小滴玻璃化法超低温保存海岛棉XH33胚性细胞的研究
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摘要
目前棉花转基因技术高度依赖于体细胞胚胎发生方式,而在体细胞胚发生过程中的胚性细胞保存和利用需要继代培养,可能引起保存材料发生遗传变异或导致物种退化,不利于资源保存和利用,而超低温保存能最大限度地保持材料的遗传稳定性。本研究以海岛棉(Gossypium barbadense) XH33胚性细胞为材料,分别从预培养、玻璃化溶液及处理时间、化冻方法以及恢复培养方法等方面进行优化,建立小滴玻璃化法超低温保存新疆海岛棉胚性细胞的方法;进一步通过流式细胞术、SSR分子标记和生理指标检测等方法验证小滴玻璃化法超低温保存后的胚性细胞遗传稳定性。结果表明,4℃条件下,XH33胚性细胞在0.3、0.5、0.7 mol/L梯度浓度蔗糖培养基上预培养3 d,然后在植物玻璃化溶液(plant vitrification solutions 2,PVS2)中装载20 min和脱水20 min;液氮保存12 h后,经40℃水浴解冻,并以0.5、0.7、1.0 mol/L梯度浓度蔗糖溶液洗涤;暗培养3 d后正常培养,细胞存活率高达55.55%。流式细胞术和SSR分子标记结果表明,超低温冷冻并未影响细胞的遗传稳定性;生理指标检测结果显示,冷冻后细胞的生理活性并未发生显著变化。本研究建立了海岛棉胚性细胞小滴玻璃化法超低温保存的技术体系,为海岛棉胚性细胞的长期保存提供了参考依据。
At present, cotton transgenic technology highly depends on somatic embryogenesis, and preservation of the cultured tissues needs subculture which might cause genetic variation or species degradation of preserved materials so that it is not conducive to the preservation and utilization of resources.Ultra-low temperature preservation can maximize the genetic stability of preserved materials. To establish the method of cryopreservation of Gossypium barbadensee XH33 embryonic cells by droplet vitrification, the present study optimized the system from preculture, vitrification solution and processing time, defrost method,and recovery cultivation, respectively. And the genetic stability of embryonic cell after cryopreservation was checked through the methods of flow cytometry, SSR molecular markers and physiological index detection.The results showed that XH 33 embryogenic cells were pre-cultured in 0.3、0.5、0.7 mol/L sucrose medium in sequence at 4 ℃ for 3 d, and then loaded and dehydrated for 20 min in the modified PVS2(plant vitrification solutions 2). After stored in liquid nitrogen for 12 h, the cells were thawed in 40 ℃ water bath and washed with 0.5, 0.7, 1.0 mol/L gradient concentration of sucrose solution and then cultured in darkness for 3 d. The cell survival rate was as high as 55.55%. The results of flow cytometry and SSR molecular markers showed that cryopreservation did not affect the genetic stability of cells, and there were no significant changes in the physiological activities of cells after cryopreservation. This study established the cryopreservation system for embryogenic cells of G.barbadensehave by vitrification which provides a reference for long-term preservation of embryogenic cells in island cotton.
At present, cotton transgenic technology highly depends on somatic embryogenesis, and preservation of the cultured tissues needs subculture which might cause genetic variation or species degradation of preserved materials so that it is not conducive to the preservation and utilization of resources.Ultra-low temperature preservation can maximize the genetic stability of preserved materials. To establish the method of cryopreservation of Gossypium barbadensee XH33 embryonic cells by droplet vitrification, the present study optimized the system from preculture, vitrification solution and processing time, defrost method,and recovery cultivation, respectively. And the genetic stability of embryonic cell after cryopreservation was checked through the methods of flow cytometry, SSR molecular markers and physiological index detection.The results showed that XH 33 embryogenic cells were pre-cultured in 0.3、0.5、0.7 mol/L sucrose medium in sequence at 4 ℃ for 3 d, and then loaded and dehydrated for 20 min in the modified PVS2(plant vitrification solutions 2). After stored in liquid nitrogen for 12 h, the cells were thawed in 40 ℃ water bath and washed with 0.5, 0.7, 1.0 mol/L gradient concentration of sucrose solution and then cultured in darkness for 3 d. The cell survival rate was as high as 55.55%. The results of flow cytometry and SSR molecular markers showed that cryopreservation did not affect the genetic stability of cells, and there were no significant changes in the physiological activities of cells after cryopreservation. This study established the cryopreservation system for embryogenic cells of G.barbadensehave by vitrification which provides a reference for long-term preservation of embryogenic cells in island cotton.
引文
陈晓玲,张金梅,辛霞,等.2013.植物种质资源超低温保存现状及其研究进展[J].植物遗传资源学报,14(3):414-427.(Chen X L,Zhang J M,Xin X,et al.2013.Progress on cryopreservation state and research of plant germplasm resources[J].Journal of Plant Genetic Resources,14(3):414-427.)
洪森荣,尹明华,王艾平,2014.江西铅山红芽芋胚性愈伤组织小滴玻璃化法超低温保存技术的研究[J].植物研究,34(3):333-338.(Hong S R,Yin M H,Wang A P.2014.Cryopreservation technique of Jiang Xi Yanshan red bud Taro(Colocasia esculenta L.Schott var.cormosus cv.Hongyayu)embryogenic callus by droplet-vitrification[J].Bulletin of Botanical Research,34(3):333-338.)
贾莉莉,曲延英,王希东,等.2015.52份海岛棉遗传多样性SSR分析[J].新疆农业大学学报,38(4):300-305.(Jia L L,Qu Y Y,Wang X D,et al.2015.Analysis on genetic diversity of 52 sea-Island cotton cultivars[J].Journal of Xinjiang Agricultural University,38(4):300-305.)
宋萍萍.2010.怀菊花种质资源玻璃化超低温保存技术研究[D].河南师范大学,硕士学位论文,导师:赵喜亭,pp.58.(Song P P.2010.The studies on cryopreservation of huaiqing chrysanthemum(Endranthema morifolium)germplasm by vitrification[D].Thesis for M.S.,Henan Normal University,Suppervisor:Zhao X T,pp.58.)
汪艳,肖媛,刘伟,等.2015.流式细胞仪检测高等植物细胞核DNA含量的方法[J].植物科学学报,33(1):126-131.(Wang Y,Xiao Y,Liu W,et al.2015,Operation skills of fiow cytometer for detecting nuclear DNA contents in higher plant cells[J].Plant Science Journal,33(1):126-131.)
王君晖,黄纯农.1994.玻璃化法-园艺作物茎尖和分生组织超低温保存的新途径[J].园艺学报,21(3):277-282.(Wang J H,Huang C N.1994.Vitrification-new approach for cryopreservation shoot-tip and meristems of horticultura crops[J].Acta Horticulturae Sinica,21(3):277-282.)
杨敏文.2002.快速测定植物叶片叶绿素含量方法的探讨[J].光谱实验室,19(4):478-481.(Yang M W.2002.Study on rapid determination of chlorophyⅡcontent of leaves[J].Chinese Journal of Spectroscopy Labaratory,19(4):478-481.)
张延红.2013.秦艽和党参的休眠培养与玻璃化超低温保存研究[D].甘肃农业大学,博士学位论文,导师:张金文,pp.60.(Zhang Y H.2013.In vitro culture and vitrification cryopreservation of dormant duds of Gentiana straminea Maxim.and Codonopsis pilosula(Franch.)Nannf.[D].Thesis for Ph.D.,Gansa Agricultural University,Suppervisor:Zhang J W,pp.60.)
Benson E E.2008.Cryopreservation of phytodiversity:A critical application of theory practice[J].Critical Reviews in Plant Sciences,27(3):141-219.
Kong L S,von Aderkas P.2011.A noval method of crypreservation without a cryoprotectant for immature somatic embryos of conifer[J].Plant Cell Tissue and Organ Culture,106(3):115-125.
Baierski F,Stock J,Hanf B,et al.2018.ATP content and cell viability as indicators for cryostressacross the diversity of life[J].Frontiers in Physiology,9(1):921.Doi:10.3389/fphys.2018.00921
Folgado R,Sergeant K,Renaut J,et al.2014.Changes in sugar content and proteome of potato in response to cold and dehydration stress and their implications for cryopreservation[J].Journal of Proteomics,98(1):99-111.
Sakai A,Engelmann F.2007.Vitrification,encapsulation-vitrification and droplet-vitrification:A review[J].Cryo-Letters,28(3):151-172.
Salaj T,Matu?íkováL,Swennen R,et al.2012.Long-term maintenance of Pinus nigra embryogenic cultures through cryopreservation[J].Acta Physiologiae Plantarum,34(1):227-233.
Thierry C,Florin B,Petiard V,1999.Changes in protein metabolism during the acquisition of tolerance to cyopreservation of carrotsomatic embryos[J].Plant Physiology and Biochemistry,37(2):145-154.
Wang Q C,Cuellar W J.,Rajamaki M L,et al.2008.Combined thermo-therapy and cryotherapy for virus eradication:Relation of virus distribution,subcellular changes,cell survival and viral RNA degradation in shoot tips to efficient production of virus-free plants[J].Molecular Plant Pathology,9(1):237-250.
Wang Q C,Mawassi M,Li P,et al.2003.Elimination of Grapevine virus A(GVA)by cryopreservation of in vitrogrown shoot tips of Vitis vinifera L.[J].Plant Science,165(2):321-327.
Wen B,Cai C T,Wang R L,et al.2012.Cytological and physiological changes in recalcitrant Chinese fan palm(Livistona chinensis)embryos during cryopreservation[J].Protoplasma,249(1):323-335.
Wen B,Wang R L,Cheng H Y.et al.2010.Cytological and physiological changes in orthodox maize embryos during cryopreservation[J].Protoplasma,239(1):57-67.
洪森荣,尹明华,王艾平,2014.江西铅山红芽芋胚性愈伤组织小滴玻璃化法超低温保存技术的研究[J].植物研究,34(3):333-338.(Hong S R,Yin M H,Wang A P.2014.Cryopreservation technique of Jiang Xi Yanshan red bud Taro(Colocasia esculenta L.Schott var.cormosus cv.Hongyayu)embryogenic callus by droplet-vitrification[J].Bulletin of Botanical Research,34(3):333-338.)
贾莉莉,曲延英,王希东,等.2015.52份海岛棉遗传多样性SSR分析[J].新疆农业大学学报,38(4):300-305.(Jia L L,Qu Y Y,Wang X D,et al.2015.Analysis on genetic diversity of 52 sea-Island cotton cultivars[J].Journal of Xinjiang Agricultural University,38(4):300-305.)
宋萍萍.2010.怀菊花种质资源玻璃化超低温保存技术研究[D].河南师范大学,硕士学位论文,导师:赵喜亭,pp.58.(Song P P.2010.The studies on cryopreservation of huaiqing chrysanthemum(Endranthema morifolium)germplasm by vitrification[D].Thesis for M.S.,Henan Normal University,Suppervisor:Zhao X T,pp.58.)
汪艳,肖媛,刘伟,等.2015.流式细胞仪检测高等植物细胞核DNA含量的方法[J].植物科学学报,33(1):126-131.(Wang Y,Xiao Y,Liu W,et al.2015,Operation skills of fiow cytometer for detecting nuclear DNA contents in higher plant cells[J].Plant Science Journal,33(1):126-131.)
王君晖,黄纯农.1994.玻璃化法-园艺作物茎尖和分生组织超低温保存的新途径[J].园艺学报,21(3):277-282.(Wang J H,Huang C N.1994.Vitrification-new approach for cryopreservation shoot-tip and meristems of horticultura crops[J].Acta Horticulturae Sinica,21(3):277-282.)
杨敏文.2002.快速测定植物叶片叶绿素含量方法的探讨[J].光谱实验室,19(4):478-481.(Yang M W.2002.Study on rapid determination of chlorophyⅡcontent of leaves[J].Chinese Journal of Spectroscopy Labaratory,19(4):478-481.)
张延红.2013.秦艽和党参的休眠培养与玻璃化超低温保存研究[D].甘肃农业大学,博士学位论文,导师:张金文,pp.60.(Zhang Y H.2013.In vitro culture and vitrification cryopreservation of dormant duds of Gentiana straminea Maxim.and Codonopsis pilosula(Franch.)Nannf.[D].Thesis for Ph.D.,Gansa Agricultural University,Suppervisor:Zhang J W,pp.60.)
Benson E E.2008.Cryopreservation of phytodiversity:A critical application of theory practice[J].Critical Reviews in Plant Sciences,27(3):141-219.
Kong L S,von Aderkas P.2011.A noval method of crypreservation without a cryoprotectant for immature somatic embryos of conifer[J].Plant Cell Tissue and Organ Culture,106(3):115-125.
Baierski F,Stock J,Hanf B,et al.2018.ATP content and cell viability as indicators for cryostressacross the diversity of life[J].Frontiers in Physiology,9(1):921.Doi:10.3389/fphys.2018.00921
Folgado R,Sergeant K,Renaut J,et al.2014.Changes in sugar content and proteome of potato in response to cold and dehydration stress and their implications for cryopreservation[J].Journal of Proteomics,98(1):99-111.
Sakai A,Engelmann F.2007.Vitrification,encapsulation-vitrification and droplet-vitrification:A review[J].Cryo-Letters,28(3):151-172.
Salaj T,Matu?íkováL,Swennen R,et al.2012.Long-term maintenance of Pinus nigra embryogenic cultures through cryopreservation[J].Acta Physiologiae Plantarum,34(1):227-233.
Thierry C,Florin B,Petiard V,1999.Changes in protein metabolism during the acquisition of tolerance to cyopreservation of carrotsomatic embryos[J].Plant Physiology and Biochemistry,37(2):145-154.
Wang Q C,Cuellar W J.,Rajamaki M L,et al.2008.Combined thermo-therapy and cryotherapy for virus eradication:Relation of virus distribution,subcellular changes,cell survival and viral RNA degradation in shoot tips to efficient production of virus-free plants[J].Molecular Plant Pathology,9(1):237-250.
Wang Q C,Mawassi M,Li P,et al.2003.Elimination of Grapevine virus A(GVA)by cryopreservation of in vitrogrown shoot tips of Vitis vinifera L.[J].Plant Science,165(2):321-327.
Wen B,Cai C T,Wang R L,et al.2012.Cytological and physiological changes in recalcitrant Chinese fan palm(Livistona chinensis)embryos during cryopreservation[J].Protoplasma,249(1):323-335.
Wen B,Wang R L,Cheng H Y.et al.2010.Cytological and physiological changes in orthodox maize embryos during cryopreservation[J].Protoplasma,239(1):57-67.