樱桃砧木Colt离体叶片再生

以樱桃砧木Colt试管苗的叶片为外植体,通过先诱导愈伤组织分不定芽以及叶片直接分化不定芽两种途径诱导再生,结果表明,在MS附NAA1．0mg/L、KT3．0mg/L、ZT0．25mg/L培养基中,愈伤诱导率可达100％;诱导的愈伤MS附加NAA0．2mg/L、IAA0．5mg/L、6－BA0．5mg/L、KT1．0mg/L,GA0．5mg/L培养基中,不定芽分化率为21．3％,在MS附加6-BA6．0mg/L、NAA1．0mg/L、GA0．5mg/L中,叶片一叶柄不定芽诱导率可达48．3％。     

野牛草成熟胚离体培养及植株再生

1植物名称野牛草[Buchloe dactyloides(Nutt.)texoka]. 2材料类别成熟胚. 3培养条件(1)愈伤组织诱导培养基:MS 2,4-D1.5～6.0 mg·L-1(单位下同) 6-BA 0.1 脯氨酸1 000 水解酪蛋白(CH)500 谷氨酰胺500 α-酮戊二酸100 硫代硫酸银(STS)5;(2)愈伤组织继代培养基:MS 3/2MS(有机) 2,4-D 2.5 6-BA 0.1 CH1 000 聚乙烯吡咯烷酮(PVP)200或维生素C(vC)200;(3)再生培养基:不附加任何植物生长调节物质的MS基本培养基(MS0).所有培养基中均添加3%蔗糖、0.56%琼脂,pH 5.8.愈伤组织诱导及继代培养为暗培养,不定芽分化及植株再生过程中光照12 h·d-1,光照度为1 500lx,培养温度为(25±1)℃.     

中国菟丝子离体再生体系的建立

选取萌发3～5d、长度3～5cm的中国菟丝子（Cuscuta Chinensis Lam）幼苗,将其分为上、中、下3部分并作为外植体进行离体培养与植株再生研究。结果表明,其上、中部片段更适宜愈伤组织诱导;诱导培养基以添加1mg L^-1 NAA和1mg L^-1 BA的MMS培养基效果最好,此培养基也可用于愈伤组织的继代培养,愈伤组织在上述培养基中已生长一年之久。分化培养基为添加1mg L^-1 BA的MMS培养基,平均每块愈伤组织可以产生2．8株植株。     

樱桃砧木Colt离体叶片再生

以樱桃砧木Colt试管苗的叶片为外植体 ,通过先诱导愈伤组织分化不定芽以及叶片直接分化不定芽两种途径诱导再生。结果表明 :在MS附加NAA 1 0mg/L、KT3 0mg/L、ZT0 2 5mg/L培养基中 ,愈伤诱导率可达 1 0 0 % ;诱导的愈伤在MS附加NAA 0 2mg/L、IAA0 5mg/L、6 BA 0 5mg/L、KT 1 0mg/L、GA 0 5mg/L培养基中 ,不定芽分化率为 2 1 3% ;在MS附加 6 BA 6 0mg/L、NAA 1 0mg/L、GA 0 5mg/L中 ,叶片 -叶柄不定芽诱导率可达 48 3%。     

向日葵离体再生体系的建立

为了建立高效的向日葵离体再生体系,从基因差异、外植体取材、生长素和细胞分裂素浓度、附加物的添加等方面出发,对向日葵愈伤诱导、分化、生根等过程进行了系统优化。结果表明：杂交材料相对于自交材料更容易实现再生;最佳外植体是生长4 d的子叶;最佳愈伤诱导培养基是MS培养基 (MS) +2.0 mg/L 6-苄基腺嘌呤 (6-BA)+0.5 mg/L奈乙酸 (NAA)+1.0 mg/L激动素 (KT),诱导率最高可达100％;最佳分化培养基是MS+0.2 mg/L 6-BA+0.5 mg/L NAA+0.3 mg/L KT+0.3 mg/L硝酸银 (AgNO3)+0.2 g/L活性炭 (AC),芽分化率可达71％;最佳生根培养基是1/2 MS+0.6 mg/L吲哆丁酸 (IBA),生根率最高为77％。方差分析表明,材料基因型、外植体生长时间、激素、AgNO3、AC对向日葵再生呈现显著性影响。     

黄斑橡胶榕离体再生体系的建立

以黄斑橡胶榕(Ficus robusta“Yellow spot”)的顶芽为外植体进行离体培养与植株再生研究。结果表明,黄斑橡胶榕的诱导培养基以MS BA 2.0 mg L-1 NAA 0.1 mg L-1为宜;继代增殖以MS BA 1.0 mg L-1 NAA 0.05 mg L-1为宜,每个外植体可产生3个芽;生根培养基以MS IBA 0.1 mg L-1 AC 100 mg L-1为宜,生根率96.67%以上。试管苗移栽成活率达到98%以上。     

甘蓝型油菜高效离体再生体系的建立

以甘蓝型油菜(Brassica napusL.)HC8为材料,从无菌苗苗龄、预培养基激素浓度、预培养天数、6-BA及NAA的浓度等方面对影响油菜组织培养的因素进行了分析研究,建立了甘蓝型油菜品系HC8的离体再生技术体系。结果表明,该油菜组织培养的最佳苗龄为5 d;最佳预培养时间为5 d,最佳2,4-D浓度为1.0 mg/L;子叶最佳诱导培养基为MS+2.0 mg/L 6-BA+0.05 mg/LNAA+3.5 mg/L AgNO3或MS+3.0 mg/L 6-BA+0.1 mg/L NAA+3.5 mg/L AgNO3,该条件下子叶愈伤组织诱导率最高可达100%,再生频率及分化频率分别可达88.0%和108.33%;下胚轴最佳诱导培养基为MS+4.0 mg/L 6-BA+0.05 mg/L NAA+3.5 mg/L AgNO3,子叶愈伤组织诱导率最高可达95.24%,再生频率及分化频率分别可达81.82%和104.55%;最佳生根培养基为MS+0.5 mg/L NAA,生根率最高为90.0%。     

细枝木麻黄离体培养过程中愈伤组织和再生芽的细胞组织学观察

为探讨细枝木麻黄(Casuarina cunninghamianaMiq.)愈伤组织分化过程的细胞组织学,对离体培养条件下的愈伤组织进行扫描电子显微镜和石蜡切片观察,分析愈伤组织的细胞分裂、分化以及芽再生的发生过程。结果表明,新鲜外植体培养于愈伤组织诱导培养基上,伤口处的薄壁细胞开始脱分化,培养1周后形成明显的愈伤组织;继续培养2周后,胚性愈伤组织形成,且表层细胞启动分化形成芽原基;培养4周,可肉眼观察到胚性芽原基,数量增多并逐渐分化形成不定芽;培养至第6周,生成不定芽,并大量增殖和分化。因此,细枝木麻黄是通过愈伤组织分化形成胚状体的途径进行植株再生的,为建立细枝木麻黄组织培养高效再生体系提供了理论依据。     

DNA transfer by highly asymmetric somatic hybridisation in Medicago truncatula (+) Medicago rugosaand Medicago truncatula(+) Medicago scutellata

A regenerable line of Medicago truncatula (Jemalong 2HA) as a recipient species, was fused with the sexually incompatible species Medicago scutellata or Medicago rugosa. The treatments described maintain the chromosome number of the recipient but enable the transfer of small amounts of DNA of the donor species, probably by intergenomic recombination. Without a chromosome number-change fusion products can readily regenerate to produce fertile plants; and potentially a library with a diverse array of new genetic material. The selection of fused cells is based on treatment of the recipient cells with iodoacetamide (IOA), a non-regenerable donor, γ-irradiation of the donor, and regeneration on a medium favouring the recipient. DNA transfer was demonstrated by amplified fragment length polymorphism (AFLP), Southern hybridisation and changed morphology. Received: 21 December 2000 / Accepted: 5 April 2001     

Repetitive somatic embryogenesis in Medicago truncatula ssp. Narbonensis and M. truncatula Gaertn cv. Jemalong

Medicago truncatula ssp Narbonensis and four genotypes of M. truncatula Gaertn cv. Jemalong were tested for their somatic embryogenesis potential using a two-step protocol. In the first step, embryogenic callus was induced in folioles isolated from shoots grown in vitro and cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid and zeatin. In the second step, somatic embryos were allowed to develop from the induced callus in MS growth-regulator-free medium. Individual somatic embryos were then isolated and transferred again to growth regulator free medium where they formed secondary somatic embryos in repetitive cycles. Conversion of somatic embryos into plantlets was achieved by isolating late-torpedo-phase somatic embryos with distinct cotyledons and reculturing them onto MS growth regulator free medium. The system of repetitive somatic embryogenesis in M. truncatula described here represents a permanent source of embryogenic material that can be used for the genetic modification of this species. Received: 7 August 1997 / Revision received: 22 December 1997 / Accepted: 20 January 1998     

蒺藜苜蓿糖基转移酶基因SMALL AND EMERALD1的克隆和功能研究

类黄酮代谢对于植物生长发育和植物-环境互作至关重要,其中糖基转移酶介导的糖基化修饰在类黄酮代谢中发挥着重要作用。为了研究蒺藜苜蓿中糖基转移酶的生物学功能,通过定向筛选蒺藜苜蓿Tnt1逆转座子插入突变体库,获得了一类植株矮小、叶片深绿的突变体small and emerald1 (se1)。通过基因表型连锁性分析成功克隆了SE1基因,该基因编码1个糖基转移酶,与拟南芥中调控类黄酮生物合成的AtUGT84A1氨基酸同源性为52.8%。对野生型和se1突变体叶片的类黄酮含量进行测定发现类黄酮总量在se1突变体中显著降低(P<0.01)。进一步研究发现在se1突变体中类黄酮合成途径关键基因CHS、F3H和F3’H表达水平下降。亚细胞定位显示SE1可能在细胞质和细胞核中发挥生物学功能。研究表明糖基转移酶基因SE1可能参与蒺藜苜蓿类黄酮合成代谢调控,进而影响其生长发育。此外,研究还发现SE1基因对于叶绿素合成可能具有负向调控作用。     

Transformation of floral organs with GFP in Medicago truncatula

 A high frequency of embryogenesis and transformation from all parts of flowers of two lines of Medicago truncatula R-108–1 and Jemalong J5 were obtained. Using this flower system, we obtained transgenic plants expressing promoter-uidA gene fusions as well as the gfp living cell color reporter gene. Moreover, this method allows us to save time and to use a smaller greenhouse surface for the culture of donor plants. Southern hybridization showed that the internal gfp fragment had the expected size and the number of T-DNA copies integrated in the plant genome varied between one and three. These data suggest that the presence of the GFP protein has no toxic effects, since no rearrangement of the gfp reporter gene was detected in the regenerated plants. Received: 25 May 1999 / Revision received: 2 August 1999 / Accepted: 2 August 1999     

蒺藜苜蓿MtSAG113基因的转化及表达特征分析

Senescence Associated Gene 113(SAG113)基因属于PP2Cc超家族,该基因的研究主要集中在植物衰老领域.为分析蒺藜苜蓿(Medicago truncatula)MtSAG113基因的表达特征,探究MtSAG113基因的功能.该基因从蒺藜苜蓿中克隆得到,以烟草(Nicotiana tab...     

A CDPK isoform participates in the regulation of nodule number in Medicago truncatula

Medicago spp. are able to develop root nodules via symbiotic interaction with Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in various signalling pathways in plants, and we found that expression of MtCPK3, a CDPK isoform present in roots of the model legume Medicago truncatula, is regulated during the nodulation process. Early inductions were detected 15 min and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers was also present in inoculated M. truncatula dmi mutants and in wild-type roots subjected to salt stress, indicating that this rapid response is probably stress-related. In contrast, the later response was concomitant with cortical cell division and the formation of nodule primordia, and was not observed in wild-type roots inoculated with nod (-) strains. This late induction correlated with a change in the subcellular distribution of CDPK activities. Accordingly, an anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in epidermal onion cells, a localization that depends on myristoylation and palmitoylation sites of the protein, suggesting a dual subcellular localization. MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone linked to the regulation of cortical cell division and other nodulation-related responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium rhizogenes-transformed roots. Although no major phenotype was detected in these roots, when infected with rhizobia, the total number of nodules was, on average, twofold higher than in controls. This correlates with the lack of MtCPK3 induction in the inoculated super-nodulator sunn mutant. Our results suggest that CPK3 participates in the regulation of the symbiotic interaction.     

蒺藜苜蓿MtbHLH25基因克隆、亚细胞定位及表达分析

【目的】bHLH转录因子数量众多,能够广泛参与植物的生长发育和逆境胁迫等过程。本试验以蒺藜苜蓿R108为材料,初步探讨MtbHLH25基因的功能。【方法】通过PCR扩增技术从蒺藜苜蓿中克隆MtbHLH25基因和启动子,构建酵母表达载体并用LiAc转化法转移到Y2H Gold酵母菌株中进行酵母自激活检测,构建亚细胞定位载体并通过冻融法转入农杆菌EHA105,菌液注射到烟草下表皮细胞后利用SP8激光共聚焦显微镜观察,通过实时荧光定量PCR技术研究MtbHLH25基因的时空表达水平。【结果】（1）从蒺藜苜蓿中成功克隆出MtbHLH25基因和启动子,该基因总长882 bp,共编码293个氨基酸。启动子序列分析发现其包含了ABA、MeJA、GA和SA等响应元件。（2）进化树结果表明MtbHLH25蛋白与蚕豆和长柔毛野豌豆中bHLH蛋白高度同源。（3）亚细胞定位结果显示MtbHLH25蛋白定位于细胞核。（4）酵母自激活检测结果显示MtbHLH25蛋白具有自激活活性。（5）表达分析结果显示,MtbHLH25在蒺藜苜蓿根、茎、叶、花和果实中均有表达,其中在根中表达水平最高;外源SA、MeJA、ABA、GA以及盐胁迫使MtbHLH25基因表达量都呈下降趋势,推测SA、MeJA、ABA、GA以及盐胁迫对MtbHLH25基因的表达起到负调控作用。干旱胁迫能够显著诱导MtbHLH25基因表达量的上升,说明该转录因子可能在干旱胁迫中起到正调控作用。【结论】MtbHLH25基因可能对盐胁迫敏感,在干旱胁迫中可能发挥正调控作用。此外,MtbHLH25蛋白具有自激活活性,对下游启动子调控的报告基因可能具有激活作用。     

印度梨形孢促进蒺藜苜蓿生长及其提高耐盐性研究

【目的】研究盐胁迫下印度梨形孢定殖对豆科模式植物蒺藜苜蓿生长发育的影响。【方法】通过分析不同生境下植物的根长、根鲜重和茎鲜重,以及体内抗氧化物酶活性、脯氨酸含量、甜菜碱醛脱氢酶基因(BADH)的表达,确定印度梨形孢对蒺藜苜蓿生长的促进作用,并初步阐释印度梨形孢诱导植物耐盐性的机制。【结果】印度梨形孢能在蒺藜苜蓿根部定殖并能促进植物的生长发育,有效缓解盐胁迫造成的生长抑制。印度梨形孢能提高植物体内抗氧化物酶活性,增加游离脯氨酸含量并诱导BADH基因的表达。【结论】印度梨形孢作为植物生长促进因子可以用来提高植物耐盐性,实现盐碱土壤的间接改良。     

Optimizing TILLING populations for reverse genetics in Medicago truncatula

Medicago truncatula has been widely adopted as a model plant for crop legume species of the Vicieae. Despite the availability of transformation and regeneration protocols, there are currently limited tools available in this species for the systematic investigation of gene function. Within the framework of the European Grain Legumes Integrated Project ( http://www.eugrainlegumes.org ), chemical mutagenesis was applied to M. truncatula to create two mutant populations that were used to establish a TILLING (targeting induced local lesions in genomes) platform and a phenotypic database, allowing both reverse and forward genetics screens. Both populations had the same M2 line number, but differed in their M1 population size: population 1 was derived from a small M1 population (one-tenth the size of the M2 generation), whereas population 2 was generated by single seed descent and therefore has M1 and M2 generations of equal size. Fifty-six targets were screened, 10 on both populations, and 546 point mutations were identified. Population 2 had a mutation frequency of 1/485 kb, twice that of population 1. The strategy used to generate population 2 is more efficient than that used to generate population 1, with regard to mutagenesis density and mutation recovery. However, the design of population 1 allowed us to estimate the genetically effective cell number to be three in M. truncatula . Phenotyping data to help forward screenings are publicly available, as well as a web tool for ordering seeds at http://www.inra.fr/legumbase