Establishment of a transformation system in close relatives of wheat under the assistance of TaWOX5

Species closely related to wheat are important genetic resources for agricultural production,functional genomics studies and wheat improvement.In this study,a wheat gene related to regeneration,TaWOX5,was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum,hexaploid triticale,and rye(Secale cereale L.)using their immature embryos.Transgenic plants were efficiently generated.During the transformation process,the Agrobacterium infection efficiency was assessed by histochemical staining forβ-glucuronidase(GUS).Finally,the transgenic nature of regenerated plants was verified by polymerase chain reaction(PCR)-based genotyping for the presence of the GUS and bialaphos resistance(bar)genes,histochemical staining for GUS protein,and the QuickStix strip assay for bar protein.The transformation efficiency of T.monococcum genotype PI428182 was 94.4%;the efficiencies of four hexaploid triticale genotypes Lin456,ZS3297,ZS1257,and ZS3224 were 52.1,41.2,19.4,and 16.0%,respectively;and the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%.Fluorescence in situ hybridization(FISH)and genomic in situ hybridization(GISH)analyses indicated that the GUS transgenes were integrated into the distal or near centromere(proximal)regions of the chromosomes in transgenic T.monococcum and hexaploid triticale plants.In the transgenic hexaploid triticale plants,the foreign DNA fragment was randomly integrated into the AABB and RR genomes.Furthermore,the transgene was almost stably inherited in the next generation by Mendel’s law.The findings in this study will promote the genetic improvement of the three plant species for grain or forage production and the improvement of cereal species including wheat for functional genomics studies.

Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

Establishment of Protoplast Transformation System in Alternaria tenuissima Using G418 Selection Marker

[Objective] The study aimed to establish a protoplast transformation system in Alternaria tenuissima. [Method] The protoplast of A.tenuissima was firstly prepared by enzymolysis method; then the yielded protoplast was transformed by G418 resistant DNA plasmid using PEG/CaCl2 method. [Result] The growth phenotype and PCR detection showed that resistance gene had integrated into A.tenuissima genome. The transformation efficiency of this method reached per μg DNA 3-4 transformants. After subculture thrice under nonselective condition, G418 resistance could still inherit stably. [Conclusion] The transformation system of A.tenuissima was successfully established, which laid basis for studying of the gene function of Alternaria tenuissima.

Optimization of Genetic Transformation System of Tobacco K326 Mediated by Agrobacterium

[Objective]The aim was to optimize genetic transformation system in tobacco K326 mediated by Agrobacterium.[Method]The leaf of tobacco aseptic seedling was taken as explants to study the optimization of Agrobacterium-mediated genetic transformation system.[Result] The highest transformation efficiency was obtained when the explants were pre-cultured in the medium of MS ＋ 2 mg/L 6-BA ＋ 0.2 mg/L IAA for 2 d,and then infected with Agrobacterium GV3101（OD600 =0.6） for 5 min.The PCR detection proved that npt II gene had been integrated into the regenerated tobacco plants.[Conclusion]A highly efficient genetic transformation system of tobacco leaf mediated by Agrobacterium was established.

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.

Optimization of Agrobacterium Mediated Genetic Transformation System of Tomato Meifen No.1

[Objective] The aim of this study was to carry out study on the optimization of Agrobacterium mediated genetic transformation system of tomato Meifen No.1.[Method] The cotyledon of tomato cultivar Meifen No.1 was used as the explant,and the Agrobacterium mediation method was used to optimize its genetic transformation efficiency so as to establish the efficient Agrobacterium mediated genetic transformation system of tomato cotyledon.[Result] The highest transformation efficiency was obtained when the explants were cultivated for 2 d on MS ＋ 2.0 mg/L 6-BA＋ 0.5 mg/L IAA medium and then infected with Agrobacterium EHA105（OD = 0.4）for 5 min;it was proved by PCR analysis that the target nptII gene had been integrated into the genome of regenerated plants.[Conclusion] The result in this study had provided basis for the transfer of valuable genes into tomato Meifen No.1.

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.

Approaches to the Elaboration of Regeneration and Transformation Systems for Elite Kazakh Cotton Varieties

The development and wide application of genetic transformation for cotton improvement are restrained by the unresolved problem of genotype dependence in regeneration in vitro.High embryogenic and regenerative potential have been obtained for limited number of Coker type genotypes。

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize(Zea mays L.)

Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.

Establishment of A Simple and Efficient Agrobacterium-mediated Genetic Transformation System to Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Chinese cabbage,belonging to Brassica rapa species,is an important vegetable in Eastern Asia.It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low.The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.In this study,we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors,with 93.50%regeneration rate on average.Based on this,a simple and efficient Agrobacteriummediated genetic transformation methodwas established,without pre-culture procedure and concentration adjustment of hormone and AgNO_(3) in co-cultivation and selection media.Using this system,transformants could be obtained within 3.5–4.0 months.Average transformation frequency is up to 10.83%.The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.

Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1 IA

To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation(ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118.

Development of Highly Efficient Transformation System of Yeast-Like Conidia of Tremella fuciformis

Tremellafuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, which is also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. Thus, it is a good recipient cell for exogenous gene expression. In this study, the expression plasmid pAN7-1 （containing promoter gpd-An derived from Aspergillus nidulans and selectable marker gene hph conferring resistance to hygromycin B） and plasmid pLg-hph （containing promoter gpd-Le derived from Lentinula edodes and selectable marker gene hph） were transformed into the yeast-like conidia of T. fuciformis by PEG-mediated protoplast transformation, respectively. The primary putative transformants were selected by the sandwich screening method with the selective medium containing 50 μg mL^-1 hygromycin. The putative transformants were obtained from the primary putative transformants transferred on PDSA plates containing 100 μg mL^-1 hygromycin for second round selection. Experimental results showed that the optimal concentration of PEG 4000 for mediating protoplast transformation was 25%. PCR and Southern blotting confirmed that the selectable marker gene hph was integrated effectively into the genome of the yeast-like conidia of T. fuciformis with plasmid pLg-hph transformation. Its transformation efficiency was 110 transformants per μg DNA, and the hph gene was integrated into the genome of some yeast-like conidia with plasmid pAN7-1 transformation. However, its transformation efficiency was only 9 transformants per μg DNA. The presence of hph gene in the genome of transformants after 5 generations of sub- culturing on PDSB medium was confirmed by PCR, suggesting that the foreign gene hph was stable during subculture.

Establishment of genetic transformation system via Agrobacterium in tall fescue cultivar

Tall fescue （Festuca arundinacea Schreb.） is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable, and repeatable approach in transforming the grass using Agrobacterium （EHA105）, where β-glucuronidase gene （uidA） was used as a reporter and hygromycin phosphotransferase gene （hyg） as a selectable marker. An effective expression of transgene was observed in transforming 2-month-old calli derived from mature seeds （cv. Bingo） cultured on MS medium supplemented with 9 mg·L^-1 2, 4-D. A two-step solid medium selection with increasing hygromycin concentration （from 30 to 50 mg· L^-1） was used to obtain resistant calli. Transgenic plants have been produced from many independent transformed calli. The presence of functional β-glucuronidase gene （uidA） was detected in hygromycin-resistant calli. Transgenic plants were regenerated and PCR and Southern blot confirmed transgene integration in the tall fescue genome.

Main Achievements of Cotton Large-scale Transformation System

Cotton large-scale transformation methods system was established based on innovation of cotton transformation methods.It obtains 8000 transgenic cotton plants per year by combining Agrobacterium tumefaciens-mediated,pollen-tube pathway and biolistic methods together efficiently.More

Development of High Efficient in vitro Regeneration and Transformation System in Strawberry

In this paper, several factors that affect the efficiency of in vitro adventitious bud regeneration and Agrobacterium tumefaciens-mediated transformation of F. vesca were studied. The results showed that F. vesca seeds germination rate was the highest while seeds were cultured in water, and the germination rate was the lowest while seeds were cultured on MS medium supplemented with hormone; the germination rates that seeds cultured on two and three layers filter paper were higher than that seeds cultured on four and five layers filter paper. In vitro adventitious regeneration efficiency was affected by different explants types. The significant difference was existed between petioles and leaves. When using the same type explants, in vitro adventitious buds regeneration rate and the average number of buds per explant between Ruegen （RE） and Yellow Wonder （YW） had no significant difference. RE to Agrobacterium tumefaciens was more sensitive than YW. Using seedling leaves of RE and YW as materials, an efficient Agrobacterium-mediated transformation system was developed. In this system, the concentration of bacteria was OD600=0.5, the explants were immersed in bacteria broth for 9 min, the co-cultured time was 2 days, and had no pre-cultured time. The percentage of explants with resistant buds of RE and YW was compared. The putative transformed plants were confirmed by PCR.

Regeneration and gene transformation systems of Robima pseudoacacia 'Idaho' mediated by Agrobacterium tumefaciens

Robinia pseudoacacia ‘Idaho＇ is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5 mg·L^-16-BA benefitted callus proliferation and 0.25 mg·L^-1 IBA promoted shoot rooting; however, a higher IBA concentration will inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentration of it was 8 mg·L^-1.

IN VIVO-I:VITRO TRANSFORMATION SYSTEM OF RAT TRACHEAL EPITHELIAL CELLS

An in vivo-in vitro transformation system was establi-shed in primary rat tracheal epithelial cells(RTE) whichwere cultured in serum-free F12 medium with the additionof growth factors.Carcinogens were given in vivo by subcu-taneous injection or intratracheal instillation.About 7days after carcinogen exposure,RTE cells were cultured inserum-free medium and then selected in serum-containingmedium.Transformed colonies were counted 5-6 weeks afterthe plating of RTE cells.

Construction of a transformation system for the stable expression of foreign genes in Chlorella sp.

A stable transformation system for the expression of foreign genes in the unicellular green marine alga （Chlorella sp. MACC/C95）was established. Using electroporation, the alga was transformed with a plasmid containing the phytase gene under the control of CaMV35S promoter and the neomycin phosphotransferase （ npt ） as a selectable marker gene. The integration of the phytase gene into the Chlorella genome was revealed by PCR and Southern blotting analysis. RT-PCR analysis revealed the expression of phytase gene at the transcript level. The enhanced activity of phytase enzyme in the transformants confirmed the integration and successful expression of phytase gene. The introduced phytase gene and its protein expression were stably maintained for at least 30 generations in media devoid of selectable antibiotics G418. This is an important step toward the production of useful foreign proteins in Chlorella sp. MACC/C95.

Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System for Aspergillus awamori

[ Objective ] This study aimed to establish Agrobacterium tumefaciens-mediated genetic transformation system for Aspergillus awamori and investigate the feasibility of expressing heterologous proteins in A. awamori. [ Method] Appropriate A. awamori host strains were determined according to the secretory protein profile. Selectable marker was selected for genetic transformation by drug sensitivity analysis. The established A. awamori genetic transformation system was used for transformation and expression analysis of Rhizomucor miehei lipase （RML）. The feasibility of using A. awamori to express heterologous proteins was investigated by identification of transformants and property analysis. [ Result~ Based on the analysis of secretory protein profile, A. awamori strains CBS115.52 and CICC2257 were determined as the host strains for heterologous protein expression ; drug sensitivity analysis shows that hygromycin B resistance gene （ HygBr ） is an effective ge- netic seleetable marker; by using Agrobacterium tumefaciens-mediatcd transformation （ATMT） method, the plasmid pHGW-amdS containing HygBr was successfully transformed into A. awamori strain CBS115.52 to establish the genetic transformation system ofA. awamorl with HygBr as selectable marker. RML was transformed into A. awamori and its expression was validated by substrate hydrolysis test, SDS-PAGE and Western blot. [ Conclusion] This study demonstrates that the genetic transformation system of A. awamori mediated by Agrobacterium tumefaciens has potential feasibility for expression of heterologous proteins.

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.