摘要
本论文以发根农杆菌诱导的甘薯发根为技术体系,研究在发根体系上培养甘薯茎线虫可行性,同时发展了利用该体系鉴定甘薯品种抗线虫的特性以及评价杀线剂的杀线作用等研究,探索了甘薯遗传转化体系的建立。取得了以下主要结果:
1、在研究胡萝卜、甘薯发根单寄主培养无菌线虫体系的基础上,以甘薯品种徐薯18的试管苗为材料,用发根农杆菌菌株Accc10060(携带质粒RiA4b)诱导出徐薯18发根,将甘薯茎线虫接种到发根上构建发根培养线虫体系。通过该体系对甘薯茎线虫的繁殖情况进行调查。结果表明:甘薯茎线虫在MS、MSR培养基的发根上都能生长发育和繁殖,完成其生活周期;为了方便观察,选用MSR培养基发根上培养的线虫开展研究,在26℃,9cm的培养皿中,发根上线虫的适宜接种密度为100条/皿,30d后线虫可繁殖4.3倍左右,在此体系中,线虫的卵孵化时间为4d左右。
2、用病地自然诱发鉴定高感甘薯茎线虫病品种徐薯18、栗子香和高抗品种鲁78066的发根体系接种甘薯茎线虫,6周后调查发根繁殖线虫情况及线虫侵染发根情况,然后评价它们的抗线虫特性。结果表明:接种6周后,线虫在徐薯18、栗子香和鲁78066发根上繁殖倍数分别为8.82、0.76和0.70;发根染色后观察到徐薯18有多处被线虫侵入,栗子香和鲁78066被线虫侵入位点较少;基于以上结果,鉴定徐薯18为高感甘薯甘薯茎线虫病品种,栗子香和鲁78066为高抗甘薯对线虫病品种;徐薯18和鲁78066对线虫病抗性鉴定结果和病地自然诱发鉴定结果相一致,栗子香鉴定结果不同于病地自然诱发鉴定结果,可能是因为植物对线虫的抗性机制不同;本研究鉴定结果具有体系简单、直观方便、重复性好以及不受自然环境影响等优点,进一步完善有望发展为植物对线虫病抗性鉴定新的体系。
3、进一步对徐薯18和鲁78066发根游离氨基酸组成及含量的分析表明:感病品种徐薯18发根在游离氨基酸组成上较抗病品种鲁78066多出3种游离氨基酸,它们分别是:谷氨酸、丙氨酸和苯丙氨酸;接种线虫2周后,徐薯18和鲁78066发根游离氨基酸含量均有不同程度的下降,感病品种徐薯18被线虫利用的每种游离氨基酸的量均明显多于抗病品种鲁78066;在鲁78066发根培养基中补加氨基酸试验表明:在分别补加了100mg/L丙氨酸和苯丙氨酸的MSR培养基中,其发根上接种的甘薯茎线虫繁殖数量明显增加,仅3周线虫繁殖数分别是未添加氨基酸的鲁78066发根对照的2.28和1.77倍。
4、选用生产上防治甘薯茎线虫病效果较好的杀线剂:40%甲基异柳磷乳油、50%辛硫磷和0.2%红竽宝以及植物源农药0.3%印楝素乳油,用发根体系来评价它们的杀线活性。结果表明:40%甲基异柳磷乳油、50%辛硫磷和0.2%红竽宝对甘薯茎线虫都具有很强的杀线虫活性,测定结果和田间测定结果相一致;对环境友好的植物源农药0.3%EP楝素乳油对甘薯茎线虫同样具有很强的杀线虫活性;说明用发根体系评价杀线剂对甘薯茎线虫的杀线活性是可行的。
5、为了探索通过转基因途径获得抗茎线虫病的甘薯品种的可行性,建立了甘薯品种栗子香的胚性悬浮细胞培养系,用根癌农杆菌菌株C58C1对根癌农杆菌介导的甘薯遗传转化进行了系统的研究。该菌株所携带的pCB2002H质粒上含有除草剂抗性基因Bar基因。结果表明,有效的转化方法是:用甘薯栗子香胚性细胞悬浮培养技术获得大量胚性悬浮细胞,将胚性悬浮细胞团(直径小于1.0mm)转移到MS+2,4-D2.0mg/L固体培养基上预培养一周后作为受体,侵染菌液浓度OD值为0.8,在固体MS+2,4-D2.0mg/L培养基上共培养3天,在附加100mg/L羧苄青霉素和10mg/L除草剂的MS+2,4-D2.0mg/L固体选择培养基上筛选抗性愈伤组织,选择4-6周后将除草剂抗性愈伤转移到附加100mg/L羧苄青霉素和10mg/L除草剂的MS+ABA1.0mg/L固体培养基上使体细胞胚发育成熟并发芽。用此方法转化380个细胞团,共获得了除草剂抗性愈伤组织12个,愈伤组织都陆续形成了体细胞胚,体细胞胚进一步发育成熟并发芽形成植株,为下一步的研究打下基础。
In this study,we investigated the possibility of stem nematode culture based on hairy root system using the technology of inducing hairy root by Agrobacterium rhizogens for sweet potato.Also,we developed the technology to identify the character of resistance to nematode in sweet potato and evaluate the effect of nematocide using this system.At last, we constructed the genetic transformation system of sweet potato.The main results are as follows:
Based on the study of the system of Ditylenchus destructor in monoxenic cultures on hairy roots of carrot and sweet potato,the plant in vitro of Xushu 18 was used to induce hairy roots by Agrobacterium rhizogenes strain Acccl0060 transformed with vector RiA4b.Stem nematode was established on sweet potato hairy root.Ditylenchus destructor's reproductions were investigated.The results showed that Ditylenchus destructor can develop and reproduce normally on hairy roots with MS and MSR medium,and the nematode was able to complete its life cycle.In order to observe clear, the system of stem nematode in monoxenic culture on MSR medium was used,100 nematodes per ware for 9cm Disposable cultivation was fit at 26℃,the production rate of nematode was 4.3 fold after 30 days on sweet potato hairy roots.Egg hatching was 4 days in this system.
Resistance to nematode was tested through natural infection in field for Xushul 8 and Lizhixiang which were susceptible cultivars and Lu78066 which was resistant cultivar. After inoculating nematodes on hairy root systems,the resistance to nematode for sweet potato cultivars was evaluated by observing the nematode reproduction on hairy-roots and nematode infecting root tissue in 6 weeks.The production rate of D.destructor was 8.82、0.76 and 0.70 after 6 weeks on sweet potato hairy roots Xushul8,Lizhixiang and Lu78066.Nematodes were observed at many sites in Xushul 8 root tissue,but only at few sites in Lizhixiang and Lu78066 root tissue.The results show that Xushul8 is susceptible to nematode while Lizhixiang and Lu78066 are resistant to nematode.The results of evaluating nematode resistance and evalution by natural infection in field for Xushul8 and Lu78066 are consistent,but not for Lizhixiang.It may be due to different mechanism of resistance to nematode.The testing results showed that hairy root system was a versatile,easy and reproducible system for testing nematode resistance,which will be a new system to evaluate the resistance to nematode for sweet potato cultivars under some improvement.
The compositions and contents of free amino acids in Xushul8 and Lu78066 hairy roots were also investigated.There were three free amino acids in Xushul8 which was susceptible cultivar but not in Lu78066 which was resistant cultivar,which are Glu, Ala and Phe.The contents of free amino acids in these two cultivars were all reduced in different degree after 2 weeks of inoculation nematodes,and the variances of contents of free amino acids in susceptible cultivar Xushul8 were more than those in resistance cultivar Lu78066.The test of adding amino acid to medium showed that reproduction number of Ditylenchus destructor increase distinct on Lu78066 hairy roots in addition of Ala and Phe to MSR medium than on MSR medium,the production rate of nematode was 2.28 and 1.77 after 3 weeks.
In this study,we used the nematocides such as 40%Isofenphos methyl oil,50% phoxime,0.2%Hongyubao and Botanical pesticide named 0.3%Azadirachtin oil,which were very effective in prevention and cure against Ditylenchus destructor in field.,They were assessed for nematicidal activity against Ditylenchus destructor using a hairy root-feeding method in vitro.40%Isofenphos methyl oil,50%phoxime,and 0.2% Hongyubao showed strong nematicidal activity against Ditylenchus destructor,according with assessing in field.Botanical pesticide 0.3%Azadirachtin oil which was friendly to environment showed strong nematicidal activity against Ditylenchus destructor too.The results showed that assessing nematicidal activity against D.destructor for nematocide using hairy root system is feasible.
An Agrobacterium tumefaciens-mediated transformation system of sweet potato, Ipomoea batatas(L.)Lam.,was established by using embryogenic suspension cultures of the cultivar Lizixiang.A.turnefaciens strain C58C1 harboring a vector pCB2002H with Bar gene in the present study.An effective gene transfer system was established.After 7 days subculture of embryogenic suspension cells less than lmm in size were transferred into MS solid medium with 2mg/L 2,4-dichlorophenoxyacetic acid(2,4-D)were cocultivated with C58C1(OD_(600nm)=0.8)for 3 days.After cocultivation,the infected suspension cultures were transferred into MS solid medium with 2 mg/L 2,4-D,10mg/L glufosinate and 200mg/L carbencillin for the selection culture,after 4-6 weeks these formed glufosinate-resistant embryogenic calluses formed plantlets via somatic embryogenesis.Using such gene transfer system,380 Embryogenic suspension cells were transformed and 12 glufosinate-resistant embryogenic calluses were formed.Then they all formed plantlets via somatic embryogenesis in succession,which laid the groundwork for future research.
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