地被植物蔓花生再生体系建立及多倍体诱导
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摘要
蔓花生(Arachis duranensis Krapov.&W.C.Gregory)义名遍地黄金,蝶形花亚科落花生属多年生宿根草本植物,原产亚洲热带及南美洲。它既适用于做优良的牧草,又可以做绿肥和防治水土流失的植物。因为蔓花生具有良好的株型和外观效果,而且一般不用修剪,可有效节省人力及物力,是极有前途的优良地被植物。现今蔓花生在生态治理、园林绿化上得到了广泛的应用。福建、广东、海南等多个地方已大规模使用该品种作为绿化材料而且取得了良好的景观效益,但由于蔓花生抗寒性较差,不利于大面积推广。目前,国内对蔓花生的研究主要在栽培管理及园林应用方面,国外通过分子手段对蔓花生进行研究,而对其育种方面的研究较少。而在传统的育种方法中,诱导多倍体是一种快速得到新品种的方法,并且多倍体一般都表现出抗寒性增强等特性。为此本实验开展了蔓花生再生体系的建立及多倍体诱导的研究工作。通过秋水仙素结合组织培养进行多倍体诱导,从处理方式、秋水仙素浓度和处理时间对多倍体诱导的效应及多倍体鉴定方面进行了研究。
从外植体的取材、消毒、初代培养物的建立、激素的种类及浓度的筛选、芽的分化、根的诱导、移栽进行了研究,初步提供了一组用于蔓花生组织培养的培养基。在初代培养时,得出叶片为外植体时洗涤剂清洗20min,自来水冲洗30min,0.1%升汞浸泡7min,其污染率可以控制在37%,死亡率可控制在0%。将消完毒的外植体切成0.5cm×0.5cm的方块接种在MS+NAA 0.5mg/L+6-BA 3.0mg/L(pH=5.8)愈伤组织诱导率为83.33%:继代培养基为MS+NAA0.1mg/L+6-BA 1.0 mg/L(pn=5.8)有较好的诱导效果,分化率为86.67%:生根培养时,以1/2为基本培养基附加1.0mg/LNAA可使根生长的长而壮,生根率均可以达到86.67%。待根长1～2cm时移栽,8月左右,移栽成活率可达80%。
采用培养基添加法、浸泡法以及先预培养后处理法对蔓花生的叶片愈伤组织、丛生芽进行多倍体诱导。结果表明:用0.1%秋水仙碱进行48h浸泡处理可以获得较好的诱导效果。其成活率和诱导率为66.67%和75.00%。采用去壁低渗—火焰干燥法检测蔓花生染色体数目发现二倍体染色体数目为2n=2X=20,四倍体为2n=4X=40。从植株形态上看,其四倍体表现为茎秆直径增粗、叶色变深、叶片变长、变宽、变厚、叶形指数减小等:在气孔保卫细胞大小、气孔数目以及叶绿体数量上也有显著到极显著变化。
Arachis duranensis Krapov.&W.C.Gregory,originating in the tropics of Asian and South Africa, is a perennial herbaceous plant,Papilionideae Giseke,the genus Arachis.As a kind of forage,it can be used as green manure and erosion control plants also.Now,A.duranens Krapov.&W.C.Gregory has been seen as a very promising good ground cover plant for its plant form,appearance effects,no pruning and manpower saving sake.At present,the A..duranensis Krapov.&W.C.Gregory has been widely used in ecological management and landscaping.And it has been large-scale used as green materials in Fujian,Guangdong,Hainan and other areas and has reaped the harvest of landscape benefits.But as the cold resistance of A.duranensis Krapov.&W.C.Gregory is poor,its potential use ability is largely limited.At present the studies on A.duranens Krapov.&W.C.Gregory mainly concentrates in cultivation,application in garden,and molecular biology.As the risistance ability of polyploid is better than diploid.Trails were desined to own polyploid of A.duranens Krapov.&W.C.Gregory.To establish regeneration system and gain polyploidy,experiments were carried out on and the treat method,colchicine concentration and treatment time were studied.And the inducing effect and the identification were studied also.
The tissue culture and cloning propagation including the establishment of aseptic culture,bud differentiation,selection of suitable hormone,rooting and acclimatization have been studied.As the explants,leaves were treated as follow:detergent for 20 minutes,running tap water for 30 minutes and 0.1%HgCl_2 for 7 minutes.And the contaminate rate can be controlled in 37%and the death rate also can be controlled in 0%.The explants was cultured on the inducement medium MS+NAA 0.5mg/L+6-BA 3.0mg/L(pH=5.8)and the inducement rate could reach to 83.33%.The best medium for propagation is MS+NAA 0.1mg/L+6-BA 1.0 mg/L(pH=5.8)and the split rate is 86.67%.1/2MS added 1.0mg/L NAA can make the root long and strong,and the rooting rate could reach to 86.67%.When the length of root is 1～2cm,lilies can be acclimatized and the survival rate can reach to 80%.
To induce polyploid,we deal embryogenic callus and caespitose buds with different methods called mixing culture with colchicine,immersing stem segment,pre-cultivated firstly and then sprinkling colchicine respectively.As the results show that the follow method is satisfying:0.1% colchicine immerse stem segment for 48 hours.The survival rate and induce rate are 66.67%and 75.00%respectively.Chromosome identification shows that the number of chromosome of the diploid is 2n=2X=20,while that of the tetraploid is 2n=4X=40.Morphologically,the stems of the tetraploid become stronger,the leaves become longer,broader and thicker,the color gets darker than that of the diploid,and the leaf index declines.Compared with the characteristic of the stoma of the diploid,the guard cell of the tetraploid becomes longer and broader;the number of the stomas declines and the number of chloroplast increases.
从外植体的取材、消毒、初代培养物的建立、激素的种类及浓度的筛选、芽的分化、根的诱导、移栽进行了研究,初步提供了一组用于蔓花生组织培养的培养基。在初代培养时,得出叶片为外植体时洗涤剂清洗20min,自来水冲洗30min,0.1%升汞浸泡7min,其污染率可以控制在37%,死亡率可控制在0%。将消完毒的外植体切成0.5cm×0.5cm的方块接种在MS+NAA 0.5mg/L+6-BA 3.0mg/L(pH=5.8)愈伤组织诱导率为83.33%:继代培养基为MS+NAA0.1mg/L+6-BA 1.0 mg/L(pn=5.8)有较好的诱导效果,分化率为86.67%:生根培养时,以1/2为基本培养基附加1.0mg/LNAA可使根生长的长而壮,生根率均可以达到86.67%。待根长1～2cm时移栽,8月左右,移栽成活率可达80%。
采用培养基添加法、浸泡法以及先预培养后处理法对蔓花生的叶片愈伤组织、丛生芽进行多倍体诱导。结果表明:用0.1%秋水仙碱进行48h浸泡处理可以获得较好的诱导效果。其成活率和诱导率为66.67%和75.00%。采用去壁低渗—火焰干燥法检测蔓花生染色体数目发现二倍体染色体数目为2n=2X=20,四倍体为2n=4X=40。从植株形态上看,其四倍体表现为茎秆直径增粗、叶色变深、叶片变长、变宽、变厚、叶形指数减小等:在气孔保卫细胞大小、气孔数目以及叶绿体数量上也有显著到极显著变化。
Arachis duranensis Krapov.&W.C.Gregory,originating in the tropics of Asian and South Africa, is a perennial herbaceous plant,Papilionideae Giseke,the genus Arachis.As a kind of forage,it can be used as green manure and erosion control plants also.Now,A.duranens Krapov.&W.C.Gregory has been seen as a very promising good ground cover plant for its plant form,appearance effects,no pruning and manpower saving sake.At present,the A..duranensis Krapov.&W.C.Gregory has been widely used in ecological management and landscaping.And it has been large-scale used as green materials in Fujian,Guangdong,Hainan and other areas and has reaped the harvest of landscape benefits.But as the cold resistance of A.duranensis Krapov.&W.C.Gregory is poor,its potential use ability is largely limited.At present the studies on A.duranens Krapov.&W.C.Gregory mainly concentrates in cultivation,application in garden,and molecular biology.As the risistance ability of polyploid is better than diploid.Trails were desined to own polyploid of A.duranens Krapov.&W.C.Gregory.To establish regeneration system and gain polyploidy,experiments were carried out on and the treat method,colchicine concentration and treatment time were studied.And the inducing effect and the identification were studied also.
The tissue culture and cloning propagation including the establishment of aseptic culture,bud differentiation,selection of suitable hormone,rooting and acclimatization have been studied.As the explants,leaves were treated as follow:detergent for 20 minutes,running tap water for 30 minutes and 0.1%HgCl_2 for 7 minutes.And the contaminate rate can be controlled in 37%and the death rate also can be controlled in 0%.The explants was cultured on the inducement medium MS+NAA 0.5mg/L+6-BA 3.0mg/L(pH=5.8)and the inducement rate could reach to 83.33%.The best medium for propagation is MS+NAA 0.1mg/L+6-BA 1.0 mg/L(pH=5.8)and the split rate is 86.67%.1/2MS added 1.0mg/L NAA can make the root long and strong,and the rooting rate could reach to 86.67%.When the length of root is 1～2cm,lilies can be acclimatized and the survival rate can reach to 80%.
To induce polyploid,we deal embryogenic callus and caespitose buds with different methods called mixing culture with colchicine,immersing stem segment,pre-cultivated firstly and then sprinkling colchicine respectively.As the results show that the follow method is satisfying:0.1% colchicine immerse stem segment for 48 hours.The survival rate and induce rate are 66.67%and 75.00%respectively.Chromosome identification shows that the number of chromosome of the diploid is 2n=2X=20,while that of the tetraploid is 2n=4X=40.Morphologically,the stems of the tetraploid become stronger,the leaves become longer,broader and thicker,the color gets darker than that of the diploid,and the leaf index declines.Compared with the characteristic of the stoma of the diploid,the guard cell of the tetraploid becomes longer and broader;the number of the stomas declines and the number of chloroplast increases.
引文
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