柑橘品种茎段离体培养条件的优化及微芽嫁接试验
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摘要
【目的】优化柑橘品种茎段离体培养条件并开展微芽嫁接(试管内茎尖嫁接)试验,为培育无毒柑橘组培种苗提供参考依据。【方法】对温州蜜柑和纽荷尔脐橙成年态枝条进行不同浓度HgCl_2和NaClO消毒,筛选适宜组培利用的柑橘外植体消毒方式、采集季节及激素配比;开展嫁接砧木种子消毒和微芽嫁接试验,分析两个柑橘品种微芽嫁接的萌发状况。【结果】温州蜜柑外植体的最适消毒方法为70%酒精+1.0‰HgCl_2消毒6 min处理两次;纽荷尔脐橙外植体的最佳消毒方法为70%酒精+15.0%NaClO处理两次;采集成年态柑橘茎段进行离体培养的最佳时期为夏季;在MS固体培养基中添加1.0 mg/L 6-苄氨基嘌呤(6-BA)和0.1 mg/L萘乙酸(NAA)均有利于两个柑橘品种外植体腋芽萌发。砧木种子最适消毒方法为剥去内外种皮+70%酒精+1.5%NaClO溶液处理;两个柑橘品种微芽嫁接的萌发率较低,分别为10.00%和6.67%,但嫁接成功后柑橘苗长势良好。【结论】于夏季剪取温州蜜柑和纽荷尔脐橙成年态枝条带芽茎段,分别经70%酒精+1.0‰HgCl_2消毒6 min处理两次、70%酒精+15.0%NaClO处理两次,在添加1.0 mg/L6-BA+0.1 mg/L NAA的MS固体培养基中接种,可获得最佳的培养效果。利用微芽嫁接技术嫁接的柑橘苗长势良好,可在提高其嫁接萌发率基础上推广应用。
【Objective】Citrus stem culture conditions in vitro was explored and the shoot-tip grafting(stem tip grafting in tube)was carried out which could provide reference for cultivating virus-free citrus culture seedling.【Method】The mature stems of Satsuma mandarin and Newhall navel orange were disinfected with different concentrations of HgCl_2 and NaClO. The disinfection methods of citrus explants suitable for tissue culture,collecting season and hormone ratio were screened. The rootstock seed disinfection and the shoot-tip grafting were carried out to analyze germination status of grafted shoot-tips for the two varieties.【Result】The optimum disinfection method for S. mandarin explants was treated with 70% alcohol+1.0‰ HgCl_2 for 6 min for 2 times. The optimum disinfection method for Newhall navel orange explants was 70% alcohol+15.0% NaClO solution for 2 times. The optimum citrus stem collecting period for culture in vitro was summer. Added 1.0 mg/L 6-benayl aminopuring(6-BA)and 0.1 mg/L naphthylacetic acid(NAA)in MS solid medium were beneficial to the germination of axillary buds for the two citrus varieties. The optimum disinfection method for rootstock seed was removing the inner and outer seed coat+70% alcohol+1.5% NaClO solution. The germination rate of the two varieties were low,which were 10.00% and 6.67% respectively,but citrus seedlings grew well after successful grafting.【Conclusion】The mature stems with buds are harvested in summer and S. mandarin stems were disinfected with 70% alcohol+1.0‰ HgCl_2 for 6 min for 2 times;and Newhall navel orange were treated with 70% alcohol+15.0% NaClO solution for 2 times. Then they are cultured in MS solid medium supplemented with 1.0 mg/L 6-BA+0.1 mg/L NAA. This method can achieve good culture effects. The citrus seedlings grafted with shoot-tip grow well. Therefore,this method can be used after improving the graft germination rate on this basis.
【Objective】Citrus stem culture conditions in vitro was explored and the shoot-tip grafting(stem tip grafting in tube)was carried out which could provide reference for cultivating virus-free citrus culture seedling.【Method】The mature stems of Satsuma mandarin and Newhall navel orange were disinfected with different concentrations of HgCl_2 and NaClO. The disinfection methods of citrus explants suitable for tissue culture,collecting season and hormone ratio were screened. The rootstock seed disinfection and the shoot-tip grafting were carried out to analyze germination status of grafted shoot-tips for the two varieties.【Result】The optimum disinfection method for S. mandarin explants was treated with 70% alcohol+1.0‰ HgCl_2 for 6 min for 2 times. The optimum disinfection method for Newhall navel orange explants was 70% alcohol+15.0% NaClO solution for 2 times. The optimum citrus stem collecting period for culture in vitro was summer. Added 1.0 mg/L 6-benayl aminopuring(6-BA)and 0.1 mg/L naphthylacetic acid(NAA)in MS solid medium were beneficial to the germination of axillary buds for the two citrus varieties. The optimum disinfection method for rootstock seed was removing the inner and outer seed coat+70% alcohol+1.5% NaClO solution. The germination rate of the two varieties were low,which were 10.00% and 6.67% respectively,but citrus seedlings grew well after successful grafting.【Conclusion】The mature stems with buds are harvested in summer and S. mandarin stems were disinfected with 70% alcohol+1.0‰ HgCl_2 for 6 min for 2 times;and Newhall navel orange were treated with 70% alcohol+15.0% NaClO solution for 2 times. Then they are cultured in MS solid medium supplemented with 1.0 mg/L 6-BA+0.1 mg/L NAA. This method can achieve good culture effects. The citrus seedlings grafted with shoot-tip grow well. Therefore,this method can be used after improving the graft germination rate on this basis.
引文
陈洪明,刘科宏,周彦,王雪峰,周常勇,李中安.2014.柑桔黄脉病毒脱毒方法研究[J].中国南方果树,43(3):71-73.[Chen H M,Liu K H,Zhou Y,Wang X F,Zhou C Y,Li Z A.2014.Study on yellow veins virus-free method of citrus[J].Fruit Trees in Southern China,43(3):71-73.]
陈泽雄,刘奕清,娄娟.2007.利用柑橘茎段诱导不定芽改进微芽嫁接技术的研究[J].西南师范大学学报(自然科学版),32(1):57-61.[Chen Z X,Liu Y Q,Lou J.2007.Study on improving micro-bud grafting technique by inducing adventitious buds of citrus stem segments[J].Journal of Southwest China Normal University(Natural Science Edition),32(1):57-61.]
邓才生,胡启灿,陈燕,江金兰.2009.柑桔组织培养与茎尖微芽嫁接试验初探[J].福建农业科技,40(4):54-55.[Deng C S,Hu Q C,Chen Y,Jiang J L.2009.Study on tissue culture of citrus and micropropagation of shoot tip[J].Fujian Agricultural Science and Technology,40(4):54-55.]
胡选萍,陈志远.2014.耐寒柑橘愈伤组织诱导优化[J].湖北农业科学,20(18):4460-4463.[Hu X P,Chen Z Y.2014.Cold-induced citrus callus induction and optimization[J].Hubei Agricultural Sciences,20(18):4460-4463.]
李凯,陈雄庭,洪磊,甘露.2011.化州橘红成年态茎段离体培养的改良[J].热带生物学报,2(1):42-45.[Li K,Chen X T,Hong L,Gan L.2011.Improvement of in vitro culture of adult stem segments for Exocarpium citri grandis[J].Journal of Tropical Biology,2(1):42-45.]
李丽.2014.柑橘茎尖微芽嫁接脱毒技术研究[D].杭州:浙江大学.[Li L.2014.Studies on shoot-tip micrografting for virus elimination[D].Hangzhou:Zhejiang University.]
刘科宏,周彦,李中安.2016.柑橘茎尖嫁接脱毒技术研究进展[J].园艺学报,43(9):1665-1674.[Liu K H,Zhou Y,Li Z A.2016.Technical progress on shoot-tip grafting of citrus[J].Acta Horticulture Sinica,43(9):1665-1674.]
隆雨薇.2015.柑橘成年态组织培养及脱毒方法的研究[D].武汉:华中农业大学.[Long Y W.2015.In vitro cultural of mature citrus stems and studying on virus-free methods[D].Wuhan:Huazhong Agricultural University.]
罗君琴,李丽,聂振朋,柯甫志,徐建国,孙建华.2010.“439”橘橙小种子离体无菌再生培养试验初报[J].浙江柑桔,27(1):20-23.[Luo J Q,Li L,Nie Z P,Ke F Z,Xu J G,Sun J H.2010.Preliminary report on sterile regeneration of“439”orange small seeds[J].Zhejiang Citrus,27(1):20-23.]
罗君琴,李丽,聂振朋,徐建国,柯甫志,孙建华.2011.柑橘试管内茎尖微芽嫁接技术试验初报[J].浙江农业科学,2(5):1031-1032.[Luo J Q,Li L,Nie Z P,Xu J G,Ke FZ,Sun J H.2011.Preliminary report of shoot-tip grafting technique[J].Journal of Zhejiang Agricultural Sciences,2(5):1031-1032.]
罗君琴,李丽,王海琴,聂振朋,林华良.2007.温州蜜柑无菌系建立及快繁调控技术研究[J].浙江柑桔,24(4):18-21.[Luo J Q,Li L,Wang H Q,Nie Z P,Lin H L.2007.Study on establishment of aseptic strains of Wenzhou mandarin and regulation technology of fast propagation[J].Zhejiang Citrus,24(4):18-21.]
乔芬,祁鹏志,伏卉,张鹏,吴珊,姜玲.2011.柑橘衰退病和碎叶病复合感染病原的脱毒研究[J].华中农业大学学报,30(4):416-421.[Qiao F,Qi P Z,Fu H,Zhang P,Wu S,Jiang L.2011.Studying on finding new method for eradication pathogen of citrus tristeza virus and citrus tatter leaf virus[J].Journal of Huazhong Agricultural University,30(4):416-421.]
王子成,李忠爱,邓秀新.2005.柑橘成年态茎段外植体消毒方法研究[J].河南大学学报(自然科学版),35(2):57-60.[Wang Z C,Li Z A,Deng X X.2005.Study on disinfection methods of adult stem segments explants of citrus[J].Journal of Henan University(Natural Science Edition),35(2):57-60.]
徐定华,张攀,范志帷,许少华,覃礼琳,艾佳.2007.红心柚外植体无菌系建立的初步研究[J].福建果树,33(1):15-17.[Xu D H,Zhang P,Fan Z W,Xu S H,Qin L L,Ai J.2007.Establishment of regeneration system of pomelo explant in vitro[J].Fujian Fruit Tree,33(1):15-17.]
周海霞.2010.不同脱毒方法对红江橙种性变异及脱黄龙病毒效果的影响[D].湛江:广东海洋大学.[Zhou H X.2010.Different virus elimination methods in species variation of citrus Siensis osbeck cv.‘Hongjiang’and the effect of the virus of Liberobacter asiaticum[D].Zhanjiang:Guangdong Ocean University.]
Hilf M E,Lewis R S.2016.Transmission and propagation of‘candidatus liberibacter asiaticus’by grafting with individual citrus leaves[J].Phytopathology,106(5):452-458.
Murashinge T,Bitters W P,Rangan T S,Nauer E M,Roistacher C N,Holliday P B.1972.A technique of shoot apex grafting and its utilization towards recovering virus-free citrus clones[J].Hortscience,7(2):118-119
Shokrollah H,Abdullah T L,Sijam K,Sijam K,Siti Nor AA,Nur Ashikin P A.2009.Differential reaction of citrus species in Malaysia to Huanglongbing(HLB)disease using grafting method[J].American Journal of Agricultural&Biological Science,4(1):32-38.
Wu Q,Xia R.2005.Effects of AM fungi on drought tolerance of citrus grafting seedling trifoliate orange/cara cara[J].Journal of Applied Ecology,16(5):865-869.
陈泽雄,刘奕清,娄娟.2007.利用柑橘茎段诱导不定芽改进微芽嫁接技术的研究[J].西南师范大学学报(自然科学版),32(1):57-61.[Chen Z X,Liu Y Q,Lou J.2007.Study on improving micro-bud grafting technique by inducing adventitious buds of citrus stem segments[J].Journal of Southwest China Normal University(Natural Science Edition),32(1):57-61.]
邓才生,胡启灿,陈燕,江金兰.2009.柑桔组织培养与茎尖微芽嫁接试验初探[J].福建农业科技,40(4):54-55.[Deng C S,Hu Q C,Chen Y,Jiang J L.2009.Study on tissue culture of citrus and micropropagation of shoot tip[J].Fujian Agricultural Science and Technology,40(4):54-55.]
胡选萍,陈志远.2014.耐寒柑橘愈伤组织诱导优化[J].湖北农业科学,20(18):4460-4463.[Hu X P,Chen Z Y.2014.Cold-induced citrus callus induction and optimization[J].Hubei Agricultural Sciences,20(18):4460-4463.]
李凯,陈雄庭,洪磊,甘露.2011.化州橘红成年态茎段离体培养的改良[J].热带生物学报,2(1):42-45.[Li K,Chen X T,Hong L,Gan L.2011.Improvement of in vitro culture of adult stem segments for Exocarpium citri grandis[J].Journal of Tropical Biology,2(1):42-45.]
李丽.2014.柑橘茎尖微芽嫁接脱毒技术研究[D].杭州:浙江大学.[Li L.2014.Studies on shoot-tip micrografting for virus elimination[D].Hangzhou:Zhejiang University.]
刘科宏,周彦,李中安.2016.柑橘茎尖嫁接脱毒技术研究进展[J].园艺学报,43(9):1665-1674.[Liu K H,Zhou Y,Li Z A.2016.Technical progress on shoot-tip grafting of citrus[J].Acta Horticulture Sinica,43(9):1665-1674.]
隆雨薇.2015.柑橘成年态组织培养及脱毒方法的研究[D].武汉:华中农业大学.[Long Y W.2015.In vitro cultural of mature citrus stems and studying on virus-free methods[D].Wuhan:Huazhong Agricultural University.]
罗君琴,李丽,聂振朋,柯甫志,徐建国,孙建华.2010.“439”橘橙小种子离体无菌再生培养试验初报[J].浙江柑桔,27(1):20-23.[Luo J Q,Li L,Nie Z P,Ke F Z,Xu J G,Sun J H.2010.Preliminary report on sterile regeneration of“439”orange small seeds[J].Zhejiang Citrus,27(1):20-23.]
罗君琴,李丽,聂振朋,徐建国,柯甫志,孙建华.2011.柑橘试管内茎尖微芽嫁接技术试验初报[J].浙江农业科学,2(5):1031-1032.[Luo J Q,Li L,Nie Z P,Xu J G,Ke FZ,Sun J H.2011.Preliminary report of shoot-tip grafting technique[J].Journal of Zhejiang Agricultural Sciences,2(5):1031-1032.]
罗君琴,李丽,王海琴,聂振朋,林华良.2007.温州蜜柑无菌系建立及快繁调控技术研究[J].浙江柑桔,24(4):18-21.[Luo J Q,Li L,Wang H Q,Nie Z P,Lin H L.2007.Study on establishment of aseptic strains of Wenzhou mandarin and regulation technology of fast propagation[J].Zhejiang Citrus,24(4):18-21.]
乔芬,祁鹏志,伏卉,张鹏,吴珊,姜玲.2011.柑橘衰退病和碎叶病复合感染病原的脱毒研究[J].华中农业大学学报,30(4):416-421.[Qiao F,Qi P Z,Fu H,Zhang P,Wu S,Jiang L.2011.Studying on finding new method for eradication pathogen of citrus tristeza virus and citrus tatter leaf virus[J].Journal of Huazhong Agricultural University,30(4):416-421.]
王子成,李忠爱,邓秀新.2005.柑橘成年态茎段外植体消毒方法研究[J].河南大学学报(自然科学版),35(2):57-60.[Wang Z C,Li Z A,Deng X X.2005.Study on disinfection methods of adult stem segments explants of citrus[J].Journal of Henan University(Natural Science Edition),35(2):57-60.]
徐定华,张攀,范志帷,许少华,覃礼琳,艾佳.2007.红心柚外植体无菌系建立的初步研究[J].福建果树,33(1):15-17.[Xu D H,Zhang P,Fan Z W,Xu S H,Qin L L,Ai J.2007.Establishment of regeneration system of pomelo explant in vitro[J].Fujian Fruit Tree,33(1):15-17.]
周海霞.2010.不同脱毒方法对红江橙种性变异及脱黄龙病毒效果的影响[D].湛江:广东海洋大学.[Zhou H X.2010.Different virus elimination methods in species variation of citrus Siensis osbeck cv.‘Hongjiang’and the effect of the virus of Liberobacter asiaticum[D].Zhanjiang:Guangdong Ocean University.]
Hilf M E,Lewis R S.2016.Transmission and propagation of‘candidatus liberibacter asiaticus’by grafting with individual citrus leaves[J].Phytopathology,106(5):452-458.
Murashinge T,Bitters W P,Rangan T S,Nauer E M,Roistacher C N,Holliday P B.1972.A technique of shoot apex grafting and its utilization towards recovering virus-free citrus clones[J].Hortscience,7(2):118-119
Shokrollah H,Abdullah T L,Sijam K,Sijam K,Siti Nor AA,Nur Ashikin P A.2009.Differential reaction of citrus species in Malaysia to Huanglongbing(HLB)disease using grafting method[J].American Journal of Agricultural&Biological Science,4(1):32-38.
Wu Q,Xia R.2005.Effects of AM fungi on drought tolerance of citrus grafting seedling trifoliate orange/cara cara[J].Journal of Applied Ecology,16(5):865-869.