无菌猕猴桃种子采集方法研究
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摘要
无菌的猕猴桃种子是猕猴桃胚乳培养、实生苗微嫁接等技术的基础材料,利用消毒剂灭菌是常用的无菌种子采集手段,应用最广泛的消毒剂为升汞(mercuric chloride)和次氯酸钠(NaClO)。为了避免使用消毒剂,该研究提出了一种新的无菌猕猴桃种子采集方法——无菌搅拌法,同时为探索其准确性和应用性,比较了0.2%升汞灭菌20 min、10%次氯酸钠灭菌20 min、无菌搅拌法三种方式采集无菌猕猴桃种子的效果,并对种子萌发和幼苗形成的影响进行了研究。结果表明:无菌搅拌法、0.2%升汞灭菌20 min是稳定有效的无菌猕猴桃采集方式,10%次氯酸钠灭菌20 min的采集效果不稳定;在相同的时间内,无菌搅拌法的种子发芽率最高,为89.90%,但发芽势较低,均可正常成苗; 10%次氯酸钠灭菌20 min的发芽率次之,与无菌搅拌法的种子无显著差异,发芽势和成苗率最高,分别为47.47%和67.86%,且有打破猕猴桃种子休眠的作用,整体作用类似于赤霉素(GA_3)浸种的效果; 0.2%升汞灭菌20 min对猕猴桃种子的萌发有抑制作用,各项指标均显著低于无菌搅拌法种子,且生长缓慢。此外,无菌搅拌法是物理处理,对种子、操作人员、环境均无害。这证实了无菌搅拌法的实用性和优势,发现了次氯酸钠在打破猕猴桃种子休眠方面的作用,为其它同类型果实的无菌种子采集提供了参考。
Aseptic seeds of kiwifruit is fundamental tested material of endosperm culture,micro-grafting and other researches. It is a usual way to gather aseptic seeds to use disinfectant to sterilize. Among them,mercuric chloride and Na Cl O are the most widely used disinfectants. This article showed that aseptic stirring method is a new method to obtain aseptic seeds of kiwifruit. In order to explore accuracy and applicability of aseptic stirring method,kiwifruit seeds were used to study the effects of three methods,including 0.2% mercuric chloride sterilization for 20 min,10% Na Cl O sterilization for 20 min,aseptic stirring method,and their effects on seed germination and plant growth. The results showed that aseptic stirring method and 0.2% mercuric chloride sterilization for 20 min were an effective and reproductive way for gathering aseptic seeds of kiwifruit. Oppositely,gathering aseptic seeds of kiwifruit which in 10% NaClO sterilization for20 min was unstable. The results also showed that the three methods were effective,and at the same time,seed germination rate of aseptic stirring method was the highest. And the rate was 89.90%,but its germination energy was lower,sprouts could grow healthily,germination rate of seeds with 10% NaClO sterilization for 20 min was the second place,but there was no significant difference in germination rate of seeds between 10% Na ClO sterilization for 20 min and aseptic stirring method,its germination energy and seedling rate were the highest which were 47.47% and 67.86% respectively,and could give the seed a premature start and also could break effectively the dormancy of kiwifruit seeds like GA_3. And 0.2% mercuric chloride sterilization for 20 min inhibited germination of kiwifruit seeds,the various index was significantly lower than aseptic stirring method,sprouts grew slowly,most of the seeds could not develop into young seedlings within the allotted time. In addition,aseptic stirring method was a physical method,and it was harmless for seeds,workers and environment. The study indicated that the practicality and advantage of aseptic stirring method,and found the ability of NaClO to break the dormancy of kiwifruit seeds,which provided a reference for the obtaining aseptic seeds of other same type of fruits.
Aseptic seeds of kiwifruit is fundamental tested material of endosperm culture,micro-grafting and other researches. It is a usual way to gather aseptic seeds to use disinfectant to sterilize. Among them,mercuric chloride and Na Cl O are the most widely used disinfectants. This article showed that aseptic stirring method is a new method to obtain aseptic seeds of kiwifruit. In order to explore accuracy and applicability of aseptic stirring method,kiwifruit seeds were used to study the effects of three methods,including 0.2% mercuric chloride sterilization for 20 min,10% Na Cl O sterilization for 20 min,aseptic stirring method,and their effects on seed germination and plant growth. The results showed that aseptic stirring method and 0.2% mercuric chloride sterilization for 20 min were an effective and reproductive way for gathering aseptic seeds of kiwifruit. Oppositely,gathering aseptic seeds of kiwifruit which in 10% NaClO sterilization for20 min was unstable. The results also showed that the three methods were effective,and at the same time,seed germination rate of aseptic stirring method was the highest. And the rate was 89.90%,but its germination energy was lower,sprouts could grow healthily,germination rate of seeds with 10% NaClO sterilization for 20 min was the second place,but there was no significant difference in germination rate of seeds between 10% Na ClO sterilization for 20 min and aseptic stirring method,its germination energy and seedling rate were the highest which were 47.47% and 67.86% respectively,and could give the seed a premature start and also could break effectively the dormancy of kiwifruit seeds like GA_3. And 0.2% mercuric chloride sterilization for 20 min inhibited germination of kiwifruit seeds,the various index was significantly lower than aseptic stirring method,sprouts grew slowly,most of the seeds could not develop into young seedlings within the allotted time. In addition,aseptic stirring method was a physical method,and it was harmless for seeds,workers and environment. The study indicated that the practicality and advantage of aseptic stirring method,and found the ability of NaClO to break the dormancy of kiwifruit seeds,which provided a reference for the obtaining aseptic seeds of other same type of fruits.
引文
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WU Q,YAN Y,LIANG GL,2002.Callus induction and plant regeneration from endosperm culture of Averrhoa carambola[J].Chin J Trop Crops,23(2):54-57.[吴清,闫勇,梁国鲁,2002.红杨桃胚乳愈伤组织的诱导和三倍体植株再生[J].热带作物学报,23(2):54-57.]
WU XH,ZHANG YL,ZHOU Y,et al.,2013.Establishment of high frequency and direct regeneration system from leaf of‘Hayward’kiwifruit[Actinidia deliciosa(A.Chev.)C.F.Liang et A.R.Ferguson][J].Plant Physiol J,49(8):759-763.[吴秀华,张艳玲,周月,等,2013.‘海沃德’猕猴桃叶片高频直接再生体系的建立[J].植物生理学报,49(8):759-763.]
YE KY,LI JW,JIANG QS,et al.,2012.Search for proper dose of60Co-γray in Actinidia chinensis radiation breeding[J].Guihaia,32(5):694-697.[叶开玉,李洁维,蒋桥生,等,2012.猕猴桃60Co-γ射线辐射诱变育种适宜剂量的研究[J].广西植物,32(5):694-697.]
ZHANG Q,YAN Y,LIANG GL,2000.Callus induction and triploid plant regeneration from endosperm of Passiflora edulis[J].J SW Agric University,22(5):398-402.[张琴,闫勇,梁国鲁,2000.西番莲胚乳愈伤组织诱导和三倍体植株再生[J].西南农业大学学报,22(5):398-402.]
ZHAO DD,2011.Studies on endosperm culture and ploidy variations of kiwifruit[D].Hangzhou:Zhejiang University:32-34.[赵丹丹,2011.猕猴桃胚乳培养与倍性变异的研究[D].杭州:浙江大学:32-34.]
ZHENG DJ,YANG LR,YUN Y,et al.,2017.Seed dormancy mechanism and its ecological significance of endangered species Dracaena cambodiana[J].Guihaia,37(12):1551-1559.[郑道君,杨立荣,云勇,等,2017.濒危植物海南龙血树种子休眠机理及其生态学意义[J].广西植物,37(12):1551-1559.]
ZHOU LY,PAN WM,WU YY,et al.,2013.Study on morphological characteristics of tricotyledon mutant of kiwifruit[J].Guihaia,33(4):547-551.[周玲艳,潘伟明,伍宇雁,等,2013.猕猴桃三子叶突变体的形态特征研究[J].广西植物,33(4):547-551.]
ZHOU LY,QIN HM,LAI XY,et al.,2009.Establishment of regeneration system of kiwifruit seedlings[J].Guihaia,29(4):514-517.[周玲艳,秦华明,赖幸韵,等,2009.猕猴桃实生苗再生体系的建立[J].广西植物,29(4):514-517.]
ZHOU RJ,DU GQ,SHI XX,et al.,2007.Studies on factors affecting survival rate of micro-graft in vitro in trans-genic apple[J].J Fruit Sci,24(2):215-217.[周瑞金,杜国强,师校欣,等,2007.影响转基因苹果试管微嫁接苗成活的因子[J].果树学报,24(2):215-217.]
BJ?RKLUND G,DADAR M,MUTTER J,et al.,2017.The toxicology of mercury:Current research and emerging trends[J].Environ Res,159:545.
CHANG QS,ZHANG LX,LIU J,et al.,2016.Dormancy mechanism and breaking approaches of Abutilon theophrasti seeds[J].Plant Physiol J,52(6):967-974.[常青山,张利霞,刘晶,等,2016.苘麻种子休眠机理及破眠方法[J].植物生理学报,52(6):967-974.]
CHEN XJ,WU Y,LI XK,et al.,2012.Study on techniques of fast multiplication for the tissue culture of Crossostephium chinense[J].J Cent S Univ For Technol,32(7):100-104.[陈雪鹃,吴珏,李雪珂,等,2012.芙蓉菊组培快繁技术的研究[J].中南林业科技大学学报,32(7):100-104.]
HAO XY,2017.Histological study,virus movement and detection in vitro micrografting of grapevine[D].Yangling:Northwest A&F University:2-4.[郝新意,2017.葡萄试管苗微型嫁接组织学、病毒的转运及检测研究[D].杨凌:西北农林科技大学:2-4.]
HE GR,ZHANG WC,HE BZ,et al.,2017.Aseptic germination of Ormosia hosiei[J].J Fujian Agric For Univ(Nat Sci Ed),46(6):623-629.[何官榕,张文春,何碧珠,等,2017.鄂西红豆树种子的无菌萌发[J].福建农林大学学报(自然科学版),46(6):623-629.]
JIANG SJ,ZHONG Y,ZHANG F,et al.,2014.Establishment of rapid propagation for sterile seedlings of Cryptomeria fortunei by tissue culture[J].Plant Physiol J,50(7):1039-1044.[蒋时姣,钟宇,张帆,等,2014.柳杉种子无菌苗组培快繁体系的建立[J].植物生理学报,50(7):1039-1044.]
LIN F,WANG BZ,HUANG DM,et al.,2017.Management of tissue cultured plants of Dalbergia odorifera T.Chen[J].Chin J Trop Agric,37(8):26-27.[林妃,王必尊,黄东梅,等,2017.花梨木组培苗育苗管理技术[J].热带农业科学,37(8):26-27.]
LIN Y,LONG ZL,ZHANG L,et al.,2012.Optimum technological parameters for regeneration system of endosperm of Actindia chinese CV.‘JINTAO’[J].J Nucl Agric Sci,26(2):257-261.[林颖,龙自立,张璐,等,2012.猕猴桃胚乳再生植株体系的优化[J].核农学报,26(2):257-261.]
LONG ZL,2008.Study on in vitro regeneration and variation induction of Actindia[D].Hangzhou:Zhejiang University:31-33.[龙自立,2008.猕猴桃离体再生与变异诱导的研究[D].杭州:浙江大学:31-33.]
PENG XJ,WANG YQ,2002.Callus induction and plant regeneration from endosperm of loquat[J].J Sichuan Agric Univ,20(3):228-231.[彭晓军,王永清,2002.枇杷胚乳愈伤组织诱导和不定芽发生的研究[J].四川农业大学学报,20(3):228-231.]
WANG FM,LI JW,HU YK,et al.,2018.Chromosome ploidy of ten species in genus Actinidia[J].Guihaia,38(2):220-224.[王发明,李洁维,胡亚康,等,2018.猕猴桃属十个种的染色体倍性鉴定[J].广西植物,38(2):220-224.]
WANG YW,2017.Study on nutrition pot finished seedling propagation of kiwifruit in greenhouse[D].Yangling:Northwest A&F University:12-14.[王亚威,2017.猕猴桃温室营养钵成品苗繁育技术试验研究[D].杨凌:西北农林科技大学:12-14.]
WANG YY,2016,Tissue culture and rapid propagation of kiwi[D].Yangling:Northwest A&F University:22-25.[王羽悦,2016.猕猴桃组织培养快速繁育技术研究[D].杨凌:西北农林科技大学:22-25.]
WU Q,YAN Y,LIANG GL,2002.Callus induction and plant regeneration from endosperm culture of Averrhoa carambola[J].Chin J Trop Crops,23(2):54-57.[吴清,闫勇,梁国鲁,2002.红杨桃胚乳愈伤组织的诱导和三倍体植株再生[J].热带作物学报,23(2):54-57.]
WU XH,ZHANG YL,ZHOU Y,et al.,2013.Establishment of high frequency and direct regeneration system from leaf of‘Hayward’kiwifruit[Actinidia deliciosa(A.Chev.)C.F.Liang et A.R.Ferguson][J].Plant Physiol J,49(8):759-763.[吴秀华,张艳玲,周月,等,2013.‘海沃德’猕猴桃叶片高频直接再生体系的建立[J].植物生理学报,49(8):759-763.]
YE KY,LI JW,JIANG QS,et al.,2012.Search for proper dose of60Co-γray in Actinidia chinensis radiation breeding[J].Guihaia,32(5):694-697.[叶开玉,李洁维,蒋桥生,等,2012.猕猴桃60Co-γ射线辐射诱变育种适宜剂量的研究[J].广西植物,32(5):694-697.]
ZHANG Q,YAN Y,LIANG GL,2000.Callus induction and triploid plant regeneration from endosperm of Passiflora edulis[J].J SW Agric University,22(5):398-402.[张琴,闫勇,梁国鲁,2000.西番莲胚乳愈伤组织诱导和三倍体植株再生[J].西南农业大学学报,22(5):398-402.]
ZHAO DD,2011.Studies on endosperm culture and ploidy variations of kiwifruit[D].Hangzhou:Zhejiang University:32-34.[赵丹丹,2011.猕猴桃胚乳培养与倍性变异的研究[D].杭州:浙江大学:32-34.]
ZHENG DJ,YANG LR,YUN Y,et al.,2017.Seed dormancy mechanism and its ecological significance of endangered species Dracaena cambodiana[J].Guihaia,37(12):1551-1559.[郑道君,杨立荣,云勇,等,2017.濒危植物海南龙血树种子休眠机理及其生态学意义[J].广西植物,37(12):1551-1559.]
ZHOU LY,PAN WM,WU YY,et al.,2013.Study on morphological characteristics of tricotyledon mutant of kiwifruit[J].Guihaia,33(4):547-551.[周玲艳,潘伟明,伍宇雁,等,2013.猕猴桃三子叶突变体的形态特征研究[J].广西植物,33(4):547-551.]
ZHOU LY,QIN HM,LAI XY,et al.,2009.Establishment of regeneration system of kiwifruit seedlings[J].Guihaia,29(4):514-517.[周玲艳,秦华明,赖幸韵,等,2009.猕猴桃实生苗再生体系的建立[J].广西植物,29(4):514-517.]
ZHOU RJ,DU GQ,SHI XX,et al.,2007.Studies on factors affecting survival rate of micro-graft in vitro in trans-genic apple[J].J Fruit Sci,24(2):215-217.[周瑞金,杜国强,师校欣,等,2007.影响转基因苹果试管微嫁接苗成活的因子[J].果树学报,24(2):215-217.]