Development of an Agrobacterium-mediated CRISPR/Cas9 system in pea(Pisum sativum L.)

Pea(Pisum sativum L.)is an annual cool-season legume crop.Owing to its role in sustainable agriculture as both a rotation and a cash crop,its global market is expanding and increased production is urgently needed.For both technical and regulatory reasons,neither conventional nor transgenic breeding techniques can keep pace with the demand for increased production.In answer to this challenge,CRISPR/Cas9 genome editing technology has been gaining traction in plant biology and crop breeding in recent years.However,there are currently no reports of the successful application of the CRISPR/Cas9 genome editing technology in pea.We developed a transient transformation system of hairy roots,mediated by Agrobacterium rhizogenes strain K599,to validate the efficiency of a CRISPR/Cas9 system.Further optimization resulted in an efficient vector,PsU6.3-tRNA-PsPDS3-en35S-PsCas9.We used this optimized CRISPR/Cas9 system to edit the pea phytoene desaturase(PsPDS)gene,causing albinism,by Agrobacterium-mediated genetic transformation.This is the first report of successful generation of gene-edited pea plants by this route.

Agrobacterium-Mediated Transformation of Rice: Constraints and Possible Solutions

Genetic transformation of rice(Oryza sativa L.) by introducing beneficial traits is now a central research instrument in plant physiology and a practical tool for plant improvement. Many approaches are verified for stable introduction of foreign genes into the plant genome. The review examined the different constraints that limit the success of rice genetic transformation via Agrobacterium-mediated approach and suggested possible solutions. Explant identification, gene transfer technique and construct to tailor the integration, transgene expression without collateral to genetic damage and transformant selection are among the technical challenges affecting the rice transformation. Despite the contests, Agrobacteriummediated transformation system has been a better option for producing transgenic rice varieties because of its exact T-DNA processing and simple integration of low copy-number transgene. This information is necessary for improving the transformation system for recalcitrant rice varieties.

Agrobacterium-Mediated Transfer of Arabidopsis ICE1 Gene into Lemon (Citrus Limon (L.) Burm. F. cv. Eureka)

The Arabidopsis ICEI （inducer of CBF expression 1） gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon （Citrus Limon （L.） Burm. F. cv. Eureka） genome using Agrobacterium-mediated transformation method. Epicotyl segments from in vitro grown lemon seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pMVICE1, whose T-DNA region contain ICEI gene driven by 35S CaMV promoter. Among 320 epicotyl segments inoculated, 71 explants responded and regenerated 51 elongated shoots. These shoots were subjected to an extra month of kanamycin exposure. In this way, the number of escapes reduced. Thirteen of 31 survived shoots formed roots and 7 were tested positive using PCR technique. Southern blot analyses confirmed PCR results and demonstrated that more than two copies of the ICE1 gene were integrated into the lemon genome.

In planta Agrobacterium-Mediated Transformation of Rice

The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to various plant species. In this study, the optimal conditions of a floral-dip method for production of transgenic rice variety RD41 were explored. The simple and effective inoculation medium was composed of Murashige and Skoog(MS) medium, 5% sucrose, 44 nmol/L benzylaminopurine, and 0.075% surfactant Tween-20 with pH 5.7. The transformation efficiencies of Agrobacterium tumefaciens strains AGL1 and EHA105 were compared with the Agrobacterium density at OD_(600) = 0.8–1.0 and the co-cultivation at 25 ℃ for 48 h. A. tumefaciens strain EHA105 gave slightly higher transformation efficiency than AGL1, with statistically non-significant difference. The floral-drop transformation using the optimal floral-dip conditions showed higher transformation efficiency than the floral-dip method, but the dropped flowers turned brown and died within 2 d. Production of transgenic rice variety RD41 by the floral-dip method was achieved using A. tumefaciens strain EHA105 with the optimal conditions. Screening for the gus A gene by PCR using the gus A specific primers in the T_0 lines, there were 4 transgenic lines from 286 T_0 lines(1.4% transformation efficiency). However, histochemical glucuronidase(GUS) assay demonstrated that only three of four transgenic lines exhibited gus A expression. These results indicated that floral-dip transformation is a potential tool for production of the transgenic rice, which can be used for molecular breeding via genetic engineering in the future.

Effect of Phytosulfokine-α on Agrobacterium-Mediated Transformation in Rice

Phytosulfokine- α （PSK- α ）, a biologically active peptide acting as a growth factor, plays a key role in cellular differentiation and proliferation. To test if PSK- α has some influence on agrobacterium-mediated transformation in rice, PSK-α at a series of concentrations was added into co-culture medium respectively. The results showed that PSK- α indeed affected the recovery of resistant calli and the transformation frequency of rice varieties Taipei 309 and Lijiangxintuanheigu, PSK- α at the concentration of 10 nmol/L could increase induction of resistant callus and efficiency of transformation, with a 11% and 4.9% top increase, respectively than the control. However, PSK- αat 200 nmol/L could inhibit the induction of the resistant calli. Further more, the effect of PSK-α on agrobacterium-mediated transformation is related with the concentration of 2, 4-D in selection medium. Higher induction rate of resistant calli was obtained from tissues treated with PSK- α plus 2 mg/L 2, 4-D.

Agrobacterium-Mediated Transformation of Embryogenic Calli of Anliucheng and Regeneration of Plants Containing the Chimaeric Ribonuclease Gene

Anliucheng (Citrus sinensis Osbeck), a very seedy and widely spread acidless sweet orange cul-tivar in south of China, was transformed by the strain of Agrobacterium Tumefaciens EHA105 carrying pTA29-barnase gene, which will induce pollen sterility in transgenic plants. The embryogenic calli of Anliucheng were co-cultivated with Agrobacterium tumefaciens for 3 days, and then transferred to selective medium containing 50 mg I/1 basta (a kind of herbicide) for 5 weeks. The resistant calli were recovered and regenerated 118 embryoids. A total of 13 entire plants were obtained after micro-grafted on trifoliate orange. These regenerated plants were verified by PCR amplification and confirmed by PCR-Southern blotting analysis.

Efficient Regeneration and Agrobacterium-mediated Transformation of Brassica napus Cultivar Qingza No.5

Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cuhivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cuhivar Qingza No. 5 reached about 90%, which was three times that of cotyledons. The appropriate differentiation medium was MSB ＋ 5 mg/L thidiazuron （TDZ） ＋7.5 mg/L AgNO3 ＋ 0.1 mg/L NAA ＋ 2 mg/L proline （L-pro） ＋ 250 mg/L casein acid hydrolysate （CH） ＋ 3% sucrose; the appropriate growth medium was 1/2 MSB ＋ 1 mg/L IBA ＋ 2 mg/L L-pro ＋ 250 mg/L CH ＋ 1.5% sucrose; the ap- propriate rooting medium was 1/2 MSB ＋ 0.2 mg/L IAA ＋ 1.5% sucrose. On this basis, a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin. Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nu- clear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants.

Screening Chinese soybean genotypes for Agrobacterium-mediated genetic transformation suitability

The Agrobacterium-mediated transformation system is the most commonly used method in soybean transformation.Screening of soybean genotypes favorable for Agrobacterium-infection and tissue regeneration is the most important step to establish an efficient genetic transformation system.In this study,twenty soybean genotypes that originated from different soybean production regions in China were screened for transient infection,regeneration capacity,and stable transgenic efficiency.Three genotypes,Yuechun 04-5,Yuechun 03-3,and Tianlong 1,showed comparable stable transgenic efficiencies with that of the previously reported American genotypes Williams 82 and Jack in our experimental system.For the Tianlong 1,the average stable transformation efficiency is 4.59%,higher than that of control genotypes(Jack and Williams 82),which is enough for further genomic research and genetic engineering.While polymerase chain reaction(PCR),LibertyLink strips,and β-glucuronidase(GUS) staining assays were used to detect the insertion and expression of the transgene,leaves painted with 135 mg/L Basta could efficiently identify the transformants.

Agrobacterium-mediated delivery of CRISPR/Cas reagents for genome editing in plants enters an era of ternary vector systems

Lack of appropriate methods for delivery of genome-editing reagents is a major barrier to CRISPR/Cas-mediated genome editing in plants.Agrobacterium-mediated genetic transformation(AMGT)is the preferred method of CRISPR/Cas reagent delivery,and researchers have recently made great improvements to this process.In this article,we review the development of AMGT and AMGT-based delivery of CRISPR/Cas reagents.We give an overview of the development of AMGT vectors including binary vector,superbinary vector,dual binary vector,and ternary vector systems.We also review the progress in Agrobacterium genomics and Agrobacterium genetic engineering for optimal strains.We focus in particular on the ternary vector system and the resources we developed.In summary,it is our opinion that Agrobacterium-mediated CRISPR/Cas genome editing in plants is entering an era of ternary vector systems,which are often integrated with morphogenic regulators.The new vectors described in this article are available from Addgene and/or MolecularCloud for sharing with academic investigators for noncommercial research.

A Novel Approach to Functional Analysis of the Ribulose Bisphosphate Carboxylase Small Subunit Gene by Agrobacterium-Mediated Gene Silencing

A novel approach to virus-induced post-transcriptional gene silencing for studying the function of the ribulose bisphosphate carboxylase small subunit （rbcS） gene was established and optimized using potato virus X vector and Nicotiana benthamiana as experimental material. The analysis of silencing phenomena, transcriptional level, protein expression, and pigment measurement showed that the expression of the rbcS endogenous gene was inactivated by the expression of a 500-bp homologous cDNA fragment carried in the virus vector.

Development of glyphosate-tolerant transgenic cotton plants harboring the G2-aroA gene

Given that glyphosate weed control is an effective strategy to reduce costs and improve economic outcomes of agricultural production in China,the development of glyphosate-resistant cotton holds great promise.Using an Agrobacterium-mediated transformation method,a new G2-aroA gene that encodes 5-enolpyruvylshikimate-3-phosphate synthase（EPSPS）was transformed into cotton cultivar K312.The transgenic cotton plants were regenerated from a callus tissue culture via kanamycin selection.Ten regenerated cotton plants were obtained and allowed to flower normally to produce fruit.The results from polymerase chain reaction（PCR）and Southern and Western blot analyses indicated that the target gene was integrated into the cotton chromosome and was expressed effectively at the protein level.The glyphosate tolerance analysis showed that the transgenic cotton had a high resistance to glyphosate.Further,even cotton treated with 45.0 mmol L^–1 of glyphosate was able to slowly grow,bloom and seed.The transgenic cotton may be used for cotton breeding research of glyphosate-tolerant cotton.

Infection of Early and Young Callus Tissues of Indica Rice BPT5204 Enhances Regeneration and Transformation Efficiency

A rapid and reproducible method to develop transgenic plants with enhanced transformation efficiency using Agrobacterium has been developed for the elite indica rice variety BPT 5204. Different rice calli aged from 3 to 30 d were co-cultivated with pre-incubated Agrobacterium suspension culture （LBA4404： pSB1, pCAMBIA1301） and incubated in dark for 3 d. Based on the transient GUS gene expression analysis, 6-day-old young calli showed high transformation frequency followed by 21-day-old ones. Thus, both 6-and 21-day-old calli were used for assessing the stable transformation efficiency. It was observed that the 6-day-old young transformed calli showed about 2-fold higher regeneration frequency when compared with 21-day-old calli. The transformation efficiency was enhanced for young calli to 5.9% compared with 0.8% of the 21-day-old calli. Molecular and genetic analysis of transgenic plants （To） revealed the presence of 1-2 copies of T-DNA integration in transformants and it follows Mendalian ratio in T1 transgenic plants. From the present study, it was concluded that the development of transgenic rice plants in less duration with high regeneration and transformation efficiency was achieved in BPT 5204 by using 6-day-old young calli as explants.

Response of Explants of Calli Rice (<i>Oryza sativa</i>L.) Japonica cv. “Ilmi” to Gene Transformation Using <i>Agrobacterium tumefaciens</i>-Mediated

The AtBI-1 gene encoding the Arabidopsis thaliana Bax inhibitor was introduced into Japonica cultivars of rice (“Ilmi”) by Agrobacterium-mediated transformation, and a large number of transgenic plants were produced. The neomycin phosphotransferase II (NPTII) gene was used as a selectable marker. The activity of neomycin phosphotransferase could be successfully detected in transgenic rice calluses. Introduction of the AtBI-1 gene was also confirmed by PCR using AtBI-1 specific oligonucleotide primers in regenerated plants. Stable integration and expression of the AtbI-1 gene in plants were confirmed by GFP analysis.

Constructing the barley model for genetic transformation in Triticeae

Barley（Hordeum vulgare L.） is one of the oldest domesticated crops, showing dramatic adaptation to various climate and environmental conditions. As a major cereal crop, barley ranks the 4th after wheat, maize and rice in terms of planting area and production all over the world. Due to its diploid nature, the cultivated barley is considered as an ideal model to study the polyploid wheat and other Triticeae species. Here, we reviewed the development, optimization, and application of transgenic approaches in barley. The most efficient and robust genetic transformation has been built on the Agrobacterium-mediated transfer in conjunction with the immature embryo-based regeneration. We then discussed future considerations of using more practical technologies in barley transformation, such as the T-DNA/transposon tagging and the genome editing. As a cereal crop amenable to genetic transformation, barley will serve as the most valuable carrier for global functional genomics in Triticeae and is becoming the most practical model for generating value-added products.

Optimization of Agrobacterium tumefaciens-Mediated Genetic Transformation of Maize

Immature embryos of inbred maize(Zea mays)lines(H8183,H8184,and H8185)were used for Agrobacterium infection.We used theβ-glucuronidase gene(GUS)as the target gene and the glufosinate resistance gene(bar)as the selection marker.We conducted research on several aspects,such as different genotypes,coculture conditions,screening agent concentrations,and concentrations of indole-3-butytric acid(IBA),6-benzylaminopurine(6-BA),and ascorbic acid(Vc)in the differentiation medium.We optimized the genetic transformation system,and the obtained results indicated that among the three lines studied,the induction rate of H8185 was the highest at 93.2%,followed by H8184,with H8183 having the lowest induction rate(80.1%).The best coculture method was that using the N6 coculture medium layered with a sterile filter paper.Using orthogonal analysis,we found that the optimal combination of the three factors in the differentiation medium was A_(3)(1 mg mL^(−1)IBA),B_(3)C_(1)(1.6 mg mL^(−1)6-BA),and D_(3)(1.5 mg mL^(−1)Vc).Through GUS staining analysis,Bar test-strip analysis,and polymerase chain reaction,five transgenic plants were finally obtained.This study established the optimal conditions for genetic transformation in maize.

Establishment of an efficient regeneration and genetic transformation system for Malus prunifolia Borkh. ‘Fupingqiuzi’

Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research. However, its long juvenile phase and high heterozygosity are barriers to the identification of ‘Fupingqiuzi’ progeny with excellent traits. In-vitro regeneration techniques and Agrobacterium-mediated genetic transformation systems can efficiently produce complete plants and thus enable studies of gene function.However, optimal regeneration and genetic transformation systems for ‘Fupingqiuzi’ have not yet been developed.Here, we evaluated the factors that affect the in-vitro regeneration and transformation of ‘Fupingqiuzi’. The best results were obtained when transverse leaf sections were used as explants, and they were grown in dark culture for three weeks with their adaxial sides contacting the culture medium(MS basal salts, 30 g Lsucrose, 8 g Lagar, 5 mg L6-benzylaminopurine(6-BA), 2 mg Lthidiazuron(TDZ), and 1 mg L1-naphthlcetic acid(NAA), pH 5.8). A genetic transformation system based on this regeneration system was optimized: after inoculation with A. tumefaciens solution for 8 min, 4 days of co-culture, and 3 days of delayed culture, the cultures were screened with cefotaxime(150 mg L) and kanamycin(15 mg L). We thus established an efficient regeneration and genetic transformation system for ‘Fupingqiuzi’, enabling the rapid production of transgenic material. These findings make a significant contribution to apple biology research.

Transcriptome Profiling of Barley Cultivar Hua 30 MDEC in Response to Agrobacterium Infection

Agrobacterium-mediated transformation has been widely used in plants.However,the mechanism in plant cells’response to Agrobacterium infection was very complex.The mechanism of the determinants in host cell remains obscure,especially in barley,which is recalcitrant for Agrobacterium-mediated transformation.In the present study,microspore-derived embryogenic calli(MDEC)from barley elite cultivar were employed as unique subjects to characterize the mechanisms during the Agrobacterium infection process.Hua 30 MDEC can be successfully infected by Agrobacterium.RNA-sequencing at different infection points(0,2,6,12,24 hpi)was performed.The average expressional intensity of the whole genomics increased from 0 to 2 hpi,and then decreased subsequently.More upregulated than downregulated differentially expressed genes(DEGs)were counted at the same time.GO enrichment analysis showed that protein modification was significantly overrepresented in upregulated DEGs.Chromosome-related biological processes,gene expression and cellular metabolic processes were significantly overrepresented in downregulated DEGs.KEGG analysis showed that plant defense responses,phenylpropanoid biosynthesis and biosynthesis of amino acids were significantly enriched across the infection time course.Nine DEGs related to defense responses were identified.All DEGs were upregulated from 2 to 24 hpi.We speculate that these genes are possibly related to Agrobacterium infection.These findings will provide deep insights into the molecular events occurring during the process of Agrobacterium-mediated transformation.

Female reproductive system of <i>Amaranthus</i>as the target for <i>Agrobacterium</i>-mediated transformation

Agrobacterium-mediated transformation through floral dip and rapid selection process after transgenic event had become a preference as it will overcome the difficulties faced in tissue culturing procedures and lengthy time for screening transformed progenies. Therefore, in this study, three constructs, p5b5 (14,289 bp), p5d9 (15,330 bp) and p5f7 (15,380 bp) in pDRB6b vector which has hygromycin as a selectable marker gene were introduced individually into Agrobacterium tumefaciens strain (AGL1). The cell suspension was applied to Amaranthus inflorescence by drop-by-drop technique and was left to produce seeds (T1). The T1 seeds were germinated and grown to produce seedlings under non-sterile condition. Hygromycin selection on seedling cotyledon leaves results in identification of 12 putative transformants, three from p5b5, four from p5d9 and five from p5f7. All positive putative transformants that were selected at the first stage through hygromycin spraying showed positive result in leaf disk hygromycin assay and in a construct specific polymerase chain reaction-based assay. A ~750 bp amplified hygromycin gene was further verified through sequencing. Our results suggest that Amaranthus inflorescences were able to be transformed and the transformed progenies could be verified through a combination of simple and rapid methods .

Transforming the Snapdragon Aurone Biosynthetic Genes into Petunia Alters Coloration Patterns in Transgenic Flowers

Aurones belong to a small class of flavonoids that provide yellow color in some floricultural plants including snapdragon. To explore novel flower coloration, two full-length cDNAs encoding chalcone 4'-O-glucosyltransferase (designated as SRY4'CGT) and aureusidin synthase (designated as SRYAS1) in the aurone biosynthetic pathway were cloned from yellow flowers of snapdragon (Antirrhinum majus cv. Ribbon Yellow). Binary vectors were constructed and transformed separately into Petunia hybrida harboring blue flowers. Only a few flowers in 4 out of 9 transgenic SRY4'CGT plants showed variegated blue-white sectors;as time passed, amounts of variegated flowers and proportion of white sectors in the background blue color of the new-born flowers gradually increased, until finally, the petal color was completely white in all late-born flowers. In contrast, a few flowers in 3 out of 13 transgenic SRYAS1 plants showed variegated blue-white sectors;but, the amounts of variegated flowers did not increase over the whole flowering stage, and no complete white flowers were observed. RNA samples isolated from blue and white sectors of T1 transgenic SRY4'CGT plants were analyzed by reverse transcription-PCR, transgenic SRY4'CGT transcripts were detected in both sectors;however, transcripts of an upstream gene, chalcone synthase (CHS), were abundantly detected in the blue sectors but largely reduced in the white sectors, suggesting that the expression of CHS gene was suppressed in white sectors of transgenic plants. Furthermore, HPLC coupled with mass spectrometry demonstrated cyandin, malvidin and their derivatives were absent in white sectors, causing the white phenotype. Our findings may be attractive to molecular breeders.

Creation of Transgenic Bananas Expressing Human Lysozyme Gene for Panama Wilt Resistance

Human lysozyme （HL） inhibits Fusarium oxysporum （FocR4） growth in vitro. To obtain transgenic bananas （Musa spp.） that are resistant to Panama wilt （F. oxysporum）, we introduced an HL gene that is driven by a constitutive cauliflower mosaic virus 35S promoter into the banana via Agrobacteriummediated transformation. PCR confirmed that 51 transgenic plants were obtained. The development of Panama wilt symptoms were examined after the plants had been grown in pots. The non-transgenic plants developed typical fusarium symptoms 60 d after FocR4 inoculation, whereas 24 of 51 transgenic plants remained healthy. The transgenic banana plants that showed resistance to FocR4 in the pots were then planted in a field that was heavily infected with FocR4 for further investigation. Eleven of 24 plants devel- oped symptoms before bud emergence; another 11 plants showed symptoms after bud emergence and the remaining two plants, H-67 and H-144, remained healthy and were able to fruit. Northern blotting analysis demonstrated that H-67 and H-144, bearing the strongest resistance to Panama wilt, had the highest level of HL expression and that the expression of HL was well correlated with the FocR4 resistance of transgenic plants. We conclude that Agrobacterium-mediated transformation, with the assistance of particle bombardment, is a powerful approach for banana transformation and that a transgenic HL gene can cause resistance of the crop to FocR4 in the field.