湿地松及其杂种的体细胞胚胎发生与植株再生
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摘要
【目的】明确适合湿地松Pinus elliottii及其杂交种的体细胞胚胎发生条件,建立体胚成熟萌发的技术。【方法】以2016年6月采集的2个湿地松家系(EE1,EE2)、2个湿地松杂交种家系(EC,EH)的未成熟合子胚(含胚乳)为材料,从诱导、增殖、成熟到萌发配制3个系列培养基,比较外植体采集时间、家系、基本培养基对胚性愈伤组织诱导的影响。用显微镜进行胚性愈伤组织鉴别后,进一步挑选诱导形成的胚性愈伤组织进行增殖、成熟、萌发,最终获得再生植株。【结果】湿地松及其杂交种合子胚的发育进程可划分为8个阶段,阶段Ⅱ、Ⅲ为未成熟胚,适合体胚发生,EC最早出现阶段Ⅲ合子胚。外植体诱导产生具有胚性胚柄团(ESM)结构的胚性愈伤组织,可进一步增殖。诱导培养基对愈伤组织形成及胚性愈伤组织占比具有较大影响,3种诱导培养基(T1、T2和T3)产生愈伤组织效率最高的为T1培养基(49.0%),胚性愈伤组织所占愈伤组织的比例最高为T2培养基(22.4%)。培养基配方的诱导率存在基因型间的差异,T1培养基整体诱导率低;T2培养基对家系EE1诱导率最高,为5.82%;T3培养基对参试材料均能诱导成功,且平均诱导率最高,为3.75%。随采样时间延后,家系EE1、EH的体胚诱导率逐渐增加,家系EE2、EC的体胚诱导率则随采样时间延后逐渐降低。体胚诱导率与合子发育阶段结果基本一致,阶段Ⅲ合子胚均出现较晚,前期诱导率低。胚性愈伤组织继代24次以后,胚活性逐渐降低。成熟培养基T1S和T3S可完成胚的成熟,平均每克成熟培养基分别成熟23.3和15.9个子叶胚,萌发率为32.1%,移栽保存率为47.8%。【结论】诱导培养基T3对参试家系均能诱导成功,T3具有较广泛适用性,各家系在阶段Ⅲ合子胚出现时表现出较大的诱导率,阶段Ⅲ合子胚可能为体细胞胚发生的最佳诱导阶段。建立了湿地松及其杂种体细胞胚发生方法并形成了再生植株。
【Objective】 To define the conditions suitable for somatic embryogenesis of Pinus elliottii and its hybrids, and develop a mature protocol for somatic embryogenesis maturation and germination. 【Method】 The immature zygotic embryos(with endosperm) of two P. elliottii families(EE1, EE2) and two P. elliottii hybrid(EC, EH) were sampled in June 2016. Three series of medium from induction, proliferation maturation to germination were set to compare the effects of explant collection date, family and basal medium on embryogenic callus induction. The embryogenic callus were identified through microscopic observation, and choosen for proliferation, maturation and germination culture. Finally the regenerated plantlets were developed.【Result】 The zygotic embryo development processes of P. elliottii and hybrids were divided into eight stages.The immature embryos at stage Ⅱ and Ⅲ were suitable for somatic embryogenesis. The embryos at stage Ⅲfirst appeared in EC. The embryogenic callus with embryonal suspensor mass(ESM) could be further proliferated. The induction mediums(T1, T2 and T3) played vital roles in callus formation and embryogenic callus proportion. T1 medium had the highest callus induction efficiency(49.0%), and T2 medium had the highest embryogenic callus proportion(22.4%).The mediums had genotype-specific induction rates. The overall induction rate of T1 medium was low, and T2 medium had the highest induction rate(5.82%) for EE1. T3 medium was suitable for all tested materials, with the highest mean induction rate of 3.75%. The somatic embryo induction rates of EE1 and EH gradually increased with the sampling time extension, while the somatic embryo induction rates of EE2 and EC gradually decreased. The results of somatic embryo induction rate were basically corresponded with zygote development stages. The stage Ⅲ zygotic embryo appeared late, and its induction rate was low in early stage. After the embryogenic callus were subcultured for 24 times, the embryo activities progressively decreased. The maturation medium T1 S and T3 S could accomplish embryo maturation.Their average ripening efficiencies in per gram of mature medium were 23.3 and 15.9 cotyledons, respectively.The germination rate was 32.1% and the transplanting rate was 47.8%. 【Conclusion】 All tested materials can induce somatic embryo in T3 medium. T3 medium is widely applicable, and all materials have high induction rates when zygotic embryo appears at stage Ⅲ, illustrating that stage Ⅲ may be the optimal for somatic embryo induction. The experiment establishes the protocol for somatic embryogenesis and plantlet regeneration.
【Objective】 To define the conditions suitable for somatic embryogenesis of Pinus elliottii and its hybrids, and develop a mature protocol for somatic embryogenesis maturation and germination. 【Method】 The immature zygotic embryos(with endosperm) of two P. elliottii families(EE1, EE2) and two P. elliottii hybrid(EC, EH) were sampled in June 2016. Three series of medium from induction, proliferation maturation to germination were set to compare the effects of explant collection date, family and basal medium on embryogenic callus induction. The embryogenic callus were identified through microscopic observation, and choosen for proliferation, maturation and germination culture. Finally the regenerated plantlets were developed.【Result】 The zygotic embryo development processes of P. elliottii and hybrids were divided into eight stages.The immature embryos at stage Ⅱ and Ⅲ were suitable for somatic embryogenesis. The embryos at stage Ⅲfirst appeared in EC. The embryogenic callus with embryonal suspensor mass(ESM) could be further proliferated. The induction mediums(T1, T2 and T3) played vital roles in callus formation and embryogenic callus proportion. T1 medium had the highest callus induction efficiency(49.0%), and T2 medium had the highest embryogenic callus proportion(22.4%).The mediums had genotype-specific induction rates. The overall induction rate of T1 medium was low, and T2 medium had the highest induction rate(5.82%) for EE1. T3 medium was suitable for all tested materials, with the highest mean induction rate of 3.75%. The somatic embryo induction rates of EE1 and EH gradually increased with the sampling time extension, while the somatic embryo induction rates of EE2 and EC gradually decreased. The results of somatic embryo induction rate were basically corresponded with zygote development stages. The stage Ⅲ zygotic embryo appeared late, and its induction rate was low in early stage. After the embryogenic callus were subcultured for 24 times, the embryo activities progressively decreased. The maturation medium T1 S and T3 S could accomplish embryo maturation.Their average ripening efficiencies in per gram of mature medium were 23.3 and 15.9 cotyledons, respectively.The germination rate was 32.1% and the transplanting rate was 47.8%. 【Conclusion】 All tested materials can induce somatic embryo in T3 medium. T3 medium is widely applicable, and all materials have high induction rates when zygotic embryo appears at stage Ⅲ, illustrating that stage Ⅲ may be the optimal for somatic embryo induction. The experiment establishes the protocol for somatic embryogenesis and plantlet regeneration.
引文
[1]李彦杰,栾启福,沈丹玉,等.湿地松自由授粉家系松脂组分遗传变异研究[J].林业科学研究,2012,25(6):773-779.
[2]WEN X,KUANG Y,SHI M,et al.Biology of Hylobitelus xiaoi(Coleoptera:Curculionidae),a new pest of slash pine,Pinus elliottii[J].J Econ Entomol,2004,97(6):1958-1964.
[3]MEANS D B,PALIS J G,BAGGETT M.Effects of slash pine silviculture on a florida population of flatwoods salamander[J].Conserv Biol,1996,10(2):426-437.
[4]张帅楠.湿地松材性测定技术研究与多性状联合选择[D].北京:中国林业科学研究院,2017.
[5]宗亦臣,郑勇奇,马锡权,等.湿加松良种‘中林1号’[J].湖南林业科技,2017,44(1):76-77.
[6]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.
[7]姜福星,魏丕伟,寇亚平,等.探析植物体胚发生的关键基因[J].分子植物育种,2017,15(4):1304-1311.
[8]QUIROZ-FIGUEROA F R,ROJAS-HERRERA R,GALAZ-AVALOS R M,et al.Embryo production through somatic embryogenesis can be used to study cell differentiation in plants[J].Plant Cell Tissue Organ Cult,2006,86(3):285-301.
[9]KIM Y W,MOON H K.Enhancement of somatic embryogenesis and plant regeneration in Japanese red pine(Pinus densiflora)[J].Plant Biotechnol Rep,2014,8(3):259-266.
[10]PULLMAN G S,BUCALO K.Plant embryo culture[M].New York:Humana Press,2011:267-291.
[11]PULLMAN G S,MEIN J,JOHNSON S,et al.Gibberellin inhibitors improve embryogenic tissue initiation in conifers[J].Plant Cell Rep,2005,23(9):596-605.
[12]BONGA J M,PARK Y S,OVERTON C,et al.Optimized somatic embryogenesis in Pinus strobus L.[J].In Vitro Cell Dev Biol Plant,2013,37(3):392-399.
[13]BECWAR M,CLARK J,SWINTON L S,et al.Liquidbased method for producing plant embryos:US8313946B2[P].2012-11-20.
[14]GUPTA P K,HOLMSTROM D.Protocol for somaticembryogenesis in woody plants[M].Dordrecht:Springer,2005:573-575.
[15]李清清,叶建仁,吴小芹.黑松合子胚和体细胞胚发育阶段的形态特征比较[J].林业科技开发,2012,26(5):21-23.
[16]LIAO Y K,AMERSON H V.Slash pine(Pinus elliottii Engelm.)somatic embryogenesis:I:Initiation of embryogenic cultures from immature zygotic embryos[J].New Forest,1995,10(2):145-163.
[17]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.
[18]吕守芳,张守攻,齐力旺,等.日本落叶松体细胞胚胎发生的研究[J].林业科学,2005,41(2):48-52.
[19]张建伟,王军辉,李青粉,等.云杉未成熟合子胚诱导体细胞胚胎发生[J].林业科学,2014,50(4):39-46.
[20]RAMAROSANDRATANA A V,STADEN J V.Tissue position,explant orientation and naphtha aleneacetic acid(NAA)affect initiation of somatic embryos and callus proliferation in Norway spruce(Picea abies)[J].Plant Cell Tissue Organ Cult,2003,74(3):249-255.
[21]丛建民,沈海龙,李玉花,等.水曲柳体胚发生过程中不同状态类型外植体的生理生化状态[J].华南农业大学学报,2012,33(1):48-52.
[22]徐春香,PANIS B,STROSSE H,等.体胚发生途径‘大矮蕉’植株的再生[J].华南农业大学学报,2004,25(2):63-66.
[23]徐春香,PANIS B,STROSSE H,等.香蕉胚性愈伤组织的诱导及胚性细胞悬浮系的建立[J].华南农业大学学报,2004,25(1):70-73.
[24]PULLMAN G S,NAMJOSHI K,ZHANG Y.Somatic embryogenesis in loblolly pine(Pinus taeda L.):Improving culture initiation with abscisic acid and silver nitrate[J].Plant Cell Rep,2003,22(2):85-95.
[25]PULLMAN G S,BUCALO K.Pine somatic embryogen-esis:analyses of seed tissue and medium to improve protocol development[J].New Forest,2014,45(3):353-377.
[26]GUPTA P K,DURZAN D J.Shoot multiplication from mature trees of douglas-fir(Pseudotsuga menziesii)and sugar pine(Pinus lambertiana)[J].Plant Cell Rep,1985,4(4):177-179.
[27]LITVAY J D,VERMA D C,JOHNSON M A.Influence of a loblolly pine(Pinus taeda L.)culture medium and its components on growth and somatic embryogenesis of the wild carrot(Daucus carota L.)[J].Plant Cell Rep,1985,4(6):325-328.
[28]NUNES S,MARUM L,FARINHA N,et al.Somatic embryogenesis of hybrid Pinus elliottii var.elliottii×P.caribaea var.hondurensis and ploidy assessment of somatic plants[J].Plant Cell Tissue Organ Cult,2018,132(1):71-84.
[29]陈金慧,施季森,诸葛强,等.植物体细胞胚胎发生机理的研究进展[J].南京林业大学学报(自然科学版),2003,27(1):75-80.
[30]杨玲.花楸种子生物学和体细胞胚发生体系研究[D].哈尔滨:东北林业大学,2007.
[31]王高.红松体细胞胚胎发生及超低温保存技术研究[D].上海:上海交通大学,2009.
[32]NEWTON R J,TANG W,JAIN S M.Slash pine(Pinus elliottii Engelm.)[M].Berlin Heidelberg:Springer,2005:1-10.
[33]JAIN S M,DONG N,NEWTON R J.Somatic embryogenesis in slash pine(Pinus elliottii)from immature embryos cultured in vitro[J].Plant Sci,1989,65(2):233-241.
[34]唐巍,欧阳藩,郭仲琛.湿地松体细胞胚胎发生和植株再生[J].植物资源与环境学报,1997,6(2):8-11.
[2]WEN X,KUANG Y,SHI M,et al.Biology of Hylobitelus xiaoi(Coleoptera:Curculionidae),a new pest of slash pine,Pinus elliottii[J].J Econ Entomol,2004,97(6):1958-1964.
[3]MEANS D B,PALIS J G,BAGGETT M.Effects of slash pine silviculture on a florida population of flatwoods salamander[J].Conserv Biol,1996,10(2):426-437.
[4]张帅楠.湿地松材性测定技术研究与多性状联合选择[D].北京:中国林业科学研究院,2017.
[5]宗亦臣,郑勇奇,马锡权,等.湿加松良种‘中林1号’[J].湖南林业科技,2017,44(1):76-77.
[6]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.
[7]姜福星,魏丕伟,寇亚平,等.探析植物体胚发生的关键基因[J].分子植物育种,2017,15(4):1304-1311.
[8]QUIROZ-FIGUEROA F R,ROJAS-HERRERA R,GALAZ-AVALOS R M,et al.Embryo production through somatic embryogenesis can be used to study cell differentiation in plants[J].Plant Cell Tissue Organ Cult,2006,86(3):285-301.
[9]KIM Y W,MOON H K.Enhancement of somatic embryogenesis and plant regeneration in Japanese red pine(Pinus densiflora)[J].Plant Biotechnol Rep,2014,8(3):259-266.
[10]PULLMAN G S,BUCALO K.Plant embryo culture[M].New York:Humana Press,2011:267-291.
[11]PULLMAN G S,MEIN J,JOHNSON S,et al.Gibberellin inhibitors improve embryogenic tissue initiation in conifers[J].Plant Cell Rep,2005,23(9):596-605.
[12]BONGA J M,PARK Y S,OVERTON C,et al.Optimized somatic embryogenesis in Pinus strobus L.[J].In Vitro Cell Dev Biol Plant,2013,37(3):392-399.
[13]BECWAR M,CLARK J,SWINTON L S,et al.Liquidbased method for producing plant embryos:US8313946B2[P].2012-11-20.
[14]GUPTA P K,HOLMSTROM D.Protocol for somaticembryogenesis in woody plants[M].Dordrecht:Springer,2005:573-575.
[15]李清清,叶建仁,吴小芹.黑松合子胚和体细胞胚发育阶段的形态特征比较[J].林业科技开发,2012,26(5):21-23.
[16]LIAO Y K,AMERSON H V.Slash pine(Pinus elliottii Engelm.)somatic embryogenesis:I:Initiation of embryogenic cultures from immature zygotic embryos[J].New Forest,1995,10(2):145-163.
[17]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.
[18]吕守芳,张守攻,齐力旺,等.日本落叶松体细胞胚胎发生的研究[J].林业科学,2005,41(2):48-52.
[19]张建伟,王军辉,李青粉,等.云杉未成熟合子胚诱导体细胞胚胎发生[J].林业科学,2014,50(4):39-46.
[20]RAMAROSANDRATANA A V,STADEN J V.Tissue position,explant orientation and naphtha aleneacetic acid(NAA)affect initiation of somatic embryos and callus proliferation in Norway spruce(Picea abies)[J].Plant Cell Tissue Organ Cult,2003,74(3):249-255.
[21]丛建民,沈海龙,李玉花,等.水曲柳体胚发生过程中不同状态类型外植体的生理生化状态[J].华南农业大学学报,2012,33(1):48-52.
[22]徐春香,PANIS B,STROSSE H,等.体胚发生途径‘大矮蕉’植株的再生[J].华南农业大学学报,2004,25(2):63-66.
[23]徐春香,PANIS B,STROSSE H,等.香蕉胚性愈伤组织的诱导及胚性细胞悬浮系的建立[J].华南农业大学学报,2004,25(1):70-73.
[24]PULLMAN G S,NAMJOSHI K,ZHANG Y.Somatic embryogenesis in loblolly pine(Pinus taeda L.):Improving culture initiation with abscisic acid and silver nitrate[J].Plant Cell Rep,2003,22(2):85-95.
[25]PULLMAN G S,BUCALO K.Pine somatic embryogen-esis:analyses of seed tissue and medium to improve protocol development[J].New Forest,2014,45(3):353-377.
[26]GUPTA P K,DURZAN D J.Shoot multiplication from mature trees of douglas-fir(Pseudotsuga menziesii)and sugar pine(Pinus lambertiana)[J].Plant Cell Rep,1985,4(4):177-179.
[27]LITVAY J D,VERMA D C,JOHNSON M A.Influence of a loblolly pine(Pinus taeda L.)culture medium and its components on growth and somatic embryogenesis of the wild carrot(Daucus carota L.)[J].Plant Cell Rep,1985,4(6):325-328.
[28]NUNES S,MARUM L,FARINHA N,et al.Somatic embryogenesis of hybrid Pinus elliottii var.elliottii×P.caribaea var.hondurensis and ploidy assessment of somatic plants[J].Plant Cell Tissue Organ Cult,2018,132(1):71-84.
[29]陈金慧,施季森,诸葛强,等.植物体细胞胚胎发生机理的研究进展[J].南京林业大学学报(自然科学版),2003,27(1):75-80.
[30]杨玲.花楸种子生物学和体细胞胚发生体系研究[D].哈尔滨:东北林业大学,2007.
[31]王高.红松体细胞胚胎发生及超低温保存技术研究[D].上海:上海交通大学,2009.
[32]NEWTON R J,TANG W,JAIN S M.Slash pine(Pinus elliottii Engelm.)[M].Berlin Heidelberg:Springer,2005:1-10.
[33]JAIN S M,DONG N,NEWTON R J.Somatic embryogenesis in slash pine(Pinus elliottii)from immature embryos cultured in vitro[J].Plant Sci,1989,65(2):233-241.
[34]唐巍,欧阳藩,郭仲琛.湿地松体细胞胚胎发生和植株再生[J].植物资源与环境学报,1997,6(2):8-11.