Agrobacterium-mediated Transformation Method Research Articles

Drought tolerance induction in transgenic tobacco through RNA interference of BrDST71, a drought-responsive gene from Chinese cabbage

Because drought is a major environmental factor that causes serious agricultural problems, understanding the mechanisms and genetic bases underlying plant responses to drought stress is essential. Using the Brassica rapa 135 K microarray, BrDST71 gene was identified. BrDST71 expression in drought-tolerant Chinese cabbage showed an eight-fold decrease than in wild-type, and encodes a 362 amino acid protein containing a secretory peroxidase domain. Overexpression and RNAi vectors of BrDST71 were constructed and each vector was transformed into Nicotiana tabacum by the Agrobacterium-mediated transformation method. The expression level of BrDST71 and the phenotype were analyzed under drought condition. Transgenic lines with suppressed expression of BrDST71 showed more tolerance to drought stress compared to wild-type and overexpression transgenic lines. It showed that suppressing BrDST71 expression is correlated to better growth under drought conditions. Based on these results, we suggest that down-regulation of BrDST71 improves drought tolerance.

An efficient method for Agrobacterium-mediated genetic transformation of chilli pepper (Capsicum annuum L.)

In this study, we have describe a highly efficient transformation protocol for two elite Indian cultivars of chilli pepper (Capsicum annuum L.), Pusa Sadabahar and Pusa Jwala. We used hypocotyls and cotyledons derived from young seedlings as explants, standardized regeneration medium for enhanced regeneration, and optimized parameters for promoting efficient transformation in these chilli cultivars. The optimized regeneration medium consisting of MS medium augmented with 1 mg l−1 indole-3-acetic acid and 5 mg l−1 6-benzyl amino purine induced profuse shoot regeneration from the hypocotyl and cotyledon explants, with a regeneration frequency of 81% in Pusa Sadabahar and 78% in Pusa Jwala. The transformation protocol was standardized using Agrobacterium tumefaciens strain LBA4404 harbouring pCAMBIA2301 construct with GUS reporter and NPT-II marker genes. The results demonstrated that the hypocotyl explants are more amenable to transformation than cotyledonary explants in both the cultivars studied. Further, co-cultivation of the hypocotyl explants with agrobacterial cells (OD600 = 0.2–0.5) for the duration of 72 h was found to be optimum for obtaining high transformation efficiency of about 30% in both the cultivars. The transgenic status of chilli transformants recovered in the selection medium having 30 mg l−1 kanamycin was confirmed by GUS activity, PCR, Southern and RT-PCR analyses. About 85–90% of rooted transgenic plants survived hardening and acclimatization processes. Using this protocol we produced several independent transgenic lines of each chilli cultivar.

A colonized millet grain method for Agrobacterium-mediated transformation of the button mushroom Agaricus bisporus

Lack of an efficient transformation system has hampered the molecular breeding of Agaricus bisporus. Here, we describe a highly efficient Agrobacterium-mediated transformation system for A. bisporus using foxtail millet (Setaria italica L. Beauv) grains as cultivation and infection media. Mycelium-millet complexes were prepared for co-culture and treated with ultrasonication for 10 s to improve infection efficiency. After a 72-h culture period, the newly grown mycelium-surrounded millet grain was transferred to selection medium supplemented with 200 μg/mL cefotaxime and 15 μg/mL hygromycin B (hyg) to screen positive transformants. Putative transformants were analyzed for the presence of the hyg gene by polymerase chain reaction and Southern blotting. Expression of eGFP in A. bisporus transformants was detected by fluorescence imaging, and the β-glucuronidase (GUS) protein was detected by histochemical staining. Our protocol resulted in an average 53.85% transformation frequency, and over 85% of the transformants tested remained mitotically stable, even after five successive rounds of subculturing. A feasible method for A. bisporus mushroom transformation using foxtail millet as an innovative culture medium was developed, which will benefit future functional genetic studies of this mushroom.

Inulin chain length modification using a transgenic approach opening new perspectives for chicory.

Chicory capable of synthesizing long-chain inulin is of great interest. During the growing season, the sucrose-sucrose 1-fructosyltransferase (1-SST) activity is vital for production of long-chain inulin in chicory. With the purpose to increase inulin chain length, we employed Agrobacterium-mediated transformation method. Transgenic chicory plants (Cichorium intybus L. var. sativum) cv. 'Melci' has been developed to overexpress sucrose-sucrose 1-fructosyltransferase (1-SST) under the control of the CaMV 35S promoter. The integration of the T-DNA into the plant genome was confirmed by PCR on genomic DNA using gene-specific primers. Quantification of the 1-SST transcript expression level revealed that transgenic plants showed higher 1-SST expression than those in non-transgenic plants. Further analyses proved that the fructan content of the roots significantly increased in the transgenic plants. These results revealed that overexpression of the 1-SST, the key gene in inulin biosynthesis in chicory, might serve as a novel approach to develop plants with the long-chain inulin content.

AgarTrap Protocols on your Benchtop: Simple Methods for Agrobacterium-mediated Genetic Transformation of the Liverwort Marchantia polymorpha.

Agrobacterium-mediated genetic transformation is a powerful technique in plant biology. We recently developed a simplified Agrobacterium-mediated genetic transformation method for the liverwort Marchantia polymorpha, named AgarTrap (agar-utilized transformation with pouring solutions). AgarTrap is easy to perform; all procedures can be completed within a week using a single plate of solid medium, and basic operations involve simply pouring the appropriate solutions onto the solid medium. Thus far, we have developed three types of AgarTrap methods (S-AgarTrap, G-AgarTrap, and T-AgarTrap) using three different M. polymorpha tissues: sporelings, intact gemmalings, and mature thallus pieces, respectively. Each AgarTrap method can be used to transform tissues at high efficiency, thereby producing sufficient numbers of transformants for study. The ease and efficiency of these AgarTrap methods will likely prompt widespread molecular biological analyses of M. polymorpha. In this review, we describe the basic characteristics of the three AgarTrap methods and present the detailed protocols used in our laboratory.

A transient transformation system for gene characterization in upland cotton (Gossypium hirsutum)

BackgroundGenetically modified cotton accounts for 64% of the world’s cotton growing area (22.3 million hectares). The genome sequencing of the diploid cotton progenitors Gossypium raimondii and Gossypium arboreum as well as the cultivated Gossypium hirsutum has provided a wealth of genetic information that could be exploited for crop improvement. Unfortunately, gene functional characterization in cotton is lagging behind other economically important crops due to the low efficiency, lengthiness and technical complexity of the available stable transformation methods. We present here a simple, fast and efficient method for the transient transformation of G. hirsutum that can be used for gene characterization studies.ResultsWe developed a transient transformation system for gene characterization in upland cotton. Using β-glucuronidase as a reporter for Agrobacterium-mediated transformation assays, we evaluated multiple transformation parameters such as Agrobacterium strain, bacterial density, length of co-cultivation, chemicals and surfactants, which can affect transformation efficiency. After the initial characterization, the Agrobacterium EHA105 strain was selected and a number of binary constructs used to perform gene characterization studies. 7-days-old cotton seedlings were co-cultivated with Agrobacterium and transient gene expression was observed 5 days after infection of the plants. Transcript levels of two different transgenes under the control of the cauliflower mosaic virus (CaMV) 35S promoter were quantified by real-time reverse transcription PCR (qRT-PCR) showing a 3–10 times increase over the levels observed in non-infected controls. The expression patterns driven by the promoters of two G. hirsutum genes as well as the subcellular localization of their corresponding proteins were studied using the new transient expression system and our observations were consistent with previously published results using Arabidopsis as a heterologous system.ConclusionsThe Agrobacterium-mediated transient transformation method is a fast and easy transient expression system enabling high transient expression and transformation efficiency in upland cotton seedlings. Our method can be used for gene functional studies such as promoter characterization and protein subcellular localization in cotton, obviating the need to perform such studies in a heterologous system such as Arabidopsis.

Factors Influencing in Vitro Organogenesis of Chrysanthemum morifolium cv. ‘Resomee Splendid’

BackgroundChrysanthemum; also commonly known as mums or chrysanths, is one of the most important ornamental crops worldwide. Introducing desirable traits into this valuable plant by the conventional breeding has so far been faced with some restrictions due to the limited gene pool and cross-incompatibility. Therefore, breeders have decided to exploit Agrobacterium-mediated transformation methods in order to satisfy the growing market demands. However, more efficient in vitro regeneration protocols are required for this approach.ObjectivesThe objective of this research was to develop an efficient protocol for an in vitro plant regeneration by the examining the effects of various combinations and concentrations of the plant growth regulators (PGRs) and different explants types.Materials and MethodsThe leaf and petiole explants of the Chrysanthemum morifolium cv. ‘Resomee Splendid’ were collected from the in vitro grown plantlets. Murashige and Skooge (MS) medium was supplemented with different concentrations and combinations of benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA) and thidiazuron (TDZ). Thereafter, the effects of these hormonal treatments were investigated on shoot initiation percentage, the average number of shoots per explants, callogenesis, and the type of organogenesis in regard to both types of the explants.ResultsShoots were directly formed from leaf explants on the media that only contained BAP without callus formation. Amongst the other hormonal treatments, a combination of 4.5 mg.L-1 BAP plus 1 mg.L-1 NAA resulted in the direct organogenesis from the leaf explants, which was superior to the other combinations and concentrations. In regard to the petiole explants, direct shoot formation occurred in all the media except for the ones which were fortified with TDZ. In this case, considering the shoot initiation percentage and the mean shoot number per explants, the best results were achieved in the medium supplemented with 1.5 mg.L-1 BAP and 1 mg.L-1 NAA. Results showed that interaction of either BAP or TDZ with NAA was necessary for the callus induction.ConclusionsSignificant differences in shoot initiation percentage and the average number of shoots per explants were observed both in leaves and petioles grown on different media. Moreover, the callogenesis rates, as well as organogenesis types, showed some differences among the studied explants when compared on the same media.

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus.

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 106 conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

Research Progress on Agrobacterium tumefaciens-based Transgenic Technology in Brassica rapa

Brassica rapa L. is cultivated globally and consumed in many areas worldwide. Using the transgenic Agrobacterium-mediated transformation method, which is a reproducible and efficient technique, genes can be transferred into various B. rapa species. This review summarizes the processes involved in Agrobacterium-mediated transformation of B. rapa, including surface seed sterilization, co-cultivation with A. tumefaciens, induction of callus/shoot/root formation, and confirmation of transgenic plants. In addition, factors such as the Agrobacterium strain, plant genotype, explant age, transformation efficiency of the hybrid or inbred line, and the concentrations of N6-benzyl amino purine and naphthalene acetic acid, are discussed. And this review shows clearly how to do it, what to do, and what not to do in the transgenic Agrobacterium-mediated in Brassica rapa. The information presented here lays the foundation for a simple and efficient method that resolves existing problems and improves overall transgenic B. rapa production, thereby benefiting both basic and applied research.

Application of a System for Targeting Fe-Dependent Superoxide Dismutase and Choline Oxidase Enzymes to Chloroplast as a Strategy for Effective Plant Resistance to Abiotic Stresses

The plastids involved in photosynthesis and other essential metabolic processes are the cellular compartments most susceptible to abiotic stress factors with adverse effects in plants. In order to protect the chloroplast compartment, the plants of the Belyi Naliv tomato cultivar and the Darenka potato cultivar were transformed with genes for Fe-dependent superoxide dismutase and choline oxidase with the use of the method for plant genetic engineering. These target genes were equipped with signal sequences directing the proteins into the plastid compartment. The experimental results have demonstrated the optimization of the agrobacterium-mediated transformation method for the analyzed tomato and potato cultivars. The naturally growing leaves and stems of the mature plants proved to be the most effective explants to obtain the regenerants. The highest regeneration frequency in the explants was reached with the use of a medium containing 5 mg L–1 BAP, 0.1 mg L–1 NAA, and 1 mg L–1 zeatin. Augmentin and timentin were equally efficient among the antibiotics tested for the inhibition of the agrobacterium; the concentration of 200 mg L–1 in the culture medium was sufficient to eliminate the agrobacterium. In addition, they had no inhibitory effects on the shoot regeneration process. However, the cefotaxime concentration required to inhibit the agrobacterium should be 500 mg L–1 and higher. Moreover, this antibiotic at the concentration of 100 mg L–1 was proven to inhibit the regenerant growth.

Development of an Agrobacterium-mediated transformation method for Taxus suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.

A modified in planta method of Agrobacterium-mediated transformation enhances the transformation efficiency in safflower (Carthamus tinctorius L.)

Plant transformation has emerged as an important tool to integrate foreign genes in the plant genome to modify the plants for desired traits. Though many techniques of plant transformation are available; getting single copy transgenic events and cost associated remains a big challenge. Thus Agrobacterium-mediated transformation remains the method of choice due to multiple advantages. In the present work a tissue culture free protocol of Agrobacterium-mediated transformation was optimized in safflower, an oil seed crop recalcitrant to transformation. As a proof of concept we selected pCAMBIA2300 gene cassette containing Arabidopsis specific delta 15 desaturase (FAD3) downstream to truncated seed specific promoter beta-conglycinin and optimized tissue culture free protocol of Agrobacterium-mediated transformation using embryos as explants. Addition of silwet L-77, sonication treatment, vacuum infiltration in infection medium and use of paper wicks in co-cultivation period increased the transformation efficiency to 19.3%. Further, success in transformation was confirmed via product accumulation in 21 independent transgenic events wherein oil in transformed seeds showed significant accumulation of alpha-linolenic acid (ALA; 18:3; n3) which is generated from linoleic acid (LA; 18:2; n3) in a FAD3 catalyzed reaction. The present protocol can be utilized to produce transgenic safflower with different desired characters.

Research Article Efficient method for Agrobacterium-mediated genetic transformation of tobacco nodal segments

Research Article Efficient method for Agrobacterium-mediated genetic transformation of tobacco nodal segments

Increasing branch and seed yield through heterologous expression of the novel rice S-acyl transferase gene OsPAT15 in Brassica napus L.

Branching is a predominant element in the plant architecture of Brassica napus L. and represents an important determinant of seed yield. OsPAT15 (OsDHHC1), a novel DHHC-type zinc finger protein gene, was reported to regulate rice plant architecture by altering the tillering. However, whether heterologous overexpression of the OsPAT15 gene from the monocot rice into the dicot B. napus L. would have the same effect on branching or seed yield is unknown. In this study, the DHHC-type zinc finger protein gene OsPAT15 was determined to have sulfur acyl transferase activity in the akr1Δ yeast mutant in a complementation experiment. Heterologously expressing OsPAT15 transgenic B. napus L. plants were obtained using the Agrobacterium-mediated floral-dip transformation method. As anticipated, OsPAT15 transgenic plants exhibited branching and seed yield. Compared with non-transgenic plants, OsPAT15 transgenic plants had increased primary branches (1.58–1.76-fold) and siliques (1.86–1.89-fold), resulting in a significant increase in seed yield (around 2.39–2.51-fold). Therefore, overexpression of the sulfur acyl transferase gene OsPAT15 in B. napus L. could be used to increase seed yield and produce excellent varieties.

Ectopic Expression of Hawthorn SND1 Gene in Tobacco

NAC proteins are plant-specific transcription factors (TFs) and have been shown to function in plant development processes and abiotic and/or biotic stress responses. SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is one type of NAC TFs, which is a key regulator in the regulation network for secondary wall synthesis. In this study, the SND1 gene, named CpSND1 because it has a conservative N-terminal DNA-binding domain with AtSND1, was isolated from hawthorn (Crataegus pinnatifida). The full-length CDS of this gene was 1,203 bp, encoding 400 amino acids. The CpSND1 gene was transferred into tobacco (Nicotiana tobacum) by the Agrobacterium-mediated transformation method, and 20 transgenic lines were obtained. Tobacco plants overexpressing CpSND1 had typical phenotypes, including inhibited growth, upward-curling leaves. Our results provided functional information of CpSND1 for future genetic engineering.

Agrobacterium-mediated transformation of a Eucalyptus camaldulensis × E. tereticornis hybrid using peeled nodal-stem segments with yeast HAL2 for improving salt tolerance

Genetic improvements of Eucalyptus require both breeding and gene transformation to generate desirable traits for forest industry. Because most of the current Eucalyptus transformation methods rely on juvenile seedling tissues as explants, the resulting transgenics are subjected to phenotypic segregation through the use of segregated seedlings. This is unsuitable for transformation of selected Eucalyptus breeding lines, especially the interspecific hybrids. Here, we developed an Agrobacterium-mediated transformation method using peeled nodal-stem explants from in vitro-propagated plantlets of a Eucalyptus hybrid. The explant was prepared by peeling off epidermal layers around the axillary buds using a pair of forceps, before co-cultivation with Agrobacterium tumefaciens. Transgenic shoots generated from the axillary buds were then obtained through kanamycin selection. The transformation efficiency was at most 24% as confirmed by PCR and GUS assay. Transformation of a E. camaldulensis × E. tereticornis F1 hybrid clone using yeast HAL2 showed that, out of 35, 11 transgenic plantlets had an increase in salt tolerance and grew and developed roots, though at a slow rate, on the medium supplemented with NaCl up to 300 mM concentration. This work demonstrates the efficiency of this transformation method for further genetic improvement of Eucalyptus clones selected through hybridization and breeding.

Isolation, Transformation and Overexpression of Sugarcane SoP5CS Gene for Drought Tolerance Improvement

Drought is one of the very important growth-limiting factors for sugarcane production in China. Δ1-pyrroline-5-carboxylate synthase (P5CS) is the rate-limiting enzyme in proline synthesis, and it plays an important role in plant response to drought stress. In this study, sugarcane P5CS (SoP5CS) gene was cloned and submitted in GenBank with the accession number KJ546350. The gene SoP5CS was 2151 bp in length, encoding 716 amino acids with a predicted molecular weight of 77.73 kDa and an isoelectric point of 6.14. The qRT-PCR analysis showed that the expression of SoP5CS was higher in leaf than in root and stalk, and strongly induced under ABA, PEG, NaCl and 4 °C treatments. The plant excessive expression vector of SoP5CS was built and transformed into sugarcane calli by Agrobacterium-mediated transformation method, and transgenic sugarcane plants were obtained. Under drought stress, some transgenic lines with overexpressed SoP5CS showed significantly higher in SoP5CS expression level, proline accumulation, abscisic acid content, superoxide dismutase activity and relative water content but lower MDA content and chlorophyll (SPAD value) decrease as compared with the WT plants. Moreover, all the transgenic plants increased drought stress tolerance. Our findings indicated that SoP5CS plays an important role in response to abiotic stresses and overexpression of SoP5CS can improve drought tolerance in transgenic sugarcane plants.

Tissue culture-based Agrobacterium-mediated and in planta transformation methods

Gene transformation can be done in direct and indirect (Agrobacterium-mediated) ways. The most efficient method of gene transformation to date is Agrobacterium-mediated method. The main problem of Agrobacterium-method is that some plant species and mutant lines are recalcitrant to regeneration. Requirements for sterile conditions for plant regeneration are another problem of Agrobacterium-mediated transformation. Development of genotype-independent gene transformation method is of great interest in many plants. Some tissue culture-independent Agrobacterium-mediated gene transformation methods are reported in individual plants and crops. Generally, these methods are called in planta gene transformation. In planta transformation methods are free from somaclonal variation and easier, quicker, and simpler than tissue culture-based transformation methods. Vacuum infiltration, injection of Agrobacterium culture to plant tissues, pollen-tube pathway, floral dip and floral spray are the main methods of in planta transformation. Each of these methods has its own advantages and disadvantages. Simplicity and reliability are the primary reasons for the popularity of the in planta methods. These methods are much quicker than regular tissue culture-based Agrobacterium-mediated gene transformation and success can be achieved by non-experts. In the present review, we highlight all methods of in planta transformation comparing them with regular tissue culture-based Agrobacterium-mediated transformation methods and then recently successful transformations using these methods are presented.

A simple Agrobacterium tumefaciens-mediated transformation method for rapid transgene expression in Medicago truncatula root hairs

Medicago truncatula is widely used as a model legume for symbiotic and pathogenic microbial interaction studies. Although a number of Agrobacterium-mediated transformation methods have been developed for M. truncatula, a rapid root transformation system was not yet available for this model plant. Here, we describe an easy method for rapid transgene expression in root hairs of M. truncatula, using young seedlings co-cultivated with the disarmed hypervirulent A. tumefaciens strain AGL1. This method leads to efficient expression of various GUS and fluorescent reporters in M. truncatula root hairs. We showed that transgene expression is detected as soon as 2 days following co-culture, in root hairs of a particular responsive zone lying 0.5–2 cm behind the root tip. This method can be used with a variety of M. truncatula genotypes, and is particularly useful for rapid investigation of the sub-cellular localization of fluorescent fusion proteins. Moreover, combining distinct Agrobacterium strains during the initial co-culture step efficiently generates co-transformed root hairs, suitable for co-localization of different fluorescent fusion proteins in the same cell.

Agrobacterium-mediated floral dip transformation of the model polyploid species Arabidopsis kamchatica.

Polyploidization has played an important role in the speciation and diversification of plant species. However, genetic analyses of polyploids are challenging because the vast majority of the model species are diploids. The allotetraploid Arabidopsis kamchatica, which originated through the hybridization of the diploid Arabidopsis halleri and Arabidopsis lyrata, is an emerging model system for studying various aspects of polyploidy. However, a transgenic method that allows the insertion of a gene of interest into A. kamchatica is still lacking. In this study, we investigated the early development of pistils in A. kamchatica and confirmed the formation of open pistils in young flower buds (stages 8-9), which is important for allowing Agrobacterium to access female reproductive tissues. We established a simple Agrobacterium-mediated floral dip transformation method to transform a gene of interest into A. kamchatica by dipping A. kamchatica inflorescences bearing many young flower buds into a 5% sucrose solution containing 0.05% Silwet L-77 and Agrobacterium harboring the gene of interest. We showed that a screenable marker comprising fluorescence-accumulating seed technology with green fluorescent protein was useful for screening the transgenic seeds of two accessions of A. kamchatica subsp. kamchatica and an accession of A. kamchatica subsp. kawasakiana.