 0.5mg·L~(-1)PP_(333)and in the medium without PP_(333),were used to determinate the changes of endogenous hormones of GA_3,IAA,ABA and ZT by HPLC method.The results showed:
(1)At the preservation of the early stage,the contents of GA_3,IAA and ZT were rising,and the plantlets in vitro grew rapidly.AT the preservation of the late stage,the contents of GA_3,IAA and ZT were slowing down,and the pIantlets in vitro grew fast;and the ABA showed an upward trend.
(2)At the preservation of the early stage,the contents of GA_3,IAA and ZT of the plantlets in the medium adding PP333 were lower than that of control,and at the preservation of the late stage,which started to rise,and higher than that of the control.The content of ABA in the preservation of the first six months was higher than that of the control,then lower than that of the control.
(3)The ratio of IAA/ABA in the preservation of the first four months was up,and that the preservation of the first four months,the maximum,and then the ratio began to drop.At the preservation of the early stage,the ratio of IAA/ABA of the plantlets in the medium adding PP_(333) was lower than that of control,and then declined slowly,and higher than the control.
(4)At the whole preservation stage,the ratio of IAA/ZT was the upward trend.The ratio of pre-preservation of the plantlets in the medium adding PP333 changed gently,late growth rapidly;and the ratio of the control,with a growth rate slightly faster,then more gently.
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