小麦组织培养和基因抢轰击影响因素探讨

本研究就基因枪法转化小麦过程中的组织培养和轰击参数等影响因素进行了探讨。结果表明,小麦幼胚是比幼穗或成熟胚更理想的转化受体。因供试小麦品种基因型不同,幼胚愈伤组织再生频率差异明显,辽－10为7．84％,91B569为13．68％,东农7742为54．90％。小麦本身对选择剂卡那霉素有较高的天然抗性,采用G418对小麦幼胚愈伤组织进行筛选效果明显。G418的毒性作用有滞后特点,3个小麦品种对G418的敏感性依次为辽－10＞91B569＞东农7742。用G418做选择剂筛选辽－10、91B569和东农7742抗性愈伤的适合浓度分别为25mg/L、30mg/L和35mg/L。此外,不同轰击参数影响金粉分布的范围和密度。轰击距离为6cm或9cm时,内部金粉密度大而外围金粉密度小,差异极大。轰击距离为12cm时,内部和外围金密度差异小,均匀度好。     

基因枪法向小麦导入几丁质酶基因的研究

利用基因枪法,以菜豆几丁质酶基因转化小麦幼胚愈伤组织。在轰击压力1300psi,轰击距离6cm、100μg金粉/枪和轰击距离9cm、150μg金粉/枪的2种处理条件下,获得4株春小麦东农7742转化植株,转化频率分别为0.36％和0.56％。经PCR和PCR-Southern杂交分析,证实菜豆几丁质酶基因已整合到T0和T1小麦基因组中。采用氨基葡萄糖法测定几丁质酶活力,结果表明,转基因小麦的几丁质酶活力明显高于对照株;转基因植株对白粉病症状减缓,并获得一株赤霉菌接种未扩展的转基因T1植株。     

基因枪法导入Bar基因后转化春小麦的幼胚因素

东北农业大学农学院曹颖、胡尚连、李文雄等鉴于基因枪技术不受宿主限制、转化周期短的优点和在单子叶植物遗传转化中广泛应用,他们用Bar基因转化小麦植株,即利用基因枪法将抗除草剂Bar基因导入春小麦东农7742,IE47和辽10,以培养20～22天的幼胚为受体,在80毫微克金枪粉量和6厘米的轰击距离下,基因枪发挥最佳的转化效果。其中有180个幼胚再生出12个植株,有3个呈GUS(基因)阳性,经PCR分析,检测到Bar基因,还证明用高金粉量可以提GUS基因瞬时表达。但金粉密度大,易成块、不能分散,单位面积上靶组织上金粉颗粒数量多,组织易受伤,影响抗性愈…     

影响基因枪法转化小麦幼胚的几个因素的研究

近几年,利用基因枪直接转入外源DNA,已经成为禾谷类基因转化的主要方法。但在小麦中存在着DNA导入频率低且组织受轰击后再生能力降低等问题。通过实验发现,轰击前14天将小麦幼胚接种于附加0.5mg/L脱落酸(ABA)的诱导培养基上,可显著提高小麦盾片愈伤组织的再生能力。并且,在轰击前6h至轰击后18h,将愈伤组织保持在附加0.5mol/L甘露醇的培养基中,其报告基因的表达将比对照呈几倍的提高。另外,合适的轰击用金粉与DNA配比是转化的关键因素之一。轰击培养14天左右的愈伤组织,每枪用250μg金粉附着0.5μg质粒DNA较宜。     

基因枪介导小麦成熟胚遗传转化的影响因素

小麦成熟胚作为转化受体可克服小麦幼胚存在的受季节和幼胚发育阶段限制的缺点。以湖北省小麦品种‘鄂麦12’和模式品种‘Bobwhite’为材料,成熟胚为转化受体,优化基因枪转化法的轰击压力、轰击距离、选择剂等因素,建立以小麦成熟胚为转化受体的高效转化系统。结果表明:小麦成熟胚作为转化受体时,适宜轰击压力和轰击距离组合是900 psi、6 cm;成熟胚对选择剂G418的敏感性强于幼胚,轰击后需要延长恢复时间,选择剂G418的适合浓度为20～40 mg/L。在以上优化条件下小麦成熟胚转化频率达0.3%～0.9%,已初步建立基因枪介导的小麦成熟胚遗传转化系统。     

影响草地早熟禾（Poa pratensis L.）基因枪转化关键因素研究

将含有DREB1A基因的表达载体,通过基因枪法转化草地早熟禾的胚性愈伤组织,探讨了金粉沉淀剂、金粉直径等因素对转化的影响,同时通过不同浓度潮霉素（Hygromycin,简称Hy）对未转化愈伤组织的筛选,获得最佳筛选浓度。结果表明,适合于草地早熟禾基因枪轰击的条件为：Ca（NO3）2＋PEG4000包被质粒DNA;1μm金粉作为质粒DNA的载体;合适的轰击高度为6cm、轰击次数为1次、无渗透处理。采用Hy作为草地早熟禾转基因植株抗生素筛选标记时,Baron品种愈伤组织继代的临界筛选浓度为100mg/L。     

紫果猕猴桃幼胚愈伤组织诱导及植株再生

以紫果猕猴桃(Actinidia arguta var．purpurea)幼胚为外植体,诱导愈伤组织并进行植株再生。结果表明：不同的培养基和不同的培养条件对幼胚愈伤组织的诱导率及分化率不同;0．2mg／L ZT与0．5mg／L GA。配合使用有利于促进愈伤组织的诱导;7％蔗糖、600mg／L CH与400mg／L Gln都有利于促进愈伤组织的形成;在添加0．5mg／L 6-BA、0．05mg／L NAA与0．5mg／L GA3的MS培养基中植株的再生率达93．3％。     

"东湖早"枇杷的组织培养(简报)

以“东湖早”枇杷的幼胚为外植体,在B5 2,4-D 0.5 mg/L 6-BA 1.0 mg/L培养基上诱导愈伤组织,经继代培养基B5 2,4-D 0.1 mg/L 6-BA 0.5 mg/L培养后,置于B5 6-BA 1.5 mg/L培养基上诱导分化,产生不定芽。将幼苗切段转接到MS 6-BA 2.0 mg/L NAA 0.5 mg/L培养基上进行增殖培养,繁殖系数为3~5。待芽长2~3cm时再分切成单株于1/2MS NAA 0.5 mg/L培养基上,可正常生根。     

胚龄和激素对小麦幼胚组织培养的影响

以扬麦158为试验材料,通过田间取样室内培养的方法研究了胚龄和激素对小麦幼胚组织培养的作用。结果表明,幼胚组织培养最适宜的胚龄为14—16d;适宜的2,4-D浓度为1．5-2．5mg/L;适宜的IAA浓度为2．0-3．0mg/L;适宜的6-BA浓度为0．1-0．8mg/L;适宜的KT浓度为0．5—1．5mg/L。因此,胚龄和激素对于小麦幼胚组织培养具有明显的调节作用．在组织培养实践中,充分认识和综合协调这些因素对小麦幼胚组织培养的作用,可以提高组织培养效率,使其更加有利于生物学研究、遗传转化和快速育种等工作。     

用天花粉蛋白基因转化小麦获得转基因植株

取普通小麦品种京411未成熟胚诱导愈伤组织,10天左右,对820个胚性愈伤组织用含有35S启动子的天花粉蛋白（trichosanthin,TCS)基因轰击。2周后,将这些被轰击的愈伤组织转到含潮霉素50mg/L的筛选培养基上,经分化和生根,获得了33棵再生植株,经接饲毒蚜虫抗病性鉴定和PCR,Southern杂交分析,从中筛选出4株含有编码TCS的转基因小麦植株,转化频率为0．49％。     

Analysis of biochemical and physiological changes in wheat tissue culture using different germplasms and explant types

To select adequate wheat germplasms for genetic transformation, tissue culture efficiency of 21 different wheat lines (Einkorn, Emmer, Durum wheat, etc.) were compared, along with two different explants, namely, immature embryo and mature embryo. The results showed that the average differentiation rate and regeneration rate of immature embryo calli (46.5 and 20.82 %) were better than those for mature embryo calli (14.03 and 4.37 %). The best genotypes for immature embryo callus culture were ‘Ningchun 16’ and ‘Ei 15’, ‘Xiaoyan 22’, followed by ‘Durum 332’ and ‘Tr 256’. The best genotypes for mature embryo callus culture were ‘Ying 4286’, ‘Yunyin 01’, and ‘Xiaoyan 22’. To analyze how physiological and biochemical settings influence the totipotency of calli, different physiological and biochemical indices were analyzed. Differences between immature embryo callus and mature embryo callus were significant, as well as differences of most indices among different wheat types. The interaction effects between explant types and genotypes were also significant. Correlation analysis results showed that the total phenol and soluble sugar contents were significantly correlated with callus differentiation and regeneration rates.     

Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars

Amenability to tissue culture stages required for gene transfer, selection and plant regeneration are the main determinants of genetic transformation efficiency via particle bombardment into sugarcane. The technique is moving from the experimental phase, where it is sufficient to work in a few amenable genotypes, to practical application in a diverse and changing set of elite cultivars. Therefore, we investigated the response to callus initiation, proliferation, regeneration and selection steps required for microprojectile-mediated transformation, in a diverse set of Australian sugarcane cultivars. 12 of 16 tested cultivars were sufficiently amenable to existing routine tissue-culture conditions for practical genetic transformation. Three cultivars required adjustments to 2,4-D levels during callus proliferation, geneticin concentration during selection, and/or light intensity during regeneration. One cultivar gave an extreme necrotic response in leaf spindle explants and produced no callus tissue under the tested culture conditions. It was helpful to obtain spindle explants for tissue culture from plants with good water supply for growth, especially for genotypes that were harder to culture. It was generally possible to obtain several independent transgenic plants per bombardment, with time in callus culture limited to 11–15 weeks. A caution with this efficient transformation system is that separate shoots arose from different primary transformed cells in more than half of tested calli after selection for geneticin resistance. The results across this diverse cultivar set are likely to be a useful guide to key variables for rapid optimisation of tissue culture conditions for efficient genetic transformation of other sugarcane cultivars.     

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the β-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.     

In vitro plant regeneration and genetic transformation of Dichanthium annulatum

Optimization of in vitro plant regeneration and genetic transformation of apomictic species such as Dichanthium annulatum would enable transfer of desirable genes. Seven genotypes of this grass species were screened through mature seed explant for embryogenic callus induction, callus growth and quality (color and texture), and shoot induction. Genotype IG-1999, which produced highly embryogenic, rapidly growing good-quality callus capable of regenerating at a high frequency, was selected for transformation experiments. Using a binary vector (pCAMBIA1305), frequency of GUS expression was compared between two methods of transformation. Bombardment of embryogenic calli with gold particles coated with pCAMBIA1305 at a distance of 11 cm, pressure of 4 bars, and vacuum of 27 Hg passing through 100 muM mesh produced maximum GUS expression (23%). Agrobacterium infection was maximum at an optical density of 2.0 when cocultured under vacuum for 15 min and cocultivated for 3 days at 28 degrees C in constant dark on MS medium of pH 5.8 with 3 mg/l 2,4-D, and 400 muM acetosyringone. Among two binary vectors used for Agrobacterium-mediated transformation, pCAMBIA1301 showed higher frequency of GUS expression while pCAMBIA1305 recorded more of the GUS spots per callus. Supplementation of acetosyringone in the cocultivation medium was found indispensable for Agrobacterium-mediated transformation. Injuring the calli through gold particle bombardment before their cocultivation with Agrobacterium improved the transformation efficiency. Several transgenic plants were developed using the PIG method, while stable GUS-expressing calli were multiplied during selection on MS medium containing 250 mg/l cefotaxime and 50 mg/l hygromycin, incubated in constant dark. A highly significant difference was observed between two methods of transformation for both frequency of GUS expression and GUS spots per callus. PIG-mediated transformation resulted in higher GUS expression compared to the Agrobacterium method. These results demonstrate that Dichanthium annulatum is amenable to Agrobacterium-mediated genetic transformation using a binary vector.     

大麦转化体系的改进及TrxS基因的转化

以啤酒大麦品种“晋引6号”的幼胚为转化起始材料,用基因枪法将分别携带有目的基因(TrxS)和除草剂基因(筛选基因,Bar)的两个质粒进行了共转化,同时对基因转化的相关技术和植株再生的培养方案进行了优化。结果表明,受体材料宜选用预培养15d的幼胚;在培养前2周添加1mg／L ABA可抑制胚芽萌发而且有助于胚性愈伤组织的形成;1．0mg／L ZT与0．1mg／L IAA激素配比可有效促进愈伤组织的分化。利用优化的培养条件,经在含3～5mg／L筛选剂PPT的培养基上筛选、再生及生根培养。共在178块抗性愈伤组织上获得11株再生植株,再生率达到6．2％,经对T0、T1、T2代PCR、nested PCR和Southern杂交检测表明,TrxS基因已经稳定整合到大麦基因组中且遗传稳定、结构完整。     

高羊茅组织培养再生体系及GUS基因瞬间表达研究

以成熟种子为外值体,对高羊茅纰织培养和植株再生体系进行了优化,分析了不同浓度2．4-D、6-BA和激动素对高羊茅愈伤组织诱导和愈伤组织分化成苗的影响．结果表明：9．0mg／L 2．4-L)对愈伤组织的诱导效果最佳．0．2mg／L激动素是愈伤组织分化成苗的最适浓度．二者的诱导率和分化率分别达到68．08%和45．83％。在愈伤组织继代培养基中附加1．0mg／L 2．4-D、0．5mg／L 6-BA和1．25mg／L CuSO4;有利于胚性愈伤组织的形成,可以明显促进愈伤组织分化。同时．采用基因枪法将GUS基因导入高羊茅愈伤组织中,通过组织化学染色检测到了GUS瞬间表达活性;并对影响CUS基因瞬间表达的因素进行了分析．以期为提高基因枪法遗传转化效率提供参考。     

提高小麦基因枪法转化频率的研究

用基因枪法将带Ｂａｒ－ＧＵＳ双标记基因的质粒较入普通春小麦品种中－６０６３４的幼胚盾片,并获基因植株。在轰击的经预培养３－４天的３４２块幼胚片再经筛选再生的植株中,经ＰＣＲ和Ｓｏｕｔｈｅｒｎ分析表明,     

Histological peculiarities of <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. calli cultures’ morphogenesis under antibiotic Ceftriaxone influence

It was shown that, for efficient production of regenerants in bread wheat, 18-day-old calli obtained from apical meristems of 3-day-old aseptic seedlings should be used. These calli show proliferative centers, with developed vascular system elements, that support the development of meristematic centers that later produce regenerants. It was shown that regeneration from the calli occurs by direct organogenesis but not by somatic embryogenesis. Under the influence of the antibiotic Ceftriaxone, which was added to the regeneration medium in a concentration of 400 mg/L, two different bread wheat genotypes showed, firstly, changes in apical origin callus morphology; secondly, an intensification of the regeneration processes; thirdly, root development in the regenerants that allows one to avoid the additional step of plant rooting.     

Erratum to: Transformation of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) microspore-derived callus and microspores by particle bombardment

Twenty-seven bialaphos-tolerant and GUS-positive lines were produced from 2,940 callus pieces after particle bombardment of wheat microspore-derived callus. Regenerated plants were mainly of the albino type. In an attempt to avoid this problem, wheat microspores were used as target cells for particle bombardment. Pre-cultivation for a period of 3-8 days improved the frequency of GUS-expressing microspores. Helium rupture pressures between 7,584 kPa and 12,411 kPa, the amount of gold per bombardment (ranging from 37 µg to 300 µg) and particle size (0.6-1.0 µm) did not significantly affect transient expression. Microspore response measured as number of recovered embryos was not significantly affected by variations in helium pressure or amount of gold used, but response was significantly influenced by particle size. The highest number of GUS-expressing embryos was 3.5 embryos per 10⁶ microspores, which was obtained after 4 days of pre-cultivation, 9,308 kPa rupture pressure, 0.6+1.0 µm particles (1:1) and 150 µg gold particles per bombardment.