A positive experience in applying the biolistic approach to potato varieties Aksor and Nevskiy

The method of biological ballistics (biolistic transformation, genetic bombardment) of plants is one of the most modern methods used for direct gene transfer into plant cells. The main advantages of this method include the ability to simultaneously incorporate several target genes into the plant genome, carry out transfer without unnecessary agrobacterial parts and plasmid DNA sequences, and the short time needed to produce transgenic cells. For different plant objects, the efficiency of obtaining transgenic plants by the ballistic method varies from 1 to 3 %. For potato plants, the transformation efficiency is quite low at the moment and the selection of optimal conditions for biolistics is one of the pressing issues of practical biotechnology. This article presents a successful experience of introducing two genes of interest into two potato varieties using the biolistic approach. The results of biolistic transformation experiments are presented for two types of explants: potato internodes and calli of the varieties Aksor and Nevskiy. Of the 862 explants used for transformation, 56 regenerated plants were obtained. PCR screening of transformants revealed one plant with the insertion of the chitinase gene, one with the insertion of the endo-β-1,3-glucanase gene, and co-transformation by both genes was confirmed in four regenerants. The average transformation efficiency for potato explants was 0.7 %. A high number of regenerants (56) as opposed to a low number of transformants (6) reflects an attempt to increase the number of regenerants by using a lower concentration of the selective agent (kanamycin). Although this approach requires more effort, it can be used to produce potato lines with integrated genes of interest for further use in crop breeding. The lines of potato obtained in the current study by introducing two genes associated with the plant response to fungal pathogens will be further assessed for their resistance to fungal diseases and, if successful, will be used in potato crop breeding.

In vitro cultivation, aquaculture and methods of transformation of water caltrop Trapa L.

In vitro and aquarium cultures of water caltrop were obtained. Optimal conditions of cultivation were determined. The cultures were subjected to Agrobacterium rhizogenes-mediated and biolistic transformation.

Biolistic transformation of plants using CRISPR/Cas9 technology

Experiments on CRISPR/Cas9 mediated knock-in approaches in the PDS gene of various genes of interest were planned. Biolistic bombardment mediated delivery of target vectors to plant explants was suggested.

CISGENIC BIOLISTIC TRANSFORMATION FOR OBTAINING NEW FORMS OF POTATOES WITH IMPROVED RESISTANCE TO LATE BLIGHT

This article presents the results of application of cisgenic biolistic transformation for the accelerated production of new forms of potato with increased resistance to late blight. The reason for late blight development is the parasitic organism Phytophthora infestans, belonging to oomycetes (pseudo-fungi), which infects valuable agricultural plants. In this study, with the aim of combating P. infestans, a number of experiments on the biolistic transformation of the most common potato varieties Aksor and Nevskiy were carried out in Kazakhstan. Two potato genes – Rpi-vnt1.1 and StREM1.3 – were selected as targets for introduction. Expression of the first gene should be activated, and the expression of the REMORIN1.3 gene should be suppressed. Rpi-vnt1.1 was under the control of Solanum tuberosum polyubiquitin gene promotor (Pat) and Arabidopsis thaliana polyubiquitin 5 gene terminator (ubq5). Knock-down double stranded RNA-hairpin gene construction for StREM1.3 silencing was under the control of Solanum tuberosum phytochrome B gene promotor (phyB) and Arabidopsis thaliana hot-shock protein 18.2 terminator (HSP18.2). Three series of biolistic transformation were carried out, as a result of which 636 regenerated plants of potato varieties Aksor and Nevskiy were obtained. DNA was extracted from the plant material of potato transformant plants in the quality and quantity suitable for PCR analysis for the presence of an insert. PCR analysis was carried out, revealing 52 plants carrying the VNT insert. StREM1.3 silencing gene construction was detected in plant lines by qPCR, based on comparative analysis of of gene expression level and revealed 6 lines with reliably lower StREM1.3 expression level in comparison with wild-type plants.

Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation

Background: Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. Objective: In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. Methods: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. Results: PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. Conclusion: The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.

Highly Efficient Biolistic Transformation of Embryogenic Cell Suspensions of Banana Via a Liquid Medium Selection System

An efficient biolistic transformation system of banana combined with a liquid medium selection system was developed during this study. An embryogenic cell suspension (ECS) of Musa acuminata cv. Baxi (AAA) was bombarded with a particle delivery system. After 7 days of restoring culture in liquid M2 medium, embryogenic cells were transferred to a liquid selection M2 medium supplemented with 10 μg/mL hygromycin for resistance screening. The untransformed cell clusters were inhibited or killed, and a small number of transformants proliferated in the liquid selection medium. After the 0th, first, second, and third generation of antibiotic screening, there were 0, 65, 212, and 320, respectively, vitality-resistant buds obtained from a 0.5-mL packed cell volume (PCV) of embryogenic cell suspension. The β-glucuronidase (GUS) staining, polymerase chain reaction (PCR) analysis, and Southern blot hybridization results all demonstrated a 100% positive rate of regenerated resistant seedlings. Interestingly, the number of buds obtained through third-generation screening was almost equal to that obtained from the original ECS in M2 medium without antibiotics. These results suggested that the liquid medium selection system facilitated the proliferation of a positive transgenic ECS, which significantly improved the regeneration rate of transformants. This protocol is suitable for the genetic transformation of all banana genotypes and is highly advantageous to varieties with low callusing potential.