‘宁杞8号’体细胞胚胎发生体系建立
|
摘要
以宁夏枸杞新品种‘宁杞8号’幼嫩叶片为外植体,探讨激素组合及添加物对‘宁杞8号’体细胞胚胎诱导、体胚增殖、萌发和植株再生的影响,并建立高效稳定的体细胞胚胎发生体系。结果表明:通过对6-BA、2,4-D和IAA进行正交分析,筛选出‘宁杞8号’体细胞胚诱导最优激素组合:6-BA 1.0 mg·L~(-1)+2,4-D0.3 mg·L~(-1)+IAA 0.4 mg·L~(-1),体胚诱导率达88.67%。极差分析比较各激素间的影响,6-BA对体胚诱导影响最显著。当高浓度的生长素及低浓度细胞分裂素浓度适宜的配比时,才能诱导产生形态正常数量多的‘宁杞8号’体细胞胚。在体胚增殖培养中,6-BA的浓度过高易导致玻璃化,不利于‘宁杞8号’体胚增殖生长。随着激素浓度的增高,体胚增殖倍数增加,玻璃化率也越高,综合分析得到‘宁杞8号’最佳体胚增殖培养基为6-BA 0.4 mg·L~(-1)+NAA 0.6 mg·L~(-1)。当添加IBA 0.3 mg·L~(-1)+GA_30.4 mg·L~(-1)+蔗糖10 g·L~(-1)时,‘宁杞8号’体胚萌发率最高,萌发率达89.17%。添加GA_3及低浓度蔗糖能促进成熟的体胚萌发。对‘宁杞8号’体萌发的影响程度依次是IBA>蔗糖>GA_3。活性炭能有效提高‘宁杞8号’体胚再生植株率,同时对萌发体胚的根的发育也有促进作用,当IBA 0.1 mg·L~(-1)+KT 0.4 mg·L~(-1)+活性炭1 g·L~(-1)时,‘宁杞8号’体胚再生植株效果最佳,体胚再生率达91.67%。
In order to establish a high-frequency regeneration system for Lycium barbarum.The leaf of L.barbarum‘Ningqi-8’was selected as material to explore the crucial effects of hormone combinations and additives on the induction of somatic embryogenesis,somatic embryo proliferation and germination and plant regeneration.The results showed that by orthogonal analysis of 6-BA,2,4-D and IAA,the optimal hormone combination of L.barbarum‘Ningqi-8’somatic embryos was 6-BA 1.0 mg·L~(-1)+2,4-D 0.3 mg·L~(-1)+IAA 0.4 mg·L~(-1),the highest induction rate could get to 88.67%.Range analysis showed that 6-BA had the most significant effect on somatic embryogenesis.Using suitable proportion of hormone combination was possible to induce somatic embryogenesis of L.barbarum‘Ningqi-8’,which had a large number of normal morphology.In the culture of somatic embryos,the high concentration of 6-BA was easy to cause vitrification,which was not conducive to the growth and proliferation of L.barbarum‘Ningqi-8’somatic embryos.The proliferation rate of somatic embryos increased with the increase of hormone concentration,and the vitrification rate increased,too.Then the most efficient proliferation medium for L.barbarum ‘Ningqi-8’ was 6-BA 0.4 mg·L~(-1)+NAA 0.6mg·L~(-1),and the highest germination rate of L.barbarum‘Ningqi-8’somatic embryo was obtained when IBA 0.3 mg·L~(-1)+GA_30.4 mg·L~(-1)+sucrose 10 g·L~(-1)was added.The highest germination rate could get to 89.17%,and the degree of influence on the germination of L.barbarum‘Ningqi-8’was IBA>sucrose>GA_3.Adding GA_3and low concentration sucrose could promote the mature somatic embryo germination.Activated carbon could effectively improve the regeneration rate of L.barbarum‘Ningqi-8’somatic embryo,and simultaneously promote the development of the root of germination embryo.IBA 0.1 mg·L~(-1)+KT 0.4 mg·L~(-1)+activated carbon 1 g·L~(-1) was found to be the suitable medium for regeneration in which L.barbarum ‘Ningqi-8’ demonstrated the highest multiplying capacity,and the regeneration rate could get to 91.67%.
In order to establish a high-frequency regeneration system for Lycium barbarum.The leaf of L.barbarum‘Ningqi-8’was selected as material to explore the crucial effects of hormone combinations and additives on the induction of somatic embryogenesis,somatic embryo proliferation and germination and plant regeneration.The results showed that by orthogonal analysis of 6-BA,2,4-D and IAA,the optimal hormone combination of L.barbarum‘Ningqi-8’somatic embryos was 6-BA 1.0 mg·L~(-1)+2,4-D 0.3 mg·L~(-1)+IAA 0.4 mg·L~(-1),the highest induction rate could get to 88.67%.Range analysis showed that 6-BA had the most significant effect on somatic embryogenesis.Using suitable proportion of hormone combination was possible to induce somatic embryogenesis of L.barbarum‘Ningqi-8’,which had a large number of normal morphology.In the culture of somatic embryos,the high concentration of 6-BA was easy to cause vitrification,which was not conducive to the growth and proliferation of L.barbarum‘Ningqi-8’somatic embryos.The proliferation rate of somatic embryos increased with the increase of hormone concentration,and the vitrification rate increased,too.Then the most efficient proliferation medium for L.barbarum ‘Ningqi-8’ was 6-BA 0.4 mg·L~(-1)+NAA 0.6mg·L~(-1),and the highest germination rate of L.barbarum‘Ningqi-8’somatic embryo was obtained when IBA 0.3 mg·L~(-1)+GA_30.4 mg·L~(-1)+sucrose 10 g·L~(-1)was added.The highest germination rate could get to 89.17%,and the degree of influence on the germination of L.barbarum‘Ningqi-8’was IBA>sucrose>GA_3.Adding GA_3and low concentration sucrose could promote the mature somatic embryo germination.Activated carbon could effectively improve the regeneration rate of L.barbarum‘Ningqi-8’somatic embryo,and simultaneously promote the development of the root of germination embryo.IBA 0.1 mg·L~(-1)+KT 0.4 mg·L~(-1)+activated carbon 1 g·L~(-1) was found to be the suitable medium for regeneration in which L.barbarum ‘Ningqi-8’ demonstrated the highest multiplying capacity,and the regeneration rate could get to 91.67%.
引文
ABDOULAYE T,MARK J,2006.Effects of carbon source and explants type on somatic embryogenesis of four cacao genotypes[J].Hortic Sci,41(3):753-758.
CAO YL,JIA YJ,LUO Q,et al,1997.Somatic embryogenesis and plant regeneration from Lycium barbarum L.suspension pith cell[J].J Ningxia Agric Coll,18(3):47-52.[曹有龙,贾勇炯,罗青,等,1997.枸杞髓部细胞悬浮培养及胚状体发生[J].宁夏农学院学报,18(3):47-52.]
EVANS DA,1983.Handbok of plant cell culture:Techniques for propagation and breeding[M].New York:Macmillan:83-113.
HE J,CAO YL,LI XY,et al,2006.On breeding of Lycium barbarum[J].Shaanxi J Agric Sci,(5):68-70.[何军,曹有龙,李晓莺,等,2006.枸杞育种研究进展[J].陕西农业科学,(5):68-70.]
JIANG Q,XIONG FQ,TANG XM,et al,2013.Effect of gibberellins light and genotype on peanut somatic embryogenesis and plantlet regeneration[J].J S Agric,44(6):903-908.[蒋菁,熊发前,唐秀梅,等,2013.赤霉素、光照及基因型对花生体细胞胚诱导和植株再生的影响[J].南方农业学报,44(6):903-908.]
LU Q,ZHANG B,LI YL,et al,2016.Wolfberry anther culture to obtain haploid plants[J].Ningxia J Agric For Sci Technol,57(6):17-19.[罗青,张波,李彦龙,等,2016.枸杞花药离体培养获得单倍体植株[J].宁夏农林科技,57(6):17-19.]
MA HP,LI Y,DI L,et al,2005.Somatic embryogenesis of Lycium barbarum and plant regeneration[J].J Gansu Agric Univ,40(1):26-30.[马和平,李毅,邸利,等,2005.枸杞体细胞胚胎发生及植株再生的研究[J].甘肃农业大学学报,40(1):26-30.]
NAN XX,WANG JX,CHANG HY,et al,2014.A new variety of Lycium barbarum‘Ningqi 8’[J].Sci Silv Sin,50(12):170.[南雄雄,王锦秀,常红宇,等,2014.枸杞新品种‘宁杞8号’[J].林业科学,50(12):170.]
PAN J,WU XQ,XU JX,et al,2018.Early stage observation and conditional optimization for proliferation of resistant Pinus densiflora somatic embryogenesis[J].Plant Physiol J,54(4):686-692.[潘珺,吴小芹,许建秀,等,2018.抗性赤松体胚发生细胞结构观察与增殖条件的优化[J].植物生理学报,54(4):686-692.]
WANG ZB,MA YD,ZHU Y,et al,2014.Establishment of a high rate of somatic embryogenesis system for Lycium barbarum L.[J].J Lanzhou Univ,50(1):95-99.[王兆滨,马义德,祝英,等,2014.枸杞体细胞胚高频发生体系的建立[J].兰州大学学报,50(1):95-100.]
WANG L,ZHANG HL,2016.External factors affecting somatic embryos maturation plants[J].Shaanxi For Sci Technol,(5):73-76.[王丽,张焕玲,2016.植物体胚成熟发育外在影响因素研究进展[J].陕西林业科技,(5):73-76.]
WANG XC,LIU SQ,JI J,et al,2004.The affect factor on the germination frequency of somatic embryos of soybean[J].Soybean Sci,23(2):151-154.[王晓春,刘尚前,季静,等,2004.影响大豆体细胞胚萌发率的因素[J].大豆科学,23(2):151-154.]
YUAN S,JIA YJ,LIN HH,2003.Several physiological factors inducing somatic embryogenesis of plant[J].Plant Physiol J,39(5):508-512.[袁澍,贾勇炯,林宏辉,2003.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,39(5):508-512.]
ZHANG XN,SHEN XD,WANG JX,1996.Morphological analysis and resolution of cross abortion between tetroploid and diploid of Chinese wolfberry[J].Ningxia J Agric For Sci Technol,(4):16-18.[张新宁,沈效东,王锦秀,1996.枸杞四倍体同二倍体杂交败育的形态分析及解决方法[J].宁夏农林科技,(4):16-18.]
ZHAO PJ,2009.Study on somatic embrygensis and callus of Acacia podalyriifolia[D].Fuzhou:Fujian Agriculture and Forestry University.[赵苹静,2009.珍珠相思体细胞胚胎发生及愈伤组织培养[D].福州:福建农林大学.]
ZHANG T,2007.Histological studies of somatic embryogenesis in Eruca sativa Mill.[J].Acta Hortic Sin,34(1):131-134.[张涛,2007.芸芥体细胞胚胎发生的组织细胞学研究[J].园艺学报,34(1):131-134.]
ZHANG B,LUO Q,ZHANG XY,et al,2016.Embryoid induction of Lyciun bararum L.anther culture[J].N Hortic,(9):105-108.[张波,罗青,张曦燕,等,2016.宁夏枸杞花药培养胚状体的诱导[J].北方园艺,(9):105-108.]
CAO YL,JIA YJ,LUO Q,et al,1997.Somatic embryogenesis and plant regeneration from Lycium barbarum L.suspension pith cell[J].J Ningxia Agric Coll,18(3):47-52.[曹有龙,贾勇炯,罗青,等,1997.枸杞髓部细胞悬浮培养及胚状体发生[J].宁夏农学院学报,18(3):47-52.]
EVANS DA,1983.Handbok of plant cell culture:Techniques for propagation and breeding[M].New York:Macmillan:83-113.
HE J,CAO YL,LI XY,et al,2006.On breeding of Lycium barbarum[J].Shaanxi J Agric Sci,(5):68-70.[何军,曹有龙,李晓莺,等,2006.枸杞育种研究进展[J].陕西农业科学,(5):68-70.]
JIANG Q,XIONG FQ,TANG XM,et al,2013.Effect of gibberellins light and genotype on peanut somatic embryogenesis and plantlet regeneration[J].J S Agric,44(6):903-908.[蒋菁,熊发前,唐秀梅,等,2013.赤霉素、光照及基因型对花生体细胞胚诱导和植株再生的影响[J].南方农业学报,44(6):903-908.]
LU Q,ZHANG B,LI YL,et al,2016.Wolfberry anther culture to obtain haploid plants[J].Ningxia J Agric For Sci Technol,57(6):17-19.[罗青,张波,李彦龙,等,2016.枸杞花药离体培养获得单倍体植株[J].宁夏农林科技,57(6):17-19.]
MA HP,LI Y,DI L,et al,2005.Somatic embryogenesis of Lycium barbarum and plant regeneration[J].J Gansu Agric Univ,40(1):26-30.[马和平,李毅,邸利,等,2005.枸杞体细胞胚胎发生及植株再生的研究[J].甘肃农业大学学报,40(1):26-30.]
NAN XX,WANG JX,CHANG HY,et al,2014.A new variety of Lycium barbarum‘Ningqi 8’[J].Sci Silv Sin,50(12):170.[南雄雄,王锦秀,常红宇,等,2014.枸杞新品种‘宁杞8号’[J].林业科学,50(12):170.]
PAN J,WU XQ,XU JX,et al,2018.Early stage observation and conditional optimization for proliferation of resistant Pinus densiflora somatic embryogenesis[J].Plant Physiol J,54(4):686-692.[潘珺,吴小芹,许建秀,等,2018.抗性赤松体胚发生细胞结构观察与增殖条件的优化[J].植物生理学报,54(4):686-692.]
WANG ZB,MA YD,ZHU Y,et al,2014.Establishment of a high rate of somatic embryogenesis system for Lycium barbarum L.[J].J Lanzhou Univ,50(1):95-99.[王兆滨,马义德,祝英,等,2014.枸杞体细胞胚高频发生体系的建立[J].兰州大学学报,50(1):95-100.]
WANG L,ZHANG HL,2016.External factors affecting somatic embryos maturation plants[J].Shaanxi For Sci Technol,(5):73-76.[王丽,张焕玲,2016.植物体胚成熟发育外在影响因素研究进展[J].陕西林业科技,(5):73-76.]
WANG XC,LIU SQ,JI J,et al,2004.The affect factor on the germination frequency of somatic embryos of soybean[J].Soybean Sci,23(2):151-154.[王晓春,刘尚前,季静,等,2004.影响大豆体细胞胚萌发率的因素[J].大豆科学,23(2):151-154.]
YUAN S,JIA YJ,LIN HH,2003.Several physiological factors inducing somatic embryogenesis of plant[J].Plant Physiol J,39(5):508-512.[袁澍,贾勇炯,林宏辉,2003.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,39(5):508-512.]
ZHANG XN,SHEN XD,WANG JX,1996.Morphological analysis and resolution of cross abortion between tetroploid and diploid of Chinese wolfberry[J].Ningxia J Agric For Sci Technol,(4):16-18.[张新宁,沈效东,王锦秀,1996.枸杞四倍体同二倍体杂交败育的形态分析及解决方法[J].宁夏农林科技,(4):16-18.]
ZHAO PJ,2009.Study on somatic embrygensis and callus of Acacia podalyriifolia[D].Fuzhou:Fujian Agriculture and Forestry University.[赵苹静,2009.珍珠相思体细胞胚胎发生及愈伤组织培养[D].福州:福建农林大学.]
ZHANG T,2007.Histological studies of somatic embryogenesis in Eruca sativa Mill.[J].Acta Hortic Sin,34(1):131-134.[张涛,2007.芸芥体细胞胚胎发生的组织细胞学研究[J].园艺学报,34(1):131-134.]
ZHANG B,LUO Q,ZHANG XY,et al,2016.Embryoid induction of Lyciun bararum L.anther culture[J].N Hortic,(9):105-108.[张波,罗青,张曦燕,等,2016.宁夏枸杞花药培养胚状体的诱导[J].北方园艺,(9):105-108.]