薄荚相思和流苏相思离体培养与人工种子制作
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摘要
本研究以薄荚相思(Acacia leptocawa)组培苗为材料,以流苏相思(A.fimbriata)未成熟种子、成熟种子为外植体,进行2种相思树的离体培养与人工种子制作研究。主要研究了如下内容:①薄荚相思高效离体再生体系的建立;②薄荚相思愈伤组织诱导与再生;③流苏相思高效离体再生体系的建立;④薄荚相思和流苏相思人工种子的制作;⑤薄荚相思愈伤组织再生试管苗,人工种子再生试管苗RAPD检测等。主要研究结果如下:
1.薄荚相思高效离体再生体系的建立。以薄荚相思无菌试管苗为材料,对薄荚相思增殖、生根壮苗与移栽方面进行研究,结果表明:薄荚相思在MS+0.3 mg.L~(-1)BA+0.15 mg·L~(-1)NAA的培养基上增殖效果最好,增殖系数达到3.75,且玻璃化率也控制在最低水平(18.75%)。薄荚相思生根壮苗适宜的培养基为1/2MS+0.3 mg·L~(-1)NAA+0.5 g·L~(-1)活性炭+100g·L~(-1)椰乳,生根率达到97.56%,苗长势较好,苗高为4.68。沙子:蛭石:泥炭土(2∶1∶1)的基质最适宜薄荚相思移栽,成活率达到93.33%。
2.薄荚相思愈伤组织的诱导与再生。以薄荚相思组培苗的幼嫩茎段为材料,研究愈伤组织的诱导与再生,结果表明:MS+0.1～0.15 mg·L~(-1)NAA+1.0～2.0 mg·L~(-1)BA的培养基较利于薄荚相思愈伤组织的诱导,出愈率都达到100%,且愈伤组织质量较好。愈伤组织在MS+0.1 mg·L~(-1)NAA+1.0 mg·L~(-1)BA的培养基上再生率达到50%,且再生试管苗长势较好。
3.预处理对流苏相思未成熟和成熟种子萌发的影响。以流苏相思未成熟和成熟种子为材料,对流苏相思种子无菌萌发进行研究,结果表明:在远胚端种脊处对未成熟进行切口处理,种子的萌发率可达100%。单独使用浓硫酸处理流苏相思成熟种子15min,种子的萌发率可达100%。
4.流苏相思高效离体再生体系的建立。以流苏相思种子离体萌发的试管苗为材料,对流苏相思增殖、生根以及移栽方面进行研究,结果表明:MS+0.1mg·L~(-1)NAA+0.3 mg·L~(-1)IBA+0.3 mg·L~(-1)BA+30g·L~(-1)的白糖+6g·L~(-1)的琼脂较利于薄荚相思增殖培养,增值系数为3.22。单独使用0.3 mg·L~(-1)的IBA有利于流苏相思生根培养,生根率达到了94.17%。沙子:蛭石:泥炭土(2∶1∶1)的基质最适宜流苏相思移栽,成活率达到90.48%。
5.薄荚相思与流苏相思人工种子的制作。以薄荚相思和流苏相思微芽为繁殖体,对两种相思非体胚人工种子进行研究,结果表明:采用海藻酸钙法包埋薄荚相思微芽时,2.0%海藻酸钠交换20 min最好,萌发率高到85.93%,转株率为79.69%;对流苏相思来说,3.0%的海藻酸钠交换15min则更有利于人工种子的萌发,萌发率为85.71%。中空海藻酸钙包埋法在两种相思人工种子萌发方面都不如海藻酸钙包埋法。在薄荚相思人工胚乳中添加0.05mg·L~(-1)NAA、0.1mg·L~(-1)BA使得人工种子的萌发率提高了2.89%;通过对人工种子在自然条件下失水与复水的研究确立了双层海藻酸钙包埋法、涂抹丙三醇、添加聚乙烯吡咯啉酮为较好的保水包埋方法;薄荚相思人工种子适宜的贮藏温度为4℃;薄荚相思和流苏相思适宜贮藏的包埋方法为双层海藻酸钠法(4℃下贮藏60 d后的萌发率分别为39.58%、30.0%);薄荚相思有菌萌发适宜的包埋方法为MS+0.1%聚乙烯吡咯啉酮+0.05%ALCl_3+0.05%MgCl_2+0.05%COCl_2+0.2%的多菌灵+003%的苯甲酸钠的处理,适宜的播种基质为沙子:蛭石:泥炭土(2∶1∶1),60 d后成苗率达到了25.56%。
6.薄荚相思愈伤再生苗、人工种子再生植株的RAPD检测。对同一个外植体来源的薄荚相思组培苗、愈伤组织再生试管苗、人工种子再生植株进行RAPD分析。结果表明:薄荚相思愈伤组织再生苗与组培苗之间的RAPD图谱存在着遗传差异,说明愈伤组织再生途径在遗传上比较不稳定;人工种子再生植株与组培苗之间的RAPD图谱不存在遗传差异,说明人工种子具有较高的遗传稳定性。
总之,本研究建立了薄荚相思高效离体再生体系;首次采用薄荚相思无菌苗茎段诱导愈伤并获得再生植株;摸索出了流苏相思未成熟和成熟种子高频率萌发技术;建立了2种相思树人工种子技术体系;采用RAPD技术分析了薄荚相思愈伤再生试管苗和人工种子再生试管苗的遗传稳定性,对相思树离体快繁和人工种子应用具有重要意义。
In this experiment the tissue culture seedlings were used as materials in A.leptocarpa and the immature and mature seeds were used as explants in A.fimbriata for in vitro culture and artificial seeds production of the two Acacia species.The main contents of the experiment were as follows:①establishment of the system of the high-effeciency regeneration in vitro in A.leptocarpa;②induction and regeneration of callus in A.leptocarpa;③establishment of the system of the high-effeciency regeneration in vitro in A.fimbriata;④production of artificial seeds in A. leptocarpa and A.fimbriata;⑤RAPD analyses of the regeneration plantlet of the callus and the regeneration plantlet of artificial seeds in A.leptocarpa.The main results were described as follows:
1.Establishment of the system of the high-effeciency regeneration in vitro in A.leptocarpa. The tissue culture seedlings were used as materials to studying the proliferation,rooting and strong seedling and transplantation in A.leptocarpa.The results showed that the proliferative effect was the best on the MS medium supplemented with 0.3 mg·L~(-1)BA and 0.15 mg·L~(-1)NAA, the propagation coefficient was up to 3.75,and the rate of vitrification was also controlled on the lowest level(18.75%);the optimum medium of rooting and shoot growth was the 1/2MS supplemented with 0.3 mg·L~(-1)NAA and 0.5 g·L~(-1)AC and 100 g·L~(-1)coconut milk,the rooting rate was 97.56%,the growth vigor of tissue culture seedling was better;the surival rate was up to 93.33%after transfer into the mixture of sand and vermiculite and peat soil(2:1:1)which was suitable for transplantation.
2.Induction and regeneration of callus in A.leptocarpa.Tender stem segments of the tissue culture seedling were used as materials to callus induction in A.leptocarpa.The results showed that the MS medium supplemented with 0.1～0.15 mg·L~(-1)NAA and 1.0～2.0 mg·L~(-1)BA were faliciated to induce callus,rates of inducing callus were all up to 100%,and the quality of callus was better;the regeneration rate was up to 50%on the MS medium supplemented with 0.1 mg·L~(-1)NAA and 1.0 mg·L~(-1)BA,and the regeneration plantlets were all better in terms of growth vigor.
3.The effect of different pre-treatments on the germination of the immature and mature seeds in A.fimbriata.The immature and mature seeds were used as explants to studying the aseptic germination in A.fimbriata.The results showed that the germination rate reached 100%when doing the incision treatment at the raphe far from embryo.Under the pre-treatment of the thick vitriol for 15 min on mature seeds in A caciafimbriata,the germination rate reached 100%.
4.Establishment of the system of the high-effeciency regeneration in vitro in A.fimbriata. The tissue culture seedlings germinated of seeds in viro were used as materials to studying the proliferation,rooting and transplantation in A.fimbriata.The results showed the best formula of proliferation was the MS medium supplemented with 0.1 mg·L~(-1)NAA and 0.3 mg·L~(-1)IBA and 0.3 mg·L~(-1)6-BA and 30 g·L~(-1)sucrose and 6 g·L~(-1)agar,the propagation coefficient was 3.22;0.3 mg·L~(-1)IBA was faliciated to rooting culture,rooting rate reached 3.75;the surival rate was up to 90.48%after transfer into the mixture of sand and vermiculite and peat soil(2:1:1)which was suitable for transplantation.
5.Production of artificial seeds in A.leptocarpa and A.fimbriata.The shoot cuts of A. leptocarpa and A.fimbriata were used as encapsulating materials to study the non-embryo artificial seeds.The results showed that the 2.0%sodium alginate encapsulated 20 min was the best when using the method of calcium alginate to encapsulating the shoot cuts in A.leptocarpa, germination rate was 85.93%,plantlet formation rate was 79.69%;3.0%sodium alginate encapsulated 15 min was more suitable for gennination and regeneration of artificial seeds in A. fimbriata,germination rate was 85.71%;the calcium alginate hollow method was less than calcium alginate method in terms of germination;the rate of germination was improved 2.89%by adding 0.05 mg·L~(-1)NAA,0.1 mg·L~(-1)BA to artificial endosperm in A.leptocarpa;it was found that the double calcium alginate method,smearing glycerol,addition polyvinyl pyrrolidone were the better investing methods of water retention by studying dehydration and rehydration of artificial seeds on natural conditions;4℃was suitable for the storage of the artificial seeds in A. leptocarpa;the double calcium alginate methord was suitable for the storage of the artificial seeds in A.leptocarpa and A.fimbriata(germination rates were 39.58%and 30.3%after 4℃storage for 60 days);the investing method of additing polyvinyl pyrrolidone with 0.05%ALCl_3,0.05% MgCl_2,0.05%CoCl_2,0.2%carbendazim and 0.3%sodium benzoate was suitable for germination of the artificial seeds in A.leptocarpa under natural conditions,the mixture of sand and vermiculite and peat soil(2:1:1)was suitable for sowing,the germination of 60 days of artificial seeds was 25.56%.
RAPD analyses in the regeneration plantlet of the callus and the regeneration plantlet of artificial seeds in A.leptocarpa.The tissue culture seedling,the plantlet regenerations from callus and the plantlet regenerations artificial seeds from the same plants were used as materials in A. leptocarpa.The result indicated that there were significant differences in the patterns among original tissue culture seedling and the regenerated plants of callus,which suggested that the regenerated plants of callus were relatively unstable in heredity.There was no significant difference in the patterns among original tissue culture seedling and the regenerated plants of callus,which suggested that the regenerated plants of artificial seeds were hgihly stable in heredity.
As a whole,in this study the system of the high-effeciency regeneration in vitro was established in A.leptocarpa;Tender stem segments of the tissue culture seedling were first used to inducing callus and obtained regeneration in A.leptocarpa;the technique of high-frequency gerlnination of immature and mature seeds was developed in A.fimbriata;the technique of the production of artificial seeds was first developed in the two Acacia species.;and analyzed the hereditary stability of the regenerated plants of callus and the regenerated explants of artificial seeds by the method of RAPD,which would be of great importance for in vitro multiplication and artificial seeds application in Acacia.
1.薄荚相思高效离体再生体系的建立。以薄荚相思无菌试管苗为材料,对薄荚相思增殖、生根壮苗与移栽方面进行研究,结果表明:薄荚相思在MS+0.3 mg.L~(-1)BA+0.15 mg·L~(-1)NAA的培养基上增殖效果最好,增殖系数达到3.75,且玻璃化率也控制在最低水平(18.75%)。薄荚相思生根壮苗适宜的培养基为1/2MS+0.3 mg·L~(-1)NAA+0.5 g·L~(-1)活性炭+100g·L~(-1)椰乳,生根率达到97.56%,苗长势较好,苗高为4.68。沙子:蛭石:泥炭土(2∶1∶1)的基质最适宜薄荚相思移栽,成活率达到93.33%。
2.薄荚相思愈伤组织的诱导与再生。以薄荚相思组培苗的幼嫩茎段为材料,研究愈伤组织的诱导与再生,结果表明:MS+0.1～0.15 mg·L~(-1)NAA+1.0～2.0 mg·L~(-1)BA的培养基较利于薄荚相思愈伤组织的诱导,出愈率都达到100%,且愈伤组织质量较好。愈伤组织在MS+0.1 mg·L~(-1)NAA+1.0 mg·L~(-1)BA的培养基上再生率达到50%,且再生试管苗长势较好。
3.预处理对流苏相思未成熟和成熟种子萌发的影响。以流苏相思未成熟和成熟种子为材料,对流苏相思种子无菌萌发进行研究,结果表明:在远胚端种脊处对未成熟进行切口处理,种子的萌发率可达100%。单独使用浓硫酸处理流苏相思成熟种子15min,种子的萌发率可达100%。
4.流苏相思高效离体再生体系的建立。以流苏相思种子离体萌发的试管苗为材料,对流苏相思增殖、生根以及移栽方面进行研究,结果表明:MS+0.1mg·L~(-1)NAA+0.3 mg·L~(-1)IBA+0.3 mg·L~(-1)BA+30g·L~(-1)的白糖+6g·L~(-1)的琼脂较利于薄荚相思增殖培养,增值系数为3.22。单独使用0.3 mg·L~(-1)的IBA有利于流苏相思生根培养,生根率达到了94.17%。沙子:蛭石:泥炭土(2∶1∶1)的基质最适宜流苏相思移栽,成活率达到90.48%。
5.薄荚相思与流苏相思人工种子的制作。以薄荚相思和流苏相思微芽为繁殖体,对两种相思非体胚人工种子进行研究,结果表明:采用海藻酸钙法包埋薄荚相思微芽时,2.0%海藻酸钠交换20 min最好,萌发率高到85.93%,转株率为79.69%;对流苏相思来说,3.0%的海藻酸钠交换15min则更有利于人工种子的萌发,萌发率为85.71%。中空海藻酸钙包埋法在两种相思人工种子萌发方面都不如海藻酸钙包埋法。在薄荚相思人工胚乳中添加0.05mg·L~(-1)NAA、0.1mg·L~(-1)BA使得人工种子的萌发率提高了2.89%;通过对人工种子在自然条件下失水与复水的研究确立了双层海藻酸钙包埋法、涂抹丙三醇、添加聚乙烯吡咯啉酮为较好的保水包埋方法;薄荚相思人工种子适宜的贮藏温度为4℃;薄荚相思和流苏相思适宜贮藏的包埋方法为双层海藻酸钠法(4℃下贮藏60 d后的萌发率分别为39.58%、30.0%);薄荚相思有菌萌发适宜的包埋方法为MS+0.1%聚乙烯吡咯啉酮+0.05%ALCl_3+0.05%MgCl_2+0.05%COCl_2+0.2%的多菌灵+003%的苯甲酸钠的处理,适宜的播种基质为沙子:蛭石:泥炭土(2∶1∶1),60 d后成苗率达到了25.56%。
6.薄荚相思愈伤再生苗、人工种子再生植株的RAPD检测。对同一个外植体来源的薄荚相思组培苗、愈伤组织再生试管苗、人工种子再生植株进行RAPD分析。结果表明:薄荚相思愈伤组织再生苗与组培苗之间的RAPD图谱存在着遗传差异,说明愈伤组织再生途径在遗传上比较不稳定;人工种子再生植株与组培苗之间的RAPD图谱不存在遗传差异,说明人工种子具有较高的遗传稳定性。
总之,本研究建立了薄荚相思高效离体再生体系;首次采用薄荚相思无菌苗茎段诱导愈伤并获得再生植株;摸索出了流苏相思未成熟和成熟种子高频率萌发技术;建立了2种相思树人工种子技术体系;采用RAPD技术分析了薄荚相思愈伤再生试管苗和人工种子再生试管苗的遗传稳定性,对相思树离体快繁和人工种子应用具有重要意义。
In this experiment the tissue culture seedlings were used as materials in A.leptocarpa and the immature and mature seeds were used as explants in A.fimbriata for in vitro culture and artificial seeds production of the two Acacia species.The main contents of the experiment were as follows:①establishment of the system of the high-effeciency regeneration in vitro in A.leptocarpa;②induction and regeneration of callus in A.leptocarpa;③establishment of the system of the high-effeciency regeneration in vitro in A.fimbriata;④production of artificial seeds in A. leptocarpa and A.fimbriata;⑤RAPD analyses of the regeneration plantlet of the callus and the regeneration plantlet of artificial seeds in A.leptocarpa.The main results were described as follows:
1.Establishment of the system of the high-effeciency regeneration in vitro in A.leptocarpa. The tissue culture seedlings were used as materials to studying the proliferation,rooting and strong seedling and transplantation in A.leptocarpa.The results showed that the proliferative effect was the best on the MS medium supplemented with 0.3 mg·L~(-1)BA and 0.15 mg·L~(-1)NAA, the propagation coefficient was up to 3.75,and the rate of vitrification was also controlled on the lowest level(18.75%);the optimum medium of rooting and shoot growth was the 1/2MS supplemented with 0.3 mg·L~(-1)NAA and 0.5 g·L~(-1)AC and 100 g·L~(-1)coconut milk,the rooting rate was 97.56%,the growth vigor of tissue culture seedling was better;the surival rate was up to 93.33%after transfer into the mixture of sand and vermiculite and peat soil(2:1:1)which was suitable for transplantation.
2.Induction and regeneration of callus in A.leptocarpa.Tender stem segments of the tissue culture seedling were used as materials to callus induction in A.leptocarpa.The results showed that the MS medium supplemented with 0.1～0.15 mg·L~(-1)NAA and 1.0～2.0 mg·L~(-1)BA were faliciated to induce callus,rates of inducing callus were all up to 100%,and the quality of callus was better;the regeneration rate was up to 50%on the MS medium supplemented with 0.1 mg·L~(-1)NAA and 1.0 mg·L~(-1)BA,and the regeneration plantlets were all better in terms of growth vigor.
3.The effect of different pre-treatments on the germination of the immature and mature seeds in A.fimbriata.The immature and mature seeds were used as explants to studying the aseptic germination in A.fimbriata.The results showed that the germination rate reached 100%when doing the incision treatment at the raphe far from embryo.Under the pre-treatment of the thick vitriol for 15 min on mature seeds in A caciafimbriata,the germination rate reached 100%.
4.Establishment of the system of the high-effeciency regeneration in vitro in A.fimbriata. The tissue culture seedlings germinated of seeds in viro were used as materials to studying the proliferation,rooting and transplantation in A.fimbriata.The results showed the best formula of proliferation was the MS medium supplemented with 0.1 mg·L~(-1)NAA and 0.3 mg·L~(-1)IBA and 0.3 mg·L~(-1)6-BA and 30 g·L~(-1)sucrose and 6 g·L~(-1)agar,the propagation coefficient was 3.22;0.3 mg·L~(-1)IBA was faliciated to rooting culture,rooting rate reached 3.75;the surival rate was up to 90.48%after transfer into the mixture of sand and vermiculite and peat soil(2:1:1)which was suitable for transplantation.
5.Production of artificial seeds in A.leptocarpa and A.fimbriata.The shoot cuts of A. leptocarpa and A.fimbriata were used as encapsulating materials to study the non-embryo artificial seeds.The results showed that the 2.0%sodium alginate encapsulated 20 min was the best when using the method of calcium alginate to encapsulating the shoot cuts in A.leptocarpa, germination rate was 85.93%,plantlet formation rate was 79.69%;3.0%sodium alginate encapsulated 15 min was more suitable for gennination and regeneration of artificial seeds in A. fimbriata,germination rate was 85.71%;the calcium alginate hollow method was less than calcium alginate method in terms of germination;the rate of germination was improved 2.89%by adding 0.05 mg·L~(-1)NAA,0.1 mg·L~(-1)BA to artificial endosperm in A.leptocarpa;it was found that the double calcium alginate method,smearing glycerol,addition polyvinyl pyrrolidone were the better investing methods of water retention by studying dehydration and rehydration of artificial seeds on natural conditions;4℃was suitable for the storage of the artificial seeds in A. leptocarpa;the double calcium alginate methord was suitable for the storage of the artificial seeds in A.leptocarpa and A.fimbriata(germination rates were 39.58%and 30.3%after 4℃storage for 60 days);the investing method of additing polyvinyl pyrrolidone with 0.05%ALCl_3,0.05% MgCl_2,0.05%CoCl_2,0.2%carbendazim and 0.3%sodium benzoate was suitable for germination of the artificial seeds in A.leptocarpa under natural conditions,the mixture of sand and vermiculite and peat soil(2:1:1)was suitable for sowing,the germination of 60 days of artificial seeds was 25.56%.
RAPD analyses in the regeneration plantlet of the callus and the regeneration plantlet of artificial seeds in A.leptocarpa.The tissue culture seedling,the plantlet regenerations from callus and the plantlet regenerations artificial seeds from the same plants were used as materials in A. leptocarpa.The result indicated that there were significant differences in the patterns among original tissue culture seedling and the regenerated plants of callus,which suggested that the regenerated plants of callus were relatively unstable in heredity.There was no significant difference in the patterns among original tissue culture seedling and the regenerated plants of callus,which suggested that the regenerated plants of artificial seeds were hgihly stable in heredity.
As a whole,in this study the system of the high-effeciency regeneration in vitro was established in A.leptocarpa;Tender stem segments of the tissue culture seedling were first used to inducing callus and obtained regeneration in A.leptocarpa;the technique of high-frequency gerlnination of immature and mature seeds was developed in A.fimbriata;the technique of the production of artificial seeds was first developed in the two Acacia species.;and analyzed the hereditary stability of the regenerated plants of callus and the regenerated explants of artificial seeds by the method of RAPD,which would be of great importance for in vitro multiplication and artificial seeds application in Acacia.
引文
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