收藏本站
收藏 | 手机打开
二维码
手机客户端打开本文

Agrobacterium Tumefaciens-Mediated Transformation in Citrus and Characterization of Transgenic Regenerants

EHSAN ULLAH KHAN  
【摘要】:Citrus is one of the most widely grown and economically important fruit crops in the world. Development of an efficient direct regeneration system from leaf explants of citrus has been attempted in this study. The effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Shoot regeneration medium consisted of MT salts plus 0.5 mg/L BA,0.5 mg/L Kinetin,0.1 mg/L NAA,3% sucrose and 0.8% agar, pH 5.8, proved to be the most effective medium for direct induction of shoots from leaf explants among the three media tested. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants excised from fully developed leaves resulted in better shoot regeneration capacity compared to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and RAPD analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data generated in this study revealed that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future. Attempts were made to develop an efficient Agrobacterium tumefaciens-mediated transformation system to produce transgenic Valencia sweet orange (Citrus sinensis L. Osbeck) plants from leaf discs via direct organogenesis in the current study. Various factors affecting T-DNA delivery including explant age, Agrobacterium concentration, immersion time, inoculation and co-cultivation media, co-cultivation period and temperature, kanamycin, acetosyringone and antibiotic concentrations were optimized based on GFP gene expression and recovery of kanamycin resistant shoots following cocultivation as indicators. Shoots regenerated on medium containing MT salts supplemented with 0.1 mg/L NAA,0.5 mg/L BA and 0.5 mg/L kinetin arose directly from leaf margins in the form of protuberances without any callus intervening phase. Leaf explants from 3 months old in vitro seedlings had positive effects on transformation efficiency. Infection in Agrobacterium suspension of OD600:0.6 for 10 min and co-culturing on medium containing 25 mg/L acetosyringone for 3 days at 25℃yielded efficient transient GFP expression, shoots regeneration response and transformation efficiency. The transformation efficiency (12.33%) and mean number of transformed shoots per explant (5.4) were obtained after transfer of 6-8 weeks on selection medium (MT salts augmented with 0.5 mg/L BA,0.5 mg/L Kinetin,0.1 mg/L NAA,50 mg/L kanamycin and 250 mg/L cefotaxime). Integration of GFP gene in transgenic plants was confirmed by PCR analysis. Flow cytometry analysis revealed that all the transgenic plants produced in this system were of diploid nature and morphologically identical with their donor plants. This protocol in the present study is the first of its kind ever developed to transform and regenerate citrus plants using leaf direct regeneration system. Development of this highly efficient regeneration transformation protocol paved the way for generating a T-DNA mutant collection of citrus that will in turn significantly impacts functional genomic research and gene discovery in Rutaceae and other fruit crops. Cold is one of the most important environmental factors that adversely affect the growth and productivity of citrus plants. In an effort to induce cold tolerance in a citrus cultivar'GWZ'(Gailiangcheng orange x Weizhang Satsuma mandarin), PtrICE gene was introduced in cell suspensions cultures through transformation mediated by Agrobacterium. After about 8 weeks of transfer into selection medium (MT salts+50 mg/L kanamycin+400 mg/L cefatoxime), whitish kanamycin resistance calli formed from transformed cells, whereas, non-transformed cells grew slowly in the form of dead brownish layer. Six kanamycin-resistant cell lines were isolated and proliferated. The integration of transgene in all cell lines was confirmed by PCR, and RT-PCR analysis. In comparison to the non-transformed cell lines, the transformed ones exhibited a higher level of antioxidant enzymes (SOD, POD, and CAT) activities under stress conditions, which enabled them to inhibit the production of ROS. In addition, the viability of transformed cell lines determined via TTC reduction assay and Evans Blus staining method was more pronounced compared to non-transformed ones in this study. Over-expression of PtrICE gene reduced electrolyte lakage levels (EL), Malondialdehyde (MDA) contents and H2O2 accumulation in transgenic cell lines in all stress treatments. Transgenic cell lines exhibited enhanced tolerance under cold stress is being used for regeneration and future breeding purposes. Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature. In this work, TfGA2-ox2 gene was introduced into callus lines of 'Bingtangcheng'Sweet orange(Citrus sinensis L. Osbeck) by Agrobacterium-mediated transformation with the aim to decrease the amounts of bioactive GA levels in these plants and thereby reducing their stature. To optimize the transformation conditions, several factors were assessed, including co-cultivation period, duration of pre-culture in darkness and the infection time of Agrobacterium tumefaciens. PCR analysis confirmed the successful integration of TfGA2-ox2 gene in kanamycin-resistant calluses. The average percentage of kanamycin-resistant calli on selection medium was 11.71%. Maximum transformation efficiency of resistant callus lines based on polymerase chain reaction detection was 69.23%. This approach may provide an alternative to the application of chemical growth retardants used for reducing the stature of plants.


知网文化
【相似文献】
中国期刊全文数据库 前20条
1 梁容;;秘鲁:三月至九月出口柑桔十三万吨[J];中国果业信息;2017年04期
2 LI Fang;DAI Su-ming;DENG Zi-niu;LI Da-zhi;LONG Gui-you;LI Na;LI Yi;Alexandra Gentile;;Evaluation of parameters affecting Agrobacterium-mediated transient expression in citrus[J];Journal of Integrative Agriculture;2017年03期
3 GUAN Xin;TAN Si;Günther Buchholz;Peter Nick;ZHOU Zhi-qin;;A method to evaluate the bioactive function of fruit extracts of Chinese wild Citrus with microtubular activity[J];Journal of Integrative Agriculture;2017年04期
4 ;小柠檬大妙用[J];时代英语(高三);2017年02期
5 ;Preliminary Studies on CPG/Hinf Ⅰ RFLP Groups of Citrus tristeza virus Infected Sweet Oranges in China[J];Agricultural Sciences in China;2006年01期
6 ;Cloning and Characterization of a Novel cDNA Encoding Late Embryogenesis-Abundant Protein 5 Like (LEA-5) Gene from Cara Cara Navel Orange Fruit (Citrus sinensis Osbeck)[J];Agricultural Sciences in China;2006年04期
7 ;Physiological Mechanism on the Difference of Sugar Accumulation in Four Citrus Varieties[J];Agricultural Sciences in China;2003年12期
8 李思经;;柑桔砧木的改良[J];农业科技通讯;1988年06期
9 胡柳伯;切制香圆也会灼伤皮肤[J];中国中药杂志;1989年01期
10 徐建国;试论“橘逾淮而北为枳”之“枳”[J];中国农史;1989年01期
11 P Panigrahi;A K Srivastava;;Water and nutrient management effects on water use and yield of drip irrigated citrus in vertisol under a sub-humid region[J];Journal of Integrative Agriculture;2017年05期
12 LI Ding-li;XIAO Xuan;GUO Wen-wu;;Production of Transgenic Anliucheng Sweet Orange(Citrus sinensis Osbeck) with Xa21 Gene for Potential Canker Resistance[J];Journal of Integrative Agriculture;2014年11期
13 ;Study on the Role in Scavenging Reactive Oxygen Free Radicals of Citrus Flavonoides in Vitro[J];Medicinal Plant;2010年07期
14 ;Preliminary Studies on Species and Distribution of Citrus Viroids in China[J];Agricultural Sciences in China;2008年09期
15 Derong YANG;Zhiwei ZENG;Long ZHOU;Jinping LI;Chen XU;Lingxiang ZHOU;Fang WANG;;Identification and Control of HLB Disease in Citrus grandis[J];Asian Agricultural Research;2019年03期
16 ;Genetic Diversity and Global Distribution of Citrus tristeza virus (CTV) Strains[J];Journal of Northeast Agricultural University(English Edition);2012年02期
17 ;Construction and Evaluation of the Theoretical Model of Citrus Cooperative Organization[J];Asian Agricultural Research;2010年11期
18 ;Analysis of SSR in Citrus Sequences from EMBL Database[J];Agricultural Sciences in China;2005年07期
19 ;In vitro Plant Regeneration from the Mature Tissue of Navel Orange (Citrus sinensis L. Osbeck) by Direct Organogenesis[J];Agricultural Sciences in China;2005年03期
20 Dirceu Mattos Jr;Franz W R Hippler;Rodrigo M Boaretto;Eduardo S Stuchi;José A Quaggio;;Soil boron fertilization: The role of nutrient sources and rootstocks in citrus production[J];Journal of Integrative Agriculture;2017年07期
中国重要会议论文全文数据库 前10条
1 MEI Peng-ying;ZHOU Chang-yong;;NADPH oxidase might negatively feedback regulate the generation of H_2O_2 in citrus[A];中国植物病理学会2015年学术年会论文集[C];2015年
2 ;Molecular Identification of Seven Citrus Viroids from China[A];中国植物病理学会2009年学术年会论文集[C];2009年
3 ;Establishment of transformation system for precocious trifoliate orange and seedless molecular breeding in Citrus[A];第六届中国植物逆境生理学与分子生物学学术研讨会论文摘要汇编[C];2010年
4 ;Genome-wide analysis of the polyamine oxidase gene family in Citrus sinensis[A];从植物科学到农业发展——2012全国植物生物学大会论文集[C];2012年
5 Manosh Kumar Biswas;XU Qiang;ZHANG Xian-long;DENG Xiu-xin;;Microsatellite Markers in Citrus sinensis:Development,Characterization and Utility in Other Relatives[A];中国园艺学会2012年学术年会论文摘要集[C];2012年
6 ;Isolation of subprotoplasts via ultracentrifugation and creation of hybrid callus via cytoplast-protoplast fusion in Citrus[A];全国“植物生物技术及其产业化”研讨会论文摘要集[C];2007年
7 刘继红;;Citrus Germplasm Enhancement via Cell Engineering and Gene Engineering[A];2008年园艺植物染色体倍性操作与遗传改良学术研讨会论文摘要集[C];2008年
8 ;Citrus Germplasm Enhancement Via Cell Engineering and Genetic Engineering[A];2008园艺学进展(第八辑)——中国园艺学会第八届青年学术讨论会暨现代园艺论坛论文集[C];2008年
9 Shahzad Munir;Li Yongmei;He Pengfei;Wu Yixin;He Pengjie;Cui Wenyan;何月秋;;Different strategies to properly manage the citrus huanglongbing in China[A];中国植物病理学会2018年学术年会论文集[C];2018年
10 ;Characterization of a new natural graft chimera of citrus 'Zaohong navel orange' with potential for the industry[A];湖北省遗传学会、江西省遗传学会2006年学术年会暨学术讨论会论文摘要集[C];2006年
中国博士学位论文全文数据库 前10条
1 EHSAN ULLAH KHAN;[D];华中农业大学;2010年
2 卡罗斯(JUAN CARLOS CORONA SEGURA);委内瑞纳和中国江西省柑橘鲜果需求的比较分析[D];华中农业大学;2013年
3 Manosh Kumar Biswas;[D];华中农业大学;2010年
4 Matolo Luyolo;南非与其他金砖国家的柑橘贸易研究[D];中国农业科学院;2016年
5 TOUQEER AHMAD;[D];华中农业大学;2013年
6 Chofong Gilbert Nchongboh;[D];华中农业大学;2014年
7 M.N.R.Baig;[D];华中农业大学;2009年
8 Sagheer Atta;[D];西南大学;2011年
9 Elsayed Mohamed(Elsayed Nishawy Saleh);利用转基因番茄验证柚转录因子CgDREB对果实成熟性状的影响[D];华中农业大学;2014年
10 赵雪梅;胡柚皮化学成分及其活性研究[D];浙江大学;2003年
中国硕士学位论文全文数据库 前10条
1 熊光明;应用AFLP分子标记对柑橘属(Citrus)植物进行鉴别与系统分类研究[D];西南农业大学;2002年
2 周春丽;海藻糖合酶基因转化佛手(Citrus medica L.var.sarcodactylis)方法的研究[D];西北农林科技大学;2005年
3 艾米娜(AMINA SALMA);[D];华中农业大学;2016年
4 金帅;脐橙(Citrus sinensis Osbeck)果实油斑病的代谢组学分析[D];华中农业大学;2013年
5 刘萍;发汗和热处理改善温州蜜柑(Citrus unshiu Marc)果实贮藏特性的机理[D];华中农业大学;2010年
6 杨丽;代代花化学成分的研究[D];华南理工大学;2010年
7 Chen Kaloantsimo Sarah;马达加斯加Amber山国家保护区的植被多样性与恢复[D];北京林业大学;2011年
8 吴方方;打蜡对温州蜜柑(Citrus unshiu Marc)果实异味物质积累的影响[D];华中农业大学;2010年
9 黄训才;湖南主要柑桔病毒病的分子检测及其毒系分型研究[D];湖南农业大学;2006年
10 KEITA OUALY;[D];华中师范大学;2018年
中国重要报纸全文数据库 前10条
1 《网络世界》记者 周源;思杰:25年的坚持与创新[N];网络世界;2014年
2 本报记者 晓瑗;Orange:连接从细处做大[N];人民邮电;2018年
3 本报记者 晓瑗;Orange初尝银行业务甜头[N];人民邮电;2019年
4 本报记者 梓茎;运营商开银行是要闹哪样?[N];人民邮电;2017年
5 本报记者 晓瑗;移动金融 运营商不差钱 差什么?[N];人民邮电;2017年
6 本报记者 晓镜;运营商拼内容 拼分发还是自创?[N];人民邮电;2017年
7 本报记者 高超 赵妍 崔亮亮 党博文;全球主要运营商半年业绩解读[N];通信产业报;2017年
8 赵媛;Orange:为云服务注入电信基因[N];人民邮电;2014年
9 关文;Orange云计算成为“征服2015”重要支柱[N];人民邮电;2013年
10 李智鹏;Orange与微软扩大统一通信合作[N];计算机世界;2009年
 快捷付款方式  订购知网充值卡  订购热线  帮助中心
  • 400-819-9993
  • 010-62982499
  • 010-62783978