长穗偃麦草成熟种胚高频再生体系
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摘要
以长穗偃麦草(Elytrigia elongata)成熟胚为外植体,在MS基本培养基的基础上,附加不同浓度的2,4-D、6-BA和NAA等植物生长调节剂,开展对其愈伤组织诱导、绿苗分化和生根等的试验研究。结果表明,适宜愈伤组织诱导的培养基为MC (MS+30 g·L~(–1)麦芽糖+1 g·L~(–1) CH+200×5 mL·L~(–1) VB+0.5 g·L~(–1) L-Pro+3 g·L~(–1)植物凝胶)+3 mg·L–12,4-D+0.025 mg·L~(–1) 6-BA,诱导率达77.78%,4周后可见淡黄色愈伤组织;最佳分化培养基为MC (MS+30 g·L~(–1)麦芽糖+1 g·L~(–1) CH+200×5 mL·L~(–1) VB+0.5 g·L~(–1) L-Pro+3 g·L~(–1)植物凝胶)+0.1 mg·L~(–1) 2,4-D+3 mg·L~(–1) 6-BA,分化率达66.67%,4周后出现芽点,同时伴随根毛发生;最佳生根培养基为MR(1/2MS+15 g·L~(–1)麦芽糖+3 g·L~(–1)植物凝胶)+0.5 mg·L~(–1) NAA,移栽后全部成活。从而建立了一套从长穗偃麦草"成熟种胚–诱导愈伤组织–绿苗分化–生根–移栽"的组培再生体系,为进一步研究其抗逆分子机制奠定了重要基础。
The mature embryos of Elytrigia elongata seeds were used as explants and MS was used as the basic medium to carry out research on callus induction, green plantlet differentiation, and rooting by adding different growth regulator media.The results showed that the suitable medium for callus induction was MC(MS + 30 g·L~(–1) Maltose + 1 g·L~(–1) CH + 5 mL·L~(–1)200× VB + 0.5 g·L~(–1) L-Pro + 3 g·L~(–1) Phytagel) + 3 mg·L~(–1) 2,4-D + 0.025 mg·L~(–1) 6-BA. The induction rate was 77.78%, and the callus was visible after 4 weeks. The best differentiation medium was MC(MS + 30 g·L~(–1) Maltose + 1 g·L~(–1) CH +5 mL·L~(–1) 200× VB + 0.5 g·L~(–1) L-Pro + 3 g·L~(–1) Phytagel) + 0.1 mg·L~(–1) 2,4-D + 3 mg·L~(–1) 6-BA, and the differentiation rate was 66.67%. The budding point appeared after 4 weeks, accompanied by root hair development. The best rooting medium was MR(1/2 MS + 15 g·L~(–1) Maltose + 3 g·L~(–1) Phytagel) + 0.5 mg·L~(–1) NAA. All plantlets survived after transplanting.Therefore, differentiation tissue culture regeneration system for E. elongata was established, including mature embryosinducing callus-green plantlet differentiation-rooting-transplantation, which lays an important foundation for further study of its molecular mechanism of resilience.
The mature embryos of Elytrigia elongata seeds were used as explants and MS was used as the basic medium to carry out research on callus induction, green plantlet differentiation, and rooting by adding different growth regulator media.The results showed that the suitable medium for callus induction was MC(MS + 30 g·L~(–1) Maltose + 1 g·L~(–1) CH + 5 mL·L~(–1)200× VB + 0.5 g·L~(–1) L-Pro + 3 g·L~(–1) Phytagel) + 3 mg·L~(–1) 2,4-D + 0.025 mg·L~(–1) 6-BA. The induction rate was 77.78%, and the callus was visible after 4 weeks. The best differentiation medium was MC(MS + 30 g·L~(–1) Maltose + 1 g·L~(–1) CH +5 mL·L~(–1) 200× VB + 0.5 g·L~(–1) L-Pro + 3 g·L~(–1) Phytagel) + 0.1 mg·L~(–1) 2,4-D + 3 mg·L~(–1) 6-BA, and the differentiation rate was 66.67%. The budding point appeared after 4 weeks, accompanied by root hair development. The best rooting medium was MR(1/2 MS + 15 g·L~(–1) Maltose + 3 g·L~(–1) Phytagel) + 0.5 mg·L~(–1) NAA. All plantlets survived after transplanting.Therefore, differentiation tissue culture regeneration system for E. elongata was established, including mature embryosinducing callus-green plantlet differentiation-rooting-transplantation, which lays an important foundation for further study of its molecular mechanism of resilience.
引文
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[2]万千,张秀荣,赵兰飞,刘风珍,王宏伟,孔令让.小麦TaNF-YB6基因亚细胞定位和非生物胁迫表达分析.分子植物育种,2016,14(11):2904-2911.WAN Q, ZHANG X R, ZHAO L F, LIU F Z, WANG H W, KONG L R. Subcellular localization of TaNF-YB6 in wheat and its expression analysis under abiotic stress. Molecular Plant Breeding, 2016, 14(11):2904-2911.
[3] GORCZYCA A, OLEKSY A, GALA-CZEKAJ D, URBANIAK M, LASKOWSKA M, WASKIEWICZ A, STEPIEN L. Fusarium head blight incidence and mycotoxin accumulation in three durum wheat cultivars in relation to sowing date and density. The Science of Nature, 2018, 105(21/2):170-175.
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[5]默韶京,桂茹,郎明林.长穗偃麦草DREB类基因EeAP2.2的克隆与序列分析.植物遗传资源学报,2011,12(5):764-769.MO S J, GUI R, LANG M L. Cloning and sequence analysis of DREB-like gene EeAP2.2 in Thinopyrum ponticum. Journal of Plant Genetic Resources, 2011, 12(5):764-769.
[6] MENG L, ZHANG L, GUO Q, LI S, MAO P, Tian X. Cloning and transformation of EeHKT1;4 gene from Elytrigia elongata.Protein and Peptide Letters, 2016, 23(5):488-494.
[7]王际睿,颜泽洪,魏育明,郑有良.长穗偃麦草Y型高分子量谷蛋白基因的鉴定与分子克隆.农业生物技术学报,2004, 12(2):143-146.WANG J R, YAN Z H, WEI Y M, ZHENG Y L. Identification and molecular cloning of y-type high-molecular-weight glutenin subunit genes from Elytrigia elongata(Host)Nevski. Journal of Agricultural Biotechnology, 2004, 12(2):143-146.
[8]鲍芫,米福贵.长穗偃麦草与中间偃麦草杂种成熟胚离体培养与再生体系的建立.草地学报,2007, 24(11):348-351.BAO W, MI F G. A study on induction of emdryogenic callus and plant regeneration from mature embryos of Elytrigia hybrid(E.elongata x E. intermedia). Acta Agrestia Sinica, 2007, 24(11):348-351.
[9] SHAPIRO J. Biological action in Read-Write genome evolution. Interface Focus, 2017, 7(5):20160115.
[10] WANG Z,MA D,ZHU Z,MU Y,YANG Y, FENG L, YANG H,LIANG J,LIU Y, LIU L,LU H. Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts. World Journal of Gastroenterology, 2017, 23(48):8512-8525.
[11]张辉,魏建华,霍秀文,徐春波,米福贵,云锦风.蒙农杂种冰草成熟胚愈伤组织的诱导及植株再生.草地学报,2005, 13(1):30-33.ZHANG H, WEI J H, HUO X W, XU C B, MI F G, YUN J F. A Study on the induction of embryogenic callus and plant regeneration from mature embryos of Agropyron cristatum×Agropyron desertorum cv. Mengnong. Acta Agrestia Sinica, 2005,13(1):30-33.
[12]段龙飞,郭邦利,陈国爱,刘婷.花魔芋实生种子组织培养研究初探.山西农业大学学报(自然科学版),2016, 36(9):639-643.DUAN L F, GUO B L, CHEN G A, LIU T. Study on tissue culture of Amorphophallus konjac seed. Journal of Shanxi Agricultural University(Natural Science Edition), 2016, 36(9):639-643.
[13]郑茜子,袁柳祥,赵冰,申惠翡,徐静静,张冬林.秦岭美容杜鹃种子组培快繁技术研究.北方园艺,2015(24):75-79.ZHENG Q Z, YUAN L X, ZHAO B, SHEN H F, XU J J, ZHANG D L. Study on rapid propagation technology of seeds tissue culture of Rhododendron calophytum Franch in Qinling. Northern Horticulture, 2015(24):75-79.
[14]张微,王良群,刘勇,郝艳芳,杨伟,白鸿雁,武擘.玉米不同部位愈伤组织诱导及植株再生的比较研究.核农学报,2017, 31(11):2135-2144.ZHANG W, WANG L Q, LIU Y, HAO Y F, YANG W, BAI H Y, WU B. Comparative study on callus induction and plant regeneration from different maize explants. Journal of Nuclear Agricultural Sciences, 2017, 31(11):2135-2144.
[15]赵彦,陈雪英,云锦凤,刘湘萍.蒙古冰草茎尖愈伤组织及其再生植株诱导.北方农业学报,2016, 44(2):18-22.ZHAO Y, CHEN X Y, YUN J F, LIU X P. Induction of callus and plant regeneration from shoot tip in Agropyron mongolicum Keng. Journal of Northern Agriculture, 2016, 44(2):18-22.
[16] MUDAU S, STEENKAMP P, PIATER L, DE PALMA M, TUCCI M, MADALA N, DUBERY I. Metabolomics-guided investigations of unintended effects of the expression of the hydroxycinnamoyl quinate hydroxycinnamoyltransferase(hqtl)gene from Cynara cardunculus var. scolymus in Nicotiana tabacum cell cultures. Plant Physiology and Biochemistry, 2018, 127:287-298.
[17]吴桂胜,胡鸢雷,宋福平,郭晓燕,徐尔尼.剪股颖愈伤组织诱导与植株再生.生物技术通报,2006, 4(22):16-21.WU G S, HU Y L, SONG F P, GUO X Y, XU E N. Study on callus induction and plantlet regeneration of creeping bentgrass(Agrostis palustris Huds.). Biotechnology Bulletin, 2006, 4(22):16-21.
[18]何勇,田志宏,郑用琏.高羊茅成熟胚离体培养及高频植株再生.草业科学,2005, 22(6):28-34.HE Y, TIAN Z H, ZHENG Y L. High frequency plant regeneration from mature embryos in vitro culture of tall fescue. Pratacultural Science,2005, 22(6):28-34.
[19]王万军,王文芳,曹建军,白守信.小麦叶片愈伤组织及其再生植株的诱导.西北植物学报,1998, 18(3):90-94.WANG W J, WANG W F, CAO J J, BAI S X. The induction of callus and plantlet regeneration from the leaf of wheat(Triticum aestivum L.). Acta Botanica Boreali-Occidentalia Sinica, 1998, 18(3):90-94.
[20] LIM S, SUBRAMANIAM S, ZAMZURI I, AMIR H G. Biotization of in vitro calli and embryogenic calli of oil palm(Elaeis guineensis Jacq.)with diazotrophic bacteria Herbaspirillum seropedicae(Z78). Plant Cell, Tissue and Organ Culture, 2016, 127(1):251-262.
[21] HUANG F, WANG M, LI Z. Caucasian clover(Trifolium ambiguum Bieb.)×white clover(T. repens L.)-interspecific hybrids developed through tissue culture. Legume Research, 2017, 40(5):830-835.
[2]万千,张秀荣,赵兰飞,刘风珍,王宏伟,孔令让.小麦TaNF-YB6基因亚细胞定位和非生物胁迫表达分析.分子植物育种,2016,14(11):2904-2911.WAN Q, ZHANG X R, ZHAO L F, LIU F Z, WANG H W, KONG L R. Subcellular localization of TaNF-YB6 in wheat and its expression analysis under abiotic stress. Molecular Plant Breeding, 2016, 14(11):2904-2911.
[3] GORCZYCA A, OLEKSY A, GALA-CZEKAJ D, URBANIAK M, LASKOWSKA M, WASKIEWICZ A, STEPIEN L. Fusarium head blight incidence and mycotoxin accumulation in three durum wheat cultivars in relation to sowing date and density. The Science of Nature, 2018, 105(21/2):170-175.
[4]胡志昂,张亚平.中国动植物的遗传多样性.杭州:浙江科学技术出版社,1997.HU Z A, ZHANG Y P. The Genetic Diversity of Animals and Plants in China. Hangzhou:Zhejiang Scientific and Technology Press, 1997.
[5]默韶京,桂茹,郎明林.长穗偃麦草DREB类基因EeAP2.2的克隆与序列分析.植物遗传资源学报,2011,12(5):764-769.MO S J, GUI R, LANG M L. Cloning and sequence analysis of DREB-like gene EeAP2.2 in Thinopyrum ponticum. Journal of Plant Genetic Resources, 2011, 12(5):764-769.
[6] MENG L, ZHANG L, GUO Q, LI S, MAO P, Tian X. Cloning and transformation of EeHKT1;4 gene from Elytrigia elongata.Protein and Peptide Letters, 2016, 23(5):488-494.
[7]王际睿,颜泽洪,魏育明,郑有良.长穗偃麦草Y型高分子量谷蛋白基因的鉴定与分子克隆.农业生物技术学报,2004, 12(2):143-146.WANG J R, YAN Z H, WEI Y M, ZHENG Y L. Identification and molecular cloning of y-type high-molecular-weight glutenin subunit genes from Elytrigia elongata(Host)Nevski. Journal of Agricultural Biotechnology, 2004, 12(2):143-146.
[8]鲍芫,米福贵.长穗偃麦草与中间偃麦草杂种成熟胚离体培养与再生体系的建立.草地学报,2007, 24(11):348-351.BAO W, MI F G. A study on induction of emdryogenic callus and plant regeneration from mature embryos of Elytrigia hybrid(E.elongata x E. intermedia). Acta Agrestia Sinica, 2007, 24(11):348-351.
[9] SHAPIRO J. Biological action in Read-Write genome evolution. Interface Focus, 2017, 7(5):20160115.
[10] WANG Z,MA D,ZHU Z,MU Y,YANG Y, FENG L, YANG H,LIANG J,LIU Y, LIU L,LU H. Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts. World Journal of Gastroenterology, 2017, 23(48):8512-8525.
[11]张辉,魏建华,霍秀文,徐春波,米福贵,云锦风.蒙农杂种冰草成熟胚愈伤组织的诱导及植株再生.草地学报,2005, 13(1):30-33.ZHANG H, WEI J H, HUO X W, XU C B, MI F G, YUN J F. A Study on the induction of embryogenic callus and plant regeneration from mature embryos of Agropyron cristatum×Agropyron desertorum cv. Mengnong. Acta Agrestia Sinica, 2005,13(1):30-33.
[12]段龙飞,郭邦利,陈国爱,刘婷.花魔芋实生种子组织培养研究初探.山西农业大学学报(自然科学版),2016, 36(9):639-643.DUAN L F, GUO B L, CHEN G A, LIU T. Study on tissue culture of Amorphophallus konjac seed. Journal of Shanxi Agricultural University(Natural Science Edition), 2016, 36(9):639-643.
[13]郑茜子,袁柳祥,赵冰,申惠翡,徐静静,张冬林.秦岭美容杜鹃种子组培快繁技术研究.北方园艺,2015(24):75-79.ZHENG Q Z, YUAN L X, ZHAO B, SHEN H F, XU J J, ZHANG D L. Study on rapid propagation technology of seeds tissue culture of Rhododendron calophytum Franch in Qinling. Northern Horticulture, 2015(24):75-79.
[14]张微,王良群,刘勇,郝艳芳,杨伟,白鸿雁,武擘.玉米不同部位愈伤组织诱导及植株再生的比较研究.核农学报,2017, 31(11):2135-2144.ZHANG W, WANG L Q, LIU Y, HAO Y F, YANG W, BAI H Y, WU B. Comparative study on callus induction and plant regeneration from different maize explants. Journal of Nuclear Agricultural Sciences, 2017, 31(11):2135-2144.
[15]赵彦,陈雪英,云锦凤,刘湘萍.蒙古冰草茎尖愈伤组织及其再生植株诱导.北方农业学报,2016, 44(2):18-22.ZHAO Y, CHEN X Y, YUN J F, LIU X P. Induction of callus and plant regeneration from shoot tip in Agropyron mongolicum Keng. Journal of Northern Agriculture, 2016, 44(2):18-22.
[16] MUDAU S, STEENKAMP P, PIATER L, DE PALMA M, TUCCI M, MADALA N, DUBERY I. Metabolomics-guided investigations of unintended effects of the expression of the hydroxycinnamoyl quinate hydroxycinnamoyltransferase(hqtl)gene from Cynara cardunculus var. scolymus in Nicotiana tabacum cell cultures. Plant Physiology and Biochemistry, 2018, 127:287-298.
[17]吴桂胜,胡鸢雷,宋福平,郭晓燕,徐尔尼.剪股颖愈伤组织诱导与植株再生.生物技术通报,2006, 4(22):16-21.WU G S, HU Y L, SONG F P, GUO X Y, XU E N. Study on callus induction and plantlet regeneration of creeping bentgrass(Agrostis palustris Huds.). Biotechnology Bulletin, 2006, 4(22):16-21.
[18]何勇,田志宏,郑用琏.高羊茅成熟胚离体培养及高频植株再生.草业科学,2005, 22(6):28-34.HE Y, TIAN Z H, ZHENG Y L. High frequency plant regeneration from mature embryos in vitro culture of tall fescue. Pratacultural Science,2005, 22(6):28-34.
[19]王万军,王文芳,曹建军,白守信.小麦叶片愈伤组织及其再生植株的诱导.西北植物学报,1998, 18(3):90-94.WANG W J, WANG W F, CAO J J, BAI S X. The induction of callus and plantlet regeneration from the leaf of wheat(Triticum aestivum L.). Acta Botanica Boreali-Occidentalia Sinica, 1998, 18(3):90-94.
[20] LIM S, SUBRAMANIAM S, ZAMZURI I, AMIR H G. Biotization of in vitro calli and embryogenic calli of oil palm(Elaeis guineensis Jacq.)with diazotrophic bacteria Herbaspirillum seropedicae(Z78). Plant Cell, Tissue and Organ Culture, 2016, 127(1):251-262.
[21] HUANG F, WANG M, LI Z. Caucasian clover(Trifolium ambiguum Bieb.)×white clover(T. repens L.)-interspecific hybrids developed through tissue culture. Legume Research, 2017, 40(5):830-835.