Development of Efficient, Reproducible and Stable Agrobacterium-Mediated Genetic Transformation of Five Potato Cultivars

The developments in transformation technology have enabled the scientists to incorporate, mutate or substitute gene(s) leading to a particular trait; advancing it to a point where only few technical limitations remain. Genotype dependency and explant types are important factors affecting transformation efficiency in potato. In the present study, a rapid, reproducible and stable Agrobacterium-mediated transformation procedure in potato was developed by a combination of different plant growth regulators. Leaf discs and internodal explants of five cultivars of potato, i.e. Lady Olympia, Granola, Agria, Désirée and Innovator were infected with Agrobacterium tumefaciens strain LBA4404 containing pBIN19 expression vector with β-glucuronidase gusA gene under the control of 35S CaMV promoter. Kanamycin was used as plant selectable marker for screening of primary transformants at concentration of 100 mg/L. Both explants responded positively; internode being more suitable explant for better transformation efficiency. Based on GUS histochemical assay, the transformation efficiency was 22, 20, 18.6, 15 and 10 % using the internodal explant, and 15, 12, 17, 8 and 6 % using leaf discs as explant in Lady Olympia, Granola, Agria, Désirée and Innovator respectively. Furthermore, PCR assays confirmed the presence of gusA and nptII genes in regenerated plants. The molecular analysis in succeeding progeny showed proper integration and expression of both genes. The results suggest Lady Olympia as the best cultivar for future transformation procedures. Overall, the short duration, rapidity and reproducibility make this protocol suitable for wider application of transgenic potato plants.

Addition of Multiple Introns to a Cas9 Gene Results in Dramatic Improvement in Efficiency for Generation of Gene Knockouts in Plants

The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by stably transforming a Cas9 expression construct into the plant genome. The efficiency with which mutations are obtained in genes of interest can vary considerably depending on specific features of the constructs, including the source and nature of the promoters and terminators used for expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency with which mutations could be obtained in target genes in Arabidopsis thaliana with the Cas9 nuclease, we have investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLS) and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on Cas9 activity and that two NLSs work better than one. However, the most important impact on the efficiency of the constructs was obtained by addition of 13 introns into the Cas9 coding sequence, which dramatically improved editing efficiencies of the constructs; none of the primary transformants obtained with a Cas9 lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of primary transformants generated with intronized Cas9 displayed mutant phenotypes. The intronized Cas9 was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

Translocation and duplication from CRISPR-Cas9 editing in Arabidopsis thaliana

Cut DNA ends in plants may recombine to form novel molecules. We asked whether CRISPR-Cas9 expression in plants could induce nonhomologous recombination between diverse and heterologous broken DNA ends. We induced two breaks separated by 2.3 or by 8.5 kilobases leading to duplication of the intervening DNA and meiotic transmission of the 2.3kb duplication. Two or more dsDNA breaks in nonhomologous chromosomes led to ligation of breakpoints consistent with chromosome arm translocations. Screening 881 primary transformants we obtained 195 PCR products spanning independent, expected translocation junctions involving ends produced by cutting different loci. Sequencing indicated a true positive rate of 84/91 and demonstrated the occurrence of different junction alleles. A majority of the resulting structures would be deleterious and none were transmitted meiotically. Ligation of interchromosomal, heterologous dsDNA ends suggest that the CRISPR-Cas9 can be used to engineer plant genes and chromosomes in vivo.Significance StatementWe explored how genome editing tools such as CRISPR-Cas9 could provide new ways to tailor novel genomic combinations and arrangements. We show that distant cut ends often precisely come together, that cuts in different chromosomes can result in translocations, and that two cuts within a chromosome often result in the duplication of the intervening segment. Formation of multiple structures with precise junctions will enable engineered rearrangements that can be predicted with accuracy.

Development of insect-resistant cotton lines with targeted expression of insecticidal gene

In order to address biosafety concerns regarding the constitutive expression of foreign genes in crops, we applied a strategy aimed at confining foreign gene expression in insect wounding sites of cotton. For this purpose, a plant expression construct was designed by cloning the AoPR1 promoter (pathogenesis-related protein gene isolated from Asparagus officinalis) upstream from the insecticidal gene cry1Ac. The Turkish cotton cultivar cv. STN-468 was transformed using the Agrobacterium tumefaciens strain LBA4404 containing the recombinant binary vector pRD400 harboring cry1Ac under a wound-inducible promoter. The neomycin phosphotransferase (nptII) gene was used as a selectable marker at a concentration of 100 mg/L. The primary transformants were analyzed for T-DNA integration and expression using standard molecular approaches. The efficacy of insecticidal gene control of the AoPR1 promoter was investigated using leaf bioassays with 2nd instar larvae of Helicoverpa armigera and Spodoptera littoralis. Positive primary transformants from T0 progeny were further raised under greenhouse conditions to obtain progeny (T1). The introduced gene was properly inherited and expressed in T1 progeny. The mechanical wounding of plants resulted in increased cry1Ac protein levels during 0-48 h of the wounding period. The transgenic lines exhibited appreciable levels of resistance against targeted insect pests in the leaf bioassays. The use of a wound-inducible promoter to drive insecticidal gene expression is a valuable insect resistant management strategy as gene expression will remain limited to the insect biting sites of plant and crop, food and environmental concerns can be minimized.

Transgenic expression of sorghum DREB2 in rice improves tolerance and yield under water limitation

SUMMARYPlant-specific transcription factors belonging to the dehydration response element binding (DREB)/C-repeat binding factor (CBF) subfamily of the AP2/EREBP family specifically interact with dehydration-responsive elements (DRE)/C-repeat (CRT) and control the expression of many stress-inducible genes in plants. Two major subgroups of DREB proteins are represented by DREB1 and DREB2, which are induced specifically under cold and drought/salt stress, respectively. A DREB2 transcription factor gene from sorghum, SbDREB2 was identified and cloned in binary vectors, such that it was driven either by a constitutive CaMV35S promoter or a stress-inducible rd29A promoter. These gene constructs were transferred into rice through Agrobacterium tumefaciens-mediated transformation. Expression patterns of the native DREB gene (OsDREB2) and the transgene (SbDREB2) were similar. Both genes showed induction at 1 h exposure to drought, after which expression gradually dropped to basal levels by 24 h. Constitutive expression of SbDREB2 led to pleiotropic effects in rice and these transgenics did not set seed. The rd29A: SbDREB2 rice plants set seed and the grains collected from primary transformants were sown to raise T1 plants. The drought-stressed rd29A: SbDREB2 transgenics showed a significantly higher number of panicles as compared to the wild-type rice plants. Other phenological and agronomic traits were not affected in wild-type and rd29A: SbDREB2 transgenic rice.

Improved Cucumber Transformation by a Modified Explant Dissection and Selection Protocol

Agrobacterium-mediated transformation of Cucumis sativus L. cotyledons was investigated, to identify important factors that affect transformation efficiency. The factors evaluated were initial explant preparation, including preculture, pricking of the explant, inoculation time and co-cultivation regime. We also modified the selection regime, compared to previously published protocols. Our results show that dissecting the proximal half of the cotyledon by a V-shaped cut resulted in a higher transformation rate, compared to two other methods of dissection, as indicated by transient β-glucuronidase gene expression. Selection on 100 mg·L-1 kanamycin resulted in the early development of nontransformed shoots, whereas a gradual increase of kanamycin concentration up to 200 mg·L-1 resulted in the subsequent formation of transgenic shoots on the same medium. The overall transformation frequency in these experiments, expressed as the number of rooted, confirmed transgenic plants per initial number of explants, was 1.7%. The stable integration of T-DNA was confirmed in the primary transformants and their progeny. Abbreviations: ABA = abscisic acid; AdS = adenine sulfate; AS = acetosyringone; BA = 6-benzylaminopurine; GA3 = gibberellic acid; IBA = indolebutyric acid.

Pathogen-derived Resistance to Tomato Spotted Wilt Virus in Transgenic Tomato and Tobacco Plants

This study was undertaken to remedy significant yield losses in commercial tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana tabacum L.) production caused by tomato spotted wilt virus (TSWV). One of the possible sources of resistance can be incorporation into the host plant of a viral nucleoprotein (N) gene by Agrobacterium-mediated transformation. Twelve primary transformants of tomato and 141 of tobacco were analyzed for the expression of the N gene and for resistance to the TSWV infection. The tests have demonstrated that transgenic plants were protected against virus infection irrespective of whether or not they contained detectable levels of the translational product.

p16Ink4a Interferes with Abelson Virus Transformation by Enhancing Apoptosis

ABSTRACT Pre-B-cell transformation by Abelson virus (Ab-MLV) is a multistep process in which primary transformants are stimulated to proliferate but subsequently undergo crisis, a period of erratic growth marked by high levels of apoptosis. Inactivation of the p53 tumor suppressor pathway is an important step in this process and can be accomplished by mutation of p53 or down-modulation of p19Arf, a p53 regulatory protein. Consistent with these data, pre-B cells from either p53 or Ink4a/Arf null mice bypass crisis. However, the Ink4a/Arf locus encodes both p19Arf and a second tumor suppressor, p16Ink4a, that blocks cell cycle progression by inhibiting Cdk4/6. To determine if p16Ink4a plays a role in Ab-MLV transformation, primary transformants derived from Arf −/− and p16 Ink4a−/− mice were compared. A fraction of those derived from Arf −/− animals underwent crisis, and even though all p16 Ink4a−/− primary transformants experienced crisis, these cells became established more readily than cells derived from +/+ mice. Analyses of Ink4a/Arf −/− cells infected with a virus that expresses both v-Abl and p16Ink4a revealed that p16Ink4a expression does not alter cell cycle profiles but does increase the level of apoptosis in primary transformants. These results indicate that both products of the Ink4a/Arf locus influence Ab-MLV transformation and reveal that in addition to its well-recognized effects on the cell cycle, p16Ink4a can suppress transformation by inducing apoptosis.

The Absence of Msh2 Alters Abelson Virus Pre-B-Cell Transformation by Influencing p53 Mutation

ABSTRACT Defects in DNA mismatch repair predispose cells to the development of several types of malignant disease. The absence of Msh2 or Mlh1, two key molecules that mediate mismatch repair in eukaryotic cells, increases the frequency of mutation and also alters the response of some cells to apoptosis and cell cycle arrest. To understand the way these changes contribute to cancer predisposition, we examined the effects of defective mismatch repair on the multistep process of pre-B-cell transformation by Abelson murine leukemia virus. In this model, primary transformants undergo a prolonged apoptotic crisis followed by the emergence of fully transformed cell lines. The latter event is correlated to a loss of function of the p53 tumor suppressor protein and down-modulation of the p53 regulatory protein p19Arf. Analyses of primary transformants from Msh2 null mice and their wild-type littermates revealed that both types of cells undergo crisis. However, primary transformants from Msh2 null animals recover with accelerated kinetics, a phenomenon that is strongly correlated to the appearance of cells that have lost p53 function. Analysis of the kinetics with which p53 function is lost revealed that this change provides the dominant stimulus for emergence from crisis. Therefore, the absence of mismatch repair alters the molecular mechanisms involved in transformation by affecting a gene that controls apoptosis and cell cycle progression, rather than by affecting these processes directly.

Cloning maize telomeres by complementation in Saccharomyces cerevisiae

Maize telomeric restriction fragments were cloned by virtue of their ability to function as telomeres on a linear plasmid in Saccharomyces cerevisiae. Nine maize telomeric YAC transformants (MTYs) were selected by hybridization to the Arabidopsis telomere repeat (CCCTAAA) from a pool of 1537 primary transformants. Bal31 digestion of MTY3 and MTY9 DNA indicated that the telomere hybridizing tracts are located at the terminus of the linear chromosome and therefore function as telomeres in yeast. Subclones generated for pMTY7 (pMTY7SC1) and pMTY9 (pMTY9ER) hybridized to Bal31 sensitive restriction fragments in maize DNA, indicating that maize telomeric restriction fragments had been cloned. Both pMTY7SC and pMTY9ER detected telomeric RFLPs, allowing the endpoints of seven chromosome arms to be determined. Additionally, pMTY7ER mapped to the centromeric regions of chromosomes 2 and 3, suggesting a relationship between centromeric and telomeric sequences. DNA sequencing of pMTY7SC and pMTY9ER revealed that both subclones contained CA-rich regions with sporadic occurrences of the telomere repeat and its degenerate repeats. Key words : maize, telomere, RFLP, telomeric.