白刺花胚性愈伤组织诱导和植株再生体系建立
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摘要
本文探讨了不同因素对白刺花(Sophora davidii)下胚轴、子叶、茎段三种外植体胚性愈伤组织的诱导和体细胞胚发生的影响。以MS为基本培养基,研究了2,4-二氯苯氧乙酸(2,4-D)对胚性愈伤组织诱导的影响,蔗糖对胚性细胞状态的调整,6-苄氨基腺嘌呤(6-BA)和噻苯隆(TDZ)对体细胞胚形成的调控,以及脱落酸(ABA)对体细胞胚萌发的影响。白刺花不同外植体接种于愈伤组织诱导培养基上,能形成胚性、部分胚性和非胚性三种不同类型的愈伤组织,下胚轴和茎段最佳胚性愈伤组织诱导培养基为MS+2.5 mg·L~(-1) 2,4-D+0.5 mg·L~(-1)6-BA+0.5 mg·L~(-1) TDZ+40 g·L~(-1)蔗糖+3 g·L~(-1)植物凝胶+100 mg·L~(-1)谷氨酰胺+10 g·L~(-1)香蕉泥,诱导率分别为73.8%和41.5%;子叶最佳胚性愈伤组织诱导培养基为MS+2.0 mg·L~(-1) 2,4-D+0.5 mg·L~(-1) 6-BA+0.5 mg·L~(-1) TDZ+40 g·L~(-1)蔗糖+3 g·L~(-1)植物凝胶+100 mg·L~(-1)谷氨酰胺+10 g·L~(-1)香蕉泥,诱导率为57.1%。2.0 mg·L~(-1) 6-BA时,下胚轴体胚发生率最高,为66.67%,总胚数为74个·g~(-1);子叶体胚发生率为57.65%,总胚数为61个·g~(-1);茎段体胚发生率49.12%,总胚数为44个·g~(-1)。1.0 mg·L~(-1) TDZ时以下胚轴和茎段为外植体的体胚发生率最高分别为80.81%和69.18%,获得总胚数分别为113和69个·g~(-1);TDZ浓度为1.5 mg·L~(-1)时,子叶体胚发生率最高,为71.77%,总胚数87个·g~(-1)。0.5～1.0 mg·L~(-1) ABA时,体细胞胚萌发率相对较高,幼苗生长比较健壮。
In this paper,the effects of different factors on embryogenic callus induction and somatic embryogenesis from hypocotyls,cotyledon and stem of Sophora davidii were discussed.MS medium was used as basal medium which was added with 2,4-dichlorophenoxyacetic acid(2,4-D)to study the induction of embryo callus.The state of embryonic cells was investigated using sucrose as osmotic regulator.The regulations of 6-benzylaminopurine(6-BA)and thidiazuron(TDZ)on somatic embryo formation and the effect of abscisic acid(ABA)on somatic embryo germination were studied.The results show that three kinds of calli were obtained in callus induction medium,and they were called embryonic,partially embryonated and non-embryonal calli,respectively.The optimal medium for embryogenic callus induction was MS+2.5 mg·L~(-1) 2,4-D+0.5 mg·L~(-1) 6-BA+0.5mg·L~(-1) TDZ+40 g·L~(-1) sucrose+3 g·L~(-1) plant gel+100 mg·L~(-1) glutamine+10 g·L~(-1) banana slurry,and the rate of induction reached to 73.8% and 41.5% ,respectively,from hypocotyl and cotyledon on this medium.The optimal medium for embryogenic callus induction was MS+2.0 mg·L~(-1) 2,4-D+0.5 mg·L~(-1) 6-BA+0.5 mg·L~(-1)TDZ+40 g·L~(-1) sucrose+3 g·L~(-1) plant gel+100 mg·L~(-1) glutamine+10 g·L~(-1 )banana slurry from stem,and the callus induction rate of cotyledon could reach 57.1% .When the concentration of 6-BA was 2.0 mg·L~(-1),the hypocotyl somatic embryo rate was as high as 66.67% ,and the total amount of embryos was 74 g~(-1 )from hypocotyl;the cotyledon embryo body rate was 57.65% ,and total amount of embryos was 61 g~(-1) from cotyledon;the stem somatic embryo rate was 49.12% ,and the total amount of embryos was 44 g~(-1 )from stem.When the concentration of TDZ was 1.0 mg·L~(-1),the rate of somatic embryogenesis was as high as 80.81% when the explant was the hypocotyl,and 69.18% when the explant was the stem,and the total amounts of embryos were 113 and 69 g~(-1) from hypocotyl and stem,respectively.When TDZ concentration was 1.5 mg·L~(-1),the rate of somatic embryogenesis was 71.77% ,and total amount of embryos from cotyledon was 87 g~(-1).When ABA was added to 0.5–1.0 mg·L~(-1),the rate of somatic embryo germination was relatively high,and the seedling was relatively robust.
In this paper,the effects of different factors on embryogenic callus induction and somatic embryogenesis from hypocotyls,cotyledon and stem of Sophora davidii were discussed.MS medium was used as basal medium which was added with 2,4-dichlorophenoxyacetic acid(2,4-D)to study the induction of embryo callus.The state of embryonic cells was investigated using sucrose as osmotic regulator.The regulations of 6-benzylaminopurine(6-BA)and thidiazuron(TDZ)on somatic embryo formation and the effect of abscisic acid(ABA)on somatic embryo germination were studied.The results show that three kinds of calli were obtained in callus induction medium,and they were called embryonic,partially embryonated and non-embryonal calli,respectively.The optimal medium for embryogenic callus induction was MS+2.5 mg·L~(-1) 2,4-D+0.5 mg·L~(-1) 6-BA+0.5mg·L~(-1) TDZ+40 g·L~(-1) sucrose+3 g·L~(-1) plant gel+100 mg·L~(-1) glutamine+10 g·L~(-1) banana slurry,and the rate of induction reached to 73.8% and 41.5% ,respectively,from hypocotyl and cotyledon on this medium.The optimal medium for embryogenic callus induction was MS+2.0 mg·L~(-1) 2,4-D+0.5 mg·L~(-1) 6-BA+0.5 mg·L~(-1)TDZ+40 g·L~(-1) sucrose+3 g·L~(-1) plant gel+100 mg·L~(-1) glutamine+10 g·L~(-1 )banana slurry from stem,and the callus induction rate of cotyledon could reach 57.1% .When the concentration of 6-BA was 2.0 mg·L~(-1),the hypocotyl somatic embryo rate was as high as 66.67% ,and the total amount of embryos was 74 g~(-1 )from hypocotyl;the cotyledon embryo body rate was 57.65% ,and total amount of embryos was 61 g~(-1) from cotyledon;the stem somatic embryo rate was 49.12% ,and the total amount of embryos was 44 g~(-1 )from stem.When the concentration of TDZ was 1.0 mg·L~(-1),the rate of somatic embryogenesis was as high as 80.81% when the explant was the hypocotyl,and 69.18% when the explant was the stem,and the total amounts of embryos were 113 and 69 g~(-1) from hypocotyl and stem,respectively.When TDZ concentration was 1.5 mg·L~(-1),the rate of somatic embryogenesis was 71.77% ,and total amount of embryos from cotyledon was 87 g~(-1).When ABA was added to 0.5–1.0 mg·L~(-1),the rate of somatic embryo germination was relatively high,and the seedling was relatively robust.
引文
Che YF,Zhang CR,Huang X,et al(2006).Effect of TDZ on somatic embryogenesis of plant.Plant Physiol Commun,42(1):127-133(in Chinese with English abstract)[陈云凤,张春荣,黄霞等(2006).TDZ对植物体细胞胚胎发生的作用.植物生理学通讯,42(1):127-133]
Fan J,Gui M,ZhaoT,et al(2005).Study on the nutritional components of wild Sophora davidii Rom ex Pavol.Chin Wild Plant Resour,24(1):23-25(in Chinese with English abstract)[樊建,桂明英,赵天瑞等(2005).野生苦刺花食用价值研究.中国野生植物资源,24(1):23-25]
Gairi A,Rashid A(2004).TDZ-induced somatic embryogenesis in non-responsive caryopses of rice using a short treatment with 2,4-D.Plant Cell Tiss Org,76(1):29-33
Gupta PK,Durzan DJ(1986).Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos of Picea abies(Norway spruce).In Vitro Cell DevPl,22(11):685-688
K?rk?nen A(2000).Anatomical study of zygotic and somatic embryos of Tilia cordata.Plant Cell Tiss Org,61(3):205-214
Kong D,Shen H,Feng D,et al(2006).Cytological investigation of somatic and zygotic embryogenesis of Fraxinus mandshurica.Sci Silv Sin,42(12):130-134(in Chinese with English abstract)[孔冬梅,沈海龙,冯丹丹等(2006).水曲柳体细胞胚与合子胚发生的细胞学研究.林业科学,42(12):130-134]
Lin CS,Lin CC,Chang WC(2004).Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of bamboo Bambusa edulis.Plant Cell Tiss Org,76(1):75-82
Mao X,Wen M,Jiang X(2009).Research on water decoction partial pharmacodynamics of Sophora viciifolia.Yunnan Tradit Chin Med J,30(2):44-46(in Chinese)[毛晓健,温敏,蒋孝悝(2009).白刺花水煎剂的部分药效学研究.云南中医中药杂志,30(2):44-46]
Mathew MM,Philip VJ(2003).Somatic embryogenesis versus zygotic embryogenesis in Ensete superbum.Plant Cell Tiss Org,72(3):267-275
Visser C,Qureshi JA,Gill R,et al(1992).Morphoregulatory role of thidiazuron.Substitution of auxin and cytokinin requirement for the induction of somatic embryogenesis in geranium hypocotyl cultures.Plant Physiol,99(4):1704-1707
Wang QL,Wang M,Shi HR(2004).Hormones regulation on cotton somatic embryogenesis.Lett Biotech,15(6):577-579(in Chinese with English abstract)[王清连,王敏,师海荣(2004).植物激素对棉花体细胞胚胎发生的诱导及调节作用.生物技术通讯,15(6):577-579]
Wei YR,Yang H,Huang BZ,et al(2007).Effects of picloram,ABA and TDZ on somatic embryogenesis of banana.Acta Hortic Sin,34(1):81-86(in Chinese with English abstract)[魏岳荣,杨护,黄秉智等(2007).Picloram,ABA和TDZ对香蕉体细胞胚胎发生的影响.园艺学报,34(1):81-86]
Wu L,Lu W,Wei X,et al(2014).Effect of different treatment methods on hard seed germination characteristics for Sophora davidii.Mod Anim Husb,2(6):47-49(in Chinese)[吴丽芳,陆伟东,魏晓梅等(2014).不同处理方法对白刺花硬实种子萌发特性的影响研究.当代畜牧,2(6):47-49]
Wu LF,Wei XM,Lu WD,et al(2018).Dormancy mechanism and breaking methods for hard seeds of Sophora davidii.South Agr J,49(5):944-949(in Chinese with English abstract)[吴丽芳,魏晓梅,陆伟东等(2018).白刺花硬实种子的休眠机制及休眠解除.南方农业学报,49(5):944-949]
Xiao W,Huang XL,Huang X,et al(2007).Plant regeneration from protoplasts of Musa acuminata cv.Mas(AA)via somatic embryogenesis.Plant Cell Tiss Org,90(2):191-200
Xin W,Xu B,Wang G,et al(2006).Somatic embryogenesis and plant regeneration of Anthurium andraeanum.Acta Hortic Sin,33(6):1281-1286(in Chinese with English abstract)[辛伟杰,徐彬,王广东等(2006).花烛体细胞胚胎发生及植株再生.园艺学报,33(6):1281-1286]
Fan J,Gui M,ZhaoT,et al(2005).Study on the nutritional components of wild Sophora davidii Rom ex Pavol.Chin Wild Plant Resour,24(1):23-25(in Chinese with English abstract)[樊建,桂明英,赵天瑞等(2005).野生苦刺花食用价值研究.中国野生植物资源,24(1):23-25]
Gairi A,Rashid A(2004).TDZ-induced somatic embryogenesis in non-responsive caryopses of rice using a short treatment with 2,4-D.Plant Cell Tiss Org,76(1):29-33
Gupta PK,Durzan DJ(1986).Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos of Picea abies(Norway spruce).In Vitro Cell DevPl,22(11):685-688
K?rk?nen A(2000).Anatomical study of zygotic and somatic embryos of Tilia cordata.Plant Cell Tiss Org,61(3):205-214
Kong D,Shen H,Feng D,et al(2006).Cytological investigation of somatic and zygotic embryogenesis of Fraxinus mandshurica.Sci Silv Sin,42(12):130-134(in Chinese with English abstract)[孔冬梅,沈海龙,冯丹丹等(2006).水曲柳体细胞胚与合子胚发生的细胞学研究.林业科学,42(12):130-134]
Lin CS,Lin CC,Chang WC(2004).Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of bamboo Bambusa edulis.Plant Cell Tiss Org,76(1):75-82
Mao X,Wen M,Jiang X(2009).Research on water decoction partial pharmacodynamics of Sophora viciifolia.Yunnan Tradit Chin Med J,30(2):44-46(in Chinese)[毛晓健,温敏,蒋孝悝(2009).白刺花水煎剂的部分药效学研究.云南中医中药杂志,30(2):44-46]
Mathew MM,Philip VJ(2003).Somatic embryogenesis versus zygotic embryogenesis in Ensete superbum.Plant Cell Tiss Org,72(3):267-275
Visser C,Qureshi JA,Gill R,et al(1992).Morphoregulatory role of thidiazuron.Substitution of auxin and cytokinin requirement for the induction of somatic embryogenesis in geranium hypocotyl cultures.Plant Physiol,99(4):1704-1707
Wang QL,Wang M,Shi HR(2004).Hormones regulation on cotton somatic embryogenesis.Lett Biotech,15(6):577-579(in Chinese with English abstract)[王清连,王敏,师海荣(2004).植物激素对棉花体细胞胚胎发生的诱导及调节作用.生物技术通讯,15(6):577-579]
Wei YR,Yang H,Huang BZ,et al(2007).Effects of picloram,ABA and TDZ on somatic embryogenesis of banana.Acta Hortic Sin,34(1):81-86(in Chinese with English abstract)[魏岳荣,杨护,黄秉智等(2007).Picloram,ABA和TDZ对香蕉体细胞胚胎发生的影响.园艺学报,34(1):81-86]
Wu L,Lu W,Wei X,et al(2014).Effect of different treatment methods on hard seed germination characteristics for Sophora davidii.Mod Anim Husb,2(6):47-49(in Chinese)[吴丽芳,陆伟东,魏晓梅等(2014).不同处理方法对白刺花硬实种子萌发特性的影响研究.当代畜牧,2(6):47-49]
Wu LF,Wei XM,Lu WD,et al(2018).Dormancy mechanism and breaking methods for hard seeds of Sophora davidii.South Agr J,49(5):944-949(in Chinese with English abstract)[吴丽芳,魏晓梅,陆伟东等(2018).白刺花硬实种子的休眠机制及休眠解除.南方农业学报,49(5):944-949]
Xiao W,Huang XL,Huang X,et al(2007).Plant regeneration from protoplasts of Musa acuminata cv.Mas(AA)via somatic embryogenesis.Plant Cell Tiss Org,90(2):191-200
Xin W,Xu B,Wang G,et al(2006).Somatic embryogenesis and plant regeneration of Anthurium andraeanum.Acta Hortic Sin,33(6):1281-1286(in Chinese with English abstract)[辛伟杰,徐彬,王广东等(2006).花烛体细胞胚胎发生及植株再生.园艺学报,33(6):1281-1286]