Efficient Genetic Transformation Protocol Research Articles

An efficient transformation method for genome editing of elite bread wheat cultivars.

An efficient genetic transformation protocol is necessary to edit genes for trait improvement directly in elite bread wheat cultivars. We used a protein fusion between a wheat growth-regulating factor 4 (GRF4) and its interacting factor (GIF1) to develop a reproducible genetic transformation and regeneration protocol, which we then used to successfully transform elite bread wheat cultivars Baj, Kachu, Morocco, Reedling, RL6077, and Sujata in addition to the experimental cultivar Fielder. Immature embryos were transformed with the vector using particle bombardment method. Transformation frequency increased nearly 60-fold with the GRF4-GIF1-containing vectors as compared to the control vector and ranged from ~5% in the cultivar Kachu to 13% in the cultivar RL6077. We then edited two genes that confer resistance against leaf rust and powdery mildew directly in the aforementioned elite cultivars. A wheat promoter, TaU3 or TaU6, to drive the expression of guide RNA was effective in gene editing whereas the OsU3 promoter failed to generate any edits. Editing efficiency was nearly perfect with the wheat promoters. Our protocol has made it possible to edit genes directly in elite wheat cultivars and would be useful for gene editing in other wheat varieties, which have been recalcitrant to transformation thus far.

Efficient Genetic Transformation of Rice for CRISPR/Cas9 Mediated Genome-Editing and Stable Overexpression Studies: A Case Study on Rice Lipase 1 and Galactinol Synthase Encoding Genes

Rice is a staple food crop for almost half of the world’s population, especially in the developing countries of Asia and Africa. It is widely grown in different climatic conditions, depending on the quality of the water, soil, and genetic makeup of the rice cultivar. Many (a)biotic stresses severely curtail rice growth and development, with an eventual reduction in crop yield. However, for molecular functional analysis, the availability of an efficient genetic transformation protocol is essential. To ensure food security and safety for the continuously increasing global population, the development of climate-resilient crops is crucial. Here, in this study, the rice transformation protocol has been effectively optimized for the efficient and rapid generation of rice transgenic plants. We also highlighted the critical steps and precautionary measures to be taken while performing the rice transformation. We further assess the efficacy of this protocol by transforming rice with two different transformation constructs for generating galactinol synthase (GolS) overexpression lines and CRISPR/Cas9-mediated edited lines of lipase (Lip) encoding the OsLip1 gene. The putative transformants were subjected to molecular analysis to confirm gene integration/editing, respectively. Collectively, the easy, efficient, and rapid rice transformation protocol used in this present study can be applied as a potential tool for gene(s) function studies in rice and eventually to the rice crop improvement.

Genetic Transformation of Apomictic Grasses: Progress and Constraints.

The available methods for plant transformation and expansion beyond its limits remain especially critical for crop improvement. For grass species, this is even more critical, mainly due to drawbacks in in vitro regeneration. Despite the existence of many protocols in grasses to achieve genetic transformation through Agrobacterium or biolistic gene delivery, their efficiencies are genotype-dependent and still very low due to the recalcitrance of these species to in vitro regeneration. Many plant transformation facilities for cereals and other important crops may be found around the world in universities and enterprises, but this is not the case for apomictic species, many of which are C4 grasses. Moreover, apomixis (asexual reproduction by seeds) represents an additional constraint for breeding. However, the transformation of an apomictic clone is an attractive strategy, as the transgene is immediately fixed in a highly adapted genetic background, capable of large-scale clonal propagation. With the exception of some species like Brachiaria brizantha which is planted in approximately 100 M ha in Brazil, apomixis is almost non-present in economically important crops. However, as it is sometimes present in their wild relatives, the main goal is to transfer this trait to crops to fix heterosis. Until now this has been a difficult task, mainly because many aspects of apomixis are unknown. Over the last few years, many candidate genes have been identified and attempts have been made to characterize them functionally in Arabidopsis and rice. However, functional analysis in true apomictic species lags far behind, mainly due to the complexity of its genomes, of the trait itself, and the lack of efficient genetic transformation protocols. In this study, we review the current status of the in vitro culture and genetic transformation methods focusing on apomictic grasses, and the prospects for the application of new tools assayed in other related species, with two aims: to pave the way for discovering the molecular pathways involved in apomixis and to develop new capacities for breeding purposes because many of these grasses are important forage or biofuel resources.

Establishment of Codonopsis pilosula (Franch.) Nannf. transformed roots, influence of the culture conditions on root growth and production of essential oil

Codonopsis pilosula (Franch.) Nannf. is a species whose radix has long been used in traditional folk medicine in China, Japan and Korea. Codonopsis Radix is obtained from cultivations mainly in Gansu, Shanxi, Sichuan, and Guizhou Provinces of China. This study describes an efficient protocol for genetic transformation of C. pilosula by Rhizobium rhizogenes and the obtainment of transformed roots of this species with valuable medicinal properties. It was found that both the bacterial strain (A4 and ATCC 15834) and acetosyringone (AcS) addition influenced the efficiency of transformation. The highest frequency of root induction (69 %) was observed on the Murashige and Skoog (MS) agar medium supplemented with AcS after transformation with A4 strain. The transformation was confirmed by PCR analysis. Of the tested media (MS, Schenk and Hildebrandt - SH, Gamborg - B5, and Woody Plant - WPM) those with full macro- and microsalt concentrations elicited significantly greater C. pilosula transformed root (CP2) biomass accumulation than the half-strength media, apart from MS medium. The greatest CP2 root biomass was observed in SH medium (1.65 g flask−1 DW). Among the tested sucrose concentrations (10−70 g L−1), the best result was obtained by using 30 g L−1 sucrose. Essential oils isolated by hydrodistillation from CP2 transformed roots and commercially available raw material, C. pilosula roots, had very similar qualitative composition. The most important group of common constituents of both oils were sesquiterpene hydrocarbons. The main among twenty two compounds in this group possessed skeletons of barbatane, thujopsane and bisabolane; the predominant ones being α- and β-barbatene, followed by β-chamigrene and thujopsene. This study presented that C. pilosula transformed roots can be a potential source for the industrial production of the raw material from species of the genus Codonopsis used in traditional Chinese medicine.

Assessment of biolistic and Agrobacterium-mediated genetic transformation methods in Cenchrus ciliaris

Cenchrus ciliaris, commonly known as buffelgrass, is an apomictic perennial range grass usually grown in arid/semi-arid regions. Because of the difficulties faced in conventional breeding of this polymorphic polyploid grass, the development of an efficient protocol for genetic transformation is warranted. Such a protocol would enable functional genomic studies needed to elucidate the mechanism of apomixis in this species. The embryogenic calli for genetic transformation experiments were obtained by in vitro culture of the immature inflorescences of buffelgrass cv. IGFRI-3108. Four developmental stages of immature inflorescences were compared for callus induction, and the most mature stage produced most callus. Embryogenic calli were bombarded at three distances, 6 cm (L1), 9 cm (L2), or 12 cm (L3) with a marker gene uid A present in pCAMBIA1301, under the same vacuum (85 kPa) and at constant pressure (900 psi). The Agrobacterium-mediated genetic transformation was also performed using the same construct. Transient and stable expression as well as PCR amplification of the GUS gene was used for comparative analysis as well as for validation of the transformants. Transient GUS expression was present in a significantly higher percentage of bombarded calli (56.33%) than of Agrobacterium treated calli (11.17%), but the number of GUS positive cells per callus was similar. Among the three different bombardment distances, transient GUS expression was highest at L2, but stable GUS expression was highest at L1. Shoot development from the bombarded calli could be accomplished, which failed from the Agrobacterium-mediated transformed calli. Thus, the results indicate that C. ciliaris cv. IGFRI-3108 can be successfully transformed through Biolistic particle bombardment, while Agrobacterium-mediated transformation requires further optimization of transformation protocols.

Genetic Transformation in Peach (Prunus persica L.): Challenges and Ways Forward.

Almost 30 years have passed since the first publication reporting regeneration of transformed peach plants. Nevertheless, the general applicability of genetic transformation of this species has not yet been established. Many strategies have been tested in order to obtain an efficient peach transformation system. Despite the amount of time and the efforts invested, the lack of success has significantly limited the utility of peach as a model genetic system for trees, despite its relatively short generation time; small, high-quality genome; and well-studied genetic resources. Additionally, the absence of efficient genetic transformation protocols precludes the application of many biotechnological tools in peach breeding programs. In this review, we provide an overview of research on regeneration and genetic transformation in this species and summarize novel strategies and procedures aimed at producing transgenic peaches. Promising future approaches to develop a robust peach transformation system are discussed, focusing on the main bottlenecks to success including the low efficiency of A. tumefaciens-mediated transformation, the low level of correspondence between cells competent for transformation and those that have regenerative competence, and the high rate of chimerism in the few shoots that are produced following transformation.

Development of a Virus-Induced Gene Silencing System for Dioecious Coccinia grandis.

Coccinia grandis is an interesting model system to understand dioecy in Cucurbitaceae family. Recent transcriptomics and proteomics studies carried out to understand the sex expression in C. grandis have resulted in identification of many candidate sex-biased genes. In absence of an efficient genetic transformation protocol for C. grandis, virus-induced gene silencing (VIGS) would be a powerful tool to enable gene functional analysis. In current study, we explored the apple latent spherical virus (ALSV) for gene knockdown in C. grandis. The viral infection was achieved through mechanical inoculation of ALSV-infected Chenopodium quinoa leaf extract onto the cotyledons of C. grandis. ALSV-VIGS mediated knockdown of CgPDS gene was successfully achieved in C. grandis by mechanical inoculation method resulting in characteristic photobleaching. Subsequently, we developed agroinfiltration compatible vectors for direct infection of C. grandis and shortened the time-frame by skipping viral propagation in C. quinoa. Typical yellow-leaf phenotype was observed in C. grandis plants agroinfiltrated with ALSV-CgSU constructs, indicating robust silencing of CgSU gene. In addition, we improved the infection efficiency of ALSV by co-infiltration of P19 viral silencing suppressor. These results suggest that ALSV-VIGS is suitable for characterization of gene function in dioecious C. grandis and it can help us understand the mechanism of sex expression.

Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes.

Eucalypts are the most planted hardwoods worldwide. The availability of the Eucalyptus grandis genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-related genes: Cinnamoyl-CoA Reductase1 (CCR1), a key lignin biosynthetic gene and IAA9A an auxin dependent transcription factor of Aux/IAA family. Almost all transgenic hairy roots were edited but the allele-editing rates and spectra varied greatly depending on the gene targeted. Most edition events generated truncated proteins, the prevalent edition types were small deletions but large deletions were also quite frequent. By using a combination of FT-IR spectroscopy and multivariate analysis (partial least square analysis (PLS-DA)), we showed that the CCR1-edited lines, which were clearly separated from the controls. The most discriminant wave-numbers were attributed to lignin. Histochemical analyses further confirmed the decreased lignification and the presence of collapsed vessels in CCR1-edited lines, which are characteristics of CCR1 deficiency. Although the efficiency of editing could be improved, the method described here is already a powerful tool to functionally characterize eucalypts genes for both basic research and industry purposes.

Biotechnological Approaches for Biomass and Cardenolide Production in Digitalis purpurea L.

Digitalis purpurea L. is one of the main economically viable sources of cardenolides (cardiac glycosides) for the pharmaceutical industry. Nevertheless, production of cardenolides in plants grown by traditional agriculture is not always an efficient process and can be affected by biotic and abiotic factors. This chapter provides two biotechnology strategies for biomass and cardenolide production in D. purpurea. Firstly, we report biomass production using a temporary immersion system (TIS), combined with cardenolide extraction and quantification. Secondly, an efficient protocol for genetic transformation via Agrobacterium tumefaciens is provided. These strategies can be used independently or combined in order to increase the content of cardiac glycosides in D. purpurea and to unravel biosynthetic pathways associated to cardiac glycoside production.

High-efficiency Agrobacterium-mediated transformation in Quercus robur: selection by use of a temporary immersion system and assessment by quantitative PCR

An efficient protocol for genetic transformation of somatic embryos of Quercus robur by selection in a temporary immersion system is reported. The transformation frequency was 5 times higher than achieved by conventional culture on semi-solid medium, ranging between 6 and 26 % for the four genotypes evaluated. Clumps of globular or torpedo somatic embryos were precultured for 7–10 days, inoculated with Agrobacterium tumefaciens strain EHA105:p35SGUSINT and cocultivated for 4 days before being cultured for 4 weeks on semi-solid selection medium supplemented with 25 mg L−1 kanamycin. Explants were transferred to RITA® bioreactors and subjected to a two-step selection protocol involving immersion in liquid medium supplemented with 25 mg L−1 kanamycin, for 18 weeks, and then with 75 mg L−1 kanamycin. Putatively transformed explants appeared after serial transfer to selection medium over 12–16 weeks. The presence of neomycin phosphotransferase II and β-glucuronidase genes in the plant genome was confirmed by histochemical and molecular analysis, and the copy number was determined by Southern blotting and real-time quantitative polymerase chain reaction. Transformed somatic embryos were germinated and transferred to soil for acclimatization, approximately 8 months after inoculation of the original tissue with bacteria. As the limiting factor for recovery of plants from oak embryogenic lines is the low embryo conversion rate, axillary shoot lines were established from transformed germinated embryos. Transformed embryos and shoots were cultured in medium with or without kanamycin and the responses to several morphogenetic processes (recovery after cryopreservation, germination, shoot proliferation, and rooting) were evaluated.

Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata.

A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of T0 and T1 plants. Rooted T0 and T1 shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting.

Optimization of electrotransformation conditions for Leuconostoc mesenteroides subsp. mesenteroides ATCC8293

To establish an efficient genetic transformation protocol for Leuconostoc species, methods for competent-cell preparation and electroporation conditions were optimized. Leuconostoc mesenteroides subsp. mesenteroides ATCC8293 cells were sequentially treated with penicillin G and lysozyme, and the plasmid pLeuCM was subsequently transformed into the cells. Our results demonstrated that transformation efficiencies were significantly increased (100-fold), and increased electric field strength also contributed to enhance transformation efficiency. Maximum transformation efficiency (1×10(4) or more transformants per μg DNA) was achieved when cells were grown in De Man, Rogosa, Sharpe (MRS) media containing 0·25 mol l(-1) sucrose and 0·8 μg ml(-1) penicillin G, followed by treatment with 600 U ml(-1) lysozyme and electroporation at a field strength of 10 kV cm(-1). When this protocol was used to transform pLeuCM into Leuc. mesenteroides, Leuconostoc gelidum, Leuconostoc fallax and Leuconostoc argentinun, successful transformations were obtained in all cases. Furthermore, this procedure was applicable to species belonging to other genera, including Lactobacillus plantarum, Pediococcus pentosaceus and Weissella confusa. The results demonstrate that the transformation efficiency for Leuconostoc spp. could be increased via optimization of the entire electroporation procedures. These optimized conditions can be used for the extensive genetic study and the metabolic engineering of not only Leuconostoc spp. but also different species of lactic acid bacteria.

Agrobacterium tumefaciens-mediated transformation of rutabaga (Brassica napus var. napobrassica) cultivar “American Purple Top Yellow”

An efficient protocol for genetic transformation of rutabaga (Brassica napus var. napobrassica) cultivar “American Purple Top Yellow” was developed by optimizing several factors influencing gene delivery and plant regeneration. A two-step regeneration protocol, adapted from canola, was optimal for rutabaga regeneration using hypocotyl explants. Transient expression studies monitored by histochemical β-glucuronidase (GUS) assays indicated that several factors, including Agrobacterium tumefaciens strain, cocultivation time, and cocultivation medium, affected gene delivery. For stable transformation, precultured hypocotyl explants were cocultivated with Agrobacterium cells on sterilized filter paper overlaid on callus induction medium containing 100 μM acetosyringone for 6 d under a 16-h photoperiod. Selection and regeneration of transformed cells were conducted on media containing 50 mg l−1 kanamycin and 250 mg l−1 Timentin. Using this protocol, GUS- and PCR-positive transformants were obtained from 3.2 to 4.2 % of hypocotyl explants inoculated with each of the three Agrobacterium strains after 3–5 mo. Most transformants exhibited a normal phenotype. Southern blot analysis confirmed stable integration of the gusA transgene in T0 plants.

Establishment of bispyribac selection protocols for <i>Agrobacterium tumefaciens</i>- and <i>Agrobacterium rhizogenes</i>-mediated transformation of the oil seed plant <i>Jatropha curcas</i> L.

Cultivation of the biofuel plant Jatropha (Jatropha curcas L.) has spread around the world because of its drought resistance, high seed oil content, and adaptability to different environmental conditions. Because of these attributes, Jatropha has the potential to be one of the main resources for next-generation biodiesel fuel. To improve the productivity of Jatropha biomass, it is important to understand the molecular functions of key Jatropha genes, and to modify various agronomic traits of Jatropha via molecular breeding. A reliable and efficient protocol for genetic transformation of Jatropha is a prerequisite for molecular biology research and breeding on this plant. Here, we developed a system in which the herbicide bispyribac sodium salt, which inhibits acetolactate synthase, was used as the selection agent, and a two-point-mutated acetolactate synthase gene (mALS) was used to confer resistance upon transformants. Application of this system significantly improved the efficiency of Agrobacterium tumefaciens-mediated stable transformation of the high-yielding elite Jatropha population, IP-2P. The bispyribac-mALS system was also successfully applied in the Agrobacterium rhizogenes-mediated hairy roots system, which allowed integration of a foreign gene and expression in Jatropha root tissues within 2 weeks. The new protocols described here are powerful tools not only for functional studies on endogenous genes, but also for the molecular breeding of Jatropha to develop elite varieties.

Transformed Hairy Roots of Discaria trinervis: A Valuable Tool for Studying Actinorhizal Symbiosis in the Context of Intercellular Infection

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.

Transformed Hairy Roots of the actinorhizal shrub Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection

Background Nitrogen is a major limiting factor of plant growth in many ecosystems. Root nodule symbiosis (RNS) is one of the most efficient adaptations allowing plants to cope with nitrogen deficiency by establishing a symbiotic association with diazotrophic bacteria able to produce ammonium from atmospheric nitrogen. Nevertheless RNS is restricted to two groups of plants: legumes and Parasponia (Celtidaceae), that interact with a group of gram-negative proteobacteria collectively called rhizobia, and actinorhizal plants, a group of 220 species, mostly shrubs and trees distributed in the orders Fagales, Cucurbitales and Rosales, that interact with gram-positive actinomycetes of the genus Frankia[1]. All these plants belong to the Rosid I clade, suggesting a common origin for the ability to establish RNS [2]. In recent decades a strong research effort focused on model legumes lead to the identification of key molecular actors involved in nodulation, including the bacterial signalling molecules, the Nod factors and several genes involved in the symbiotic signalling pathways [3]. Much less is known in non model legumes and actinorhizal plants, particularly in species that are not infected like model legumes through root hairs but show more ancestral infection mechanisms like crack entry or intercellular infection. Yet important cues regarding the diversity and evolution of RNS are being found precisely in these more primitive non-model systems [4,5]. Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized [6,7]. Intercellular infection has been described in Discaria trinervis, an actinorhizal shrub belonging to the Rosales order [8]. To decipher the molecular mechanisms underlying intercellular infection with Frankia, we set up an efficient genetic transformation protocol for D. trinervis based on A. rhizogenes.

The Establishment of an Agrobacterium-Mediated Transformation Platform for the Non-Embryogenic Calli of Vitis vinifera L.

Non-embryogenic calli (NEC) was inevitably and heavily produced when grape embryogenic calli (EC) was induced from explants or during the subculture of EC. A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC. In this research, a vector pA5 containing a chitinase signal sequence fused to gfp (green fluorescent protein) and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L. cv. Chardonnay NEC. It was determined that NEC 10 d after subculture was the best target tissue; 30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L −1 acetosyringone (AS) was optimized as protocol. The use of bacterial densities as 1.0 at OD 600 did not result in serious tissue hypersensitive reaction and it had higher efficiency. Kanamycin at 200 mg L −1 was picked for positive expression selection. The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques (RT-PCR) and fluorescent microscopy after three sub-cultures of the selected cell line. Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC. This transformation platform could facilitate the verification of candidate somatic embryogenesis (SE) decisive genes, and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products, as NEC not only proliferates fast, but also keeps in a rather stable condition.

Development of highly efficient genetic transformation protocols for table grape Sugraone and Crimson Seedless at low Agrobacterium density

Highly efficient genetic transformation protocols and the regeneration of transgenic plants of Sugraone and Crimson Seedless grapevines (Vitis vinifera L.) were achieved from embryogenic calli co-cultured with low Agrobacterium tumefaciens densities. The sensitivity of embryogenic cultures to kanamycin, as well as the effect of Agrobacterium strains, C58(pMP90) or EHA105, and the bacterial concentration (0.06 or 0.2 at Optical Density OD600) on transformation efficiency were studied. Embryogenic cultures showed different kanamycin sensitivities and the total suppression of embryo differentiation at 20 and 50 mg/l kanamycin for Crimson Seedless and Sugraone, respectively. sgfp gene expression was evaluated in callus co-cultured with each bacterial strain. Although GFP transient expression was higher with A. tumefaciens EHA105 in both cultivars at the beginning of the culture, there were no significant differences 28 days post-inoculation. However, the concentration of Agrobacterium did affected transformation efficiency: 0.06 OD600 being more effective for the transformation of Crimson Seedless and 0.2 OD600 for Sugraone. By following the optimised procedure, 21 and 26 independent transgenic plants were generated from Sugraone and Crimson Seedless respectively, three to five months post-infection. PCR analyses were carried out to verify the integration of the sgfp and nptII genes into grapevine genome and the stable integration of the sgfp gene was confirmed by Southern blot.

Genetic Transformation of Russian Wheat Cultivars

ABSTRACTWheat (Triticum aestivum L.) ranks first among cereal crops cultivated in Russia. During last years efficient genetic transformation protocol has been developed at the ‘Biotron’, for number of Russian spring cultivars. To produce wheat transgenic plants resistant to different biotic and abiotic environmental stress, researchers worked out on the biolistic transformation procedure, inheritance of transgenes in progenies and field evaluation of homozygous plants.

An efficient protocol for genetic transformation and shoot regeneration of turmeric (Curcuma longa L.) via particle bombardment

Turmeric (Curcuma longa L.) is an important spice crop plant that is sterile and cannot be improved by conventional breeding. An efficient method for stable transformation for turmeric, C. longa L., was developed using particle bombardment. Callus cultures initiated from shoots were bombarded with gold particles coated with plasmid pAHC25 containing the bar and gusA genes each driven by the maize ubiquitin promoter. Transformants were selected on medium containing glufosinate. Transgenic lines were established on selection medium from 50% of the bombarded calluses. Transgenic shoots regenerated from these were multiplied and stably transformed plantlets were produced. Polymerase chain reaction (PCR) and histochemical GUS assay confirmed the stable transformation. Transformed plantlets were resistant to glufosinate.