苜蓿单倍体、纯合四倍体的诱导及愈伤组织DNA含量变异的研究
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摘要
获得苜蓿(Medicago sativa L.)单倍体可为获得纯合四倍体以及建立加倍单倍体群体(DH)提供原始材料。花药愈伤组织遗传变异的研究对于保证其遗传稳定性和完整性具有重要的意义。在细胞学鉴定方面,以往使用的压片法,准确性高,但费时费力难以完成大批量的测定,流式细胞仪的使用为苜蓿倍性检测,提供了一条准确、快捷的方法。
本试验的主要目的是利用花药培养获得苜蓿单倍体及纯合四倍体,在此过程中涉及的主要研究结果如下:
1、苜蓿不同基因型之间花药愈伤组织诱导能力存在显著差异。低浓度2.4-D(0.5 mg/L)与KT、BA配合使用有利于愈伤组织及芽的形成,愈伤组织诱导培养基组合NB+2.4-D 0.5mg/L+NAA 0.3mg/L+6-BA 0.5 mg/L+KT 3.0 mg/L+蔗糖6%,适合愈伤组织诱导。对花药30℃高温处理48h～72h、4℃低温处理24h有利于提高花药愈伤组织的诱导率。BA和NAA配合使用有利于诱导愈伤组织分化,分化培养基MS+BA 0.2mg/L+NAA 2mg/L(3mg/L),适合苜蓿花药愈伤组织分化。MS+2%蔗糖+0.5%琼脂,是苜蓿花培植株继代和复壮适宜的培养基。1/2 MS(MS)+NAA 0.1mg/L+1%蔗糖+0.5%琼脂,是花培苗适宜的生根培养基。
2、愈伤组织中染色体变异是非常普遍的,通过SPSS10.0分析软件分析得出愈伤组织的培养时间与DNA含量的变异系数正相关:继代0～12周DNA含量的变异百分率增加迅速,之后增加速度逐渐趋于平缓、稳定。
3、KT、NAA、6-BA对苜蓿愈伤组织DNA含量没有明显的诱变作用。2,4-D是可以引起DNA含量变异的,处理时间是关键因素。
4、愈伤组织的生长速度和胚状体的分化能力与基因型有关。分析两者相关性得到相关系数r=-0.3261(p<0.01),说明愈伤组织的生长速度与分化能力之间存在相关性。蔗糖和乳糖适合于苜蓿花药愈伤组织的生长与分化。蔗糖浓度20g/L、40g/L、60g/L三个水平对花药愈伤组织分化的影响不存在明显差异。对愈伤组织进行低温处理有利于提高愈伤组织的分化能力,4℃处理48h效果最突出,8℃的处理不利于提高愈伤组织的分化能力。
5、苜蓿愈伤组织对秋水仙素的耐受能力较强。在悬浮培养基中秋水仙素低浓度处理时,300mg/L的秋水仙素处理12周效果最好。高浓度处理时1g/L、2g/L处理48小时效果较好。通过愈伤组织不同时间段S期细胞百分率的分析,得到继代3周为细胞的集中分裂期,为秋水仙素最佳处理时期。
6、流式细胞仪检测,苜蓿花药培养再生植株为染色体倍性水平不同的混合体,50株苜蓿花药再生植株幼苗中42株四倍体,2n=4x与2n=2x的混合体为5株,单倍体为2株,三倍体为1株
Get haploid alfalfa (Medicago sativa L.) can provide raw materials for homozygous tetraploid and the creation of doubled haploid population (DH). Study on anther culture of genetic variation have a great significance for the genetic stability and the integrity. Cytological identification, in the past using the squash method have high accuracy, but complete the determination of volume was very consuming time. Flow-cytometric was used for alfalfa testing, provides a precise and fast method.
The main purpose of this study was anther culture homozygous tetraploid, the process involved the major aspects are as follows:
1. Between different genotypes of alfalfa callus induction capacity vary considerably. Relatively low concentration of 2.4-D (0.5 mg / L) and (KT, BA) with the use of callus and bud formation, the most suitable combination of callus induction medium is NB +2.4-D 0.5mg / L + NAA 0.3mg / L +6- BA 0.5 mg / L + KT 3.0 mg / L + sucrose 6%. 30℃high temperature 48h-72h and 4℃low temperature of 24h on the best callus induction. BA and NAA when used with the callus induction of differentiation more easily, the suitable combination of differentiation medium is MS+BA 0.2mg/L+NAA 2mg/L(3mg/L); MS + sucrose 2% + agar 0.5% is the most appropriate medium for a subculture and rejuvenation of alfalfa plants. 1 / 2 MS (MS) + NAA 0.1mg / L +1% sucrose +0.5% agar is suitable for anther culture plants of the rooting medium.
2. Chromosome variation in callus is very common, and software analysis obtained through callus culture time and the coefficient of variation of DNA content was positively related ; In the following generation of 0～12 weeks of this time is increasing rapidly after that with the increase in the number of subculture DNA varied cells, the increase in the percentage rate is gradual and stable.
3. KT, NAA ,6-BA on alfalfa callus DNA mutagenic effect is not obvious. 2,4-D can cause DNA content variation and the processing time is the key factor.
4. Genotypes related to the incidence of callus growth rate and embryonic callus ability. Analysis of the correlation coefficient r =- 0.3261 (p <0.01), shows between the growth rate of callus and differentiation of a certain correlation. Sucrose and lactose are well for the growth and differentiation of alfalfa callus. 40g/L, 20g/L, 60g/L sucrose in callus differentiation there is no significant difference. Low temperature treatment on callus help to improve the differentiation of callus, 4℃48h has the most prominent effect, and therefore the low temperature 8℃is not favorable for callus formation.
5.Alfalfa callus on colchicine tolerance ability. Suspension medium low concentrations, 300mg / L colchicine for 12 weeks was the best deal. When high concentration of 1g/L、2g/L were for 48 hours is better. Through analysis of the correlation between the incubation time and the percentage of S stage cells, we get the best period of colchicines is 3 weeks concentrated for the cell division stage.
6.The results of Flow-cytometric indicated that 42 plants were tetraploid plants , 5 plants were chimerics plants of diploid plus tetraploid, 2 plants were diploids plants, 1 plants were triploid plants.
本试验的主要目的是利用花药培养获得苜蓿单倍体及纯合四倍体,在此过程中涉及的主要研究结果如下:
1、苜蓿不同基因型之间花药愈伤组织诱导能力存在显著差异。低浓度2.4-D(0.5 mg/L)与KT、BA配合使用有利于愈伤组织及芽的形成,愈伤组织诱导培养基组合NB+2.4-D 0.5mg/L+NAA 0.3mg/L+6-BA 0.5 mg/L+KT 3.0 mg/L+蔗糖6%,适合愈伤组织诱导。对花药30℃高温处理48h～72h、4℃低温处理24h有利于提高花药愈伤组织的诱导率。BA和NAA配合使用有利于诱导愈伤组织分化,分化培养基MS+BA 0.2mg/L+NAA 2mg/L(3mg/L),适合苜蓿花药愈伤组织分化。MS+2%蔗糖+0.5%琼脂,是苜蓿花培植株继代和复壮适宜的培养基。1/2 MS(MS)+NAA 0.1mg/L+1%蔗糖+0.5%琼脂,是花培苗适宜的生根培养基。
2、愈伤组织中染色体变异是非常普遍的,通过SPSS10.0分析软件分析得出愈伤组织的培养时间与DNA含量的变异系数正相关:继代0～12周DNA含量的变异百分率增加迅速,之后增加速度逐渐趋于平缓、稳定。
3、KT、NAA、6-BA对苜蓿愈伤组织DNA含量没有明显的诱变作用。2,4-D是可以引起DNA含量变异的,处理时间是关键因素。
4、愈伤组织的生长速度和胚状体的分化能力与基因型有关。分析两者相关性得到相关系数r=-0.3261(p<0.01),说明愈伤组织的生长速度与分化能力之间存在相关性。蔗糖和乳糖适合于苜蓿花药愈伤组织的生长与分化。蔗糖浓度20g/L、40g/L、60g/L三个水平对花药愈伤组织分化的影响不存在明显差异。对愈伤组织进行低温处理有利于提高愈伤组织的分化能力,4℃处理48h效果最突出,8℃的处理不利于提高愈伤组织的分化能力。
5、苜蓿愈伤组织对秋水仙素的耐受能力较强。在悬浮培养基中秋水仙素低浓度处理时,300mg/L的秋水仙素处理12周效果最好。高浓度处理时1g/L、2g/L处理48小时效果较好。通过愈伤组织不同时间段S期细胞百分率的分析,得到继代3周为细胞的集中分裂期,为秋水仙素最佳处理时期。
6、流式细胞仪检测,苜蓿花药培养再生植株为染色体倍性水平不同的混合体,50株苜蓿花药再生植株幼苗中42株四倍体,2n=4x与2n=2x的混合体为5株,单倍体为2株,三倍体为1株
Get haploid alfalfa (Medicago sativa L.) can provide raw materials for homozygous tetraploid and the creation of doubled haploid population (DH). Study on anther culture of genetic variation have a great significance for the genetic stability and the integrity. Cytological identification, in the past using the squash method have high accuracy, but complete the determination of volume was very consuming time. Flow-cytometric was used for alfalfa testing, provides a precise and fast method.
The main purpose of this study was anther culture homozygous tetraploid, the process involved the major aspects are as follows:
1. Between different genotypes of alfalfa callus induction capacity vary considerably. Relatively low concentration of 2.4-D (0.5 mg / L) and (KT, BA) with the use of callus and bud formation, the most suitable combination of callus induction medium is NB +2.4-D 0.5mg / L + NAA 0.3mg / L +6- BA 0.5 mg / L + KT 3.0 mg / L + sucrose 6%. 30℃high temperature 48h-72h and 4℃low temperature of 24h on the best callus induction. BA and NAA when used with the callus induction of differentiation more easily, the suitable combination of differentiation medium is MS+BA 0.2mg/L+NAA 2mg/L(3mg/L); MS + sucrose 2% + agar 0.5% is the most appropriate medium for a subculture and rejuvenation of alfalfa plants. 1 / 2 MS (MS) + NAA 0.1mg / L +1% sucrose +0.5% agar is suitable for anther culture plants of the rooting medium.
2. Chromosome variation in callus is very common, and software analysis obtained through callus culture time and the coefficient of variation of DNA content was positively related ; In the following generation of 0～12 weeks of this time is increasing rapidly after that with the increase in the number of subculture DNA varied cells, the increase in the percentage rate is gradual and stable.
3. KT, NAA ,6-BA on alfalfa callus DNA mutagenic effect is not obvious. 2,4-D can cause DNA content variation and the processing time is the key factor.
4. Genotypes related to the incidence of callus growth rate and embryonic callus ability. Analysis of the correlation coefficient r =- 0.3261 (p <0.01), shows between the growth rate of callus and differentiation of a certain correlation. Sucrose and lactose are well for the growth and differentiation of alfalfa callus. 40g/L, 20g/L, 60g/L sucrose in callus differentiation there is no significant difference. Low temperature treatment on callus help to improve the differentiation of callus, 4℃48h has the most prominent effect, and therefore the low temperature 8℃is not favorable for callus formation.
5.Alfalfa callus on colchicine tolerance ability. Suspension medium low concentrations, 300mg / L colchicine for 12 weeks was the best deal. When high concentration of 1g/L、2g/L were for 48 hours is better. Through analysis of the correlation between the incubation time and the percentage of S stage cells, we get the best period of colchicines is 3 weeks concentrated for the cell division stage.
6.The results of Flow-cytometric indicated that 42 plants were tetraploid plants , 5 plants were chimerics plants of diploid plus tetraploid, 2 plants were diploids plants, 1 plants were triploid plants.
引文
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