De novo organogenesis and plant regeneration in <Emphasis Type="Italic">Pongamia pinnata</Emphasis>, oil producing tree legume

Role of Thidiazuron (TDZ) in inducing adventitious organogenesis in Pongamia was studied. TDZ at different concentrations (0, 0.45, 2.27, 4.54, 6.71, 9.08, 11.35, 13.12 and 22.71 μM) were used for induction of caulogenic bud formation in deembryonated cotyledon explants. Each cotyledon was cut into three segments and identified as proximal, middle and distal. Duration of TDZ exposure, influence of the segment and orientation of the explant were studied. TDZ at 11.35 μM concentration was optimum for the induction of shoots and rapid elongation. Shoots induced at higher concentration elongated after several passages in growth regulator free medium, thereby extending the period of differentiation. Exposure of the explant for 20 days yielded more number of buds than 10 days. Proximal segment of the cotyledon was more responsive. Contact of abaxial surface in the medium was more effective and generated more buds than the adaxial side. Buds differentiated and elongated on transfer to MS basal medium for 8–12 passages of 15 days each. Rooting and elongation of shoots was achieved in charcoal supplemented half-strength MS medium. Rooted plantlets survived on transfer to sand soil mixture. The plants were hardened and transferred to green house. This is the first report on in vitro regeneration of Pongamia pinnata via adventitious organogenesis using TDZ. This protocol may find application in studies in genetic transformation, isolation of somaclonal variants and in induction of mutants. It also provides a system to study the inhibitory role of TDZ on shoot differentiation.     

利用cDNA-AFLP技术分析龙眼子叶胚基因表达差异

分别提取'红核子'龙眼(Dimdcarpus longan Lour.)谢花后35 d(早期)和50 d(晚期)子叶胚RNA,建立cDNA-AFLP分析体系,获得指纹图谱.对41个差异片段回收克隆和序列分析,表明其中21个与已知功能基因具有高度同源性,这些基因的功能主要涉及侧牛器官的离轴极性、糖酵解、能最代谢、离子转运、细胞壁伸长、细胞周期调控、RNA转录、RNA翻译和调控、蛋白磷酸化调控和降解、信号转导等;其余片段与已知基因的同源性较低或没有.     

PICKLE is a CHD subfamily II ATP-dependent chromatin remodeling factor

PICKLE plays a critical role in repression of genes that regulate development identity in Arabidopsis thaliana. PICKLE codes for a putative ATP-dependent chromatin remodeler that exhibits sequence similarity to members of subfamily II of animal CHD remodelers, which includes remodelers such as CHD3/Mi-2 that also restrict expression of developmental regulators. Whereas animal CHD3 remodelers are a component of the Mi-2/NuRD complex that promotes histone deacetylation, PICKLE promotes trimethylation of histone H3 lysine 27 suggesting that it acts via a distinct epigenetic pathway. Here, we examine whether PICKLE is also a member of a multisubunit complex and characterize the biochemical properties of recombinant PICKLE protein. Phylogenetic analysis indicates that PICKLE-related proteins in plants share a common ancestor with members of subfamily II of animal CHD remodelers. Biochemical characterization of PICKLE in planta, however, reveals that PICKLE primarily exists as a monomer. Recombinant PICKLE protein is an ATPase that is stimulated by ssDNA and mononucleosomes and binds to both naked DNA and mononucleosomes. Furthermore, recombinant PICKLE exhibits ATP-dependent chromatin remodeling activity. These studies demonstrate that subfamily II CHD proteins in plants, such as PICKLE, retain ATP-dependent chromatin remodeling activity but act through a mechanism that does not involve the ubiquitous Mi-2/NuRD complex.     

影响辣椒离体再生芽伸长因素研究(英文)

以辣椒子叶为外植体,比较不同浓度BA和IAA激素组合对辣椒再生芽诱导的差异,利用筛选出的高效芽诱导培养基为基础,研究了赤霉素、芽诱导时间、培养基有机成分、不同激素组合和品种等因素对辣椒不定芽伸长的影响。结果表明:不同基因型辣椒子叶再生能力不同,BA3.0mg·L^-1+IAA0.5mg·L^-1的激素配比对不定芽诱导频率最高;不定芽的伸长百分率随着GA₃浓度的增加而增加,GA₃的适合浓度为1.0～2.0mg·L^-1;不定芽诱导时间对不定芽的伸长有一定的影响,诱导21d的不定芽,其伸长频率明显高于诱导14d的不定芽;B5有机成分在辣椒不定芽的伸长中效果优于MS有机成分;激素组合对不定芽伸长有一定的影响,Zeatin+GA₃激素组合对伸长效果最好,BA+IAA+GA₃伸长效果较好,BA+PAA(苯乙酸,phenylaceticacid)+GA₃伸长效果次之;不同品种辣椒不定芽的伸长能力有一定差异,楚风和苏椒五号再生芽伸长能力最佳。与IAA和NAA相比,IBA对再生芽生根效果较好。     

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.     

Putrescine metabolism in excised cotyledons of Pinus radiata cultured in vitro

The metabolism of ¹⁴C-putrescine and the changes in the endogenous concentrations of putrescine, spermidine and spermine were studied when cotyledons of Pinus radiata D. Don were cultured under shoot-forming (SF, + N⁶-benzyladenine) and non-shoot-forming (NSF, - N⁶-benzyladenine) conditions. Differences in the total uptake of ¹⁴C-putrescine during a 2 h pulse feeding were not significant between the SF and NSF cotyledons except on day 3. The maximum uptake of label was on day 3 in the SF cotyledons, which released the highest amount of ¹⁴CO₂ as well. ¹⁴C from the labeled putrescine was incorporated mainly into γ-aminobutyric acid, aspartate and glutamate. High performance liquid chromatography of the endogenous polyamines indicated that spermidine was the most predominant polyamine in the cultured cotyledons of radiata pine. Spermine increased by about 60% in the SF and 25% in the NSF cotyledons between days 0 and 3 of culture.     

Recovery of plastids from photooxidative damage: Significance of a plastidic factor

Glyoxysomal citrate synthase (gCS) was purified from crude extracts of watermelon (Citrullus vulgaris Schrad.) cotyledons, yielding a homogenous protein with a subunit MW of 48 kDa. The enzyme was selectively inhibited by 5,5-dithiobis-(2-nitrobenzoic acid), allowing quantification in the presence of the mitochondrial isoenzyme (mCS). Differences were also observed with respect to inhibition by ATP (k _i=2.6 mmol · l^-1 for gCS, k _i=0.33 mmol · l^-1 for mCS). The antibodies prepared against gCS did not cross-react with mCS. The immunocytochemical localization of gCS by the indirect protein A-gold procedure was restricted to the glyoxysomal membrane or the peripheral matrix of glyoxysomes. Other compartments, e.g. the endoplasmic reticulum, were not labeled. Xenopus oocytes were used for the translation of watermelon polyadenylated RNA (poly(A)⁺RNA). A translation product with a MW of 51 kDa was immunoprecipitated by the anti-gCS antibodies. It was absent in controls without poly(A)⁺RNA or with preimmune serum. A similar translation product was also immunoprecipitated after cell-free synthesis of watermelon poly(A)⁺RNA in a reticulocyte system, in contrast to the in-vivo labeled gCS (48 kDa). It was concluded that gCS is synthesized as a higher-molecular-weight precursor.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - gCS glyoxysomal citrate synthase - gMDH glyoxysomal malate dehydrogenase - k _i inhibitor constant - mCS mitochondrial citrate synthase - OAA oxaloacetate - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Alteration of the synthesis of lipoxygenase in the early stages of soybean cotyledon culture

A detailed study of lipoxygenase (EC 1.13.11.12) synthesis in cotyledons of soybean [ Glycine max (L.) Merr. cv. Century] cultured in vitro for up to 40 h showed that synthesis of this protein, measured by in vivo [35S]-methionine labelling in connection with immunological methods and cell-free translation of mRNA, underwent a large transient reduction in the first 4 h of culturing and gradually increased in the following 36 h. Northern blot hybridizations with lipoxygenase cDNA clones showed that the decrease in translational activity was the consequence of a considerable reduction in lipoxygenase mRNA in the cotyledons. From these results we conclude that the transient decline in lipoxygenase synthesis in excised soybean cotyledons is regulated at the RNA level. Similarly judged from the analysis of patterns of uni-dimensional gel electrophoresis, the synthesis of a few other polypeptides decreased during the first 4 h of culture as well, while several others increased; in cotyledons cultured for 20 to 40 h the protein-synthesis pattern had returned to that in freshly excised cotyledons. An acclimation period of ca 1 day seems to be needed for isolated soybean cotyledons to stabilize and to resume regular RNA and protein synthesis.     

Modulation of the venation pattern of cotyledons of transgenic tobacco for the tumorigenic <Emphasis Type="Italic">6b</Emphasis> gene of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> AKE10

Neoplastic plant-tissue formation, termed crown gall disease, is induced on infection with Agrobacterium tumefaciens. The tumorous tissues develop an extensive vascular system, with a venation pattern distinct from that of native host plants. We report here that the plant-tumorigenic 6b gene of the A. tumefaciens strain AKE10 is capable of inducing extensive vein formation in transgenic tobacco seedlings with distinct pattern formation. Unlike the wild-type cotyledons, transgenic cotyledons had wavy and striate veins depending on the extent of severity of leaf morphology. Graph analysis of the transgenic cotyledonous vein patterns revealed an increase in the number of branch points of veins, end-points of veins, and areas surrounded by the veins. Histological analysis showed abnormal tissue growth on the abaxial side of the cotyledon blades and continual formation of adventitious veins. These adventitiously formed veins included inverted dorso-ventrality and formation of a radial axis.