Highly efficient Agrobacterium-mediated transformation and plant regeneration system for genome engineering in tomato

Tomato (Solanum lycopersicum L.) is an important vegetable and nutritious crop plant worldwide. They are rich sources of several indispensable compounds such as lycopene, minerals, vitamins, carotenoids, essential amino acids, and bioactive polyphenols. Plant regeneration and Agrobacterium-mediated genetic transformation system from different explants in various genotypes of tomato are necessary for genetic improvement. Among diverse plant growth regulator (PGR) combinations and concentrations tested, Zeatin (ZEA) at 2.0 mg l^?1 in combination with 0.1 mg l^?1 indole-3-acetic acid (IAA) generated the most shoots/explant from the cotyledon of Arka Vikas (36.48 shoots/explant) and PED (24.68 shoots/explant), respectively. The hypocotyl explant produced 28.76 shoots/explant in Arka Vikas and 19.44 shoots/explant in PED. In contrast, leaf explant induced 23.54 shoots/explant in Arka Vikas and 17.64 shoots/explant in PED. The obtained multiple shoot buds from three explant types were elongated on a medium fortified with Gibberellic acid (GA₃) (1.0 mg l^?1), IAA (0.5 mg l^?1), and ZEA (0.5 mg l^?1) in both the cultivars. The rooting was observed on a medium amended with 0.5 mg l^?1 indole 3-butyric acid (IBA). The transformation efficiency was significantly improved by optimizing the pre-culture of explants, co-cultivation duration, bacterial density and infection time, and acetosyringone concentration. The presence of transgenes in the plant genome was validated using different methods like histochemical GUS assay, Polymerase Chain Reaction (PCR), and Southern blotting. The transformation efficiency was 42.8% in PED and 64.6% in Arka Vikas. A highly repeatable plant regeneration protocol was established by manipulating various plant growth regulators (PGRs) in two tomato cultivars (Arka Vikas and PED). The Agrobacterium-mediated transformation method was optimized using different explants like cotyledon, hypocotyl, and leaf of two tomato genotypes. The present study could be favourable to transferring desirable traits and precise genome editing techniques to develop superior tomato genotypes.     

琼脂糖和聚丙烯酰胺凝胶电泳技术检测小麦基因组DNA RAPD扩增产物的方法学比较

通过20%(wv)的琼脂糖凝胶和5%(wv)的聚丙烯酰胺凝胶电泳对小麦白粉病抗、感特性品种基因组DNA的RAPD检测结果表明:5%聚丙烯酰胺凝胶对线性DNA分子(01～20kb)和长度相差100bp以下的DNA分子的分离较20%的琼脂糖凝胶电泳效果好。因此,我们研究出了一项利用聚丙烯酰胺凝胶电泳检测小麦白粉病抗、感特性的新技术,在工作中建立了一种适合于检测小麦基因组DNA结构差异的电泳方法。该方法主要包括:(1)丙烯酰胺和亚甲基双丙烯酰胺的新配比;(2)分离DNA片段的最佳凝胶浓度;(3)电泳条件;(4)脱色、漂洗、银染、显色过程。实验发现,该技术对于小麦白粉病抗、感特性检测中的小片段和长度相差100bp以下的线性DNAPCR扩增结果的分辨效果较好。应用该技术在抗感品种间已经发现了DNA水平上的差异。     

Loss of actin cytoskeletal function and EDS1 activity,in combination,severely compromises non-host resistance in Arabidopsis against wheat powdery mildew

Plant immunity against the majority of the microbial pathogens is conveyed by a phenomenon known as non-host resistance (NHR). This defence mechanism affords durable protection to plant species against given species of phytopathogens. We investigated the genetic basis of NHR in Arabidopsis against the wheat powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt). Both primary and appressorial germ tubes were produced from individual Bgt conidia on the surface of the Arabidopsis leaves. Attempted infection occasionally resulted in successful penetration, which led to the development of an abnormal unilateral haustorium. Inoculation of a series of Arabidopsis defence-related mutants with Bgt resulted in the attenuation of reactive oxygen intermediate (ROI) production and salicylic acid (SA)-dependent defence gene expression in eds1, pad4 and nahG plants, which are known to be defective in some aspects of host resistance. Furthermore, Bgt often developed bilateral haustoria in the mutant Arabidopsis lines that closely resembled those formed in wheat. A similar decrease in NHR was observed following treatment of the wild-type Arabidopsis plants with cytochalasin E, an inhibitor of actin microfilament polymerisation. In eds1 mutants, inhibition of actin polymerisation severely compromised NHR in Arabidopsis against Bgt. This permitted completion of the Bgt infection cycle on these plants. Therefore, actin cytoskeletal function and EDS1 activity, in combination, are major contributors to NHR in Arabidopsis against wheat powdery mildew.     

Inheritance of resistance to blackmold (Alternaria alternata (Fr.) Keissler) in two interspecific crosses of tomato (Lycopersicon esculentum ×L. cheesmanii f.typicum)

Inheritance of resistance to blackmold, a disease of ripe tomato fruit caused byAlternaria alternata, was studied in two interspecific crosses. The parents, F₁ and F₂ generations of a cross between the susceptibleLycopersicon esculentum Mill. cultivar Hunt 100 and the resistantL. Cheesmanii f.typicum Riley accession LA 422, and the parents, F₁, F₂, F₃, and BC₁ P₂ generations of a cross between the susceptibleL. Esculentum cv. VF 145B-7879 and LA 422 were evaluated. The following disease evaluation traits were used: symptom rating (a symptom severity rating based on visual evaluation of lesions), diseased fruit (the number of diseased fruits divided by the total number of fruit scored), and lesion size (a function derived from the actual lesion diameter). Generation means analysis was used to determine gene action. The data of the Hunt 100 × LA 422 cross fit an additive-dominance model for all three traits. The VF 145B-7879 × LA 422 cross data best fit a model that included the additive × additive and additive × dominance interaction components for the trait diseased fruit, whereas higher-order epistatic models would have to be invoked to fit the data for the traits symptom rating and lesion size. A minimum of one gene segregated for all three traits. Broad-sense heritability estimates ranged from 0.09 to 0.16 for all three traits, indicating that selection for improved resistance to blackmold will require selection on a family performance basis.     

The histogenesis of somaclones from tomato (Lycopersicon esculentum Mill.) cotyledons

Summary A histological study ofin vitro cultured cotyledonary expiants of tomato (Lycopersicon esculentum) was performed in order to determine the site (differentiated tissue or developing callus) and the mode of plant regeneration.Results have shown that callus develops at the excision sites of cotyledonary expiants and that shoots are formed exclusively within the unorganized callus: excision areas are the only morphogenetic sites and the proximal excision is the preferred site for plant regeneration.Shoots differentiate by organogenesis within the superficial region of the callus. Few neocambial cells cooperate in the neoformation. Origin from a single cell is highly unlikely since rarely observed single activated cells never developed into shoots.Regenerated plants may be chimeras if invitro culture induces genetic diversity in the initial cells.Abbreviations IAA Indole-3-acetic acid - c callus - d vegetative dome - s shoot - ad adaxial - ab abaxial - t tracheid - p parenchyma - S sieve tube     

兼抗白粉、条锈病小偃麦渗入系CH7124抗性遗传及细胞学鉴定

CH7124是通过八倍体小偃麦TAI8335与感病小麦杂交、回交育成的兼抗白粉病、条锈病的小偃麦种质系。利用抗性接种鉴定、细胞学和基因组原位杂交(GISH)技术相结合的方法,对CH7124的抗性来源、遗传方式及细胞学特征进行了分析和鉴定。结果表明,CH7124在苗期和成株期对条锈菌系CYR29、CYR31、CYR32、CYR33和白粉菌系E09、E20、E21、E26表现为免疫或近免疫,其抗性来自中间偃麦草,受1对显性核基因控制;CH7124的根尖细胞染色体数目为2n=42,花粉母细胞减数分裂中期I(PMC MI)绝大多数细胞内可观察到21个二价体,平均配对构型为2n=0.30 I+20.79 II+0.04 III;与普通小麦中国春、绵阳11的杂种F1中,有80%以上的花粉母细胞可观察到2n=21Ⅱ的染色体构型,其平均配对构型均为2n=21II。说明CH7124具有与普通小麦相似的染色体结构和规则的配对构型。由于利用以中间偃麦草总DNA为标记探针的原位杂交未观察到可见的外源DNA杂交信号,进一步证明CH7124是一个小麦-中间偃麦草的隐形异源渗入系。     

利用酵母双杂交系统筛选与黄瓜花叶病毒外壳蛋白互作的寄主蛋白

利用酵母双杂交系统,以黄瓜花叶病毒(Cucumber mosaic virus,CMV)的外壳蛋白(coat protein,CP)为诱饵,从番茄叶片c DNA文库中筛选与其互作的蛋白。结果显示,诱饵载体pBT3-SUC-CMV-CP均能在酵母细胞中正确表达,无自激活活性而且对酵母无毒性;通过对酵母双杂交文库的筛选和回转验证,共获得了98个阳性克隆,分别编码67个可能与CMV-CP相互作用的蛋白,分别参与植物防御反应、光合作用、物质转运、信号转导、能量代谢、氨基酸代谢、细胞壁的形态建成、植物的激素代谢等。本研究结果表明,CMV CP可同时调控寄主的多个代谢过程,在CMV的致病过程中有多重功能。     

Sources and inheritance of resistance to leaf curl virus in Lycopersicon

Summary One hundred and twenty-two varieties, lines and wild accessions of Lycopersicon were screened under three different regimes during the autumn/winter season of 1982–83 and 1983–84 for resistance to tomato leaf curl virus (TLCV). L. hirsutum f. glabratum (B6013) and L. hirsutum f. typicum (A1904) proved to be highly resistant to TLCV in all three environments. Various accessions of L. peruvianum were also highly resistant. L. pimpinellifolium (A1921) exhibited no TLCV symptoms within 90 days. Of the cultivated varieties, Acc 99 exhibited the minimim score for susceptibility; AC 142, Collection No. 2, Kalyanpur Angurlata and HS 101 had a low rating for virus incidence. The inheritance of resistance was studied in the interspecific crosses between a TLCV resistant line of L. pimpinellifolium (A1921) and five (HS 101, HS 102, HS 110, Pusa Ruby and Punjab Chhuhara) susceptible cultivars of L. esculentum. Parents, F₁, F₂ and backcross progenies were artificially inoculated with local strains of TLCV using vector the viruliferious whitefly, Bemisia tabaci (Genn.). Data indicated that the resistance of L. pimpinellifolium (A 1921) is monogenic and incompletely dominant over susceptibility.