Mutant acetolactate synthase （ALS） gene as a selectable marker for Agrobacterium-mediated transformation of soybean

Soybean is one of the crops most difficult to be manipulated in vitro. Although several soybean marker genes, all the selectable markers used were from bacteria origin. To find suitable selectable marker gene from plant origin for soybean transformation, a mutant acetolactate synthase （ALS） gene from Arabidopsis thaliana was tested for Agrobacterium-mediated soybean embryo axis transformation with the herbicide Arsenal as the selective agent. Transgenic soybean plants were obtained after the herbicide se- lection and the To transgenic lines showed resistance to the herbicide at a concentration of 100 g/ha. ALS enzyme assay of To transgenic line also showed higher activity compared to the wild type control plant. PCR analysis of the T1 transgenic lines confirmed the integration and segregation of the transgene. Taken together, our results showed that the mutant ALS gene is a suitable selectable marker for soybean transformation.

Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute(Corchorus capsularis)

Jute(Corchorus capsularis L.)is the second most important natural plant fiber source after cotton.However,developing an efficient gene editing system for jute remains a challenge.In this study,the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4,an elite jute variety widely cultivated in China.The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation(BiFC)assays.The CHLOROPLASTOS ALTERADOS 1(CcCLA1)gene,which is involved in the development of chloroplasts,was targeted for editing at two sites in Meifeng 4.Based on this hairy root transformation,the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and-GhU6.9 promoters,respectively,to assess the efficiency of gene editing.Results indicated the 50.0%(GhU6.7)and 38.5%(GhU6.9)editing events in the target 2 alleles(gRNA2),but no mutation was detected in the target 1 allele(gRNA1)in transgenic-positive hairy roots.CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation.Two CcCLA1 mutants were albinic,with a gene editing efficiency of 5.3%.These findings confirm that the CRISPR/Cas9 system,incorporating promoter GhU6.7,can be used as a gene editing tool for jute.

Establishment of the Agrobacterium-mediated Genetic Transformation System of Ginkgo biloba and the Construction of the Expression Vector of Gb-DXR

[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.

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.

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.

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.

Soybean hairy roots produced in vitro by Agrobacterium rhizogenes-mediated transformation

Soybean is one of the world's most important oil and protein crops. Efficient transformation is a key factor for the improvement of soybean by genetic modification. We describe an optimized protocol for the Agrobacterium rhizogenes-mediated transformation of soybean and the induction of hairy root development in vitro. Cotyledons with 0.5-cm hypocotyls were cut from 5-day-old seedlings and used as explants. After infection and co-cultivation,hairy roots were produced in induction culture medium after 10–12 days. Using this method, 90%–99% of the infected explants of five different cultivars produced hairy roots within one month. Observations using reporter constructs showed that 30%–60% of the hairy roots induced were transformed. Based on high transformation efficiency and short transformation period, this method represents an efficient and rapid platform for study of soybean gene function.

Co-treatment with surfactant and sonication signifi cantly improves Agrobacterium-mediated resistant bud formation and transient expression efficiency in soybean

Soybean is a widely planted genetically modified crop around the world. However, it is still one of the most recalcitrant crops for genetic transformation due to the difficulty of regeneration via organogenesis and some factors that affect the transformation efficiency. The percentages of resistant bud formation and transient expression efficiency are important indexes reflecting the regeneration and transformation efficiency of soybean. In this study, the percentages of resistant bud formation and transient expression of 13-glucuronidase （GUS） were compared after treatment with sonication or surfactant and co-treatment with both. The results showed that treatment with either sonication or surfactant increased the percentage of resistant bud formation and transient expression efficiency. The highest percentages were acquired and significantly improved when cotyledon node explants were co-treated with sonication for 2 s and surfactant at 0.02% （v：v） using two different soybean genotypes, Jack and Zhonghuang 10. The improved transformation efficiency of this combination was also evaluated by development of herbicide-tolerant soybeans with transformation efficiency at 2.5-5.7% for different geno- types, which was significantly higher than traditional cotyledonary node method in this study. These results suggested that co-treatment with surfactant and sonication significantly improved the percentages of resistance bud formation, transient expression efficiency and stable transformation efficiency in soybean.

A callus transformation system for gene functional studies in soybean

Obtaining transgenic plants is a common method for analyzing gene function. Unfortunately, stable genetic transformation is difficult to achieve, especially for plants（e.g., soybean）, which are recalcitrant to genetic transformation. Transient expression systems, such as Arabidopsis protoplast, Nicotiana leaves, and onion bulb leaves are widely used for gene functional studies. A simple method for obtaining transgenic soybean callus tissues was reported recently. We extend this system with simplified culture conditions to gene functional studies, including promoter analysis, expression and subcellular localization of the target protein, and protein-protein interaction. We also evaluate the plasticity of this system with soybean varieties, different vector constructs, and various Agrobacterium strains. The results indicated that the callus transformation system is efficient and adaptable for gene functional investigation in soybean genotype-, vector-, and Agrobacterium strain-independent modes. We demonstrated an easy set-up and practical homologous strategy for soybean gene functional studies.

Simultaneous genetic transformation and genome editing of mixed lines in soybean(Glycine max)and maize(Zea mays)

Robust genome editing technologies are becoming part of the crop breeding toolbox.Currently,genome editing is usually conducted either at a single locus,or multiple loci,in a variety at one time.Massively parallel genomics platforms,multifaceted genome editing capabilities,and flexible transformation systems enable targeted variation at nearly any locus,across the spectrum of genotypes within a species.We demonstrate here the simultaneous transformation and editing of many genotypes,by targeting mixed seed embryo explants with genome editing machinery,followed by re-identification through genotyping after plant regeneration.Transformation and Editing of Mixed Lines(TREDMIL)produced transformed individuals representing 101 of 104(97%)mixed elite genotypes in soybean;and 22 of 40(55%)and 9 of 36(25%)mixed maize female and male elite inbred genotypes,respectively.Characterization of edited genotypes for the regenerated individuals identified over 800 distinct edits at the Determinate1(Dt1)locus in samples from 101 soybean genotypes and 95 distinct Brown midrib3(Bm3)edits in samples from 17 maize genotypes.These results illustrate how TREDMIL can help accelerate the development and deployment of customized crop varieties for future precision breeding.

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.

Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride

Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.

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.

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.

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 .