云南松胚性愈伤组织诱导研究
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摘要
以未成熟云南松种子为研究材料,选择合适的消毒方法,进行合子胚发育状态观测,从外植体类型、采果母树基因型、培养基种类3方面探讨其对胚性愈伤组织诱导的影响,并鉴定了胚性愈伤组织。结果表明:75%乙醇消毒30 s,无菌水冲洗2次,2%氯酸钠浸泡15 min,无菌水冲洗4～5次为种子最佳消毒方案;不同外植体类型对出愈率影响不显著,但对胚性愈伤组织的形成具有极显著影响,取带胚乳的合子胚作为外植体的胚性愈伤组织诱导率可达34.3%,而合子胚作为外植体时诱导率为14.3%;采用14种培养基接种,平均诱导率在2.2%～17.6%之间,其中接种于MLV+2, 4-D 2.0 mg/L+6-BA 1.0 mg/L+KT 1.0 mg/L+肌醇0.1 g/L,1/2LM+2, 4-D 2.2 mg/L+6-BA 1.1 mg/L+AgNO3 3.4 mg/L+肌醇0.1 g/L,DCR+6-BA 0.63 mg/L+KT 0.61 mg/L+NAA 2.0 mg/L+肌醇0.2 g/L+麦芽糖15 g/L培养基的胚性愈伤诱导率分别为17.6%、17.6%、16.5%,表明该3种培养基适合于云南松的胚性愈伤诱导;8株云南松优株未成熟带胚乳合子胚的胚性愈伤组织平均诱导率为5.1%～19.4%,最高的是076号云南松,最低的是075号云南松,表明基因型对胚性愈伤组织诱导具有极显著的影响。
Using immature Pinus yunnanensis seeds as research materials, select appropriate disinfection methods to observe the development status of zygotic embryos.The effects of explants, genotypes and medium types on embryogenic callus induction were investigated, and embryogenic calli were identified.Results show that the best disinfection method include the following steps: using 75% alcohol to disinfect for 30 s, followed by the rinsing process with sterile water for 2-3 times, soaking in 2% sodium chlorate for 15 min, and rinsing again with sterile water for 4-5 times. Different explant types impose no significant effects on the callus yield, but have significant effects on the formation of embryonic callus. The induction rate of embryonic callus is up to 34.3% when the female gamete is used as the explant, while 14.3% when the zygotic embryo is used as the explant. The average induction rate is between 2.2% and 17.6% when inoculate with 14 different kinds of media. The embryonic callus induction rates is 17.6%, 17.6% and 16.5% when inoculated with MLV+2,4-D 2.0 mg/L+6-BA 1.0 mg/L+KT 1.0 mg/L+ myo-Inositol 0.1 g/L, 1/2 LM+2,4-D 2.2 mg/L+6-BA 1.1 mg/L +AgNO3 3.4 mg/L+ myo-Inositol0.1 g/L and DCR +6-BA 0.63 mg/L +KT 0.61 mg/L+NAA 2.0 mg/L+ myo-Inositol 0.2 g/L+ maltose 15 g/L medium respectively, which indicating that the selected 3 types of medium are suitable for embryonic callus induction of female gametophyte of P. yunnanensis. The average induction rate of embryonic callus of 8 immature female gametophytes in P. yunnanensis is 5.1%-19.4%, and genotype 076 and 075 show the highest and lowest induction rate, respectively, suggesting that the genotype have significant influence on the induction of embryonic callus.
Using immature Pinus yunnanensis seeds as research materials, select appropriate disinfection methods to observe the development status of zygotic embryos.The effects of explants, genotypes and medium types on embryogenic callus induction were investigated, and embryogenic calli were identified.Results show that the best disinfection method include the following steps: using 75% alcohol to disinfect for 30 s, followed by the rinsing process with sterile water for 2-3 times, soaking in 2% sodium chlorate for 15 min, and rinsing again with sterile water for 4-5 times. Different explant types impose no significant effects on the callus yield, but have significant effects on the formation of embryonic callus. The induction rate of embryonic callus is up to 34.3% when the female gamete is used as the explant, while 14.3% when the zygotic embryo is used as the explant. The average induction rate is between 2.2% and 17.6% when inoculate with 14 different kinds of media. The embryonic callus induction rates is 17.6%, 17.6% and 16.5% when inoculated with MLV+2,4-D 2.0 mg/L+6-BA 1.0 mg/L+KT 1.0 mg/L+ myo-Inositol 0.1 g/L, 1/2 LM+2,4-D 2.2 mg/L+6-BA 1.1 mg/L +AgNO3 3.4 mg/L+ myo-Inositol0.1 g/L and DCR +6-BA 0.63 mg/L +KT 0.61 mg/L+NAA 2.0 mg/L+ myo-Inositol 0.2 g/L+ maltose 15 g/L medium respectively, which indicating that the selected 3 types of medium are suitable for embryonic callus induction of female gametophyte of P. yunnanensis. The average induction rate of embryonic callus of 8 immature female gametophytes in P. yunnanensis is 5.1%-19.4%, and genotype 076 and 075 show the highest and lowest induction rate, respectively, suggesting that the genotype have significant influence on the induction of embryonic callus.
引文
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[12]赵晓敏,沈海龙,杨玲,等.兴安落叶松胚性愈伤组织诱导影响因子的研究[J].植物研究,2007,27(5):538-543.
[13]Egertsdotter U.Plant physiological and genetical aspects of the somatic embryogenesis process in conifers[J].Scandinavian Journal of Forest Research,2018(4):1-38.
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[16]袁澍,贾勇炯,林宏辉.诱导植物体细胞胚发生的几个生理因素[J].植物生理学通讯,2003,39(5):508-512.
[17]Duncan D R,Williams M E,Zehr B E,et al.The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes[J].Planta,1985,165(3):322-332.
[18]Park Y S,Lelu-Walter M A,Harvengt L,et al.Initiation of somatic embryogenesis in Pinus banksiana,P.sylvestris at three laboratories in Canada and France[J].Plant Cell,Tissue and Organ Culture,2006,86(1):87-101.
[2]Thompson R G,von Aderkas P.Somatic embryogenesis and plant regeneration from immature embryos of western larch[J].Plant Cell Reports,1992,11(8):379-385.
[3]季孔庶,王潘潘,王金铃,等.松科树种的离体培养研究进展[J].南京林业大学学报(自然科学版),2015,39(1):142-148.
[4]Hakman I,Fowke L C,von Arnold S,et al.The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies(Norway Spruce)[J].Plant Science,1985,38(1):53-59.
[5]黄健秋,卫志明.针叶树体细胞胚胎发生的研究进展[J].植物生理学通讯,1995,31(2):85-90.
[6]唐巍,欧阳藩,郭仲琛.湿地松体细胞胚胎发生和植株再生[J].植物资源与环境,1997,6(2):8-11.
[7]申晓辉,蒋湘宁,Park Y S,et al.红松体细胞胚胎培养技术体系的建立[J].成都大学学报(自然科学版),2005,24(1):11-14.
[8]吴涛,陈少瑜,陈芳,等.云南松合子胚发育及形态特征[J].西北林学院学报,2008,23(6):91-93.
[9]齐力旺.华北落叶松体细胞胚胎发生与遗传转化系统建立的研究[D].北京:中国林业科学研究院,2000.
[10]王伟达,李成浩,张含国,等.长白落叶松愈伤组织诱导与不定芽分化[J].东北林业大学学报,2008,36(2):5-7.
[11]白卉,王伟功,曹焱.红皮云杉胚性愈伤组织诱导影响因子的研究[J].黑龙江生态工程职业学院学报,2008,21(3):20-22.
[12]赵晓敏,沈海龙,杨玲,等.兴安落叶松胚性愈伤组织诱导影响因子的研究[J].植物研究,2007,27(5):538-543.
[13]Egertsdotter U.Plant physiological and genetical aspects of the somatic embryogenesis process in conifers[J].Scandinavian Journal of Forest Research,2018(4):1-38.
[14]Couée I,Hummel I,Sulmon C,et al.Involvement of polyamines in root development[J].Plant Cell,Tissue and Organ Culture,2004,76(1):1-10.
[15]吴丽君.湿地松、火炬松离体培养植株再生技术的研究[D].南京:南京林业大学,2008.
[16]袁澍,贾勇炯,林宏辉.诱导植物体细胞胚发生的几个生理因素[J].植物生理学通讯,2003,39(5):508-512.
[17]Duncan D R,Williams M E,Zehr B E,et al.The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes[J].Planta,1985,165(3):322-332.
[18]Park Y S,Lelu-Walter M A,Harvengt L,et al.Initiation of somatic embryogenesis in Pinus banksiana,P.sylvestris at three laboratories in Canada and France[J].Plant Cell,Tissue and Organ Culture,2006,86(1):87-101.