农杆菌介导的厚皮甜瓜遗传转化体系的建立
S652; [目的]遗传转化是进行基因功能验证的重要手段,构建较为完善、高效的厚皮甜瓜遗传转化体系,为基因功能验证和厚皮甜瓜种质改良提供技术支撑.[方法]以厚皮甜瓜B8为材料,用携带植物双元表达载体pQY002005的根癌农杆菌介导转化B8子叶诱导再生,通过探究影响甜瓜遗传转化过程中的重要因子的作用,建立以B8为基础的甜瓜遗传转化体系.[结果]以正常光周期培养3 d的无菌苗子叶节为外植体,对其进行微刷+10 s超声处理可提高农杆菌侵染效率,荧光芽获得率达29.6％;压力85 kPa的2次5 min的抽真空侵染方式(间隔1 min)侵染效果较佳;4 mg·L-1的Basta较适宜筛选抗性植株.利...
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| Published in | 果树学报 Vol. 41; no. 3; pp. 533 - 542 |
|---|---|
| Main Authors | , , , , , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
中国农业科学院郑州果树研究所,郑州 450009
2024
三亚中国农业科学院国家南繁研究院,海南三亚 572000%中国农业科学院郑州果树研究所,郑州 450009%潍坊创科种苗有限公司,山东昌乐 262400 |
| Subjects | |
| Online Access | Get full text |
| ISSN | 1009-9980 |
| DOI | 10.13925/j.cnki.gsxb.20230399 |
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| Abstract | S652; [目的]遗传转化是进行基因功能验证的重要手段,构建较为完善、高效的厚皮甜瓜遗传转化体系,为基因功能验证和厚皮甜瓜种质改良提供技术支撑.[方法]以厚皮甜瓜B8为材料,用携带植物双元表达载体pQY002005的根癌农杆菌介导转化B8子叶诱导再生,通过探究影响甜瓜遗传转化过程中的重要因子的作用,建立以B8为基础的甜瓜遗传转化体系.[结果]以正常光周期培养3 d的无菌苗子叶节为外植体,对其进行微刷+10 s超声处理可提高农杆菌侵染效率,荧光芽获得率达29.6％;压力85 kPa的2次5 min的抽真空侵染方式(间隔1 min)侵染效果较佳;4 mg·L-1的Basta较适宜筛选抗性植株.利用以上方法,单次转化120个子叶节外植体,可获得31个再生荧光芽,17株生根苗,通过PCR检测确定8株阳性苗,阳性率达58.8％,阳性植株获得率为6.7％.[结论]成功建立了以B8为材料的甜瓜高效遗传转化体系,为甜瓜关键基因功能验证和种质精准改良提供技术支持. |
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| AbstractList | S652; [目的]遗传转化是进行基因功能验证的重要手段,构建较为完善、高效的厚皮甜瓜遗传转化体系,为基因功能验证和厚皮甜瓜种质改良提供技术支撑.[方法]以厚皮甜瓜B8为材料,用携带植物双元表达载体pQY002005的根癌农杆菌介导转化B8子叶诱导再生,通过探究影响甜瓜遗传转化过程中的重要因子的作用,建立以B8为基础的甜瓜遗传转化体系.[结果]以正常光周期培养3 d的无菌苗子叶节为外植体,对其进行微刷+10 s超声处理可提高农杆菌侵染效率,荧光芽获得率达29.6％;压力85 kPa的2次5 min的抽真空侵染方式(间隔1 min)侵染效果较佳;4 mg·L-1的Basta较适宜筛选抗性植株.利用以上方法,单次转化120个子叶节外植体,可获得31个再生荧光芽,17株生根苗,通过PCR检测确定8株阳性苗,阳性率达58.8％,阳性植株获得率为6.7％.[结论]成功建立了以B8为材料的甜瓜高效遗传转化体系,为甜瓜关键基因功能验证和种质精准改良提供技术支持. |
| Abstract_FL | [Objective]Melon(Cucumis melo L.)is one of the world's top ten fresh fruits and is loved by consumers all over the world.With the rapid development of biotechnology,plant breeding technolo-gy is changing from domestication breeding,hybrid breeding and molecular marker-assisted selection to artificial intelligence breeding relying on transgenic technology.Genetic transformation has been an important method for gene function verification.At present,the genetic transformation system of melon is not perfect,and the genetic transformation methods and efficiency between different melon geno-types or materials vary differently.To construct a genetic transformation system for muskmelon with a strong stability,good reproducibility and high efficiency,the present experiment was carried out,so as to provide technical support and theoretical basis for the verification of gene function and the improve-ment of germplasm resources.[Methods]In this study,the binary expressed vector pQY002005-GFP(Green Fluorescent Protein)was used to infect the explant from the cotyledons of B8 genotype(C.melo L.subsp.melo),and all explants that infected the Agrobacterium were used to induce buds regeneration.Key factors affecting the whole genetic transformation process,including the culture of seedlings,seed-lings ages,treatment of explants,infection mode and positive buds screening way,were explored to es-tablish the genetic transformation system based on B8.[Results]The explants were first cultured dark-ly and then treated by photoculture,which was not suitable for transformation.Fluorescent buds could be obtained by dark culture treatment alone,but with D2L0,D3L0 and D4L0 treatments,the metamor-phosis rate of fluorescent buds reached 42.9％,61.5％and 66.7％,respectively,which was significantly higher than that of other treatments and was not suitable for transformation.With D1L0 and D0L3 treat-ments,the fluorescent bud acquisition rate was relatively high up to 25.6％and 26.7％,respectively,and the fluorescent bud metamorphosis rate was relatively low.The above results showed that the longer the dark culture time,the higher the metamorphosis rate of regenerated buds.D0L3 or D1L0 was a suitable sterile seedling culture method under normal photoperiod.When the seedling age was consistent,the number of bud bushes,bud fluorescence rate and fluorescence bud rate in the non-invasive treatment group were significantly lower than those in other groups,indicating that trauma could promote Agro-bacterium infection and increase the number of adventitious buds.The fluorescence bud rate increased significantly by ultrasound and microbrush+sonication treatment of D1L0 explants,but there were no significant differences in the number of bud bushes and fluorescent bud rates of 1.6,18.4％,1.8 and 23.3％,respectively,indicating that although microbrushing increased the trauma area of plants and pro-moted the infection of Agrobacterium infection,it may not improve the fluorescence bud rate due to cell damage.The number of bud plexuses of D0L3 were significantly higher than D1L0,so the best treat-ment for explants was microbrush+sonication.Furthermore,the degree of infection was high after 25 min immersion,but the number of bud plexes,the fluorescence rate of the bud plexus and the fluores-cent bud rate were significantly lower than those of the vacuum pump 85 kPa for 5 min and the vacuum pump 85 kPa for 5 min twice with an interval of 1 min.While using the needle vacuum infection for 30 s,Agrobacterium could reach the deep cells,but the number of bud bushes and the acquisition rate of fluo-rescent buds were the lowest,being only 1.4 and 4.3％,which was significantly lower than that of the vacuum pump 85 kPa for 5 min,so the vacuum of the needle for 30 s was not suitable for infection.In comparison,vacuum pump 85 kPa for 5 min twice with an interval of 1 min treatment had the highest(64.8％)fluorescent bud rate,and the fluorescence area of explants was also greater than that of one vac-uum treatment.Besides,the effect of Basta on budding status was observed in order to obtain a more suitable concentration.The analysis showed that without adding Basta,B8 had strong budding ability.When the concentration of Basta was 2 mg·L-1,the germination time was later and the budding amount was less than that without Basta,but the germination rate was still high up to 78.7％,so 2 mg·L-1 Basta could not strongly inhibit negative bud plexes.When the concentration of Basta was 4 mg·L-1,the bud-ding rate of explants was late,the number of bud bushes was small,and the budding rate was 14.3％,which was lower than that of 2 mg·L-1 treatment,indicating that the addition of 4 mg·L-1 Basta could play a role in screening resistant adventitious buds.When the Basta concentration was much more than 6 mg·L-1,the effloration rate of explants was 5.5％lower,and it even caused death of explants.The above results showed that 4 mg·L-1 of Basta was a suitable concentration for screening resistant buds.[Conclusion]The results revealed that cotyledons from sterile seedling cultured for 3 days(under light condition)with microbrush and 10 seconds ultrasonic treatment,could improve the efficiency of Agro-bacterium infection.The acquisition rate of fluorescence bud was 26.2％;the best infection system was vacuumed for 5 min twice with an interval of 1 min under the pressure of 85 kPa.The suitable concen-tration for screening resistant buds was 4 mg·L-1 Basta.Thirty-one regenerated fluorescent buds and 17 rooting seedlings were obtained in a single transformation of 120 explants,and 8 positive seedlings were identified by PCR reaction,with a positive transformation rate and seedlings rate of 58.8％and 6.7％,respectively.This study successfully established a relatively complete melon genetic transforma-tion system on B8,which provided technical support and theoretical basis for key gene function verifi-cation and germplasm improvement. |
| Author | 贺玉花 唐伶俐 李文东 田小琴 赵光伟 孔维虎 徐龙兰 张健 徐永阳 |
| AuthorAffiliation | 中国农业科学院郑州果树研究所,郑州 450009;三亚中国农业科学院国家南繁研究院,海南三亚 572000%中国农业科学院郑州果树研究所,郑州 450009%潍坊创科种苗有限公司,山东昌乐 262400 |
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| Author_FL | XU Yongyang ZHANG Jian ZHAO Guangwei XU Longlan KONG Weihu LI Wendong TIAN Xiaoqin TANG Lingli HE Yuhua |
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| DocumentTitle_FL | Establishment of genetic transformation system mediated by Agrobacteri-um in muskmelon |
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| Keywords | Muskmelon Genetic transformation Seedling age Resistant bud selec-tion 遗传转化 抗性芽筛选 Infection pathway 厚皮甜瓜 侵染方式 苗龄 |
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| Title | 农杆菌介导的厚皮甜瓜遗传转化体系的建立 |
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