The gene TaCB1 overcomes genotype dependency in wheat genetic transformation

Genotype dependency is the most important factor in wheat genetic transformation, which further limits wheat improvement by transgenic integration and genome editing approaches. The application of regeneration related genes during in vitro culture could potentially contribute to enhancement of plant transformation efficiency. In the present study, a wheat gene TaCB1 in the WUSCHEL family was identified to dramatically increase the transformation efficiencies of many wheat varieties without genotype dependency after its over-expression. The expression of TaCB1 in wheat calli did not prohibit shoot differentiation and root development. The application of TaCB1 can lighten the requirement to wheat immature embryo for plant regeneration. Transgenic wheat plants can be clearly recognized by the visible phenotype of wide flag leaves. The promise function of TaCB1 on improving transformation efficiency was also tested in T. monococcum, triticale, rye, barley, and maize.

Construction of Potato DM1-3-516-R44 (DM) Transgenic System Based on Agrobacterium Transformation

Background: The sequencing potato DM1-3-516-R44 played an irreplaceable role in the study of gene function. So far, no one research the transformation system about DM. Therefore, our experiment was studied from three aspects: plant regeneration system, optimization of agrobacterium infection conditions and the effect of hygromycin on DM. Results: A relatively suitable method for genetic transformation of DM was obtained: 1) The stem callus induction medium was MS + IAA 0.5 mg/L + 6-BA 2.0 mg/L, the leaf callus induction medium was MS + NAA 1.0mg/L + 6-BA 0.5mg/L and the shoot differentiation medium was MS + 6-BA 3.0 mg/L + ZT 0.5 mg/L. 2) The specific transformation condition was the agrobacterium concentration kept the OD600 = 0.3, and co-culture time consisted 3 days in the dark. The hygromycin concentration chose 8 mg/L to screen the transgenic plants. 3) Using hygromycin to screen about 100 transgenic shoots, 75 shoots were obtained and 53 strains were identified had target stripe by PCR technology. Conclusion: The efficiency of the transformation system we created was over 50%. It provided a good basis for the study of potato gene function.

Identification of Shoot Differentiation-Related Genes in Populus euphratica Oliv.

De novo shoot regeneration is one of the important manifestations of cell totipotency in organogenesis, which reflects a survival strategy organism evolved when facing natural selection. Compared with tissue regeneration, and somatic embryogenesis, de novo shoot regeneration denotes a shoot regeneration process directly from detatched or injured tissues of plant. Studies on plant shoot regeneration had identified key genes mediating shoot regeneration. However, knowledge was derived from Arabidopsis; the regeneration capacity is hugely distinct among species. To achieve a comprehensive understanding of the shoot regeneration mechanism from tree species, we select four genetic lines of Populus euphratica from a natural population to be sequenced at transcriptome level. On the basis of the large difference of differentiation capacity, between the highly differentiated (HD) and low differentiated (LD) groups, the analysis of differential expression identified 4920 differentially expressed genes (DEGs), which were revealed in five groups of expression patterns by clustering analysis. Enrichment showed crucial pathways involved in regulation of regeneration difference, including “plant hormone signal transduction”, “cell differentiation”, "cellular response to auxin stimulus", and “auxin-activated signaling pathway”. The expression of nine genes reported to be associated with shoot regeneration was validated using quantitative real-time PCR (qRT-PCR). For the specificity of regeneration mechanism with P. euphratica, large amount of DEGs involved in "plant-pathogen interaction", ubiquitin-26S proteosome mediated proteolysis pathway, stress-responsive DEGs, and senescence-associated DEGs were summarized to possibly account for the differentiation difference with distinct genotypes of P. euphratica. The result in this study helps screening of key regulators in mediating the shoot differentiation. The transcriptomic characteristic in P. euphratica further enhances our understanding of key processes affecting the regeneration capacity of de novo shoots among distinct species.

Plantlet Regeneration from Seedling Explants of Sweet Gourd

To regenerate plantlets both directly and indirectly, different explants (cotyledon, hypocotyl, root tip and shoot tip) of sweet gourd (Cucurbita moschata) were cultured on medium supplemented with different concentrations and combinations of NAA (0.0, 0.2, 0.5 and 1.0 mg/L) and BAP (0.0, 0.5, 2.0 and 5.0 mg/L). Cotyledon explants performed best in callus induction. The combination of 5 mg/L BAP and 0.2 mg/L NAA produced the highest callus frequency in cotyledon (90.0%). The calli derived from cotyledon, hypocotyl and root tip were cultured in MS medium supplemented with different concentrations of kinetin, BAP and/or NAA for shoot induction. Regeneration via callus was achieved only from cotyledon calli at a frequency of 65.0% on 2 mg/L BAP and 0.2 mg/L NAA. Shoot tips cultured for direct regeneration, in the same media containing 1 mg/L BAP or 1 mg/L BAP + 0.2 mg/L NAA resulted in 100.0% shoot differentiation. The regenerated shoots rooted on MS medium with or without 0.1 mg/L NAA (100%).DOI: http://dx.doi.org/10.3329/pa.v18i2.18093 Progress. Agric. 18(2): 61 - 66, 2007

Utilization of Aseptic Seedling Explants for In vitro Propagation of Indian Red Wood

Micropropagation has been advocated as one of the most viable biotechnological tool for ex situ conservation of rare, endangered endemic medicinal plants germplasm. Rapid clonal micropropagation protocol for large-scale multiplication of an endemic medicinal plant Soymida febrifuga (Meliaceae) was established from 15-day aseptic seedling cotyledonary node and shoot tip explants. High frequency of sprouting and shoot differentiation was observed from cotyledonary node explants compared to shoot tip, on Murashige and Skoog (MS) medium fortified with BA, KN, 2-iP and CM. Of the cytokinins used, BA (3.0 mgl-1) supported highest average number and maximum multiple shoot differentiation (16.6). In vitro proliferated shoots were multiplied rapidly by culturing nodal segments as microcuttings, further subcultured on the same media for elongation. Elongated shoots upon transfer to MS medium fortified with IBA showed rooting within two weeks of culture. Rooted plantlets were successfully hardened and 75% of rooted shoots successfully survived on establishment to the soil. Plants looked healthy with no visually detectable phenotypic variations. This protocol provides a successful and rapid technique that can be used for ex situ conservation minimizing the pressure on wild populations and contributes to the conservation of this endemic medicinally potent flora.

In vitro Regeneration of Mungbean (Vigna radiata L.) from Different Explants

To regenerate plantlets both directly and indirectly, different explants (cotyledon, hypocotyls, root tip and shoot tip) of mungbean were cultured on medium supplemented with different concentrations and combinations of BAP (0, 1.0 and 5.0 mgL-1) and NAA (0, 0.5 and 2.5 mgL-1)). Cotyledon explants performed best in callus induction (90.0%) at the combination of 1 mgL-1) BAP and 2.5 mgL-1)NAA for both the varieties (BINA mung 5 and BINA mung 7). The calli derived from cotyledon, hypocotyls and root tip were cultured in MS medium supplemented with different concentrations of Kn, BAP and/or NAA for shoot induction. Regeneration was achieved only from cotyledon calli at a frequency of 62.50% on 5 mgL-1) BAP and 0.05 mgL-1) NAA. Shoot tip cultured for direct regeneration in the same media containing 5 mgL-1) BAP and 0.05 mgL-1) NAA resulted in 90.0% shoot differentiation in BINA mung 7. The regenerated shoots rooted on MS medium with 0.2 mgL-1) NAA (90.0).DOI: http://dx.doi.org/10.3329/pa.v19i2.16908 Progress. Agric. 19(2): 13 - 19, 2008