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

The recent advances in genome-wide transcriptome analysis have enabled researchers to identify new genes and manipulate the genome through novel genetic engineering methods to improve plant tolerance to different stresses. We conducted a genome-wide transcriptome analysis to determine tomato genes affecting the salt stress response. The results showed that among the 5784 genes that were responsive to salt stress, 103 genes (1.8%) encode transcription factors, of which 69 (1.2%) were upregulated and 34 (0.6%) were downregulated. The largest group of genes upregulated in response to salt stress (17 genes) is related to the ethylene response transcription factors (ERF) family. Specifically, it was found that the gene JERF1, encoding ERF in tomato, is upregulated in response to salt stress. In the present study we developed a new Agrobacterium-mediated transformation method to deliver the JERF1 gene to mature wheat embryos. It was investigated whether the factors vacuum infiltration, Agrobacterium cell density (OD600 1.0 and its dilutions 1:20, 2:20 and 3:20), the acetosyringone concentration (0, 200 and 400 μM) and the interaction between these factors affect transformation. To this end, Agrobacterium carrying pGWB14-JERF1 was injected into soaked wheat seeds, and T0 transgenic plants were obtained in the greenhouse. Molecular analysis of T0 plants was performed. The highest transformation efficiency was obtained under vacuum infiltration, 200 μM acetosyringone and 1:20 dilution. Notably, we used the in planta transformation method to overcome some of the problems of traditional wheat transformation methods e.g., sterile conditions, recalcitrant regeneration, time-constraints and somaclonal variations.

Agrobacterium-mediated in planta transformation of horticultural plants: Current status and future prospects

Genetic transformation is a reverse genetics tool for validation of target genes and crop improvement. However, due to its low efficiency and genotype dependency, wheat is considered a recalcitrant plant for genetic transformation. During the last 20 years, various in vitro and in planta transformation methods have been reported in wheat. Until now, biolistic particle and Agrobacterium-mediated wheat transformation methods using immature embryos as explants have been the two major transformation approaches. In addition to immature embryos, other explant types, such as mature embryos, anther-derived calli, inflorescences, apical meristems, and other floral organs, have been employed; however, they need further optimization. In addition to the common marker genes, such as bar, hpt and gus, other effective markers, ALS, AtMYB12 and pmi, have been successfully used for selection of positive transgenic plants. Numerous agronomic trait genes such as biotic stress resistance or tolerance genes have been transferred into wheat plants. Future prospects, such as recipient wheat cultivars and explants, marker free issues, and transgene silencing, are discussed. The objective of this review is to summarize current successful techniques for wheat transformation and stimulate further research into long-term wheat improvement by genetic engineering approaches.

In this review, the application of CRISPR/Cas9 plant genome editing using alternative transformation methods is discussed. Genome editing by the CRISPR/Cas9 system is usually implemented via the generation of transgenic plants carrying Cas9 and sgRNA genes in the genome. Transgenic plants are usually developed by in vitro regeneration from single transformed cells, which requires using different in vitro culture-based methods. Despite their common application, these methods have some disadvantages and limitations. Thus, some methods of plant transformation that do not depend on in vitro regeneration have been developed. These methods are known as “in planta” transformation. The main focus of this review is the so-called floral dip in planta transformation method, although other approaches are also described. The main features of in planta transformation in the context of CRISPR/Cas9 genome editing are discussed. Furthermore, multiple ways to increase the effectiveness of this approach and to broaden its use in different plant species are considered.

In planta transformation methods have opened up the possibility of transforming plant species for which no regeneration protocols currently exist. In this study, the suitability of the germ-line transformation method developed for Arabidopsis thaliana was examined for four taxa in the Brassicaceae that have not been previously transformed: Arabidopsis griffithiana, Arabidopsis lasiocarpa, Arabidopsis petraea and Capsella bursa-pastoris. Numerous transformants were obtained for A. lasiocarpa. Transformation of A. lasiocarpa was confirmed at the phenotypic and molecular levels for stably transformed lines and for backcrossed lines segregating the T-DNA insert. Parameters affecting transformation efficiency of A. lasiocarpa were also explored. As with A. thaliana, sucrose and surfactant in the inoculation medium are required for high levels of transformation, although the suitable concentrations of these are different for A. lasiocarpa. Other components present in earlier versions of the inoculation medium had little effect on transformation efficiency. Vacuum infiltration (rather than simple floral dipping) led to higher rates of transformation and did not seriously affect seed production in A. lasiocarpa. Identification of species susceptible to germ-line transformation will aid in determining the factors important for applying this technology to more recalcitrant species.

Chapter 2 - The scope of transformation and genome editing for quantitative trait improvements in rice

BackgroundRecent studies indicate that farmers are facing several challenges due to biotic and abiotic stresses like diseases, drought, cold, and soil salinity which are causing declined Citrus production. Thus, it is essential to improve these varieties which would be resistant against biotic and abiotic stresses as well as high yielding. The transformation of abiotic stress tolerant genes in Citrus species is essential for using areas affected by abiotic stresses. This study was aimed to improve resistance of Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck to abiotic stresses by transferring PsCBL and PsCIPK genes through Agrobacterium-mediated transformation. ResultsAbiotic stress tolerant PsCBL and PsCIPK genes isolated from Pisum sativum were transformed into two Citrus species, Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck, through Agrobacterium-mediated transformation method. Mature seed-derived calli of two Species were infected with Agrobacterium tumefaciens LBA4404 harboring PsCBL and PsCIPK genes. The infected calli were co-cultured in dark condition and later on washed with antibiotic solution and transferred to selection medium. Preliminary resistant calli were recovered and regenerated to plantlets. Maximum regeneration rate was 61.11 ± 1.35% and 55.55 ± 1.03%, respectively. The genetic transformation was confirmed by performing β glucuronidase (GUS) assays and subsequent PCR amplification of the GUS gene. The transformation rates of the two cultivated species were higher than previous reports. Maximum transformation frequencies were found when bacterial OD600 was 0.5 and concentration of acetosyringone was 150 μM. In-vitro evaluation of drought and salt tolerance of transgenic plantlets were done, and transgenic plantlets showed better performance than the control plants. ConclusionsThe present study demonstrates that transformation of Citrus plants with PsCBL and PsCIPK genes result in improved abiotic stress tolerance.

Highly efficient Agrobacterium-mediated transformation and plant regeneration system for genome engineering in tomato

Groundnut (Arachis hypogaea L.) is an industrial crop used as a source of edible oil and nutrients. In this study, an efficient method of regeneration and Agrobacterium-mediated genetic transformation is reported for a local cultivar GG-20 using de-embryonated cotyledon explant. A high regeneration 52.69 ± 2.32 % was achieved by this method with 66.6 μM 6-benzylaminopurine (BAP), while the highest number of shoot buds per explant, 17.67 ± 3.51, was found with 20 μM BAP and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The bacterial culture OD, acetosyringone and L-cysteine concentration were optimized as 1.8, 200 μM and 50 mg L(-1), respectively, in co-cultivation media. It was observed that the addition of 2,4-D in co-cultivation media induced accumulation of endogenous indole-3-acetic acid (IAA). The optimized protocol exhibited 85 % transformation efficiency followed by 14.65 ± 1.06 % regeneration, of which 3.82 ± 0.6 % explants were survived on hygromycin after selection. Finally, 14.58 ± 2.95 % shoots (regenerated on survived explants) were rooted on rooting media (RM3). In grafting method, regenerated shoots (after hygromycin selection) were grafted on the non-transformed stocks with 100 % survival and new leaves emerged in 3 weeks. The putative transgenic plants were then confirmed by PCR, Southern hybridization, reverse transcriptase PCR (RT-PCR) and β-glucuronidase (GUS) histochemical assay. The reported method is efficient and rapid and can also be applied to other crops which are recalcitrant and difficult in rooting.

In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.

In vitro plant regeneration was established in Echinacea pallida, a plant that is commonly used as a folk medicine to treat the common cold, fevers, inflammation and so on. Conditions for callus induction, lateral root and shoot regeneration were determined. Subsequently, two vectors pCHS and pOSAG78, carrying different selection marker genes resistant to kanamycin and hygromycin, respectively, were independently used to transform leaf explants of E. pallida using an Agrobacterium-mediated method. Genomic PCR analysis confirmed the presence of the transgene and selection marker gene in obtained transgenic lines. Southern hybridization indicated that the T-DNA insertion in some transgenic E. pallida was single copy. Among them, transformants carrying Petunia chalcone synthase (CHS) were selected for further study. CHS is a key enzyme in the biosynthesis of diverse flavonoids including anthocyanin pigmentation. Here, we analyzed the roles and compared the gene expression of two clusters of CHSs, EpaCHS-A and EpaCHS-B (EpaCHS-B1 and EpaCHS-B2), isolated from E. pallida. Two of the genes, EpaCHS-A and EpaCHS-B1, were abundantly expressed in petals, whereas EpaCHS-B2 was expressed at high levels in leaves. The expression of EpaCHSs remained constant in leaves and roots of Petunia CHS transformants, while EpaCHS-B2 expression was changed in flowers of transgenic plants. The biosynthesis of caffeic acid derivatives, cichoric acid and caftaric acid, was increased in leaves and roots of CHS transformants, respectively, while the amount of echinacoside in roots of transgenic plants was decreased. This is the first report on genetic engineering of E. pallida. The information contained herein can be used as a tool for further study of the biological pathways and secondary metabolism of specific compounds from medicinal Echinacea species.

Lettuce genetic transformation is genotype-dependent. In the present study, we have successfully developed an optimized Agrobacterium-mediated transformation protocol for elite lettuce cultivars, which belong to the romaine, leaf, and butterhead cultivar types. We optimized the type and concentration of plant growth regulators (PGRs) and selection antibiotics and found that the use of 1-naphthaleneacetic acid (NAA; 0.10 mg/L) and 6-benzyladenine (BA; 0.25 mg/L) as plant growth regulators, the use of hygromycin (15 mg/L) for transgenic plant selection, and the use of cotyledons and the first true leaf as explants efficiently yielded transformed plants for seven out of the eleven tested cultivars, achieving a 24.3–100% transformation efficiency. These seven cultivars include two romaine-type cultivars, three leaf-type cultivars, and two butterhead-type cultivars, and mark the first successful genetic transformation of the romaine cultivars ‘Kahu’ and ‘Rosalita’, the leaf cultivars ‘Red Sails’ and ‘Royal Oak Leaf’, and the butterhead cultivar ‘Lollo Biondo’. We also observed that substituting hygromycin selection with kanamycin selection (40 mg/L) resulted in a 64.3% transformation efficiency in the butterhead-type ‘Mariska’, one of the remaining four cultivars. Our newly optimized protocols are applicable in elite lettuce cultivars for Agrobacterium-mediated genetic transformation and regeneration, enabling hygromycin or kanamycin selection.

Chapter 14 - Agrobacterium-mediated transformation of plants

An efficient gene transfer system without tissue culture steps was developed for kidney bean by using sonication and vacuum infiltration assisted, Agrobacterium-mediated transformation. Transgenic kidney bean with a group 3 lea (late embryogenesis abundant) protein gene from Brassica napus was produced through this approach. Among 18 combinations of transformation methods, Agrobacterium-mediated transformation combined with 5 min sonication and 5 min vacuum infiltration turned to be optimal, resulting in the highest transformation efficiency. Transgenic kidney bean plants demonstrated enhanced growth ability under salt and water deficit stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by drought stress. Transgenic lines with high level of lea gene expression showed higher stress tolerance than lines with lower expression level. Stress tolerance of transgenic kidney bean correlated much better with lea gene expression levels than with gene integration results. There is no prior report on the production of transgenic kidney bean using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation.

CRISPR-Cas9-based genome editing is a more accurate and easy-to-use tool for genome modification of plant species. Transformation of binary vectors containing Cas9 and gRNA expression cassettes into Agrobacteriumstrains is most widely used for a wide range of plant species. In addition, CRISPR constructs can be used in transformants required for Agrobacterium-mediated gene transformation such as callus, leaves, or floral organs of plants. Agrobacterium-mediated gene transformation is more efficient than previously shown; however, due to the inherent mechanisms of plant interference of foreign RNA with plant antiviral activity, Cas9 expression levels will be suppressed by the plant, reducing the efficiency of the system. A vector system derived from tomato bushy stunt virus (TBSV) containing the suppressor protein p19, which interferes with the plant immune system, introduced together with the Cas9 protein, could improve the efficiency of genome editing. Previously obtained a TBSV strain TBSV_GFPΔCP with a GFP reporter gene, which can increase the expression level of the foreign gene and does not affect the symptomatology of the plant. In this study, TBSV_GFPΔCP were usedtogether with Cas9 introduced by Agrobacterium-mediated gene transformation and controlled the expression level of Cas9 using different constructs. Co-infiltration with TBSV_GFPΔCP showed 12-15% higher efficiency depending on the gRNA