石榴试管嫁接技术研究
|
摘要
【目的】尝试建立高效的石榴试管嫁接技术体系,尝试解决根癌农杆菌介导的石榴遗传转化中再生的转化不定芽难以生根顺利发育成完整植株的问题。【方法】以‘突尼斯软籽’石榴试管实生苗为接穗‘,豫大籽’石榴试管实生苗为砧木,设置不同的砧穗组合进行嫁接;在添加不同浓度6-BA的培养基中进行嫁接,对嫁接成活的试管苗进行驯化移栽,统计嫁接成活率和移栽成活率。【结果】进行试管嫁接时适宜采用的接穗类型为带有4片叶片的茎尖,砧木类型为不带子叶的根和下胚轴连接体,嫁接成活率可达到73.33%;将四叶一心的‘突尼斯软籽’石榴嫁接至不带叶片的‘豫大籽’石榴,嫁接苗在添加1.5 mg·L~(-1)6-BA和0.1 mg·L~(-1)NAA的MS培养基上的生长效果最好,移栽成活率最高可达75.86%。【结论】研究所建立的石榴试管嫁接体系,为解决石榴遗传转化过程中不定芽生根困难提供了一条新的途径,并为通过基因工程手段进行石榴种质改良奠定基础。
【Objective】Pomegranate(Punica granatum L.) originates from central Asia, is an economically important deciduous fruit crop, cultivates for its delicious edible fruits. In addition, pomegranate fruit has higher levels of antioxidants than the other fruit crops, such as orange(Citrus sinensis), apple(Malus domestica), grape(Vitis vinifera) and kiwifruit(Actinidia chinensis). Antioxidants are potentially beneficial in preventing cardiovascular disease, diabetes, and prostate cancer.‘Tunisa soft-seed'was introduced to China from Tunisa was popular with customers. While low temperature is a limited factor as the‘Tunisa soft-seed'cultivation in northern China. Cold which impaired plant growth and development and reduced productivity is one of the most devastating abiotic stresses in‘Tunisa soft-seed'.Therefore, enhancement of cold tolerance becomes a major subject of considerable research interest over a long period. Some manual measures could be used to enhance cold tolerance, while creating coldresistant cultivars can be an optimal choice. The improvement of cold-resistance of‘Tunisa soft-seed'by traditional breeding techniques has been constrained by complex biology and long period. Genetic engineering as a supplementation for traditional breeding has been proven to be an effective approach for creating novel germplasms with elevated stress tolerance. Establishment of an efficient and stable regeneration and transformation system is basis of genetic engineering. However, in the pomegranate transformation system, the regeneration of adventitious buds was limited due to the influence of Agrobacterium tumefaciens, antibiotics and other factors involved in the transformation process. Moreover,the induction of adventitious roots was also difficult. In this study, in vitro grafting method was attempted to establish an efficient pomegranate test-tube grafting system in order to solve the problem which the regenerated adventitious buds were difficult to regenerate adventitious roots and ultimately obtain the whole transgenic plantlets.【Methods】In this experiment, the seedlings obtained from seeds of‘Tunisa soft-seed'and‘Yudazi'were used as experimental materials. Different combinations of rootstock and scion were conducted to test the effect on the rate of the grafting survival. The seedlings of‘Yudazi'as rootstocks were divided into two types, including with and without cotyledons. While the seedlings of‘Tunisa soft-seed'as scions were divided into four types, including without leaf, two-leaf,four-leaf and six-leaf tips. Stem tip without leaf blade was as control. In order to obtain the optimal mixture of hormone in MS for pomegranate tube grafting in vitro, the grafted seedlings were then placed in MS supplemented with different concentrations of 6-Benzylaminopurine(6-BA) and 0.1 mg·L-11-Naphthaleneacetic(NAA). The grafted-seedlings were transplanted and acclimatized. The survival rate of grafting and transplanting were analyzed, respectively.【Results】In the study of the effects of different treat combinations of stock and scion on survival rate of grafting plants in vitro, the results showed that the survival rate of grafting with the stem tip with leaf blade was higher than without leaf blade. The survival rate of grafting was increased with increasing the number of stem tip with leaf blades. And the highest survival rate of grafting was obtained when the number of the scion attached blades was four,then the survival rate of grafting decreased with the increasing of the number of blades in both two types of rootstocks. When the scion was the four-leaf stem tip type, the survival rate of grafting with non-cotyledon rootstock was obviously higher than that of cotyledon rootstock. Therefore, the optimal combination of pomegranate tube grafting which the survival rate of grafting was reached 73.33% was to use the stem tip with four blades as scion and the seedlings without cotyledon as rootstock. The effects of 6-BA on survival rate of grafting was significantly different in different treatments. The results indicated that the survival rate of grafting with 6-BA added to the MS medium was higher than the culture medium without 6-BA. And the survival rate of grafting increased with the increasing the concentration of 6-BA. The highest of the survival rate of grafting was reached 96.67% when 1.5 mg · L-16-BA was supplemented into the MS medium. And then the survival rate of grafting decreased when 2.0 mg·L-16-BA was supplemented into the MS medium. The same phenomenon was also observed in the process of acclimatization and transplantation. The survival rate of transplanting increased with the increasing of the 6-BA concentration, and the highest survival rate of transplanting was reached 75.86% when 1.5 mg · L-16-BA was supplemented into the MS medium. Moreover, the grafted seedlings grew well and healthy. Therefore, the optimal grafting and growth medium after transplanting were both MS supplemented 1.5 mg · L-16-BA and 0.1 mg · L-1 NAA.【Conclusion】In this study, the grafting system of pomegranate tube in vitro was established. It provided a new way to solve the transforming adventitious buds difficult to regenerate adventitious roots in the transformation process of pomegranate. Meanwhile, it provided technical support for germplasm improvement through genetic engineering in pomegranate.
【Objective】Pomegranate(Punica granatum L.) originates from central Asia, is an economically important deciduous fruit crop, cultivates for its delicious edible fruits. In addition, pomegranate fruit has higher levels of antioxidants than the other fruit crops, such as orange(Citrus sinensis), apple(Malus domestica), grape(Vitis vinifera) and kiwifruit(Actinidia chinensis). Antioxidants are potentially beneficial in preventing cardiovascular disease, diabetes, and prostate cancer.‘Tunisa soft-seed'was introduced to China from Tunisa was popular with customers. While low temperature is a limited factor as the‘Tunisa soft-seed'cultivation in northern China. Cold which impaired plant growth and development and reduced productivity is one of the most devastating abiotic stresses in‘Tunisa soft-seed'.Therefore, enhancement of cold tolerance becomes a major subject of considerable research interest over a long period. Some manual measures could be used to enhance cold tolerance, while creating coldresistant cultivars can be an optimal choice. The improvement of cold-resistance of‘Tunisa soft-seed'by traditional breeding techniques has been constrained by complex biology and long period. Genetic engineering as a supplementation for traditional breeding has been proven to be an effective approach for creating novel germplasms with elevated stress tolerance. Establishment of an efficient and stable regeneration and transformation system is basis of genetic engineering. However, in the pomegranate transformation system, the regeneration of adventitious buds was limited due to the influence of Agrobacterium tumefaciens, antibiotics and other factors involved in the transformation process. Moreover,the induction of adventitious roots was also difficult. In this study, in vitro grafting method was attempted to establish an efficient pomegranate test-tube grafting system in order to solve the problem which the regenerated adventitious buds were difficult to regenerate adventitious roots and ultimately obtain the whole transgenic plantlets.【Methods】In this experiment, the seedlings obtained from seeds of‘Tunisa soft-seed'and‘Yudazi'were used as experimental materials. Different combinations of rootstock and scion were conducted to test the effect on the rate of the grafting survival. The seedlings of‘Yudazi'as rootstocks were divided into two types, including with and without cotyledons. While the seedlings of‘Tunisa soft-seed'as scions were divided into four types, including without leaf, two-leaf,four-leaf and six-leaf tips. Stem tip without leaf blade was as control. In order to obtain the optimal mixture of hormone in MS for pomegranate tube grafting in vitro, the grafted seedlings were then placed in MS supplemented with different concentrations of 6-Benzylaminopurine(6-BA) and 0.1 mg·L-11-Naphthaleneacetic(NAA). The grafted-seedlings were transplanted and acclimatized. The survival rate of grafting and transplanting were analyzed, respectively.【Results】In the study of the effects of different treat combinations of stock and scion on survival rate of grafting plants in vitro, the results showed that the survival rate of grafting with the stem tip with leaf blade was higher than without leaf blade. The survival rate of grafting was increased with increasing the number of stem tip with leaf blades. And the highest survival rate of grafting was obtained when the number of the scion attached blades was four,then the survival rate of grafting decreased with the increasing of the number of blades in both two types of rootstocks. When the scion was the four-leaf stem tip type, the survival rate of grafting with non-cotyledon rootstock was obviously higher than that of cotyledon rootstock. Therefore, the optimal combination of pomegranate tube grafting which the survival rate of grafting was reached 73.33% was to use the stem tip with four blades as scion and the seedlings without cotyledon as rootstock. The effects of 6-BA on survival rate of grafting was significantly different in different treatments. The results indicated that the survival rate of grafting with 6-BA added to the MS medium was higher than the culture medium without 6-BA. And the survival rate of grafting increased with the increasing the concentration of 6-BA. The highest of the survival rate of grafting was reached 96.67% when 1.5 mg · L-16-BA was supplemented into the MS medium. And then the survival rate of grafting decreased when 2.0 mg·L-16-BA was supplemented into the MS medium. The same phenomenon was also observed in the process of acclimatization and transplantation. The survival rate of transplanting increased with the increasing of the 6-BA concentration, and the highest survival rate of transplanting was reached 75.86% when 1.5 mg · L-16-BA was supplemented into the MS medium. Moreover, the grafted seedlings grew well and healthy. Therefore, the optimal grafting and growth medium after transplanting were both MS supplemented 1.5 mg · L-16-BA and 0.1 mg · L-1 NAA.【Conclusion】In this study, the grafting system of pomegranate tube in vitro was established. It provided a new way to solve the transforming adventitious buds difficult to regenerate adventitious roots in the transformation process of pomegranate. Meanwhile, it provided technical support for germplasm improvement through genetic engineering in pomegranate.
引文
[1] NAIK S K,CHAND P K. Tissue culture-mediated biotechnological intervention in pomegranate:a review[J]. Plant Cell Reports,2011,30(5):707-21.
[2] CHAUHAN R,KANWAR K. Biotechnological advances in pomegranate(Punica granatum,L.)[J]. In Vitro Cellular&Developmental Biology-Plant,2012,48(6):579-594.
[3]苑兆和,尹燕雷,朱丽琴,李云,侯乐峰.石榴保健功能的研究进展[J].山东林业科技,2008,38(1):91-93.YUAN Zhaohe,YIN Yanlei,ZHU Liqin,LI Yun,HOU Lefeng.Research progress on neutraceutical functions of pomegranate[J].Shandong Forestry Science and Technology,2008,38(1):91-93.
[4]杨莉,徐昌杰,陈昆松.果树转基因研究进展与产业化展望[J].果树学报,2003,20(5):331-337.YANG Li,XU Changjie,CHEN Kunsong. Progresses in research and industrialization of transgenic fruit crops[J]. Journal of Fruit Science,2003,20(5):331-337.
[5] OMURA M,MATSUTA N,MORIGUCHI T,KOZAKI I. Adventitious shoot and plantlet formation from cultured pomegranate leaf explants[J]. Hortscience,1987,22:133-134.
[6] MORIGUCHI T,OMURA M,MATSUTA N,KOZAKI I. In vitro adventitious shoot formation from anthers of pomegranate[J].Hortscience,1987,22(5):947-948.
[7] DEEPIKA R,KANWAR K. In vitro regeneration of Punica granatum L. plants from different juvenile explants[J]. Journal of Fruit&Ornamental Plant Research,2010,18(1):5-22.
[8] KANWAR K,JOSEPH J,DEEPIKA R. Comparison of in vitro regeneration pathways in Punica granatum L.[J]. Plant Cell Tissue&Organ Culture,2010,100(2):199-207.
[9] KANWAR,RACHNA,KASHYAP A. In vitro propagation of wild pomegranate(Punica granatum L.)[M]//In:Jindal,Bawa(eds)Intellectual property rights. Shiva Offset,Dehradun,2004:209-215.
[10] AMIN M N,ISLAM M N,AZAD M A K. Regeneration of plantlets in vitro from the seedling explants of pomegranate(Punica granatum)[J]. Plant Tissue Culture,1999,9(1):53-61.
[11] SHARON M,SINHA S. Plant regeneration from cotyledonary node of Punica granatum L[J]. Indian Journal of Plant Physiology,2000,5(4):344-348.
[12] TERAKAMI S,MATSUTA N,YAMAMOTO T,SUGAYA S,GEMMA H,SOEJIMA J. Agrobacterium-mediated transformation of the dwarf pomegranate(Punica granatum L. var. nana)[J]. Plant Cell Reports,2007,26(8):1243-1251.
[13] VERMA V,KANWAR K,TUFCHI M,KASHYAP M. Agrobacterium-mediated Cry1A(b),gene transfer in Punica granatum L.cv. Kandhari Kabuli using different In Vitro,regeneration pathways[J]. Journal of Crop Science&Biotechnology,2014,17(1):1-10.
[14]李跃霞.‘突尼斯软籽’石榴遗传转化体系的构建和ISSR-PCR反应体系的优化[D].郑州:河南农业大学,2012.LI Yuexia. Construction of genetic transformation system and optimization of ISSR-PCR system of'Tunisia Soft-seed'pomegranate[D]. Zhengzhou:Henan Agricultural University,2012.
[15]赵玉洁.‘突尼斯软籽’石榴遗传转化体系建立及转化ICE1基因的研究[D].郑州:河南农业大学,2017.ZHAO Yujie. Establishment of genetic transformation system and transformed ICE1 gene into‘Tunisia Soft-seed’pomegranate[D]. Zhengzhou:Henan Agricultural University,2017.
[16]陈延惠,谭彬,李洪涛,胡青霞,叶霞,孟海军,冯建灿.石榴2种外植体再生方法在遗传转化研究中的优势比较[J].果树学报,2012,29(4):598-604.CHEN Yanhui,TAN Bin,LI Hongtao,HU Qingxia,YE Xia,MENG Haijun,FENG Jiancan. Comparison of advantages of two kinds of explant regeneration methods for studies on genetic transformation in pomegranate[J]. Journal of Fruit Science,2012,29(4):598-604.
[17]连红可.‘突尼斯软籽’石榴再生体系和GFP报告基因的瞬时表达研究初报[D].郑州:河南农业大学,2011.LIAN Hongke. Preliminary study on the regeneration system and the reporter gene GFP instantaneous expression of Soft-seed Pomegranate variety‘Tunisia’[D]. Zhengzhou:Henan Agricultural University,2011.
[18] ESTRADA-LUNA A A,LóPEZ-PERALTA C,CáRDENAS-SORIANO E. In vitro micrografting and the histology of graft union formation of selected species of prickly pear cactus(Opuntia,spp.)[J]. Scientia Horticulturae,2002,92(3):317-327.
[19]吴雅琴,章德明.用离体微嫁接法快速检测苹果潜隐病毒[J].落叶果树,2001,33(2):4-6.WU Yaqin,ZHANG Deming. Rapidly detecting apple chlorotic leaf spot virus apple stem grooving virus and apple stem pitting virus by micrografting in vitro[J]. Deciduous Fruits,2001(2):4-6.
[20]洪树荣,吴立廉.试管苗嫩枝顶端嫁接技术的研究[J].植物科学学报,1992(3):292-294.HONG Shurong,WU Lilian. Study on tip grafting in vivo by using in vitro plantlets[J]. Journal of Wuhan Botanical Research,1992,10:292-294.
[21]王国平,李晓梅,陈秋芳.核桃无根试管苗嫁接影响因素的研究[J].河北林果研究,2006,21(2):182-184.WANG Guoping,LI Xiaomei,CHEN Qiufang. Studies on the factors affecting microshoot grafting survival of walnut[J]. Hebei Journal of Forestry and Orchard Research,2006,21:182-184.
[22]李文彬.葡萄试管内外嫁接技术及其特性研究[D].兰州:甘肃农业大学,2005.LI Wenbin. Study on grape tube inside and outside grafting technique and characteristics[D]. Lanzhou:Agricultural University of Gansu,2005.
[23]郭文武.一种将柑橘离体芽苗嫁接于温室砧木的高效方法[J].果树学报,2003,20(3):242.GUO Wenwu. An efficient and simple technique to graft citrus in vitro shoots onto greenhouse rootstock seedlings[J]. Journal of fruit science,2003,20:242.
[24]郝军虹,宋涛,侯献波.赤霉素对‘突尼斯软籽’石榴种子发芽率的影响[J].中国果菜,2012(1):24-26.HAO Junhong,SONG Tao,HOU Xianbo. Effect of gibberellinon seed germination of Tunisia soft-seed Pomegranate[J]. China Fruit&Vegetable,2012(1):24-26.
[25]李洪涛,李东伟,陈海燕,胡青霞,陈延惠. GA3对突尼斯软籽石榴种子发芽的影响[J].河南农业科学,2014,43(5):154-155.LI Hongtao,LI Dongwei,CHEN Haiyan,HU Qingxia,CHEN Yanhui. Effect of GA3on seed germination of Tunisia soft-seed Pomegranate[J]. Journal of Henan Agricultural Sciences,2014,43(5):154-155.
[26] NAVARRO L,ROISTACHER C N,MURASHIGE T. Improvement of shoot-tip grafting in vitro for virus-free citrus[J]. 1992,5(5):471-479.
[27] PALMA B,VOGT G F,NEVILLE P. A combined in vitro/in vivo method for improved grafting of Acacia senegal(L.)Wild[J].Journal of Pomology&Horticultural Science,1996,71(3):379-381.
[28]宋瑞琳,吴如健,柯冲.茎尖嫁接脱除柑桔主要病原的研究[J].植物病理学报,1999,29(3):275-279.SONG Ruilin,WU Rujian,KE Chong. Elimination of the main citrus virus and virus-like diseases by shoot-tip grafting[J]. Acta Phytopathologica Sinica,1999,29(3):275-279.
[29]赵巍巍,陈蕾,曹后男,宗成文,姜淑苓.梨的试管微嫁接技术研究[J].安徽农业科学,2010,38(2):624-626.ZHAO Weiwei,CHEN Lei,CAO Hounan,ZONG Chengwen,JIANG Shuling. Study on micro-grafting in vitro of tube Pear[J].Journal of Anhui Agricultural Sciences,2010,38(2):624-626.
[30]周瑞金,杜国强,师校欣,马宝焜.影响转基因苹果试管微嫁接苗成活的因子[J].果树学报,2007,24(2):215-217.ZHOU Ruijin,DU Guoqiang,SHI Xiaoxin,MA Baokun. Studies on factors affecting survival rate of micro-graft in vitro in transgenic apple[J]. JournaI of Fruit Science,2007,24(2):215-217.
[31] JONARD R. Micrografting and its applications to tree improvement[M]. Biotechnology in Agriculture and Forestry,1986:31-48.
[32]董高峰,黄涛,李耿光,张兰英.成年沙田柚树试管嫁接及微繁殖的研究[J].生态科学,2001,20(3):20-25.DONG Gaofeng,HUANG Tao,LI Gengguang,ZHANG Lanying. Studies on the tube-grafting and micro-propagation of matures Shatian pomelo[J]. Ecological Science,2001,20(3):20-25.
[33]田忠景.采用茎尖嫁接法获得柑桔体细胞融合体的研究[D].成都:四川农业大学,2003.TIAN Zhongjing. Study on obtaining somatic hybrids by means of shoot-tip grafting[D]. Chengdu:Sichuan Agricultural University,2003.
[34]刘雪松.长寿花(Kalanchoe blossfeldiana V. Poelln.)试管微嫁接技术的系统研究[D].重庆:西南大学,2008.LIU Xuesong. Study on the micrografting techniques of Kalanchoe blossfeldiana V. poelln[D]. Chongqing:Southwest University,2008.
[2] CHAUHAN R,KANWAR K. Biotechnological advances in pomegranate(Punica granatum,L.)[J]. In Vitro Cellular&Developmental Biology-Plant,2012,48(6):579-594.
[3]苑兆和,尹燕雷,朱丽琴,李云,侯乐峰.石榴保健功能的研究进展[J].山东林业科技,2008,38(1):91-93.YUAN Zhaohe,YIN Yanlei,ZHU Liqin,LI Yun,HOU Lefeng.Research progress on neutraceutical functions of pomegranate[J].Shandong Forestry Science and Technology,2008,38(1):91-93.
[4]杨莉,徐昌杰,陈昆松.果树转基因研究进展与产业化展望[J].果树学报,2003,20(5):331-337.YANG Li,XU Changjie,CHEN Kunsong. Progresses in research and industrialization of transgenic fruit crops[J]. Journal of Fruit Science,2003,20(5):331-337.
[5] OMURA M,MATSUTA N,MORIGUCHI T,KOZAKI I. Adventitious shoot and plantlet formation from cultured pomegranate leaf explants[J]. Hortscience,1987,22:133-134.
[6] MORIGUCHI T,OMURA M,MATSUTA N,KOZAKI I. In vitro adventitious shoot formation from anthers of pomegranate[J].Hortscience,1987,22(5):947-948.
[7] DEEPIKA R,KANWAR K. In vitro regeneration of Punica granatum L. plants from different juvenile explants[J]. Journal of Fruit&Ornamental Plant Research,2010,18(1):5-22.
[8] KANWAR K,JOSEPH J,DEEPIKA R. Comparison of in vitro regeneration pathways in Punica granatum L.[J]. Plant Cell Tissue&Organ Culture,2010,100(2):199-207.
[9] KANWAR,RACHNA,KASHYAP A. In vitro propagation of wild pomegranate(Punica granatum L.)[M]//In:Jindal,Bawa(eds)Intellectual property rights. Shiva Offset,Dehradun,2004:209-215.
[10] AMIN M N,ISLAM M N,AZAD M A K. Regeneration of plantlets in vitro from the seedling explants of pomegranate(Punica granatum)[J]. Plant Tissue Culture,1999,9(1):53-61.
[11] SHARON M,SINHA S. Plant regeneration from cotyledonary node of Punica granatum L[J]. Indian Journal of Plant Physiology,2000,5(4):344-348.
[12] TERAKAMI S,MATSUTA N,YAMAMOTO T,SUGAYA S,GEMMA H,SOEJIMA J. Agrobacterium-mediated transformation of the dwarf pomegranate(Punica granatum L. var. nana)[J]. Plant Cell Reports,2007,26(8):1243-1251.
[13] VERMA V,KANWAR K,TUFCHI M,KASHYAP M. Agrobacterium-mediated Cry1A(b),gene transfer in Punica granatum L.cv. Kandhari Kabuli using different In Vitro,regeneration pathways[J]. Journal of Crop Science&Biotechnology,2014,17(1):1-10.
[14]李跃霞.‘突尼斯软籽’石榴遗传转化体系的构建和ISSR-PCR反应体系的优化[D].郑州:河南农业大学,2012.LI Yuexia. Construction of genetic transformation system and optimization of ISSR-PCR system of'Tunisia Soft-seed'pomegranate[D]. Zhengzhou:Henan Agricultural University,2012.
[15]赵玉洁.‘突尼斯软籽’石榴遗传转化体系建立及转化ICE1基因的研究[D].郑州:河南农业大学,2017.ZHAO Yujie. Establishment of genetic transformation system and transformed ICE1 gene into‘Tunisia Soft-seed’pomegranate[D]. Zhengzhou:Henan Agricultural University,2017.
[16]陈延惠,谭彬,李洪涛,胡青霞,叶霞,孟海军,冯建灿.石榴2种外植体再生方法在遗传转化研究中的优势比较[J].果树学报,2012,29(4):598-604.CHEN Yanhui,TAN Bin,LI Hongtao,HU Qingxia,YE Xia,MENG Haijun,FENG Jiancan. Comparison of advantages of two kinds of explant regeneration methods for studies on genetic transformation in pomegranate[J]. Journal of Fruit Science,2012,29(4):598-604.
[17]连红可.‘突尼斯软籽’石榴再生体系和GFP报告基因的瞬时表达研究初报[D].郑州:河南农业大学,2011.LIAN Hongke. Preliminary study on the regeneration system and the reporter gene GFP instantaneous expression of Soft-seed Pomegranate variety‘Tunisia’[D]. Zhengzhou:Henan Agricultural University,2011.
[18] ESTRADA-LUNA A A,LóPEZ-PERALTA C,CáRDENAS-SORIANO E. In vitro micrografting and the histology of graft union formation of selected species of prickly pear cactus(Opuntia,spp.)[J]. Scientia Horticulturae,2002,92(3):317-327.
[19]吴雅琴,章德明.用离体微嫁接法快速检测苹果潜隐病毒[J].落叶果树,2001,33(2):4-6.WU Yaqin,ZHANG Deming. Rapidly detecting apple chlorotic leaf spot virus apple stem grooving virus and apple stem pitting virus by micrografting in vitro[J]. Deciduous Fruits,2001(2):4-6.
[20]洪树荣,吴立廉.试管苗嫩枝顶端嫁接技术的研究[J].植物科学学报,1992(3):292-294.HONG Shurong,WU Lilian. Study on tip grafting in vivo by using in vitro plantlets[J]. Journal of Wuhan Botanical Research,1992,10:292-294.
[21]王国平,李晓梅,陈秋芳.核桃无根试管苗嫁接影响因素的研究[J].河北林果研究,2006,21(2):182-184.WANG Guoping,LI Xiaomei,CHEN Qiufang. Studies on the factors affecting microshoot grafting survival of walnut[J]. Hebei Journal of Forestry and Orchard Research,2006,21:182-184.
[22]李文彬.葡萄试管内外嫁接技术及其特性研究[D].兰州:甘肃农业大学,2005.LI Wenbin. Study on grape tube inside and outside grafting technique and characteristics[D]. Lanzhou:Agricultural University of Gansu,2005.
[23]郭文武.一种将柑橘离体芽苗嫁接于温室砧木的高效方法[J].果树学报,2003,20(3):242.GUO Wenwu. An efficient and simple technique to graft citrus in vitro shoots onto greenhouse rootstock seedlings[J]. Journal of fruit science,2003,20:242.
[24]郝军虹,宋涛,侯献波.赤霉素对‘突尼斯软籽’石榴种子发芽率的影响[J].中国果菜,2012(1):24-26.HAO Junhong,SONG Tao,HOU Xianbo. Effect of gibberellinon seed germination of Tunisia soft-seed Pomegranate[J]. China Fruit&Vegetable,2012(1):24-26.
[25]李洪涛,李东伟,陈海燕,胡青霞,陈延惠. GA3对突尼斯软籽石榴种子发芽的影响[J].河南农业科学,2014,43(5):154-155.LI Hongtao,LI Dongwei,CHEN Haiyan,HU Qingxia,CHEN Yanhui. Effect of GA3on seed germination of Tunisia soft-seed Pomegranate[J]. Journal of Henan Agricultural Sciences,2014,43(5):154-155.
[26] NAVARRO L,ROISTACHER C N,MURASHIGE T. Improvement of shoot-tip grafting in vitro for virus-free citrus[J]. 1992,5(5):471-479.
[27] PALMA B,VOGT G F,NEVILLE P. A combined in vitro/in vivo method for improved grafting of Acacia senegal(L.)Wild[J].Journal of Pomology&Horticultural Science,1996,71(3):379-381.
[28]宋瑞琳,吴如健,柯冲.茎尖嫁接脱除柑桔主要病原的研究[J].植物病理学报,1999,29(3):275-279.SONG Ruilin,WU Rujian,KE Chong. Elimination of the main citrus virus and virus-like diseases by shoot-tip grafting[J]. Acta Phytopathologica Sinica,1999,29(3):275-279.
[29]赵巍巍,陈蕾,曹后男,宗成文,姜淑苓.梨的试管微嫁接技术研究[J].安徽农业科学,2010,38(2):624-626.ZHAO Weiwei,CHEN Lei,CAO Hounan,ZONG Chengwen,JIANG Shuling. Study on micro-grafting in vitro of tube Pear[J].Journal of Anhui Agricultural Sciences,2010,38(2):624-626.
[30]周瑞金,杜国强,师校欣,马宝焜.影响转基因苹果试管微嫁接苗成活的因子[J].果树学报,2007,24(2):215-217.ZHOU Ruijin,DU Guoqiang,SHI Xiaoxin,MA Baokun. Studies on factors affecting survival rate of micro-graft in vitro in transgenic apple[J]. JournaI of Fruit Science,2007,24(2):215-217.
[31] JONARD R. Micrografting and its applications to tree improvement[M]. Biotechnology in Agriculture and Forestry,1986:31-48.
[32]董高峰,黄涛,李耿光,张兰英.成年沙田柚树试管嫁接及微繁殖的研究[J].生态科学,2001,20(3):20-25.DONG Gaofeng,HUANG Tao,LI Gengguang,ZHANG Lanying. Studies on the tube-grafting and micro-propagation of matures Shatian pomelo[J]. Ecological Science,2001,20(3):20-25.
[33]田忠景.采用茎尖嫁接法获得柑桔体细胞融合体的研究[D].成都:四川农业大学,2003.TIAN Zhongjing. Study on obtaining somatic hybrids by means of shoot-tip grafting[D]. Chengdu:Sichuan Agricultural University,2003.
[34]刘雪松.长寿花(Kalanchoe blossfeldiana V. Poelln.)试管微嫁接技术的系统研究[D].重庆:西南大学,2008.LIU Xuesong. Study on the micrografting techniques of Kalanchoe blossfeldiana V. poelln[D]. Chongqing:Southwest University,2008.