继代培养时间对抗性黑松体胚发生的影响
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摘要
以继代培养0.5、1.5 a和3.5 a的抗性黑松37#家系胚性愈伤组织为材料,观察不同培养时间胚性细胞的形态和体胚成熟的过程,并对愈伤组织的增殖率、每克愈伤形成的体胚数量及体胚萌发率和植株成活率进行测定。结果表明:在继代培养过程中,胚性胚柄细胞团(ESM)结构逐渐疏松,胚柄细胞呈现缩短、增粗的现象;培养时间对抗性黑松愈伤组织的增殖、分化、体胚萌发及植株成活率均有显著影响。3种不同培养时间愈伤组织的增殖率分别为200.37%、182.47%和111.63%,每克胚性愈伤组织形成成熟体细胞胚分别为77、31、5个,萌发率分别为84.43%、51.10%、11.13%;植株成活率分别为73.00%、50.00%、6.70%,均呈下降趋势;3种愈伤体胚成熟的时间也存在显著差异,继代培养时间越长,体胚分化成熟过程越慢,愈伤组织形成子叶胚的时间最短为46 d,最长为74 d。因此,抗性黑松胚性愈伤组织在继代培养过程中胚性逐步降低,体胚发生和植株再生能力下降,短期培养的胚性愈伤组织(0.5 a)为抗性黑松体胚发生较为理想的初始材料,且愈伤组织继代培养时间不宜超过1.5 a。
The callus of 37#family used for the experimentwas cultured for 0.5 years,1.5 years and 3.5 years,respectively. During this period, the cell morphology and the maturation stage of somatic embryos were observed.In addition, the proliferation rate, the number of somatic embryos per gram of embryonic callus, the normal germination rate and survival rate of plants were measured. The results revealed that embryonic somatic mass(ESM)gradually became loose and cell stalk was short, thick and deformed in the long-term subculture process. The time of subculture had significant effects not only on the proliferation and differentiation of callus but also somatic embryo germination and survival rate of plants. The proliferation rate of 3 kinds callus was 200.37%, 182.47% and111.63%, the number of somatic embryos was 77, 31 and 5, the normal germination rate was 84.43%, 51.10% and11.13%, and the survival rate of plants was 73.00%, 50.00% and 6.70%, respectively, all showing a downward trend. Additionally, there were significant differences in the time of embryo maturation among 3 kinds callus. The callus formed the cotyledon embryo for a minimum of 46 days and a maximum of 74 days. As the subculture time increased, the process of somatic embryo differentiation became slower and slower. It can be seen that the potential of embryogenic callus of nematode-resistant Pinus thunbergii was gradually reduced and the abilities of somatic embryogenesis and plants regeneration were decreased during the subculture. And the short-term cultured embryogenic callus(0.5 years) was an ideal starting material for somatic embryogenesis of nematode-resistant P.thunbergii, and the callus culture time should be less than 1.5 years.
The callus of 37#family used for the experimentwas cultured for 0.5 years,1.5 years and 3.5 years,respectively. During this period, the cell morphology and the maturation stage of somatic embryos were observed.In addition, the proliferation rate, the number of somatic embryos per gram of embryonic callus, the normal germination rate and survival rate of plants were measured. The results revealed that embryonic somatic mass(ESM)gradually became loose and cell stalk was short, thick and deformed in the long-term subculture process. The time of subculture had significant effects not only on the proliferation and differentiation of callus but also somatic embryo germination and survival rate of plants. The proliferation rate of 3 kinds callus was 200.37%, 182.47% and111.63%, the number of somatic embryos was 77, 31 and 5, the normal germination rate was 84.43%, 51.10% and11.13%, and the survival rate of plants was 73.00%, 50.00% and 6.70%, respectively, all showing a downward trend. Additionally, there were significant differences in the time of embryo maturation among 3 kinds callus. The callus formed the cotyledon embryo for a minimum of 46 days and a maximum of 74 days. As the subculture time increased, the process of somatic embryo differentiation became slower and slower. It can be seen that the potential of embryogenic callus of nematode-resistant Pinus thunbergii was gradually reduced and the abilities of somatic embryogenesis and plants regeneration were decreased during the subculture. And the short-term cultured embryogenic callus(0.5 years) was an ideal starting material for somatic embryogenesis of nematode-resistant P.thunbergii, and the callus culture time should be less than 1.5 years.
引文
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[21]许建秀,吴静,叶建仁,等.抗松材线虫病赤松胚性愈伤组织的维持与稳定增殖[J].南京林业大学学报(自然科学版),2017,41(1):49-54.
[22]Gupta P K,Durzan D J.Shoot multiplication from mature trees of Douglas-fir(Pseudotsuga menziesii)and sugar pine(Pinus lambertiana)[J].Plant Cell Reports,1985,4(4):177-179.
[23]Arnold S,Eriksson T.A revised medium for growth of pea mesophyll protoplasts[J].Physiologia Plantarum,2010,39(4):257-260.
[24]Pullman G S,Johnson S,Tassel S V,et al.Somatic embryogenesis in loblolly pine(Pinus taeda)and Douglas fir(Pseudotsuga menziesii):improving culture initiation and growth with MES pH buffer,biotin,and folic acid[J].Plant Cell Tissue&Organ Culture,2005,80(1):91-103.
[25]许建秀,吴小芹,叶建仁,等.抗松材线虫病赤松体细胞胚的发育和成熟萌发[J].林业科学,2017,53(12):41-49.
[26]Schwartz B W,Vernon D A,Meinke D W.Development of thesuspensor:differentiation,communication,and programmed cell death during plant embryogenesis[M]//Larkins B A,Vasil I K.Cellular and molecular biology of plant seed development,vol 4,Advances in cellular and molecular biology of plants.Dordrecht:Springer,1997:53-72.
[27]都晓龙,孙春玉,张美萍,等.继代次数对吉粳88愈伤组织生理生化指标及细胞形态的影响[J].安徽农业科学,2016,44(5):155-158.
[28]Fowke L C,Attree S M.Applied and basic studies of somatic embryogenesisin white spruce(Picea glanca)and black spruce(Picea mariana)[C]//Woong Y S,Advances in Developmental Biology and Biotechnology of Higher Plants.The Korean Sociely of Plant Tissue Culture,1993:5-7.
[29]Gupta P K,Timmis R,Carlson W C.Somatic embryogenesis:A possible tool for large-scale propagation of forestry species[C]//Woong Y S.Advances in Developmental Biology and Biotechnology of Higher Plants.The Korean Society of Plant Tissue Culture,1993:18-37.
[30]耿菲菲,肖丰坤,吴涛,等.思茅松成熟体细胞胚的胚性愈伤组织诱导与增殖[J].东北林业大学学报,2015,43(5):59-63.
[2]王斐,申荷丽.日本的抗松材线虫育种研究[J].世界林业研究,2004,10(6):44-46.
[3]李清清,叶建仁,朱丽华,等.抗松材线虫病赤松组培苗的抗病性测定[J].东北林业大学学报,2013,41(7):45-47.
[4]Zhu L H,Chu X F,Sun T Y,et al.Micropropagation of Pinus densiflora,and the evaluation of nematode resistance of regenerated microshoots in vitro[J].Journal of Forestry Research,2018(2):1-10.
[5]梁芬.抗松材线虫病黑松体细胞胚胎发生及植株再生体系研究[D].南京:南京林业大学,2016.
[6]孙婷玉,叶建仁,吴小芹,等.抗松材线虫病黑松胚性愈伤组织诱导[J].东北林业大学学报,2015,43(9):96-99.
[7]刘玲梅,汤浩茹,刘娟.试管苗长期继代培养中的形态发生能力与遗传稳定性[J].生物技术通报,2008,4(5):22-27.
[8]薛美凤,郭余龙,李名扬,等.长期继代对棉花胚性愈伤组织体胚发生能力及再生植株变异的影响[J].西南农业学报,2002,15(4):19-21.
[9]Lai K L,Liu L F.International congress of plant tissue and cell culture[C]//Minn USA Abstrcts.University Minnesota Minneapolis,1986:186.
[10]及华,张海新,赵玉芬,等.梨S系矮化砧组培苗增殖系数和质量的影响因子[J].河北农业科学,2004,8(2):40-44.
[11]续九如,李春立,孙建设.毛叶枣组培快繁技术研究[J].北京林业大学学报,2003,25(3):28-32.
[12]苏秀城.杉木无性系不同继代代数组培苗差异研究[J].福建林学院学报,2000,20(4):353-356.
[13]刘福平,林清洪,章宁.球根海棠继代愈伤组织分化潜力丧失研究(简报)[J].亚热带植物科学,2003,32(4):64-51.
[14]Lelu M A,Bastien C,Klimaszewska K,et al.An improved method for somatic plantlet production in hybrid larch(Larix×leptoeuropaea):Part 1.Somatic embryo maturation[J].Plant Cell,Tissue and Organ Culture,1994,36(1):107-115.
[15]Tautorus T E,Fowke L C,Dunstan D I.Somatic embryogenesis in conifers[J].Canadian Journal of Botany,1991,69(9):1873-1899.
[16]杨金玲,桂耀林,郭仲琛.白杄胚性愈伤组织长期继代培养中的分化能力及染色体稳定性研究[J].西北植物学报,2000,20(1):44-47,158.
[17]杨映根,桂耀林,唐巍,等.青杄愈伤组织在继代培养中的分化能力及染色体稳定性研究[J].植物学报,1994,36(12):934-939,981.
[18]王喆之,李克勤,张大力,等.陆地棉胚性愈伤组织的变异及高频胚胎发生[J].植物学报,1994,36(5):331-408.
[19]张宝红,李凤莲,李付广,等.棉花胚性愈伤组织的长期保持技术[J].中国棉花,1996,25(4):26.
[20]向凤宁,张举仁,陈惠民,等.玉米胚性愈伤组织的长期继代及其染色体分析[J].西北植物学报,1994,14(3):157-163.
[21]许建秀,吴静,叶建仁,等.抗松材线虫病赤松胚性愈伤组织的维持与稳定增殖[J].南京林业大学学报(自然科学版),2017,41(1):49-54.
[22]Gupta P K,Durzan D J.Shoot multiplication from mature trees of Douglas-fir(Pseudotsuga menziesii)and sugar pine(Pinus lambertiana)[J].Plant Cell Reports,1985,4(4):177-179.
[23]Arnold S,Eriksson T.A revised medium for growth of pea mesophyll protoplasts[J].Physiologia Plantarum,2010,39(4):257-260.
[24]Pullman G S,Johnson S,Tassel S V,et al.Somatic embryogenesis in loblolly pine(Pinus taeda)and Douglas fir(Pseudotsuga menziesii):improving culture initiation and growth with MES pH buffer,biotin,and folic acid[J].Plant Cell Tissue&Organ Culture,2005,80(1):91-103.
[25]许建秀,吴小芹,叶建仁,等.抗松材线虫病赤松体细胞胚的发育和成熟萌发[J].林业科学,2017,53(12):41-49.
[26]Schwartz B W,Vernon D A,Meinke D W.Development of thesuspensor:differentiation,communication,and programmed cell death during plant embryogenesis[M]//Larkins B A,Vasil I K.Cellular and molecular biology of plant seed development,vol 4,Advances in cellular and molecular biology of plants.Dordrecht:Springer,1997:53-72.
[27]都晓龙,孙春玉,张美萍,等.继代次数对吉粳88愈伤组织生理生化指标及细胞形态的影响[J].安徽农业科学,2016,44(5):155-158.
[28]Fowke L C,Attree S M.Applied and basic studies of somatic embryogenesisin white spruce(Picea glanca)and black spruce(Picea mariana)[C]//Woong Y S,Advances in Developmental Biology and Biotechnology of Higher Plants.The Korean Sociely of Plant Tissue Culture,1993:5-7.
[29]Gupta P K,Timmis R,Carlson W C.Somatic embryogenesis:A possible tool for large-scale propagation of forestry species[C]//Woong Y S.Advances in Developmental Biology and Biotechnology of Higher Plants.The Korean Society of Plant Tissue Culture,1993:18-37.
[30]耿菲菲,肖丰坤,吴涛,等.思茅松成熟体细胞胚的胚性愈伤组织诱导与增殖[J].东北林业大学学报,2015,43(5):59-63.