Overexpression Populus d-Type Cyclin Gene PsnCYCD1;1 Influences Cell Division and Produces Curved Leaf in Arabidopsis thaliana

d-type cyclins (CYCDs) are a special class of cyclins and play extremely important roles in plant growth and development. In the plant kingdom, most of the existing studies on CYCDs have been done on herbaceous plants, with few on perennial woody plants. Here, we identified a Populus d-type cyclin gene, PsnCYCD1;1, which is mainly transcribed in leaf buds and stems. The promoter of PsnCYCD1;1 activated GUS gene expression and transgenic Arabidopsis lines were strongly GUS stained in whole seedlings and mature anthers. Moreover, subcellular localization analysis showed the fluorescence signal of PsnCYCD1;1-GFP fusion protein is present in the nucleus. Furthermore, overexpression of the PsnCYCD1;1 gene in Arabidopsis can promote cell division and lead to small cell generation and cytokinin response, resulting in curved leaves and twisted inflorescence stems. Moreover, the transcriptional levels of endogenous genes, such as ASs, KNATs, EXP10, and PHB, were upregulated by PsnCYCD1;1. Together, our results indicated that PsnCYCD1;1 participates in cell division by cytokinin response, providing new information on controlling plant architecture in woody plants.

In Planta Genetic Transformation of Mungbean (Vigna radiata (L.) Wilczek) with Marker Gene

In genetic improvement of mungbean much success has not been achieved due to its recalcitrant nature towards in vitro regeneration. An attempt was made to develop an Agrobacterium-mediated in planta genetic transformation protocol for a locally grown mungbean variety BARI Mung-3 using a screenable marker gene. Two minutes of vacuum infiltration followed by 60 minutes of incubation period in Agrobacterium suspension of Winans’ AB medium containing wounded tobacco leaf extract was found most suitable towards genetic transformation in pricked de-coated half seed explants. An optical density (OD600) of 0.7 was found most effective for transient gus gene expression. Chimeric GUS expression was observed in the root and leaf tissues from the successfully transformed plantlets obtained through in planta transformation. This methodology of genetic transformation was found more suitable, easier and less time consuming than tissue culture based genetic transformation, which may be used for the genetic improvement of mungbean.

Transformation and confirmation of GUS gene expression in Solanum melongena L. of PLR 1 cultivar

In the present study GUS gene transformation was carried out in eggplant using Agrobacterium strain with pBAL2 vector harboring gus gene and nptII as selection marker gene. The factors which are affecting (enhancing) the frequency of transient gus gene expression are different physical and biochemical variables has been carried out. It is observed that the 4 day precultured explants showed the minimum survival rate in the medium when compared with 2-day co cultivated medium. The explants which had undergone co-cultivation for 4 to 5 days showed GUS activity, the tissues were adversely affected due to the overgrowth of bacteria. The gene specific primers for nptII and gus gene were used for amplification and it has given 680bp and 1.9 kb amplified fragments respectively and recorded. The band was detected in the selected plants, but it was absent from the negative control (non-transformed) plant in the Southern hybridization. Our experiment showed 0.80-1.60 percentage of efficiency in transformation. With a total of 849 infected shoots were undergone confirmation tests which results 9 PCR positives (1.06% efficiency). The Transformant kept in the Environmental Growth Chamber and transferred to field condition subsequently.

OPTIMIZATION OF FACTORS AFFECTING THE Agrobacterium tumefaciens- MEDIATED TRANSFORMATION OF Eucalyptus saligna

ABSTRACT This study aimed to evaluate the effect of factors that may affect the genetic transformation of cotiledonary explants of Eucalyptus saligna mediated by EHA105 strain of Agrobacterium tumefaciens. The vector pBI121 carrying gus gene under control of 35S CaMV promoter was used. The effect of the following factors was evaluated: explant pre-culture, use of different antibiotics and presence of acetosyringone (AS) in co-culture media. An antioxidant solution was also used during excision, containing ascorbic acid (250mg.L-1), citric acid (25mg.L-1) and PVP-40 (1g.L-1). Pre-culture of the explants before the co-culture with bacteria was done over a 4-day period in MS culture medium supplemented with 4.4µM BAP and 2.7ìM NAA. After theco-culture period, three concentrations of kanamycin (12.5;25 and 50mg.L-1) combined with 300mg.L-1 Augmentin® in the culture medium were tested The influence of the antibiotic was also evaluated by keeping the explants in a medium containing 50mg.L-1 Km and 300mg.L-1 Augmentin® or 500mg.L-1 cefotaxime. It was concluded that Augmentin® stimulates organogenesis, that a Km concentration of 12.5mg.L-1 allows selection of explants transformed with gus gene and, finally, the addition of AS (50ìM) to the liquid and solid co-culture media has a positive effect on gus gene expression. Moreover, the use of an antioxidant solution during cotyledon excision is dispensable and the pre-culture of the explants has no effect on bud regeneration or gus gene expression. A transformation efficiency of 1.5% was reached.

Genetic transformation of Populus nigra X P. deltoides (black poplar, clone Gradizka)

Aim. To carry out genetic transformation of poplar Populus nigra x P. deltoides clone Gradizka with the model gene construct pCB002 carrying selective gene of kanamycin resistance and marker gene of β-glucuronidase. Methods. Genetic transformation was performed with the using leaf, stem and petiole poplar explants. Transformants were selected on the medium with kanamycin, and transgene was identified by polymerase chain reaction (PCR) and histochemical GUS assay. Results. Successful transformants selected on kanamycin media were confirmed by the presence of PCR-product for the gene nptII with the length 700 bp, and gus gene expression was also observed. Conclusions. Protocol for genetic transformation of P. nigra x P. deltoides clone Gradizka established here will be used for poplar genetic modification to create new clones with commercially important traits. Keywords: genetic transformation, Populus sp., microclonal propagation.

Transformasi Gen Antisens ACC Oksidase pada Pepaya dengan Teknik Penembakan Partikel

Papaya (Carica papaya L.) is a climacteric fruit that exhibit a<br />very fast ripening rate. Ethylene controls the ripening event<br />in the papaya fruit. 1-aminocyclopropane-1-carbocxylic acid<br />(ACC) oxidase gene encodes a specific enzyme for ethylene<br />biosynthesis. The gene had become a target for manipulation<br />to make a gene construct of an antisense ACC oxidase<br />to regenerate transgenic papaya that has a characteristic of<br />delayed ripening. The objective of the experiment is to engineer transgenic papaya that has a delayed ripening characteristic by transforming papaya with the antisense ACC oxidase gene through particle bombardment technique. The immature embryos of papaya variety Burung<br />were used for the explants. Antisense ACC oxidase and reporter (gus) genes were co-transformed to papaya calli. Four hundreds eighteen calli were bombarded by the antisense ACC oxidase gene. The transformation experiment resulted 25 putatives transgenic plants out of fifty plants<br />acclimatized in a greenhouse. Gus gene expression assay observed at 9 days after bombardment showed that the papaya explants bombarded twice at 9 cm shoot distance had 53.3% transformation rate of gus positive and 5.25 blue spots number in average. The results of PCR analysis showed that four out of 25 transgenic putative papaya plants (TR6, TR9, TR20, dan TR24), indicated a positive PCR of the antisense ACC oxidase gene with the amplified fragment DNA size of 800 base pair.

Expression and Promoter Analysis of Six Heat Stress-Inducible Genes in Rice

During the long evolutionary process, plant gradually formed a series of strategies and mechanisms to cope with stress environment such as drought, heat, cold, and high salinity. Six highly heat responsive genes were identified in rice by microarray data analysis. The qRT-PCR analysis confirmed that the expression of these six genes were highly heat inducible and moderately responded to salt stress, polyethylene glycol, and abscisic acid treatment, but little affected by cold treatment. Promoters of the three highly heat-inducible genes (OsHsfB2cp, PM19p, and Hsp90p) were used to drive GUS gene expression in rice. The results of the GUS gene expression, histochemical staining, and GUS activities in panicles and flag leaves of the transgenic rice plants confirmed high heat-induced GUS activities and moderate drought-induced activities. The three promoters exhibited similar high activity lever in rice leaf under heat, but OsHsfB2cp and PM19p showed much higher activities in panicles under heat stress. Our work confirmed that the OsHsfB2c and PM19 promoters were highly heat inducible and further characterization and reconstruction ofcis-elements in their promoters could lead to the development of highly effective heat-inducible promoters for plant genetic engineering.