Agrobacterium-mediated Transformation Protocol Research Articles

An efficient protocol for Agrobacterium-mediated transformation and regeneration of Campanula medium (Canterbury bells) based on leaf disc explants

Campanula medium is an important ornamental plant known for its blue and bell-shaped flowers and a good model for studying flower colour formation and modification. The major outstanding obstacle preventing the developing of genetic modification and improvements in C. medium is a missing transformation method. For the first time, an effective and reliable in vitro regeneration and Agrobacterium-mediated transformation system has been established in this study. Different types and combinations of plant growth regulators (PGRs) were investigated for shoot regeneration by using leaf explants from three different cultivars. A high regeneration rate (50% for cv. ‘Blue double’, 58% for cv. ‘White double’ and 44% for cv. ‘Pink double’) was achieved on MS-based medium supplemented with 11.5 μM thidiazuron (TDZ) and 2.3 μM 1-naphthaleneacetic acid (NAA). In total, the Agrobacterium strains ABI, AGL1 and GV3101:pMP90, transformed with a binary plasmid harbouring NPTII and GFP gene cassettes, were evaluated for plant transformation, while various factors affecting the transformation efficiency were also identified. The highest transformation rate of about 22% was obtained from explants pre-cultured on regeneration medium and infected with AGL1. However, transgenic shoots were produced by all three Agrobacterium strains studied and confirmed by PCR and GFP fluorescence microscopy. Campanula medium regeneration and genetic transformation methods were established while various factors affecting the transformation efficiency were identified such as pre-culture treatment of leaf explants and different Agrobacterium strains.

Feasible regeneration and agro bacterium-mediated transformation of Brassica juncea with Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene.

In the present study an effort has been made to optimize the in vitro regeneration protocol for Agrobacterium-mediated transformation of Brassica juncea, because of its importance as oilseed crops. The highest callus induction frequency of 87% was observed on MS (Murashige and Skoog, 1962) medium supplemented with 4 µM 6-benzyladenine (BA) after four weeks of culture period. Subculturing of organogenic calli in MS media with a similar hormonal composition resulted in shoot organogenesis after six weeks of culture cultivation. The highest shoot induction frequency (92%) was recorded on MS medium containing 4 µM BA in combination with 1 µM of α-naphthalene acetic acid (NAA). Further, well-developed roots were formed in MS media augmented with 6 µM of Indole acetic acid (IAA) in combination with 1 µM Kinetin (Kn). Cotyledon explants were exploited in vitro for the successful transformation of B. juncea. A binary vector comprised of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene under the transcriptional control of a glycinin promoter and with a basta selection marker was introduced into A. tumefaciens strain GV3101 via electroporation. EaDAcT gene is responsible for unusual triacylglycerol’s production where the sn-3 position is esterified with acetate instead of the long-chain fatty acid found in the triacylglycerol’s. The highest regeneration frequency (100%) of transgenic shoots was observed on MS medium supplemented with 4 µM BA plus 1 µM NAA in the presence of 25 mg l−1 basta and 160 mg l−1 timintin. The efficiency of stable transformation was found to be approximately 7% in the transgenic plants. Moreover, the transformed regenerated shoots were confirmed by PCR analysis using EaDAcT gene-specific primers.

CRISPR/Cas9-Based Gene Editing in Soybean.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean, a number of laboratories have obtained mutants by CRISPR/Cas9 system; however, there has been not yet a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single guide RNA) transgenes.

TransgenicMiscanthus lutarioripariusthat co-expresses theCry 2Aa#andBargenes

Miscanthus lutarioriparius, a domestic species of China, belongs to the genus of perennial rhizomatous Miscanthus and is a promising energy crop for biomass production. To establish an Agrobacterium tumefaciens transformation protocol and obtain transgenic plants co-expressing the Cry 2Aa#and Bar genes, an Agrobacterium-mediated transformation method for M. lutarioriparius was developed in this study. To inhibit tissue browning, optimized callus induction and regeneration media were developed. Embryogenic calli were efficiently induced from immature inflorescences on Murashige & Skoog (MS) medium containing 0.2 g L−1citric acid. Agrobacterium tumefaciens EHA105 carrying pC3300 with the Cry 2Aa#and Bar genes were introduced into embryogenic calli. Six transgenic lines were selected by Basta screening and confirmed by genomic PCR. The herbicide tolerance of M. lutarioriparius transgenic plants was verified by spraying with glufosinate and expression of the Cry 2Aa#protein by ELISA. Therefore, an Agrobacterium-mediated transformation protocol for M. lutarioriparius was established and the co-expression of herbicide tolerance and Cry 2Aa#was successfully introduced with the system.

Optimization of Mature Embryo-Based Tissue Culture and Agrobacterium-Mediated Transformation in Model Grass Brachypodium distachyon.

Agrobacterium-mediated genetic transformation is well established in the model grass Brachypodium distachyon. However, most protocols employ immature embryos because of their better regenerative capacity. A major problem associated with the immature embryo system is that they are available only during a limited time window of growing plants. In this study, we have developed an optimized Agrobacterium-mediated genetic transformation protocol that utilizes mature embryos. We have adopted seed shearing and photoautotrophic rooting (PR) in callus induction and root regeneration, respectively, with evident significant improvement in these aspects. We have also revealed that the newly developed chemical inducer Fipexide (FPX) had the ability to induce callus, shoots, and roots. By comparison, we have demonstrated that FPX shows higher efficiency in shoot generation than other frequently used chemicals in our mature embryo-based system. In addition, we demonstrated that the age of embryogenetic callus severely affects the transformation efficiency (TE), with the seven-week-old embryogenetic callus having the highest TE reaching 52.6%, which is comparable with that in immature embryo transformation. The new methodologies reported here will advance the development and utilization of Brachypodium as a new model system for grass genomics.

Transgenic potato lines expressing CP4-EPSP synthase exhibit resistance against glyphosate

Potato crops are particularly vulnerable to weed competition from emergence to canopy closure and are subject to significant yield loss. Glyphosate is broad spectrum herbicide used to control weeds worldwide. In order to incorporate glyphosate resistant trait in four potato cultivars (Lady Olympia, Desiree, Agria and Granola), an efficient, cost effective, reproducible and stable Agrobacterium-mediated genetic transformation protocol was performed using leaf and internodal explants. Agrobacterium strain LBA4404 harboring newly modified recombinant binary vector pCAMHE-EPSPS containing EPSP synthase gene under the control of Cauliflower mosaic virus 35S promoter was used to infect explants. The overall transformation efficiency was 26.4%. Of the 280 plants transferred to greenhouse, 74 plants were found to be PCR positive with gene of interest. GUS histochemical, Southern blot, RT-qPCR, lateral flow dipstick assays confirmed integration and expression of EPSPS in primary transformants. The putative transgenic plants developed from these cultivars possessed enhanced resistance to glyphosate applications in T0 and first tuber generation. These transgenic potato lines could be used as source of germplasm for an efficient potato breeding program. The article reports the development of potato transgenic lines with enhanced tolerance against glyphosate.

Agrobacterium–mediated Transformation of Kalanchoe laxiflora

Plants with crassulacean acid metabolism (CAM) generally utilize water 20%–80% more efficiently than non-CAM plants. The whole genomes of several CAM plants have been sequenced or are being sequenced. For effective genome characterization and genome editing of CAM plants, an efficient transformation system is essential. In this study, we developed an Agrobacterium-mediated transformation protocol for Kalanchoe laxiflora, an obligate CAM plant,by optimizing several factors affecting the transformation efficiency. Agrobacterium strains AGL1, C58, EHA105, and GV3101 were all suitable for K. laxiflora transformation. Fifty-nine percent of the leaf explants yielded kanamycin-resistant and GUS-positive shoots. Polymerase chain reaction and quantitative real-time PCR (qRT-PCR) using gusA-, gusPlus-, nptII- and hpt-specific primers confirmed that the transgenes were integrated into K. laxiflora genome and expressed. This efficient transformation system will allow effective functional characterization of genes through over- or down-expression, knockout, or genome editing.

Genetic engineering of PPV resistance in plum rootstock ‘Elita’: an initial study

The present study describes an Agrobacterium-mediated transformation protocol for plum rootstock 'Elita' ((Prunus pumila L. × P. salicina Lindl.) × (P. cerasifera Ehrh.)) using leaf explants. The system enables an efficient plant regeneration with the retention of the traits of the original rootstock. The transformation experiments were conducted using a genetic construct containing the self-complementary sequences of a fragment of PPV-CP gene separated by an intron for inducing Plum pox virus (PPV) resistance through the mechanism of post-transcriptional gene silencing. Transgenic plum plants' rootstocks were produced from organogenic callus developed from leaf explants through a 6-month period culture after inoculation. PCR-analysis confirmed the transgenic status of the produced plants by amplification of the fragments of “hairpin”-PPV-CP construct and hpt gene. To our knowledge, this is the first report of a successful attempt to produce transgenic plum rootstock.

Effect of different factors on regeneration and transformation efficiency of tomato (Lycopersicum esculentum) hybrids

The current study aimed to produce rootstock material through micropropagation by developing efficient regeneration and Agrobacterium-mediated transformation protocols for three high quality commercial tomato hybrids (Felina, Siena and Don Jose) to overexpress the GmGSTU4 gene from Glycine max L. previously shown to enhance antioxidant activity. We investigated the plant growth regulators zeatin (Z) and 3-idoleacetic acid (IAA) to determine their best combination for an efficient regeneration protocol for each hybrid. The highest regeneration efficiency was observed in Felina (94.4%) with 1.0 mg/l Z and 0.1 mg/l IAA. In contrast, Don Jose (92.5%) and Siena (83.3%) performed better with 0.5 mg/l Z and 0.1 mg/l IAA. The three hybrids did not differ in micropropagation index, however, Felina showed the highest number of in vitro rooted and in vivo acclimatized plants. Factors such as the age of explant, days in pre- and co-culture and the concentrations of acetosyringone and thiamine on Agrobacterium-mediated genetic transformation were assessed. The transformation indices were 37.04% for the Felina, 13.8% for Siena and 8.33% for Don Jose. We conclude that targeted genotype-specific regeneration protocols will provide an efficient and cost effective genetic transformation system for rootstock production and further incorporation into micropropagation and transgrafting systems.

Development of a rapid and highly efficient Agrobacterium-mediated transformation system for pigeon pea [Cajanus cajan (L.) Millsp

ABSTRACTAn efficient genetic transformation system is a prerequisite for studying gene functions, molecular breeding program, and introducing new traits. Agrobacterium tumefaciens-mediated genetic transformation is a widely preferred and accepted method for many plants, including pigeon pea. However, the efficiency of transformation of pigeon pea using the existing protocols is low and time-consuming. In the present study, we developed a rapid and highly efficient transformation system of pigeon pea, using embryonic axis-attached cotyledons as explants. We systematically investigated the influence of varying optical densities of Agrobacterium suspension, duration of incubation, and co-cultivation on the transformation efficiency. In our system, a transformation efficiency of approximately 83% was achieved using Agrobacterium cells at an optical density (OD600) of 0.25, infection time of 15 min, and co-culturing with explants for 72 h in the light with 100µM acetosyringone. The entire procedure, starting from seed to establishment of transformed plants in soil, was achieved in 35–40 days. This is a rapid and highly efficient protocol for Agrobacterium-mediated transformation of pigeon pea, which could potentially be a useful reference, not only for the genetic improvement of pigeon pea but also for other recalcitrant leguminous plants.

Improvement of Agrobacterium-mediated transformation for tannin-producing sorghum.

Sorghum (Sorghum bicolor L.) ranks as the fifth most widely planted cereal in the world and is used for food as well as a biomass plant for ethanol production. Use of the TX430 non-tannin sorghum variety has enhanced Agrobacterium-mediated sorghum transformation. These protocols could not be applied, however, to other tannin producing sorghum varieties such as the BTx623 model cultivar for sorghum with full genome information of sorghum. Here we report an improved protocol for Agrobacterium-mediated genetic transformation of tannin-producing sorghum variety BTx623. We successfully developed modification of root regeneration condition for generation of transgenic plant of BTx623. We inoculated immature embryos with Agrobacterium tumefaciens strain EHA105 harboring pMDC32-35S-GFP to generate transgenic plants. In the root regeneration step, we found that regeneration from transformed calli was affected by tannin. For root regeneration, shoots that appeared were not transferred to agar plate, but instead transferred to vermiculite in a plastic pod. Direct planting of regenerated shoots into vermiculite prevented the toxic effect of tannin. Root regeneration efficiency from calli emerged shoots in vermiculite was 78.57%. Presence of sGFP transgene in the genome of transgenic plants was confirmed by PCR and sGFP expression was confirmed in transgenic plants. This improved protocol of Agrobacterium-mediated transformation for tannin-producing sorghum BTx623 could be a useful tool for functional genomics using this plant.

Genetic Transformation of Oat Mediated by Agrobacterium is enhanced with Sonication and Vacuum Infiltration

BackgroundOat (Avena sativa) with high nutritive value and fiber content is used as the main food grain in many countries for human diet as well as animal feed. Recently, it became difficult to transfer new genes through the conventional breeding due to the lack of desirable traits.ObjectivesThe current study aimed at achieving a standardized protocol for Agrobacterium-mediated transformation in oat.Materials and MethodsFor oat transformation, mature seeds were sterilized, germinated, and used for explants generation. Agrobacterium tumefaciens GV3101 with the binary vector pCAMBIA 1305.1, which carries gus as reporter gene, was utilized in the transformation. The co-cultivation treatment assisted with sonication, and vacuum infiltration, and their combination was employed for transformation with different incubation periods of 48, 72, and 96 hours under the dark conditions.ResultsAmong the different transformation treatments, the vacuum treatment with 72 hours dark incubation had the best results. Vacuum infiltration of the cultures from leaf base produced a maximum of 25% hygromycin-resistant explants. These explants upon GUS assay and PCR analysis revealed 21.85% and 19.04% transformation efficiency, respectively.ConclusionsIt could be concluded that vacuum infiltration assisted Agrobacterium-mediated transformation is the most efficient method to conduct the genetic improvement of the oat using transformation protocol.

Effective Agrobacterium-mediated transformation protocols for callus and roots of halophyte ice plant (Mesembryanthemum crystallinum)

BackgroundIce plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants. Many salt stress-responsive ice plant genes have been identified with molecular and biochemical approaches. However, no further functional characterization of these genes in host plant due to lack of easy and effective transformation protocols.ResultsTo establish efficient transformation system of ice plants, three types of ice plant materials, hypocotyl-derived callus, aseptically-grown seedlings and pot-grown juvenile plants, were used to develop Agrobacterium-mediated transformation protocols. The highest transient transformation efficiency was with 5-day-old ice plant callus co-incubated with an Agrobacterium tumefaciens at 2.5 × 109 cells mL−1 for 48 h. The 3-day-old ice plant seedlings with root tip removed were successfully infected with A. tumefaciens or A. rhizogenes, and obtained 85% and 33–100% transient transformation rates, respectively. The transient transformation assays in ice plant callus and seedlings demonstrated that the concentrations of Agrobacteria, the durations of co-incubation time, and the plant growth stages were three important factors affecting the transient transformation efficiencies. Additionally, pot-grown juvenile plants were syringe-injected with two A. rhizogenes strains A8196 and NCPPB 1855, to establish transformed roots. After infections, ice plants were grown hydroponically and showed GUS expressions in transformed roots for 8 consecutive weeks.ConclusionsOur Agrobacterium-mediated transformation protocols utilized hypocotyl-derived callus and seedlings as plant materials, which can be easily obtained in large quantity. The average successful transient transformation rates were about 2.4–3.0% with callus and 33.3–100.0% with seedlings. We also developed a rapid and efficient protocol to generate transgenic roots by A. rhizogenes infections without laborious and challenging tissue culture techniques. This protocol to establish composite ice plant system demonstrates excellent improvements in efficiency, efficacy, and ease of use over previous ice plant transformation protocols. These Agrobacterium-mediated transformation protocols can be versatile and efficient tools for exploring gene functions at cellular and organ levels of ice plants.

A plant regeneration platform to apply new breeding techniques for improving disease resistance in grapevine rootstocks and cultivars

Worldwide grapevine cultivation is based on the use of elite cultivars, in many cases strictly linked to local important wine brands. Most ofVitis viniferacultivars have high susceptibility to fungal and viral diseases therefore, new breeding techniques (e.g. Cisgenesis, RNAi and gene editing) offer the possibility to introduce new clones of the main cultivars with increased diseases resistance, in order to reduce environmental impact and improve quality in the intensive wine grape industry. This study is finalized to develop efficientin vitroregeneration and transformation protocols to extend the application of these technologies in wine grape cultivars and rootstocks. With this aim,in vitroregeneration protocols based on the production of meristematic bulks (Mezzetti et al., 2002) were optimized for different grapevine cultivars (Glera, Vermentino, Sangiovese, Thompson Seedless) and rootstocks (1103 Paulsen, and 110 Richter). The meristematic bulks were then used as explants forAgrobacteriummediated genetic transformation protocols, by comparing the use of NPTII and e-GFP as marker genes. Results confirmed the efficiency of meristematic bulks as the regenerating tissue to produce new modified plants in almost all the above genotypes. The highest regeneration efficiency in some genotypes allowed the selection of stable modified lines/calli with only the use of e-GFP marker gene. This protocol can be applied in the use of MYB marker gene for the production of cisgenic lines. Genotypes having the highest regeneration and transformation efficiency were also used for transformation experiments using a hairpin gene construct designed to silence the RNA-dependent RNA polymerase (RpRd) of the GFLV and GLRaV3, which would induce multiple virus resistances, and the Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2 to controlB. cinereainfection.

Protocol for Agrobacterium-Mediated Transformation and Transgenic Plant Production of Switchgrass.

Switchgrass (Panicum virgatum L.) is one of the most important bioenergy crops for lignocellulose ethanol production. Molecular breeding provides a powerful tool to supplement conventional switchgrass breeding by introducing or editing genes of interest. In this chapter, we describe Agrobacterium tumefaciens-mediated transformation protocols for lowland tetraploid switchgrass cultivar Alamo.

Poplar Transformation.

First publications of successful Agrobacterium-mediated transformation of tobacco were published more than 30 years ago. Protocols for Agrobacterium-based transformation as well as biolistic bombardment and PEG transformation of protoplasts are available for more than 150 plant species from various plant families. Also for many Populus species and hybrids, adapted transformation protocols have been published. The standard protocol for Agrobacterium-mediated transformation of different Populus genotypes is the leaf-disc method. Here, we first describe the transfer of genes into poplar by using the Agrobacterium-based leaf disc methods. In addition, alternative basic transformation methods, namely, biolistic bombardment and PEG transformation of protoplasts, are also described. Further, we present improved poplar transformation protocols by simplifying the transformation procedure and optimizing tissue preparation and plant regeneration.

A Unified Agrobacterium-Mediated Transformation Protocol for Alfalfa (Medicago sativa L.) and Medicago truncatula.

Simplification of transformation procedures greatly improves work efficiency. In this chapter, we introduce a unified Agrobacterium-mediated transformation protocol that is used for both alfalfa (Medicago sativa L., Regen SY4D) and Medicago truncatula (ecotype R108). Whole trifoliates or leaflets are used as explants. Sonication is applied to enhance Agrobacterium infection and cytokinins are added to the medium to facilitate shoot regeneration. More than 90% transformation efficiency is achieved for alfalfa, while the relatively lower efficiency of up to 60% is obtained for M. truncatula, which depends on different selectable markers in the gene constructs. Transgenic plants are produced within 4-8months with average timeline of 6months. Using this unified protocol, the same types of media are used for both species which results in significant saving in time and resources.

Efficient plant regeneration and Agrobacterium-mediated transformation via somatic embryogenesis in purslane (Portulaca oleracea L.): an important medicinal plant

Portulaca oleracea is an important medicinal plant, which is a source of pharmacologically active molecules such as β-Carotene, ascorbic acid, and Omega-3 fatty acids. The present research focuses on the development of an efficient protocol for micropropagation and Agrobacterium-mediated genetic transformation of P. oleracea. Callus induction, somatic embryogenesis, and plant regeneration from stem and leaf explants were investigated at various concentrations of kinetin (Kin) and 6-Benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid, 1-Naphthaleneacetic acid and 2,4-Dichlorophenoxyacetic acid (2,4-D). Direct differentiation of somatic embryos from leaf explants occurred on the MS medium supplemented with 1.5 mg/L BAP under dark conditions. The embryos were transferred to the same medium without growth regulators under 16 h light/8 h dark cycles. In this medium, germinated somatic embryos rapidly developed into healthy plantlets with shoots and roots. Several parameters such as pre-culture of explants, co-cultivation period, wounding of explants, type of explants and bacterial strains were studied to optimize transformation efficiency. Different kanamycin concentrations were assessed for the selection of transgenic plants. Agrobacterium tumefaciens strains LBA4404 and GV3101, harbouring the GUS gene on pBI121 binary vector, were used for plant transformation and strain LBA4404 was found to be more efficient. The results indicated that use of leaf as explant, pre-culture of explants for 7 days, co-cultivation period for 4 days at 25 ± 2 °C and wounding of leaf explants produced the best transformation results. Expression, integration and inheritance of GUS reporter gene were confirmed by histochemical and molecular analyses.

Nuclear dynamics in the homothallic ascomycete Sordaria fimicola

BackgroundThe coprophilous ascomycete Sordaria fimicola usually reproduces sexually. Sexual differentiation in S. fimicola is accompanied by cellular and morphological changes, followed by multicellular tissue development to complete the sexual cycle. Although the morphological features of the sexual reproductive structure in S. fimicola have been well characterized, little is known about the nuclear dynamics and organization during these processes. Therefore, in this study, we successfully developed an Agrobacterium-mediated protoplast transformation protocol and generated histone H2B-mCherry-labeled S. fimicola strains. The life cycle of S. fimicola begins with germination of the ascospore and ends with the formation and discharge of new ascospores from the mature black sexual fruiting bodies, the so-called perithecia. The nuclear dynamics of the fluorescently labeled strains were examined during ascospore germination, hyphal elongation, and hyphal fusion using fluorescent microscopy.ResultsLive imaging revealed that the nuclei in the germlings and fusion hyphae during the pre-contact interaction are located adjacent to the tip.ConclusionsThis is the first report of the application of a fluorescence labeling technique in S. fimicola. This application will help researchers gain a better understanding of nuclear distribution and investigate the protein–protein interaction networks during fruiting body formation for advanced molecular genetic studies in S. fimicola.

Development of a stable Agrobacterium-mediated transformation protocol for Sorghum bicolor Tx430.

Sorghum is a recalcitrant crop for Agrobacterium-mediated genetic transformation. Several parameters related to Agrobacterium-mediated transformation were tested to optimize sorghum transformation frequencies. In this study, we evaluated pretreatment of sorghum variety Tx430 immature embryos using Agrobacterium strain GV2260. Pretreatment of immature embryos with heat (43°C) treatment for 15 or 21 min, and centrifugation resulted in a transformation efficiency of up to 1.9% of immature embryos treated. Although further optimization to enhance transformation efficiency is required, this study contributes to the genetic validation of genes of interest and molecular breeding in sorghum plants.