小偃22幼胚体细胞胚性无性系的诱导

采用田间取样和室内培养的方法,研究了不同幼胚发育时期、盾片接种方式和状态、4℃预处理时间对小偃22幼胚体细胞胚性无性系诱导的影响。结果表明:(1)小偃22未成熟种子长度占颖壳长度比例为60%~80%时,幼胚处于半透明向淡黄色过渡状态,其愈伤组织诱导率达到95%以上,胚性愈伤组织诱导率达到46.1%~49.4%,此时是幼胚最佳培养时期。(2)未成熟籽粒在4℃冰箱保湿保存3 d内可陆续接种,幼胚愈伤组织诱导率在95%以上,胚性愈伤组织发生率达到34%~50%,接种时应使盾片向上并保持完整状态。     

体胚发生途径‘大矮蕉''植株的再生

‘大矮蕉'Musa AAA cv. Grande Naine的胚性细胞悬浮系(ECS)在M2液体培养基中预培养1周和2周后,分别将其接种在RD1或M3培养基上,于光照或黑暗条件下进行体胚的再生. 再生体胚在接种后3周左右开始出现,经8周的培养后,在不同预培养时间、再生培养基种类及培养条件下,胚性细胞(团)质量增长了约5～18倍,从沉积细胞体积(SCV)为1 mL的ECS获得的再生体胚数为0.71×105～3.07×105. 植株的再生率及从SCV为1 mL的ECS获得的再生植株数也因上述体胚再生条件的不同而异.     

Induction and Genetic Identification of Embryogenic Calli from Hybrids of Shatian Pummelo

Shatian pummelo （Citrus grandis L. Osbeck cv. Shatian） is an elite variety in China, and the regeneration of the embryogenic callus is difficult. Diploid Shatian pummelo was used as the female and crossed with the allotetraploid somatic hybrid NS （Nova Tangelo ＋ Succari Sweet orange）, [ （ C reticulata Blanco x C. paradisi Macf.） cv. Nova ＋ C sinensis L. Osbeck cv. Succari]. About 90 days after pollination, the embryos obtained from crosses were cultured on the solid media of MT ＋ ME （malt extraction, 500 mg L^-1） and MT ＋ GA3 （1 mg L^-1）. The embryogenic callus was initiated from the embryoids and plantlets＇ hypocotyls and could be subcultured. Flow cytometry and SSR analysis verified that the callus was from the triploid hybrids. The callus had embryogenesis capacity and produced a large number of embryoids on MT ＋Lactose （50 g L^-1） medium after being subcultured for two years. It is comparatively easier to obtain the callus from the hybrid embryo than from Shatian pummelo itself. The callus is valuable for the conservation and utilization of Shatian pummelo.     

宽皮橘蕉柑的胚性愈伤组织诱导、悬浮细胞系建立及植株再生

取蕉柑花后 5周幼果的幼胚接种于含不同浓度BA、KT与IAA组合的MT培养基上 ,结果表明 ,暗培养条件是蕉柑胚性愈伤组织诱导成功的关键 .胚状体在后期发育中畸形胚的比例很高 ,经切割后 ,在MT +BA2 .0mg·L-1+IAA0 5mg·L-1+蔗糖 0 0 3kg·L-1培养基上可诱导产生丛芽 ,诱导率为 2 6 7% .幼芽在 1/ 2MT +NAA 1 0mg·L-1培养基上生根率达到了 87 0 % .蕉柑胚性愈伤组织在MT液体培养基中不断地继代培养 ,可形成生长稳定的胚性悬浮细胞系 ,体胚诱导实验证明悬浮细胞系具有很强的体细胞胚胎发生能力 .     

Plant regeneration from protoplasts isolated from primary calli using mature embryos of two Brazilian rice cultivars

A plant regeneration system from rice protoplasts using calli derived from mature embryos was established for the two Brazilian modern rice cultivars IAC-201 and IAC-165. After 30 to 40 days of in vitro culture it was possible to obtain on average 6 million protoplasts per gram of callus. Microscopic selection of embryogenic calli was a key step for protoplast isolation. The production of embryogenic calli increased when L-proline and casein hydrolysate were used in the callus induction medium. The Oc or IR52 nurse cell lines were essential for protoplast division. Different regeneration media were studied and 139 plants were regenerated which set seed. Some of the regenerated plants showed morphological variation such as the presence of awns in spite of the short time of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transient gene expression in transformed banana (Musa cv. Bluggoe) protoplasts and embryogenic cell suspensions

Summary In order to introduce currently-available genes with agronomical value into banana, two genetic transformation protocols have been optimized.Firstly, regenerable protoplasts isolated from embryogenic cell suspensions of the cultivar Bluggoe have been used for the introduction of several chimaeric uidA gene constructs by electroporation. With the inclusion of polyethylene glycol and heat shock, the frequency of transiently expressing protoplasts reached 1.8% as shown by an in situ -glucuronidase assay. A duplicated 35S promoter with an alfalfa mosaic virus leader sequence (pBI-426) induced the highest expression rate among the constructs tested.Embryogenic cell suspensions of cv. Bluggoe have also been bombarded with accelerated particles coated with a high expression uidA gene construct (pEmuGN) using a biolistic gun. After a partial optimization of the procedure, transient GUS assays reproducibly demonstrated the presence of 400 blue foci in 30 l of settled cell volume (approximately 25 mg cells). Selection and characterization of antibiotic-resistant transformed cultures is in progress.Abbreviations AMV alfalfa mosaic virus - GUS -glucuronidase - TGE transient GUS expression - uidA gene for -glucuronidase     

High-efficiency transformation of Gossypium hirsutum embryogenic calli mediated by Agrobacterium tumefaciens and regeneration of insect-resistant plants

A simple protocol of transformation of cotton (Gossypium hirsutum L.) at a high frequency has been developed via Agrobacterium mediation, coupled with the use of embryogenic calli as explants. Embryogenic calli derived from only one to two somatic embryogenic calli lines of two Chinese cotton cultivars, the cvs. Ekang 9 and Jihe 321 which have low embryogenic potency were first inoculated with the A. tumefaciens strain LBA4404 harbouring binary vector pBin438 carrying a synthetic Bacillus thuringiensis‐active Cry1Ac and API‐B chimeric gene. Infected embryogenic calli were co‐cultivated for 48 h and were then moved on to the selection medium with kanamycin (100 mg/l) for 7‐8 weeks. Then, the kanamycin‐resistant calli (Km¹) subcultured in proliferation medium would re‐differentiate to form somatic embryos in 30 days. Cotyledon embryos were transferred to 100‐ml Erlenmeyer flasks for germination and regeneration. Putative transformants were confirmed by polymerase chain reaction and Southern blot analysis. Forty‐five regenerated plants were successfully transferred to soil, of which 12 proved to have the active Cry1Ac and API‐B chimeric gene. Insect resistance was tested by bioassay. The transgenic plants were highly resistant to cotton bollworm (Heliothis armigera) larvae, with mortality (insect resistance) ranging from 95.8 to 100%. In comparison with the methods used in Agrobacterium‐mediated transformation of cotton hypocotyls or cotyledons, about 6 months are saved by using the method presented in this paper to obtain a large number of transgenic plants.     

籼稻不同品种的遗传转化和植株再生

用携带水稻蜡质基因反义 RNA片段的 p1 3 W4质粒的根癌农杆菌 EHA1 0 5 ,分别感染籼稻品种龙特甫 B、协青早B、珍汕 97B和 -3 2 B的幼胚愈伤组织 ,经共培养和在潮霉素 2 5～ 5 0 mg/L浓度下的二次筛选后 ,获得了对潮霉素表现抗性的愈伤组织 ,抗性愈伤组织的频率在 2 0 .1 %～ 2 7.1 %之间 ;抗性愈伤组织经分化培养 ,分化频率在 1 2 .0 %～ 2 1 .2 %。品种间绿菌分化情况存在较大差异 ,龙特甫 B的抗性愈伤组织分化成绿苗的频率为 8.8% ;珍汕 97B的抗性愈伤组织虽能分化 ,但是 ,无法获得绿苗。对转基因植株分别进行了 GUS活性测定和 PCR检测 ,结果显示 :获得的转基因植株 ,92 %的植株GUS检测呈阳性反应 ,并能正常结实 ,在 T1种子的胚乳中也能检测到 GUS染色的分离 ,证明外源基因确已导入这些植株中 ,而且 ,能正常遗传。在抗性愈伤组织的分化中 ,试验了潮霉素对分化的影响 ,结果显示 :不添加潮霉素 ,能明显地提高绿苗频率 ,但转基因植株的可靠性也随之下降。     

羊草胚性愈伤组织的形成及植株再生

以羊草幼嫩根茎和种子为外植体,在MS、B_5和8114三种培养基上,配合使用生长素2.4-D的浓度水平为1,2,4mg/L,诱导愈伤组织以B_5或MS+2.4-D 4mg/L效果最佳。在继代培养中降低2.4-D浓度,相应提高蔗糖和肌醇含量,能有效地促进愈伤组织质地转变,形成致密的胚性愈伤组织。通过研究证明,羊草种子是羊草组织培养的最佳外植体,其愈伤组织诱导率高,质地优良且幼苗分化率好,并具有易于操作的优点。     

农杆菌介导玉米愈伤遗传转化研究

以玉米自交系R18-599和齐319幼胚诱导的胚性愈伤为转化受体,对农杆菌浸染并共培养3 d后对GUS基因的瞬时表达率进行检测,优化遗传转化体系。结果表明:愈伤组织的生长状态对转化效率有很大影响,以愈伤组织继代两次、预培养7 d左右为浸染的最佳时期;另外,EHA105菌株对玉米自交系齐319愈伤的浸染效果要好于对R18-599,其平均GUS瞬时表达率分别为31.20%和23.74%,经t测验达显著水平(t<0.05)。