不同基因型樱桃植株再生体系的研究
摘要
本研究旨在进一步优化和拓宽樱桃不同基因型组织培养和快速繁殖体系,以离体叶片、离体叶柄、节间、根等多种外植体为试材,探讨并丰富樱桃遗传转化体系的再生系统。
以樱桃杂种优系‘F8’(Prunus.avium L.×Prunus . pseudocerasus L.)、‘F10’(Prunus.avium L.×Prunus. pseudocerasus L.)、‘H8’(Prunus. cerasus L×Prunus.pseudocerasus L.)和‘H10’(Prunus. cerasus L×Prunus.pseudocerasus L.)的茎尖为材料,MS和F14为基本培养基,通过添加不同浓度的6-苄基氨基腺嘌呤(6-BA)、3-吲哚丁酸(IBA)、赤霉素(GA3)进行杂种樱桃再生体系的构建研究,结果表明:F14+6-BA (0.3~0.5)mg/L+GA30.1 mg/L适合各个杂种优系初代培养诱导出植株,F14+6-BA (0.5~1.0)mg/L+IBA(0.1~0.3)mg/L +GA30.1 mg/L能够很好的促进各个杂种优系分化不定芽,1/2MS+IBA0.5 mg/L(或NAA 0.8 mg/L)是各个杂种优系的最佳生根培养基。
以甜樱桃(Prunus avium L.)品种‘meizao’和‘Summit’、杂种优系‘F8’、‘F10’、‘H8’和‘H10’等不同基因型的试管苗为材料,进行樱桃离体叶片、叶柄、子叶、节间、根再生试验。结果表明:甜樱桃品种‘meizao’的离体叶片在WPM+6-BA 2.0 mg/L +IAA 2.0mg/L +GA30.1 mg/L培养基上,不定芽再生率可达到90%,‘H10’、‘Summit’、‘F10’、‘F8’在6-BA2~2.5 mg/L、IAA 1.0~2.0 mg/L的激素配比范围内,也获得了76.0%、86.0%、80.0%、64.0%的再生率。离体叶片接种后暗培养1周、远轴面向下接种和选择试管苗上部的幼嫩叶片,均有利于叶片再生。优系‘H10’叶柄接种于改良MS+6-BA2.0 mg/L+IBA0.1 mg/L +2,4-D0.5 mg/L的培养基,可获得40.0%的再生率,其他品种仅有H8的叶柄获得了少量的再生植株。选择带一小块叶片的叶柄和5mg/L的硝酸银处理有利于叶柄再生;优系‘F10’的幼嫩节间接种于LS+6-BA1.0 mg/L+NAA2.0 mg/L+CH100 mg/L培养基,最高再生率为40.0%,在6-BA1~2mg/L和NAA1~2mg/L的激素配比范围内,‘H8’、‘H10’的离体节间再生率最高分别为25.0%和30.0%。选择试管苗黄化处理的节间,再生率要低于未经过黄化处理的节间;优系‘H8’整体根在MS+6-BA1.0 mg/L +2,4-D2.0 mg/L + IBA0.05 mg/L + NAA 0.05 mg/L的培养基上,达到了96.7%的再生率,在6-BA 0.5~2 mg/L和2,4-D 0.5~2.0 mg/L的激素配比范围内,‘H10’、‘F8’、‘F10’离体根再生率分别为93.3%、96.7%和93.3%。另外,根切段接种没有整体根再生频率高。
以樱桃杂种优系‘H8’试管苗的离体节间和中国樱桃‘对樱’的试管苗离体叶片为材料,MS为基本培养基,通过调整植物激素种类和浓度,筛选适合‘H8’试管苗离体节间和‘对樱’离体叶片再生不定根的培养基配方,结果表明:‘H8’离体节间和‘对樱’离体叶片,均在培养基MS+NAA1.0 mg/L上获得了最大的不定根再生率64.0%和80.0%,NAA浓度过大或过低均不利于不定根再生。离体叶片和离体节间再生不定根的成功,从多角度、多层次建立樱桃离体再生不定芽提供了新途径。
As In Vitro leaf,leafstalk,internodes and root the material ,the study is to optimize and open up tissue culuture and rapid propagation system in different gene cherry,and discuss regeneration system of cherry genetic transformation.
In Vitro construction of regeneration system for different selections of cherry plant hybrid including‘F8’(Prunus cerasus L×Prunus.pseudocerasus L.),‘F10’(Prunus. avium L.×Prunus. pseudocerasus L.),‘H8’(Prunus. cerasus L×Prunus. pseudocerasus L.and‘H10’(Prunus cerasus L×Prunus pseudocerasus L.) were studied.It was showed that by taking MS and F14 basic medium supplemented with different combinations of plant growth regulators such as IBA and GA3,the micropropagation technologies including preliminary culture, subculture and rooting culture were successfully established.Medium F14+6-BA(0.3~0.5)mg/L+GA3 0.1mg/L is suitable for preliminary culture of the tested selections,F14+6-BA(0.5~1.0)mg/L+IBA0.1mg/L+ GA30.1mg/L is an optimal subculture medium for proliferation of the In Vitro shoots, satisfact ory rooting response can be obtained on medium 1/2 MS+IBA 0.5 mg/L(or NAA0.8 mg/L).
The patterns of plant regeneration in different genotypes of cherry plants,including sweet cherry cultivars‘meizao’and‘Summit’,hybrid lines‘F8’、‘F10’、‘H8’and‘H10’were comparatively studied, especially focused on the regeneration technology of the several hybrid lines.It was showed that cherry plants with different genetic background take specifically patterns and routes for plant regeneration. Satisfactory regenerations can be obtained in different materials by chose In Vitro leaves, leafstalks, internodes and root systems as explants,and by taking special induction and culture manipulations, including the basic mediums, levels and combinations of plant growth regulators.The primary results is as follows:above 90.0% of In Vitro leaves of sweet cherry cultivar‘Meizao’regenerate adventitious buds on WPM+6-BA2.0 mg/L+IAA2.0mg/L+GA30.1mg/L‘,H10’‘,Summit’‘,F10’、‘F8’can also get 76.0%,86.0%, 80.0%,64.0% regeneration respectively in 6-BA(2~2.5mg/L) and IAA(1.0~2.0mg/L) In addition, choosing young leaves at upper part of the tube plantlet and culture in darkness for 1 week, with the axial surface contact with the medium benefit to adventitious bud regeneration.when taken In Vitro leafstalks of hybrid‘H10’, 40.0% regeneration can be obtained on modified MS+6-BA2.0 mg/L+IBA0.1mg/L +2,4-D0.5 mg/L,only‘H8’lesfstalk get a small quantity of regeneration plant in others. In addition, leafstalk with a piece of leaf section and treatment with 5mg/L of AgNO3 improved adventitious shoot regeneration In Vitro leafstalk explants.while the medium LS+6-BA 1.0 mg/L+NAA2.0 mg/L+CH100 mg/L is optimal for regeneration of hybrid‘F10’,40.0% regeneration can be obtained, etiolation treatment internodes gave less adventitious shoots than that of un-treated ones.As high as 96.7% of intact root system of hybrid H8 regenerate buds on MS+6-BA1.0 mg/L +2,4-D2.0 mg/L +IBA0.05mg/L+NAA0.05 mg/L. H8,H10 are 25.0% and 30.0% in intro internode regeneration respectively, while‘H10’‘,F8’‘,F10’are 93.3%,96.7%, 93.3% in intro root regeneration respectively in other concentration. More adventitious shoots regeneration can be obtained on intact root system than that of root sections.
Adventitious root regeneration was also tested by taking In Vitro internodes of‘H8’and leaves of‘duiying’as materials. It was showed that on MS basic medium supplement with NAA1.0 mg/L,64.0% and 80.0% root regeneration can be obtained respectively, concentration of NAA, higher or lower than 1.0 mg/L decreased root induction. The result indicated that plant regeneration can be indirectly achieved via adventitious root regarding to the materials in which direct adventitious bud regeneration is not easy to be obtained.
以樱桃杂种优系‘F8’(Prunus.avium L.×Prunus . pseudocerasus L.)、‘F10’(Prunus.avium L.×Prunus. pseudocerasus L.)、‘H8’(Prunus. cerasus L×Prunus.pseudocerasus L.)和‘H10’(Prunus. cerasus L×Prunus.pseudocerasus L.)的茎尖为材料,MS和F14为基本培养基,通过添加不同浓度的6-苄基氨基腺嘌呤(6-BA)、3-吲哚丁酸(IBA)、赤霉素(GA3)进行杂种樱桃再生体系的构建研究,结果表明:F14+6-BA (0.3~0.5)mg/L+GA30.1 mg/L适合各个杂种优系初代培养诱导出植株,F14+6-BA (0.5~1.0)mg/L+IBA(0.1~0.3)mg/L +GA30.1 mg/L能够很好的促进各个杂种优系分化不定芽,1/2MS+IBA0.5 mg/L(或NAA 0.8 mg/L)是各个杂种优系的最佳生根培养基。
以甜樱桃(Prunus avium L.)品种‘meizao’和‘Summit’、杂种优系‘F8’、‘F10’、‘H8’和‘H10’等不同基因型的试管苗为材料,进行樱桃离体叶片、叶柄、子叶、节间、根再生试验。结果表明:甜樱桃品种‘meizao’的离体叶片在WPM+6-BA 2.0 mg/L +IAA 2.0mg/L +GA30.1 mg/L培养基上,不定芽再生率可达到90%,‘H10’、‘Summit’、‘F10’、‘F8’在6-BA2~2.5 mg/L、IAA 1.0~2.0 mg/L的激素配比范围内,也获得了76.0%、86.0%、80.0%、64.0%的再生率。离体叶片接种后暗培养1周、远轴面向下接种和选择试管苗上部的幼嫩叶片,均有利于叶片再生。优系‘H10’叶柄接种于改良MS+6-BA2.0 mg/L+IBA0.1 mg/L +2,4-D0.5 mg/L的培养基,可获得40.0%的再生率,其他品种仅有H8的叶柄获得了少量的再生植株。选择带一小块叶片的叶柄和5mg/L的硝酸银处理有利于叶柄再生;优系‘F10’的幼嫩节间接种于LS+6-BA1.0 mg/L+NAA2.0 mg/L+CH100 mg/L培养基,最高再生率为40.0%,在6-BA1~2mg/L和NAA1~2mg/L的激素配比范围内,‘H8’、‘H10’的离体节间再生率最高分别为25.0%和30.0%。选择试管苗黄化处理的节间,再生率要低于未经过黄化处理的节间;优系‘H8’整体根在MS+6-BA1.0 mg/L +2,4-D2.0 mg/L + IBA0.05 mg/L + NAA 0.05 mg/L的培养基上,达到了96.7%的再生率,在6-BA 0.5~2 mg/L和2,4-D 0.5~2.0 mg/L的激素配比范围内,‘H10’、‘F8’、‘F10’离体根再生率分别为93.3%、96.7%和93.3%。另外,根切段接种没有整体根再生频率高。
以樱桃杂种优系‘H8’试管苗的离体节间和中国樱桃‘对樱’的试管苗离体叶片为材料,MS为基本培养基,通过调整植物激素种类和浓度,筛选适合‘H8’试管苗离体节间和‘对樱’离体叶片再生不定根的培养基配方,结果表明:‘H8’离体节间和‘对樱’离体叶片,均在培养基MS+NAA1.0 mg/L上获得了最大的不定根再生率64.0%和80.0%,NAA浓度过大或过低均不利于不定根再生。离体叶片和离体节间再生不定根的成功,从多角度、多层次建立樱桃离体再生不定芽提供了新途径。
As In Vitro leaf,leafstalk,internodes and root the material ,the study is to optimize and open up tissue culuture and rapid propagation system in different gene cherry,and discuss regeneration system of cherry genetic transformation.
In Vitro construction of regeneration system for different selections of cherry plant hybrid including‘F8’(Prunus cerasus L×Prunus.pseudocerasus L.),‘F10’(Prunus. avium L.×Prunus. pseudocerasus L.),‘H8’(Prunus. cerasus L×Prunus. pseudocerasus L.and‘H10’(Prunus cerasus L×Prunus pseudocerasus L.) were studied.It was showed that by taking MS and F14 basic medium supplemented with different combinations of plant growth regulators such as IBA and GA3,the micropropagation technologies including preliminary culture, subculture and rooting culture were successfully established.Medium F14+6-BA(0.3~0.5)mg/L+GA3 0.1mg/L is suitable for preliminary culture of the tested selections,F14+6-BA(0.5~1.0)mg/L+IBA0.1mg/L+ GA30.1mg/L is an optimal subculture medium for proliferation of the In Vitro shoots, satisfact ory rooting response can be obtained on medium 1/2 MS+IBA 0.5 mg/L(or NAA0.8 mg/L).
The patterns of plant regeneration in different genotypes of cherry plants,including sweet cherry cultivars‘meizao’and‘Summit’,hybrid lines‘F8’、‘F10’、‘H8’and‘H10’were comparatively studied, especially focused on the regeneration technology of the several hybrid lines.It was showed that cherry plants with different genetic background take specifically patterns and routes for plant regeneration. Satisfactory regenerations can be obtained in different materials by chose In Vitro leaves, leafstalks, internodes and root systems as explants,and by taking special induction and culture manipulations, including the basic mediums, levels and combinations of plant growth regulators.The primary results is as follows:above 90.0% of In Vitro leaves of sweet cherry cultivar‘Meizao’regenerate adventitious buds on WPM+6-BA2.0 mg/L+IAA2.0mg/L+GA30.1mg/L‘,H10’‘,Summit’‘,F10’、‘F8’can also get 76.0%,86.0%, 80.0%,64.0% regeneration respectively in 6-BA(2~2.5mg/L) and IAA(1.0~2.0mg/L) In addition, choosing young leaves at upper part of the tube plantlet and culture in darkness for 1 week, with the axial surface contact with the medium benefit to adventitious bud regeneration.when taken In Vitro leafstalks of hybrid‘H10’, 40.0% regeneration can be obtained on modified MS+6-BA2.0 mg/L+IBA0.1mg/L +2,4-D0.5 mg/L,only‘H8’lesfstalk get a small quantity of regeneration plant in others. In addition, leafstalk with a piece of leaf section and treatment with 5mg/L of AgNO3 improved adventitious shoot regeneration In Vitro leafstalk explants.while the medium LS+6-BA 1.0 mg/L+NAA2.0 mg/L+CH100 mg/L is optimal for regeneration of hybrid‘F10’,40.0% regeneration can be obtained, etiolation treatment internodes gave less adventitious shoots than that of un-treated ones.As high as 96.7% of intact root system of hybrid H8 regenerate buds on MS+6-BA1.0 mg/L +2,4-D2.0 mg/L +IBA0.05mg/L+NAA0.05 mg/L. H8,H10 are 25.0% and 30.0% in intro internode regeneration respectively, while‘H10’‘,F8’‘,F10’are 93.3%,96.7%, 93.3% in intro root regeneration respectively in other concentration. More adventitious shoots regeneration can be obtained on intact root system than that of root sections.
Adventitious root regeneration was also tested by taking In Vitro internodes of‘H8’and leaves of‘duiying’as materials. It was showed that on MS basic medium supplement with NAA1.0 mg/L,64.0% and 80.0% root regeneration can be obtained respectively, concentration of NAA, higher or lower than 1.0 mg/L decreased root induction. The result indicated that plant regeneration can be indirectly achieved via adventitious root regarding to the materials in which direct adventitious bud regeneration is not easy to be obtained.
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Dolgov SV,1999.Genetic transformation of sour cherry(Cerasus vulgaris Mill.)leaf discs.In:Bajaj YPS Biotechnology in Agricultural and Forestry[M].Transgenic trees.44:29~38.
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Feucht W,Schmid P S.1980.Effect of orthodihyd rox yphenols on growth and protein pattern of callus cultures from Prunus avium[J].Physiologia P1antarum.50:309~313.
Garin E, Grenier E , Grenier-de march G.1997.Somatic embryogenesis in wild cherry (Prunus avium)[J]. Plant Cell,Tissue and Culture.48(2):83~91
Gutierrez-Pesce P,Taylor K,Muleo R,et al.1998.Somatic embryogenesis and shoot rengenerati- on from transgenic roots of the cherry rootstock Colt(P.avium×P.pseudcoerasus)mediated by pRi855T-DNA of Agrobacterium rhizogenes[J].Plant Cell Rep.17:574~580.
Hebert D,Kikert J R,Smith F D,et al.1993.Optimization of biolistic transformation of embryogenic grape cell suspensions[J].Plant Cell Rep. 12: 585~589.
Hokanson K E,Pooler M R. 2000.Regeneration of ornamental cherry (Prunus) taxa from mature stored seed[J]. Hort Science. 35:745~748.
Holefors A,Xue Z T,Welander M. 1998.Transformation of the apple root-stock M26 with rolA gene and its influence on growth [J].Plant Sci.136:69~78.
Ilieva I, Georgiev V, Zagorska N. 1995.Organogensis bimmature embryo culture in Prunus cerasus and in Prunus cerasus×Prunus avium hybrid[J] . Rastenier DnNauki.32:115~117.
James D J,Passey A J,Malhotra S B.1984.Organogenesis in callus derived from stem and leaf tissues of apple and cherry rootstocks[J].Plant cell Tiss.org.cult.(3):333~341.
Kornova K. 1995.Micropropagation of wild cherry k-1[J].Rasteniev Dni Nauki.32(7/8):97~98.
Lane W D, Cossio F. 1986.Adventitious shoots from cotyledons of immature cherry and apricot embryos Canadian Journal of Plant[J],Science.66:953~959.
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Linsmaier E M and F. Skoog.1965.Organic growth factor requirements of tobacco tissue culture[J]. Physiol Plant. 18:100~127.
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