杉木未成熟胚胚性愈伤组织诱导影响因素探析
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摘要
该研究从基因型、6-BA浓度、外植体接种方式和合子胚发育阶段等方面,分析杉木未成熟胚胚性愈伤组织诱导的影响因素。结果表明:基因型、6-BA浓度、外植体接种方式和合子胚发育阶段均对胚性愈伤组织诱导频率有不同程度影响。6种基因型中,有3种基因型诱导出胚性愈伤组织,其中基因型S18胚性愈伤组织诱导频率最高,为11.7%。6-BA浓度在1.0~1.5 mg·L~(-1)范围内时,基因型S18的胚性组织诱导频率较高。以在去皮种子的一端切开一个小口的接种方式为最优,将合子胚剥出的方式易造成合子胚褐化死亡,将未剥皮的种子切开一个小口后直接接入培养基的方式不利于愈伤组织生成。适合胚性愈伤组织诱导的合子胚发育阶段为受精至胚器官分化阶段,合子胚进入成熟阶段后不利于胚性愈伤组织诱导,合子胚易生长成完整植株。
Cunninghamia lanceolata is a fast-growing and high-yield timber forest species endemic in China. It has many advantages of fast growth,high production,good quality and wide use,and plays an important role in the forestry production in South China. In this research,an experiment of embryogenic callus induction was carried out. In the experiment,six different genotypes of C. lanceolata improvement species were used as initial explant,and four factors affecting embryogenic callus induction were studied and analysed. These four factors were the key elements on embryogenic callus formation,and they were as follows: genotypes,6-BA combination,inoculating ways and developmental stages of immature zygotic embryos including. The results showed that the factors of genotypes,6-BA combination,inoculating ways and developmental stages of immature zygotic embryos all had significant effects on the induction rate of embryogenic callus,but the influence degree each were not identical. Three genotypes in six genotypes obtained embryogenic callus,and one of the genotype( S18) had the highest induction rate of embryogenic callus with 11.7%. When the range of 6-BA combinations was from 1.0 mg·L-1to 1.5 mg·L-1,the genotype S18 had the highest induction rate. The best method of inoculating was to peel and cut a small hole on end of seed. It was not a good inoculating way to take out zygotic embryos from endosperm,and the zygotic embryos were easy to die in that way. The inoculating method of only cutting a small hole on end of seed and non-peeling was unsuited for embryogenic callus induction. The suitable developmental stage for embryogenic callus induction was from fertilization stage to embryo organ differentiation stage,and it was difficult to obtain embryogenic callus when the zygotic embryos entered into the mature stage. The results indicated that the zygotic embryos which already entered into the mature stage were used as initial explants in the experiment of embryogenic callus induction,and they grew into a complete plant,not callus.
Cunninghamia lanceolata is a fast-growing and high-yield timber forest species endemic in China. It has many advantages of fast growth,high production,good quality and wide use,and plays an important role in the forestry production in South China. In this research,an experiment of embryogenic callus induction was carried out. In the experiment,six different genotypes of C. lanceolata improvement species were used as initial explant,and four factors affecting embryogenic callus induction were studied and analysed. These four factors were the key elements on embryogenic callus formation,and they were as follows: genotypes,6-BA combination,inoculating ways and developmental stages of immature zygotic embryos including. The results showed that the factors of genotypes,6-BA combination,inoculating ways and developmental stages of immature zygotic embryos all had significant effects on the induction rate of embryogenic callus,but the influence degree each were not identical. Three genotypes in six genotypes obtained embryogenic callus,and one of the genotype( S18) had the highest induction rate of embryogenic callus with 11.7%. When the range of 6-BA combinations was from 1.0 mg·L-1to 1.5 mg·L-1,the genotype S18 had the highest induction rate. The best method of inoculating was to peel and cut a small hole on end of seed. It was not a good inoculating way to take out zygotic embryos from endosperm,and the zygotic embryos were easy to die in that way. The inoculating method of only cutting a small hole on end of seed and non-peeling was unsuited for embryogenic callus induction. The suitable developmental stage for embryogenic callus induction was from fertilization stage to embryo organ differentiation stage,and it was difficult to obtain embryogenic callus when the zygotic embryos entered into the mature stage. The results indicated that the zygotic embryos which already entered into the mature stage were used as initial explants in the experiment of embryogenic callus induction,and they grew into a complete plant,not callus.
引文
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ZHEN RH,2008.The researches of tissue culture and somatic embryogenesis on Chinese fir duperior clones[D].Xiamen:Xiamen University:46-53.[郑仁华,2008.杉木优良无性系组培快繁及体胚发生试验研究[D].福建:厦门大学:46-53.]
CHEN JH,WANG HY,ZHU GQ,et al,2000.Technical progress of plant somatic embryogenesis[J].Chin For Sci Technol,14(3):9-11.[陈金慧,王洪云,诸葛强,等,2000.林木体胚发生技术进展[J].林业科技开发,14(3):9-11.]
FELIPE A,POUPIN MJ,MATUS JT,et al,2008.Synthetic seed production from somatic embryos of Pinus radiate[J].Biotechnol Lett,30:1847-1852.
HUANG AM,FEI BH,LIU JL,2006.Advances in research on the wood property of Chinese fir[J].World For Res,19(1):47-52.[黄安民,费本华,刘君良,2006.杉木木材性质研究进展[J].世界林业研究,19(1):47-52.]
HUANG QJ,WEI ZM,1995.Review on somatic embryogenesis of coniferous tree[J].Chin For Sci Technol,31(2):85-90.[黄健秋,卫志明,1995.针叶树体细胞胚胎发生的研究进展[J].植物生理学通讯,31(2):85-90.]
LI CH,LIU BG,KIMTD,et al,2003.Somatic embryogenesis and plant regeneration in elite genotypes of Picea koraiensis[J].Plant Biotechnol Rep,2:259-265.
LU SF,ZHANG SG,QI LW,et al,2003.Application of somatic embryognenesis in woody species[J].World For Res,16(6):6-11.[吕守芳,张守攻,齐力旺,等,2003.体细胞胚胎发生在林木树种中的应用[J].世界林业究,16(6):6-11.]
PARK YS,LELUW,HARVEGNT L,et al,2006.Initiation of somatic embryogenesis in Pinus banksiana,P.strobus,P.pinaster,and P.sylvestris at three laboratories in Canada and France[J].Plant Cell Tiss Org,86:87-101.
SALAJOVA T,SALAJ J,2005.Somatic embryogenesis in Pinus nigra:embryogenic tissue Initiation,maturation and regeneration ability of established cell lines[J].Biol Plant,49(3):33-339.
SHI JS,2000.The challenges to forestry biotechnology and tree breeding in the 21st century[J].J Nanjing For Univ,24(6):1-6.[施季森,2000.迎接21世纪现代林木生物技术育种的挑战[J].南京林业大学学报,24(6):1-6.]
WEN YF,2011.The research of initiation and subculture of embryogenesis tissue of Cunninghina lanceolata(Lamb)Hook based on the immature zygotic embryo[D].Fuzhou:Fujian Agriculture and Forestry University:5-19.[温亚峰,2011.基于未成熟合子胚的杉木胚性组织诱导和继代的研究[D].福建:福建农林大学:5-19.]
XI ML,SHI JS,2005.Organogenesis and somatic embryogenesis from cotyledons and hypocotyls of Cunninghamia lanceolata Hook[J].Mol Plant Breed,3(6):846-852.[席梦利,施季森,2005.杉木子叶和下胚轴的器官发生与体胚发生[J].分子植物育种,3(6):846-852.]
XI ML,SHI JS,2006.Organogenesis and somatic embryogenesis from mature zygotic embryos of Cunninghamia lanceolata[J].Sci Silv Sin,42(9):29-33.[席梦利,施季森,2006.杉木成熟合子胚器官发生和体胚发生[J].林业科学,42(9):29-33.]
YILDIRIM T,KAYA Z,ISIK K,2006.Induction of embryogenic tissue and maturation of somatic embryos in Pinus brutia Ten[J].Plant Cell Tiss Org,87:67-76.
ZHANG CX,LI Q,KONG LS,2007.Induction,development and maturation of somatic embryos in Bunge’s pine(Pinus bungeana Zucc.ex Endl.)[J].Plant Cell Tiss Org,91:273-280.
ZHEN RH,2008.The researches of tissue culture and somatic embryogenesis on Chinese fir duperior clones[D].Xiamen:Xiamen University:46-53.[郑仁华,2008.杉木优良无性系组培快繁及体胚发生试验研究[D].福建:厦门大学:46-53.]