香樟胚性愈伤组织遗传转化体系建立

通过根癌农杆菌介导的遗传转化方法,建立香樟胚性愈伤组织遗传转化体系.结果表明:以胚性愈伤组织为受体,能取得比较理想的转化效果;50 mg·L-1的潮霉素对胚性愈伤组织有很好的筛选效果;根癌农杆菌OD600为0.6时,能获得较高的转化率;侵染时间提高为40min时,对提高转化效率有显著的效果;共培养3 d能够得到较好的转化效率.对转基因香樟愈伤组织进行PER检测,结果表明GUS基因已整合到香樟胚性愈伤组织的基因组中.     

农杆菌介导慈竹4CL基因遗传转化梁山慈竹

以梁山慈竹2种类型成熟胚的愈伤组织为材料,采用农杆菌遗传介导的方法,将已构建好的具有降低木质素含量的PBI121-4CL-RNAi表达载体导入愈伤组织,探讨愈伤组织预培养时间、菌液浓度、侵染时间、共培养时间和温度对遗传转化的影响。研究结果表明,淡黄色、颗粒状、疏松易碎的胚性愈伤组织是较好的遗传转化材料。以在愈伤组织培养基上预培养8天的淡黄色、颗粒状、疏松易碎的胚性愈伤组织为转化受体,在菌液浓度为OD600=0.05的EHA105中侵染20min后,在25℃、黑暗条件下共培养2天(共培养基表面加一层无菌滤纸),在含有卡那霉素为55mg.L-1的抗性筛选培养基上筛选30天,抗性愈伤组织率为90%,经PCR检测,慈竹4CL基因已导入梁山慈竹愈伤组织中。抗性愈伤组织在芽诱导培养基上诱导30天,可获得丛生芽,待丛生芽长至3～5cm后,在生根培养基上经过20～30天的诱导,可产生1～8条根,获得再生植株。经PCR检测,慈竹4CL基因已导入梁山慈竹再生植株中,获得了转基因植株,转化效率为9%。RT-PCR检测结果表明,转4CL基因的梁山慈竹愈伤组织和植株的内源4CL基因的表达受到抑制,且表达量比对照明显降低。     

根癌农杆菌介导Caz基因转化烟草条件研究

利用根癌农杆菌介导Caz基因转化烟草,将外源基因导入烟草愈伤组织,建成可诱导表达细胞质和细胞核CaM/CaM结合多肽的转基因烟草．研究结果表明:植物材料的选择和培养、农杆菌的培养和稀释、植物材料与农杆菌共培养的程度及转基因植株的筛选条件是影响农杆菌转化效率的重要因素．     

马尾松幼苗茎段愈伤组织诱导的研究

以马尾松优良种子萌发后的幼苗的茎段为实验材料,研究了不同培养基类型、激素种类和激素质量浓度对愈伤组织诱导的影响.结果表明:改良GD培养基为诱导愈伤组织的最佳诱导培养基;改良GD+4 mg·L-12,4-D+2 mg·L-16-BA+500 mg·L-1CH+30 g·L-1蔗糖+8 g·L-1琼脂的组合为愈伤组织最佳增殖配方;暗堵养20 d后平均直径能达到8.5 mm.这为马尾松幼苗茎段愈伤组织分化或进行遗传转化奠定了研究基础.     

华北落叶松胚性愈伤组织诱导影响因子的研究

以成熟合子胚为外植体,用完全随机、正交设计等试验方法加之组培技术来研究影响华北落叶松(Larix princi-pis-rupprechtii)胚性愈伤组织诱导的几种主要因子。结果表明:诱导胚性愈伤组织过程中的最佳灭菌方式为2%Na-ClO灭菌30 min,剥皮、去胚乳处理。其中污染率和NaClO的灭菌时间呈负相关:NaClO灭菌时间越长,污染率越低。诱导胚性愈伤组织的最佳琼脂浓度为4 g·L-1,且过高和过低的琼脂浓度不仅降低了其诱导率,提高了愈伤组织的褐化水平,也影响了外植体的发育形态。外源激素的正交试验结果表明:2,4-D是主要影响因子,对诱导率的影响显著,其次是KT,最后是6-BA。最佳的植物外源激素配比是2,4-D 1.8 mg·L-1+6-BA 0.3 mg·L-1+KT 0.3 mg·L-1。     

油茶优良无性系叶片愈伤组织诱导研究

以油茶优良无性系湘林1号和湘林4号为材料,采用正交设计试验对其叶片进行离体培养并诱导形成愈伤组织.试验结果表明:激素种类与浓度组合对愈伤组织诱导影响很大,且两个品种表现不一致,湘林1号以IBA 0.01 mg·L-1+KT 0.5 mg·L-1+2,4-D 0.5 mg·L-1+NAA 2.0 mg·L-1最佳,湘林4号以IBA 0.1 mg·L-1+6-BA 0.5 mg·L-1+KT 1.0 mg·L-1+NAA 2.0 mg·L-1最佳,诱导率均达100%.低温预处理5～10 d有助于愈伤组织的诱导,不同光照条件以及叶片不同部位均对愈伤组织的诱导有影响.     

中国水仙花器官愈伤组织诱导、分化及组织学观察

为了建立中国水仙遗传转化体系,为转基因分子育种提供良好受体,以中国水仙‘金盏银台’品种的不同花器官为实验材料,以MS为基本培养基,研究了花药、花梗、子房、花柄愈伤组织诱导情况.对愈伤组织诱导效果较好的花梗和花药培养基中添加不同浓度6-苄基腺嘌呤(6-BA)、硫酸腺嘌呤(Ad)、萘乙酸(NAA)和2,4-二氯苯氧乙酸(2,4-D),筛选出愈伤组织诱导及鳞茎芽再生的最适培养基配方.结果表明:花梗愈伤组织诱导及鳞茎芽分化的最佳培养基为MS+1 g·L-1NaH2P04+0.003 g· L-16-BA+0.001 g· L-1NAA+0.2 g·L-1 Ad+30g· L-1蔗糖,花药愈伤组织诱导及鳞茎芽分化的最佳培养基为MS +0.002 g·L-16-BA +0.001 g·L-1 NAA +30 g·L-1蔗糖;花梗愈伤组织诱导到鳞茎芽分化的时间需要30～35 d,而花药这一过程为80～90 d.对花梗愈伤组织诱导及鳞茎芽分化过程进行组织学观察表明:本研究中产生的小鳞茎是由愈伤组织再分化得来的,表明花梗更有利于作为转基因受体,从而解决了转基因研究过程的一个瓶颈问题.     

地被菊间接体细胞胚发生途径的转基因受体体系的研究

采用三步培养法建立了地被菊花品种‘玉人面'间接体细胞胚发生途径的转基因受体体系.试验以地被菊花品种‘玉人面'茎段(节间)为外植体,研究了生长调节剂、光照强度等因子对其间接体细胞胚诱导和分化的影响,同时进行了抗生素敏感性试验.结果表明:胚性愈伤组织诱导培养基为MS KT 2.0 mg·L-1 2,4-D 2.0 mg·L-1 NAA 0.5 mg·L-1,诱导15 d后,将获得的黄绿色致密颗粒状胚性愈伤组织转入胚性愈伤组织分化培养基MS KT 2.0 mg·L-1 2,4-D 1.0 mg·L-1 NAA 0.5 mg·L-1中,诱导15 d后再转入MS KT 2.0 mg·L-1 NAA 0.5 mg·L-1的分化培养基中培养20 d,光照强度为1 000～2 000 lx,胚性愈伤组织诱导率、分化率分别为95.3%、92.7%,平均每个外植体分化的芽数为17.8个,获得的再生芽的稳定性为99.5%.抗生素敏感性试验表明:地被菊花品种‘玉人面'间接体细胞胚发生途径的转基因受体体系中,卡那霉素选择压为20 mg·L-1;头孢霉素在胚性愈伤组织诱导时的选择压为300 mg·L-1,在胚性愈伤组织分化培养时选择压为100 mg·L-1.     

热激启动子控制的FT基因诱导杨树早期开花体系的优化

为实现杨树早期开花,缩短育种周期,利用农杆菌介导的转化方法,探讨影响热激启动子控制的FT1基因转化白杨派杂种无性系的因素,优化其转化条件,并实现FT1基因的转化,采用热激诱导方法诱导2～3个月大的转基因植株早期开花。结果表明:叶盘转化前的预培养、菌液浓度、侵染时间及共培养时间对卡那霉素抗性植株再生率的影响均达到极显著水平;叶盘在愈伤组织诱导培养基上暗培养6天,然后用活化至OD600=0.5的菌液侵染60min,再在愈伤组织诱导培养基上共培养2天,该条件下FT1基因转化白杨派杂种的卡那霉素抗性植株再生率可达29.84%;37℃每天1h持续热激3周可使FT1基因顺利表达,促进转基因植株开花;热激植株大小是影响转基因植株热激诱导开花的限制因素,低于20cm的植株不能诱导开花;热激诱导可产生相对较多的正常花器,但花器变异仍然十分广泛。     

农杆菌介导的楸树遗传转化体系

【目的】以楸树胚性愈伤组织为受体,建立有效的楸树遗传转化体系,为今后楸树性状的遗传改良奠定基础。【方法】通过农杆菌EHA105介导以胚性愈伤组织作为外植体进行遗传转化,通过正交试验获得最优的遗传转化条件,进而将外源基因转入到楸树基因组中。【结果】在1/2 MS培养基中添加不同梯度浓度的卡那霉素(Kana)进行选择压力筛选,在添加了60 mg·L~(-1)Kana的1/2 MS培养基中,楸树胚性愈伤组织的分化率为0.00%,存活率仅为5.71%,因此确定60 mg·L~(-1)为遗传转化的选择压。采用正交设计L18(37)进行农杆菌介导的楸树遗传转化试验,通过GUS化学组织染色统计瞬时表达率,正交试验直观分析和单因素方差分析结果表明:在预培养时间为2天,采用农杆菌菌株EHA105、菌液浓度OD600值为0.7、添加乙酰丁香酮(AS)浓度为300μmol·L~(-1)、侵染时间为10 min,共培养时间为5天的条件下,农杆菌介导的转化效率最高,且对转化效率影响最大的2个因素是乙酰丁香酮浓度和预培养时间。对浸染后的胚性愈伤组织进行8个月的筛选培养,共获得32个抗性组织团,对其中15个增殖较多的抗性愈伤组织进行PCR检测,表明86.67%的抗性组织团中有外源基因整合到楸树基因组中。内源激素水平会对植物体细胞胚分化产生影响,细胞分裂素(CTK)和脱落酸(ABA)促进体胚发生,生长素(IAA)和赤霉素(GA)对体胚发生有抑制作用。通过测定内源激素可知,转基因的抗性组织中内源CTK和ABA水平显著低于野生型的楸树胚性愈伤组织,而内源IAA和GA则显著高于野生型胚性愈伤组织,推测内源激素水平可能是转基因抗性组织体胚分化能力比较差的原因。【结论】建立了农杆菌介导的楸树胚性愈伤组织的遗传转化体系,对筛选获得的15个抗性愈伤组织进行PCR检测,其中13个抗性愈伤组织中有外源基因的整合。内源激素水平的变化可能是导致楸树转基因抗性愈伤组织难以分化的原因。     

Establishment of a transgenic system in fast-growing black locust (Robinia pseudoacacia L.)

The AhDREB1 gene, cloned from Atriplex hortensis L., was transferred into black locust (Robinia pseudoacacia L.) by an Agrobacterium-mediated transformation. The results suggest that stems of black locust sub-cultured in vitro for 20 d are suitable for genetic transformation. The optimum concentrations of kanamycin and cefotaxime were 30 and 150 mg.L-1, respectively. Impor-tant factors affecting the transformation efficiency were studied by means of a L9(34) orthogonal design. An effective system for ge-netic transformation in black locust was developed as follows: the stems were pre-cultured for 2 d, immersed in the Agrobacterium solution (OD6oo = 0.7) with 10 mg'L-1 acetosyringone for 21 min and then co-cultured for 2 d. The selection pressures, changing from low to high, could improve transformation efficiency. The transgenic plants were identified by a PCR method. The PCR results indicated that the AhDREB1 gene had been integrated into the genome of black locust and two lines of the transgenic plants were obtained.     

Improvement in salt tolerance of Populus tomentosa from transformation by a mtl-D gene

Transgenic lines were achieved by transforming the E. coli 1-phosphate mannitol dehydrogenase gene (mtl-D) into the Populus tomentosa Carr. genome. An Agrobacterium tumefaciens strain (AGL1), constructed by cloning mtl-D into the disarmed plasmid pBin438, was used to infect leaves of the clone YW2. The infected leaf discs were cultured on a medium containing 30 mg·L-1 kanamycin and 500 mg·L-1 cefotaxime. Transgenic plantlets regenerated from the infected leaves, rooted on the medium containing 30 mg·L-1 kanamycin. PCR and a Southern blotting test verified that the exogenous mtl-D gene had integrated into the transformation plants of the P. tomentosa genome. The mannitol content in control plant was 69μg·g-1 FW, and the mannitol contents of the transgenic lines T1 to T5 ranged between 103.7 and 289.5μg·g-1 FW. Of the shoots of the control plants 20% survived; on the medium containing 0.6% NaCl, 60% and 70% of two transgenic shoots survived on a medium containing 0.8% NaCl.     

Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene

Valencia sweet orange (Citrus sinensis (L.) Osbeck) calluses were used as explants to develop a new transformation system for citrus mediated by Agrobacterium tumefaciens. Factors affecting Agrobacterium-mediated transformation efficiency included mode of pre-cultivation, temperature of cocultivation and presence of acetosyringone (AS). The highest transformation efficiency was obtained with a 4-day pre-cultivation period in liquid medium. Transformation efficiency was higher when cocultivation was performed for 3 days at 19 degrees C than at 23 or 28 degrees C. Almost no resistant callus was obtained if the cocultivation medium lacked AS. The transformation procedure yielded transgenic Valencia plants containing the pTA29-barnase gene, as verified by PCR amplification and confirmed by Southern blotting. Because male sterility is a common factor leading to seedlessness in citrus cultivars with parthenocarpic characteristics, production of seedless citrus genotypes by Agrobacterium-mediated genetic transformation is a promising alternative to conventional breeding methods.     

刺五加体细胞胚状体发生及成苗的研究

以刺五加试管苗叶片为试材,将叶片接种于含有不同种类和浓度激素的培养基上进行培养,研究不同激素对刺五加胚性愈伤组织诱导、胚状体发生及生根的影响。结果表明：诱导叶片胚性愈伤组织发生的最适培养基为MS＋6-BA 1.5 mg.L-1＋2,4-D 1.0 mg.L-1,胚状体生长发育的最适培养基为MS＋6-BA1.0 mg.L-1＋IBA0.5 mg.L-1,生根的最适培养基为1/2MS＋NAA0.4 mg.L-1。     

High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of citrus tristeza virus

In preliminary experiments on Agrobacterium-mediated transformation of citrus, we found transformation events occurring in callus formed from the cambium. Factors affecting Agrobacterium-sour orange (Citrus aurantium L.) interactions, such as culture medium, explant source and culture conditions, were studied to assess competence for transformation in such callus and to improve transformation frequency. Cell divisions and redifferentiation from the transgenic cells leading to transformation events were stimulated more by a combination of benzylaminopurine (BAP) + naphthalene-acetic acid (NAA) in the regeneration-selection medium than by BAP alone. Both age and source of the sour orange plant material affected transformation frequency. Explants from 4-month-old seedlings grown in the greenhouse showed higher transformation frequency than younger and older plant materials, indicating that they had a more suitable balance between dedifferentiated cells competent for transformation and Agrobacterium virulence. Enhancement of transformation frequency enabled us to incorporate the coat protein gene of citrus tristeza virus (CTV) in a sufficient number of sour orange plants to be able to evaluate this strategy for producing CTV-resistant plants.     

Regeneration and gene transformation systems of Robinia pseudoacacia ''Idaho'' mediated by Agrobacterium tumefaciens

Robinia pseudoacacia 'Idaho' is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentration of it was 8 mg·L-1.     

刺槐未成熟合子胚的体细胞胚胎发生和植株再生

以刺槐不同胚龄的未成熟合子胚为外植体,采用混合正交试验设计,研究幼胚胚龄和不同外源激素种类及质量浓度对刺槐胚性愈伤组织的诱导和体细胞胚分化、萌发的影响.结果表明:开花后8周(55天左右)是胚性愈伤组织和体胚诱导的最佳外植体取材时期;MS+2,4-D 5.0 mg·L-1 +BA0.5 mg·L-1是诱导胚性愈伤组织的最佳培养基,出愈率最高为95.42％±0.02％;MS +NAA0.5 mg·L-1 +BA0.5 mg·L-1+谷氨酰胺250 mg·L-1+水解酪蛋白500 mg·L-1是体细胞胚诱导和分化的最佳培养基,直接体细胞胚发生率最高为92.40％±0.12％,通过愈伤组织诱导体细胞胚发生的频率最高为90.73％ ±0.49％.一旦形成球形胚,将培养物转接到不含任何生长调节剂的MS培养基上,体细胞胚经成熟萌发可进一步形成完整小植株.     

AAT基因转化杂交构树叶片组培筛选研究

为了提高杂交构树的组培效率,以蒺藜苜蓿为研究对象,进行生物信息分析,克隆蒺藜苜蓿目的基因AAT,构建表达载体,通过农杆菌转化方法将目的载体转入杂交构树组培叶片中,优化组培相关体系配比,抗生素筛选配比,RT-PCR和定量PCR检验重组植株。结果表明:最佳灭菌体系为75%乙醇消毒20 s,0.3%HgCl 2消毒15 min,污染率低,成活率高;最佳愈伤期激素配比为6-BA 2.5 mg/L、NAA 0.2 mg/L,最佳壮苗期激素配比为6-BA 1.5 mg/L、NAA 0.2 mg/L。最佳筛选抗生素配比为噻孢霉素(Cef)300 mg/L、潮霉素(Hyg)7.5 mg/L;通过RT-PCR验证试验结果,并通过定量PCR测定AAT基因表达含量,重组植株与野生型相比提高2.5倍。试验首次建立了重组载体遗传转化杂交构树,对叶片组培及筛选方法具有重要意义。     

巨桉无性系EG5叶片高效再生体系的建立

以巨桉栽培无性系EG5无菌苗的叶片为外植体进行愈伤组织诱导与植株再生研究。结果表明:愈伤组织高效诱导和不定芽分化的最适培养基为改良MS+0.12 mg·L-1TDZ+0.25 mg·L-1NAA;硝酸铵对EG5叶片愈伤组织生长及植株分化的影响较大,最适宜质量浓度为0.412 5 g·L-1;最佳伸长培养基配方为改良MS+0.3 mg·L-16BA+0.05 mg·L-1IBA+0.3 mg·L-1IAA;暗培养10 d能促进不定芽分化,延缓愈伤组织老化和褐变速度。生根培养基为改良MS+0.4 mg·L-1NAA时生根率最高,为65.47%,移植成活率为90%以上。