Efficient Plant Transformation Research Articles

Enhancing Agrobacterium-mediated plant transformation efficiency through improved ternary vector systems and auxotrophic strains.

Agrobacterium-mediated transformation is an essential tool for functional genomics studies and crop improvements. Recently developed ternary vector systems, which consist of a T-DNA vector and a compatible virulence (vir) gene helper plasmid (ternary helper), demonstrated that including an additional vir gene helper plasmid into disarmed Agrobacterium strains significantly improves T-DNA delivery efficiency, enhancing plant transformation. Here, we report the development of a new ternary helper and thymidine auxotrophic Agrobacterium strains to boost Agrobacterium-mediated plant transformation efficiency. Auxotrophic Agrobacterium strains are useful in reducing Agrobacterium overgrowth after the co-cultivation period because they can be easily removed from the explants due to their dependence on essential nutrient supplementation. We generated thymidine auxotrophic strains from public Agrobacterium strains EHA101, EHA105, EHA105D, and LBA4404. These strains exhibited thymidine-dependent growth in the bacterial medium, and transient GUS expression assay using Arabidopsis seedlings showed that they retain similar T-DNA transfer capability as their original strains. Auxotrophic strains EHA105Thy- and LBA4404T1 were tested for maize B104 immature embryo transformation using our rapid transformation method, and both strains demonstrated comparable transformation frequencies to the control strain LBA4404Thy-. In addition, our new ternary helper pKL2299A, which carries the virA gene from pTiBo542 in addition to other vir gene operons (virG, virB, virC, virD, virE, and virJ), demonstrated consistently improved maize B104 immature embryo transformation frequencies compared to the original version of pKL2299 (33.3% vs 25.6%, respectively). Therefore, our improved Agrobacterium system, including auxotrophic disarmed Agrobacterium strains and a new ternary helper plasmid, can be useful for enhancing plant transformation and genome editing applications.

New improvements in grapevine genome editing: high efficiency biallelic homozygous knock-out from regenerated plantlets by using an optimized zCas9i

BackgroundFor ten years, CRISPR/cas9 system has become a very useful tool for obtaining site-specific mutations on targeted genes in many plant organisms. This technology opens up a wide range of possibilities for improved plant breeding in the future. In plants, the CRISPR/Cas9 system is mostly used through stable transformation with constructs that allow for the expression of the Cas9 gene and sgRNA. Numerous studies have shown that site-specific mutation efficiency can vary greatly between different plant species due to factors such as plant transformation efficiency, Cas9 expression, Cas9 nucleotide sequence, the addition of intronic sequences, and many other parameters. Since 2016, when the first edited grapevine was created, the number of studies using functional genomic approaches in grapevine has remained low due to difficulties with plant transformation and gene editing efficiency. In this study, we optimized the process to obtain site-specific mutations and generate knock-out mutants of grapevine (Vitis vinifera cv. ‘Chardonnay’). Building on existing methods of grapevine transformation, we improved the method for selecting transformed plants at chosen steps of the developing process using fluorescence microscopy.ResultsBy comparison of two different Cas9 gene and two different promoters, we increased site-specific mutation efficiency using a maize-codon optimized Cas9 containing 13 introns (zCas9i), achieving up to 100% biallelic mutation in grapevine plantlets cv. ‘Chardonnay’. These results are directly correlated with Cas9 expression level.ConclusionsTaken together, our results highlight a complete methodology for obtaining a wide range of homozygous knock-out mutants for functional genomic studies and future breeding programs in grapevine.

The Establishment of Two Efficient Transformation Systems to Manipulate and Analyze Gene Functions in Quinoa (Chenopodium quinoa Willd.)

Quinoa (C. quinoa) is considered a gluten-free food with abundant nutrients and high tolerance to multiple abiotic stresses, which is the potential to become a major crop in future. Genetic manipulation will provide powerful tools to investigate the function and mechanism of those important genes in the regulation of quinoa development and stress responses, and further improve the quinoa in the field. However, the efficient plant transformation system for quinoa has not been well developed yet. Here, we established two rapid and efficient transformation systems for quinoa by using hairy roots and agroinfiltration of leaves, which provide useful tools for quick analysis of gene function. Hairy roots were obtained from three types of explants: cotyledon-nod with hypocotyl, cotyledon itself, and hypocotyl pieces. Interestingly, explants of cotyledon-nod with hypocotyl showed the highest transformation efficiency at 67.9%, and cotyledon displayed medium efficiency at 42.2%, while hypocotyl explants with the lowest at 31.6%. We also obtained transgenic quinoa roots successfully in-vivo, which showed low efficiency but provides a potential method to test gene functions in live plants. By using young leaves for agroinfiltration, direct injection showed a better transgenic effect compared with vacuum penetration. In juxtaposition, the transformation systems using both hairy root and leaf infiltration establish an efficient and convenient way to manipulate and analyze gene functions in quinoa, and a potential strategy for transgenic quinoa.

Promoting genotype-independent plant transformation by manipulating developmental regulatory genes and/or using nanoparticles.

This review summarizes the molecular basis and emerging applications of developmental regulatory genes and nanoparticles in plant transformation and discusses strategies to overcome the obstacles of genotype dependency in plant transformation. Plant transformation is an important tool for plant research and biotechnology-based crop breeding. However, Plant transformation and regeneration are highly dependent on species and genotype. Plant regeneration is a process of generating a complete individual plant from a single somatic cell, which involves somatic embryogenesis, root and shoot organogeneses. Over the past 40years, significant advances have been made in understanding molecular mechanisms of embryogenesis and organogenesis, revealing many developmental regulatory genes critical for plant regeneration. Recent studies showed that manipulating some developmental regulatory genes promotes the genotype-independent transformation of several plant species. Besides, nanoparticles penetrate plant cell wall without external forces and protect cargoes from degradation, making them promising materials for exogenous biomolecule delivery. In addition, manipulation of developmental regulatory genes or application of nanoparticles could also bypass the tissue culture process, paving the way for efficient plant transformation. Applications of developmental regulatory genes and nanoparticles are emerging in the genetic transformation of different plant species. In this article, we review the molecular basis and applications of developmental regulatory genes and nanoparticles in plant transformation and discuss how to further promote genotype-independent plant transformation.

Development of an efficient marker-free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1.

SummaryWe have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium‐derived vir and T‐DNA‐mediated transformation. Oh H1 is a unique, non‐phytopathogenic species, categorized as a BSL‐1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1‐8 strain containing a binary vector system. Oh H1‐8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy‐. Oh H1‐8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1‐8 generated high‐quality, transgenic events with single‐copy, plasmid backbone‐free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock‐inducible excision system resulting in marker‐free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null‐segregant lines to wild‐type controls. We have demonstrated that Oh H1‐8, combined with spectinomycin selection, is an efficient, rapid, marker‐free and yield‐neutral transformation system for elite soybean.

Optimization of Protoplast Isolation from Leaf Mesophylls of Chinese Cabbage (Brassica rapa ssp. pekinensis) and Subsequent Transfection with a Binary Vector.

Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and the lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. In this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were isolated following a 4 h incubation in a solution comprising 1.5% (v/v) cellulase, 0.25% (v/v) macerozyme, 0.25% (v/v) pectinase, 0.5 M mannitol, 15 mM CaCl2, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 106 protoplasts, 78% of which were viable. The gfp reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with the 40 µg binary vector for 30 min in a solution containing 40% PEG. The presence of gusA and hptII in the protoplasts was confirmed by PCR. The methods developed in this study would be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage.

A novel method of generating RNAi libraries for high‐throughput gene function analysis of creeping bentgrass

AbstractRNA interference (RNAi) is a powerful tool for gene function analysis, particularly for outcrossing polyploid species where simultaneous mutations of multiple alleles of a gene with similar but nonidentical sequences may not be readily achieved with the novel clustered regularly interspaced short palindromic repeats/Cas9 gene editing tools. The most effective RNAi strategy in plants is to use long hairpin RNAi (lhRNAi) constructs containing inverted repeats (IRs) of target genes. Previously, we used a Phi29‐Amplified RNAi Construct method to create a single lhRNAi construct that was shown to be highly effective in gene silencing in the model grass Brachypodium distachyon (L.) P.Beauv. Here, we describe a method of creating a library of lhRNAi constructs ready for high‐throughput gene function analysis in creeping bentgrass (Agrostis stolonifera L.), an important turfgrass species. A cDNA population in which salt‐responsive genes were enriched by suppression subtractive hybridization was used as the source of IRs. Sequencing of 13 randomly selected colonies from the lhRNAi library showed that the size of IRs varied from 184 to 994 bp, which is appropriate for RNAi, and nine of the IRs were shown to be homologous to known genes in closely related grasses. In addition to the demonstration of the proof‐of‐concept of creating an expression‐ready library, the salt stress‐specific lhRNAi library represents a valuable resource for studying the functions of salt‐responsive genes in creeping bentgrass. The method described here is broadly applicable to all plant species where an efficient plant transformation platform is available.

Agrobacterium-mediated In-planta transformation of bread wheat (Triticum aestivum L.)

Agrobacterium-mediated in-planta transformation method allows efficient plant transformation without tissue culture. In the present study, a tissue culture independent protocol for floral transformation in bread wheat was developed. Three bread wheat genotypes namely, HD 2967, HD 3086 and Bobwhite were used for floral transformation. At pre-anthesis stage, clipped florets were sprayed with inoculation medium containing Agrobacterium tumefaciens strain EHA 105 culture harboring binary vector pCAMBIA1301-p35S:hptII-p35S:gus and allowed to grow till maturity. Positive transformants were confirmed through antibiotic selection, PCR analysis and southern blotting. The method led to generation of transformation events in Bobwhite and HD 2967 wheat genotypes. The transformation efficiency of T1 transgenic plants was higher in Bobwhite as compared to HD 2967. Floral spray method of transformation developed in the present study is efficient for development of transgenic wheat and can be used for improvement of wheat using transgenic approach.

A case study of using an efficient CRISPR/Cas9 system to develop variegated lettuce

The clustered, regularly interspaced, short palindromic repeat associated endonuclease 9 (CRISPR/Cas9) system has emerged as a powerful approach for precision breeding to create plants with desirable traits. However, the CRISPR/Cas9 system relies heavily on an efficient plant transformation system that is usually time-consuming and costly. Here, we have constructed a CRISPR-Cas9 vector with neomycin phosphotransferase II and green fluorescent protein (eGFP-NPTII), where the high expression of GFP during plant regeneration allowed us to minimize the positional effect on T-DNA expression and facilitate screening T-DNA-free mutants. Successful gene editing using CRISPR/Cas9 has been illustrated in different plant species, but an important aesthetic characteristic of leaf variegation remained unexplored. With the newly designed construct, we have targeted the variegation gene <i>LsVAR2</i> in lettuce. Our results indicated that <i>LsVAR2</i> is closely related to both <i>AtFtsH2</i> and <i>AtFtsH8</i>, in which homozygous mutations lead to an albino phenotype while a variegated phenotype was induced by CRISPR/Cas9 <i>de novo</i> gene editing. In conclusion, the unique design of our CRISPR/Cas9 construct could efficiently edit the target gene and ease the screening of non-TDNA mutants through detecting GFP signals during plant regeneration and progeny segregation. Additionally, the success of gene-editing of <i>LsVAR2</i> in lettuce demonstrates proof in this method to develop novel plant breeding materials for valuable horticultural plant species.

Electroporation-mediated genetic transformation of oil palm (Elaeis guineensis)

Abstract. Darmawan C, Wiendi NMA, Utomo C, Liwang T. 2020. Electroporation-mediated genetic transformation of oil palm (Elaeis guineensis). Biodiversitas 21: 3720-3726. Novel traits introduction to existing varieties may shorten the duration of oil palm genetic improvement. This approach heavily relies on efficient plant transformation and regeneration methods. Commonly used methods such as biolistic and Agrobacterium-mediated transformation still generated low efficiency for oil palm. Therefore, it is important to find alternative transformation methods that have the potency to give better results. Electroporation is usually used for delivering genes into bacteria or plant protoplast where cell wall does not exist, however, in this research, it was used to deliver plasmid into plant tissues. The aim of this research is to establish an optimum electroporation protocol for delivering genes into oil palm calli. Electric field strength of 250, 500, 750, 1,000, and 1,250 V/cm were applied for oil palm calli electroporation. Transient GUS assay was not applicable for initial detection presumably due to oil palm endogenous GUS-like protein activity after electroporation. Delivered gene could be detected in survived calli from all tested electric field strength treatments and the highest calli growth rates were observed from 250 V/cm electroporation treatment. Overall results showed that electroporation could be used to deliver specific genes into oil palm calli and might be developed further to increase its efficiency.

Plant Transformation Techniques: Agrobacterium- and Microparticle-Mediated Gene Transfer in Cereal Plants.

Biotechnological methods for targeted gene transfers into plants are key for successful breeding in the twenty-first century and thus essential for the survival of humanity. Two decades ago, genetic transformation of crop plants was not routine, and it was all but impossible with important cereals such as barley and wheat. The recent focus on crop plant genomics-yet based on the Arabidopsis toolbox-boosted the research for more efficient plant transformation protocols, thereby considerably widened the number of genetically tractable crops. Moreover, modern genome editing methods such as the CRISPR/Cas technique are game changers in plant breeding, though heavily dependent on technical optimization of plant transformation. Basically, there are two successful ways of introducing DNA into plant cells: one is making use of a living DNA vector, namely, microbes such as the soil bacterium Agrobacterium tumefaciens that infects plants and naturally transfers and subsequently integrates DNA into the plant genome. The other method uses a direct physical transfer of DNA by means of microinjection, microprojectile bombardment, or polymers such as polyethylene glycol. Both ways subsequently require sophisticated strategies for selecting and multiplying the transformed cells under tissue culture conditions to develop into a fully functional plant with the new desirable characteristics. Here we discuss practical and theoretical aspects of cereal crop plant transformation by Agrobacterium-mediated transformation and microparticle bombardment. Using immature embryos as explants, the efficiency of cereal transformation is compelling, reaching today up to 80% transformation efficiency.

A Short History and Perspectives on Plant Genetic Transformation.

Plant genetic transformation is an important technological advancement in modern science, which has not only facilitated gaining fundamental insights into plant biology but also started a new era in crop improvement and commercial farming. However, for many crop plants, efficient transformation and regeneration still remain a challenge even after more than 30years of technical developments in this field. Recently, FokI endonuclease-based genome editing applications in plants offered an exciting avenue for augmenting crop productivity but it is mainly dependent on efficient genetic transformation and regeneration, which is a major roadblock for implementing genome editing technology in plants. In this chapter, we have outlined the major historical developments in plant genetic transformation for developing biotech crops. Overall, this field needs innovations in plant tissue culture methods for simplification of operational steps for enhancing the transformation efficiency. Similarly, discovering genes controlling developmental reprogramming and homologous recombination need considerable attention, followed by understandingtheir role in enhancing genetic transformation efficiency in plants. Further, there is an urgent need for exploring new and low-cost universal delivery systems for DNA/RNA and protein into plants. The advancements in synthetic biology, novel vector systems for precision genome editing and gene integration could potentially bring revolution in crop-genetic potential enhancement for a sustainable future. Therefore, efficient plant transformation system standardization across species holds the key for translating advances in plant molecular biology to crop improvement.

SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation

Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium supplemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of A. tumefaciens in the plant tissue culture process. We generated a mutant A. tumefaciens strain GV2260 (recA-SacB/R) that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R) can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcription factor.

Agrobacterium tumefaciens-mediated transformation of axillary bud callus of Hibiscus rosa-sinensis L. ‘Ruby’ and regeneration of transgenic plants

Hibiscus rosa-sinensis L. is a popular ornamental species valued for its large brightly coloured ephemeral flowers and has a range of health-promoting properties. The value of H. rosa-sinensis could be improved even further if there were ways to prolong the display life of its short-lived flowers, and to improve its frost tolerance. Development of an efficient plant transformation and regeneration procedure that allows introduction of genes into the plant will greatly facilitate this. Here we outline a transformation and regeneration procedure that is the first to produce transformed H. rosa-sinensis plants successfully. We first optimised callus induction and shoot regeneration efficiency. The highest shoot regeneration frequency of 66.7 % was achieved in the cultivar ‘Ruby’ when callus induced from axillary buds using a basal medium supplemented with 2.22 µM benzylaminopurine and 2.47 µM β-naphthoxyacetic acid was cultured on shoot regeneration medium. The frequency of shoot regeneration from callus was lower in ‘Ben James’ and absent in ‘Bright Light’, indicating genotypic differences. When axillary bud-derived callus of ‘Ruby’ was co-cultured with Agrobacterium tumefaciens harbouring a β-glucuronidase (GUS) reporter plasmid, 49 % of calli produced shoots on selection media. All tested plantlets were confirmed as transformed based on the presence of the GUS transgene in the genomic DNA and GUS activity measurements. Roots were induced on transgenic plantlets using half-strength basal medium supplemented with 2.85 µM indole-3-acetic acid. This simple protocol can be used to improve the ornamental, agronomic and health-promoting traits of H. rosa-sinensis hitherto recalcitrant to A. tumefaciens-mediated transformation.

Maize, tropical (Zea mays L.).

Maize (Zea mays L.) is the third most important food crop globally after wheat and rice. In sub-Saharan Africa, tropical maize has traditionally been the main staple of the diet; 95 % of the maize grown is consumed directly as human food and as an important source of income for the resource-poor rural population. The biotechnological approach to engineer biotic and abiotic traits implies the availability of an efficient plant transformation method. The production of genetically transformed plants depends both on the ability to integrate foreign genes into target cells and the efficiency with which plants are regenerated. Maize transformation and regeneration through immature embryo culture is the most efficient system to regenerate normal transgenic plants. However, this system is highly genotype dependent. Genotypes adapted to tropic areas are difficult to regenerate. Therefore, transformation methods used with model genotypes adapted to temperate areas are not necessarily efficient with tropical lines. Agrobacterium-mediated transformation is the method of choice since it has been first achieved in 1996. In this report, we describe a transformation method used successfully with several tropical maize lines. All the steps of transformation and regeneration are described in details. This protocol can be used with a wide variety of tropical lines. However, some modifications may be needed with recalcitrant lines.

Expression of a hevein-like gene in transgenic Agave hybrid No. 11648 enhances tolerance against zebra stripe disease

Zebra disease, which is caused by Phytophthora nicotianae, is a serious global threat to the main cultivar Agave hybrid No. 11648 (H.11648). This disease requires a long period to obtain anti-P. nicotianae variants by crossbreeding, given that 10 years are needed for this plant to bloom. Therefore, transgenic technology could be useful for the production of new agave lines that might develop significant tolerance to P. nicotianae within a relatively short period. In this study, a tissue culture system was tested on H.11648. Results showed that the optimum culture medium for callus induction was SH + 3 mg L−1 6-BA + 0.5 mg L−1 NAA + 0.1 mg L−1 2,4-D + 30 g L−1 sucrose + 6.5 g L−1 carrageenan, with pH of 5.8. The induction medium for budding was SH + 1.5 mg L−1 6-BA + 0.5 mg L−1 NAA + 30 g L−1 sucrose + 6.5 g L−1 carrageenan at pH 5.8. The induction medium for rooting was SH + 0.1 mg L−1 IAA + 30 g L−1 sucrose + 6.5 g L−1 carrageenan. The primary factors influencing plant transformation efficiency were also investigated. Results showed that the effective time for infection was 10 min, the optimum concentration of acetosyringone was 200 μM, the optimum time for pre-culture of sisal callus was 3 days, and the optimum co-culture time for calli and Agrobacterium was 4 days. Sisal calli will die when phosphinothricin concentration reaches 2.0 mg L−1. Furthermore, a hevein-like protein gene from Pharbitis nil was introduced to H.11648. The integration stability and expression level of the transgene in the H.11648 genome were analyzed by using Southern blot and reverse transcription polymerase chain reaction. The mycelial growth rate of P. nicotianae was significantly inhibited by the crude leaf extracts from the transgenic H.11648 plants. After further study in vivo, the resistance of transgenic H.11648 plants to P. nicotianae infection was enhanced.

Effects of catechins on Agrobacterium-mediated genetic transformation of Camellia sinensis

In this study, recalcitrance of tea plant ( Camellia sinensis) to Agrobacterium-mediated genetic transformation was investigated with an emphasis on specialized compounds in tea. Chemical constituents in tea leaves and calli were extracted into liquid Luria–Bertani (LB) medium to determine their biological activities on Agrobacterium growth, virulence, and plant transformation efficiency. Compared to the control Agrobacterium grown in LB medium, tea leaf extract containing 6.5 mg mL−1 catechins resulted in an 84.6 % reduction of Agrobacterium growth, a 73–36 % suppression of expression for the six virulence (vir) genes, browning of infected tobacco explant wounds, and an absence of transient or stable transformation events. Tea callus extract, containing 0.22 mg mL−1 catechins, did not significantly affect Agrobacterium growth or tobacco transgenic hairy root generation, whereas it enhanced the expression of some vir genes. Treatment with authentic catechin mixtures (other than caffeine) dissolved in LB resulted in suppression of Agrobacterium growth, vir gene expression, and tobacco transformation efficiency. Our data suggest that catechins are the key active constituents in tea leaves. Transient transformation efficiencies of tea leaves were much lower than those of tobacco leaves as indicated by the GUS (β-glucuronidase) assay, probably a result of inhibition by the catechins present in tea leaves. Lower transformation efficiencies of tea calli suggested that additional plant factor(s) might also exert inhibitory effects on tea plant transformation. Agrobacterium rhizogenes ATCC 15834 induced transgenic roots from the tea explants with 15–20 % efficiency. Our data suggested catechins inhibition of tea gene transformation could be overcome by using optimized strains of Agrobacterium.

Manipulation of epigenetic factors and the DNA repair machinery for improving the frequency of plant transformation

Plant genetic engineering involves the introduction of foreign DNA into the plant genome in order to enhance/modify plant traits. In transgenic plants, it is difficult to achieve stable and predictable transgene expression over subsequent generations. Largely, this is due to the lack of critical understanding of plant perception and response to the artificially introduced foreign DNA. Recent reports have revealed components of the epigenetic module that may affect transgene stability at both pre- and post-integration steps. Furthermore, the integration of the transgene has been shown to be strictly dependent on the DNA repair machinery. In this review, we briefly summarize genetic and epigenetic factors whose manipulation can enhance the efficiency of plant transformation and the quality of genetically engineered transgenic plants.

Use of ex vitro composite plants to study the interaction of cowpea (Vigna unguiculata L.) with the root parasitic angiosperm Striga gesnerioides.

BackgroundCowpea (Vigna unguiculata L.) is an important grain and forage legume grown throughout sub-Saharan Africa primarily by subsistence farmers on poor, drought prone soils. Genetic improvement of the crop is being actively pursued and numerous functional genomics studies are underway aimed at characterizing gene controlling key agronomic characteristics for disease and pest resistances. Unfortunately, similar to other legumes, efficient plant transformation technology is a rate-limiting step in analysis of gene function in cowpea.ResultsHere we describe an optimized protocol for the rapid generation of transformed hairy roots on ex vitro composite plants of cowpea using Agrobacterium rhizogenes. We further demonstrate the applicability of cowpea composite plants to study gene expression involved in the resistance response of the plant roots to attack by the root parasitic weed, Striga gesnerioides. The utility of the new system and critical parameters of the method are described and discussed herein.ConclusionsCowpea composite plants offer a rapid alternative to methods requiring stable transformation and whole plant regeneration for studying gene expression in resistance or susceptibility responses to parasitic weeds. Their use can likely be readily adapted to look at the effects of both ectopic gene overexpression as well as gene knockdown of root associated defense responses and to the study of a broader range of root associated physiological and aphysiological processes including root growth and differentiation as well as interactions with other root pests, parasites, and symbionts.

Potassium chloride and rare earth elements improve plant growth and increase the frequency of the Agrobacterium tumefaciens-mediated plant transformation

Plant transformation efficiency depends on the ability of the transgene to successfully interact with plant host factors. Our previous work and the work of others showed that manipulation of the activity of host factors allows for increased frequency of transformation. Recently we reported that exposure of tobacco plants to increased concentrations of ammonium nitrate increases the frequency of both homologous recombination and plant transgenesis. Here we tested the influence of KCl and salts of rare earth elements, Ce and La on the efficiency of Agrobacterium-mediated plant transformation. We found that exposure to KCl, CeCl(3) and LaCl(3) leads to an increase in recombination frequency in Arabidopsis and tobacco. Plants grown in the presence of CeCl(3) and LaCl(3) had higher biomass, longer roots and greater root number. Analysis of transformation efficiency showed that exposure of tobacco plants to 50 mM KCl resulted in ~6.0-fold increase in the number of regenerated calli and transgenic plants as compared to control plants. Exposure to various concentrations of CeCl(3) showed a maximum increase of ~3.0-fold in both the number of calli and transgenic plants. Segregation analysis showed that exposure to KCl and cerium (III) chloride leads to more frequent integrations of the transgene at a single locus. Analysis of transgene intactness showed better preservation of right T-DNA border during transgene integration. Our data suggest that KCl and CeCl(3) can be effectively used to improve quantity and quality of transgene integrations.