The Factors of Genetic Transformation for Indica Rice Kasalath

Objective The aim was to explore conditions of genetic transformation for Indica rice Kasalath and laid a foundation for further study on molecular biology. Method With callus of Kasalath as transformation receptor, Agrobacterium tumefaciens-mediated method was used to conduct genetic transformation. The genetic transformation system was optimized from several aspects, including co-culture mode, co-culture time and the affertreatment method of co-culture. Result The results showed that two days is the best co-culture time for genetic transformation, the acquisition rate of resistant callus was up to 84.1%, and transformation rate was up to 73%. Whether callus contact to the culture medium directly or indirectly has no significant effect on transformation. [ Conclusion] Genetic transformation successfully transferred exogenous gene OsMAPk2 into the rice genome.

Studies on Genetic Transformation of NPR1 Gene into Maize by Microprojectile Bombardment

[Objective] This study aimed to explore the conditions of transformation of maize by microprojectile bombardment. [Method] Immature embryo-derived callus of maize inbred line 7239 was used as explants to study the effects of shoot distance, helium pressure, vacuum and bombardment frequency on the transformation efficien- cy in the particle bombardment system of maize. [Result] Considering the transfor- mation efficiency, particle bombardment with 100 μg/P of golden particles, at a shoot distance of 9 cm from the target cells, under helium pressure of 1 350 psi and vac- uum 25 inHg, and bombarding twice could achieve relatively ideal results. After se- lection on media supplemented with different concentration of hygromycin, some re- generated plants were obtained. The results of PCR and Southern blotting analysis demonstrated that the NPR1 gene had been integrated into the genome of trans- genic maize plants, with an average transformation efficiency of 1.76%. [Conclusion] The study laid the foundation for the cultivation and breeding of excellent resistant varieties of maize.

Establishment of a New Method for Genetic Transformation of Dunaliella salina

The exogenous gene was integrated into Dunaliella salina successfully by using LiAc/PEG mediating method for the first time. According to the results of histochemical staining, transgenic D. salina was blue, showing that the exogenous GUS gene was successfully expressed in the cells of D. salina. Meanwhile, the effects of growth state of D. salina, plasmid concentration and temperature on its transformation efficiency were studied, and the transformation conditions were optimized. The results show that the optimum conditions for the genetic transformation of D. salina are shown as follows： D. salina was in the early logarithmic phase; plasmid DNA concentration was 600 μg/ml; temperature was 29 ℃, and transformation efficiency was up to 74.8‰ under the best conditions. According to the results of PCR amplification and PCR-Southern hybridization, the target gene had been integrated into genome and was hereditary.

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.

An efficient method for constructing a random insertional mutant library for forward genetics in Nannochloropsis oceanica

Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.

Population genomic analysis reveals key genetic variations and the driving force for embryonic callus induction capability in maize

Genetic transformation has been an effective technology for improving the agronomic traits of maize.However,it is highly reliant on the use of embryonic callus(EC)and shows a serious genotype dependence.In this study,we performed genomic sequencing for 80 core maize germplasms and constructed a high-density genomic variation map using our newly developed pipeline(MQ2Gpipe).Based on the induction rate of EC(REC),these inbred lines were categorized into three subpopulations.The low-REC germplasms displayed more abundant genetic diversity than the high-REC germplasms.By integrating a genome-wide selective signature screen and region-based association analysis,we revealed 95.23 Mb of selective regions and 43 REC-associated variants.These variants had phenotypic variance explained values ranging between 21.46 and 49.46%.In total,103 candidate genes were identified within the linkage disequilibrium regions of these REC-associated loci.These genes mainly participate in regulation of the cell cycle,regulation of cytokinesis,and other functions,among which MYB15 and EMB2745 were located within the previously reported QTL for EC induction.Numerous leaf area-associated variants with large effects were closely linked to several REC-related loci,implying a potential synergistic selection of REC and leaf size during modern maize breeding.

Cloning and Genetic Transformation of OsOle1 Gene in Rice

[Objective] The cloning and transformation of rice OsOle1 gene were conducted in the research.[Method] OsOle1 gene was cloned by RT-PCR.The amplified OsOle1 was then ligased to pCAMBIA 1300 to construct GUS overexpression vector.Then the Agrobacterium-mediated method was used in rice callus transformation.[Result] The full-length of OsOle1 gene was 498 bp and it encoded 189 amino acids.The over-expression vector Ub：：OsOe1-GUS was prepared and transgenic plants were successfully obtained.[Conclusion] The transgenic lines laid the foundation for the function research of OsOle1.

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.

Effects of Ultrasonication on Genetic Transformation via the Pollen-Tube Pathway in Chinese Cabbage

[Objective] This research aimed to explore the method to increase conversion rate of pollen-tub pathway in Chinese cabbage.[Method] Chinese cabbage varieties Yuqing and No.03 were used as materials for the selection of germination buffer and parameters for ultrasonication.[Result] The optimal buffer for pollen germination of Chinese cabbage was 200 g/L sucrose ＋ 100 mg/L boric acid ＋ 200 mg/L calcium nitrate,the preferred ultrasonic processing power was 150 W,processing time was 5 s,interval time was 5 s and processing frequency was 8.Three T1-generation plants were obtained through selection with 200 mg/L kanamycin.[Conclusion] This research laid foundation for the further genetic transformation of Chinese cabbage.

Construction and Genetic Transformation of Over-Expression Vector of nad1 Gene in Rice

[Objective] The aim was to clone the mitochondrial-related gene nad1 and produce transgenic rice plants with nad1.[Method] The total RNA was extracted from rice seedlings and reverse transcripted into cDNA.Then the target gene nad1 was amplified by using the cDNA as template.The nad1 and Rf1b,a sequence of signal peptide of mitochondria,were linked to binary expression vector pCAMBIA1305.1.The recombinant plasmid was transformed into the callus by Agrobacterium-mediated approach.[Result] The target gene nad1 was 978 bp.The binary expression vector carrying nad1 and signal peptide of mitochondria was constructed successfully.In addition,a lot of transgenic plants were obtained.[Conclusion] The study will provide basis to investigate the effect of over-expression of nad1 on rice plant growth.

A New Image Watermarking Scheme Using Genetic Algorithm and Residual Numbers with Discrete Wavelet Transform

Transmission of data over the internet has become a critical issue as a result of the advancement in technology, since it is possible for pirates to steal the intellectual property of content owners. This paper presents a new digital watermarking scheme that combines some operators of the Genetic Algorithm (GA) and the Residue Number (RN) System (RNS) to perform encryption on an image, which is embedded into a cover image for the purposes of watermarking. Thus, an image watermarking scheme uses an encrypted image. The secret image is embedded in decomposed frames of the cover image achieved by applying a three-level Discrete Wavelet Transform (DWT). This is to ensure that the secret information is not exposed even when there is a successful attack on the cover information. Content creators can prove ownership of the multimedia content by unveiling the secret information in a court of law. The proposed scheme was tested with sample data using MATLAB2022 and the results of the simulation show a great deal of imperceptibility and robustness as compared to similar existing schemes.

Current status of genetic transformation technology developed in cucumber(Cucumis sativus L.)

Genetic transformation is an important technique for functional genomics study and genetic improvement of plants. Until now, Agrobacterium-mediated transformation methods using cotyledon as explants has been the major approach for cucumber, and its frequency has been up to 23%. For example, significantly enhancement of the transformation efficiency of this plant species was achieved from the cotyledon explants of the cultivar Poinsett 76 infected by Agrobacterium strains EHA105 with efficient positive selection system in lots of experiments. This review is to summarize some key factors influencing cucumber regeneration and genetic transformation, including target genes, selection systems and the ways of transgene introduction, and then to put forward some strategies for the increasing of cucumber transformation efficiency. In the future, it is high possible for cucumber to be potential bioreactor to produce vaccine and biomaterials for human beings.

Meropenem as an Alternative Antibiotic Agent for Suppression of Agrobacterium in Genetic Transformation of Orchid

A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies （PLBs） has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics （including ampicillin, carbenicillin, cefotaxime, and cefoperazone）, meropenem showed the highest activity in suppressing all tested A. tumefaciens strains （minimum inhibitory concentration [MIC] 〈 0.5 mg L^-1, which is equal to minimum bactericidal concentration [MBC]）. Meropenem, at all tested concentrations, except for 10 mg L^-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector plG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L^-1 meropenem and 25 mg L^-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation.

Genetic Transformation of Watermelon with Pumpkin DNA by Low Energy Ion Beam-mediated Introduction

The No.601 watermelon (citrullus lanatus) seeds were treated with 25 keV N+ implantation at the dosage of 7.8 ×1016 ions/cm2. After treatment, watermelon seeds were incubated with 380μg/μl pumpkin (Cucubita, maxima Duch) DNA solution at 35 ℃ for 5 hours. By two-generations of selection and resistance screening at seedling stage, one transformed material was selected out, whose rind color is similar to that of the donor pumpkin and whose size of seeds is between that of the donor and the receptor. Using AFLP (amplified fragment length polymorphism) technique, two polymorphic DNA fragments were amplified. This primarily testified that the donor DNA fragments/gene were introduced into the receptor cell and integrated into the genomic DNA of the receptor.

Construction and Genetic Transformation of Over-expression Vector for atp6 Gene from Rice Mitochondria

[Objective] This study aimed to clone the gene atp6 from rice mitochondria, construct the binary expression vector 35S ：： Rflb5＇ ：： atp6 and obtain transgenic plants with atp6. [Method] The special primers were designed according to the sequence of target gene. With the TRIzol method the total RNA was extracted from rice seedlings and reverse transcripted to cDNA. The ORF of atp6 was amplified by PCR, and ligated to binary expression vector pCAMBIA1302 which contains se- quence of signal peptide from mitochondria （Rflb5＇）. The recombinant plasmid was then transformed into rice callus mediated by Agrobacterium. [Result] The binary expression vector 35S ：： Rflb5＇ ：： atp6 was constructed, and positive transgenic plants were obtained. [Conclusion] This study lays foundation for understanding influence of atp6 gene over-expression on rice growth.

The Technique of Genetic Transformation Mediated by keV Ion Beam

The application of keV ion beam in life science started in China several decades ago. In 1986, researchers initially studied the mutagenic effect of ion beam, and successfully applied it to plant breeding. Nowadays, ion beam implantation technique has been extensively applied to many biological fields. This paper mainly introduces one of its important applications: genetic transformation mediated by keV ion beam.

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.

Plant regeneration and genetic transformation in switchgrass——A review

Switchgrass is native to the tallgrass prairie of North America. It is self-incompatible and has varied ploidy levels from diploid（2x） to dodecaploid（12x） with tetraploid and octoploid being the most common. The high yielding potential and the ability to grow well in marginal lands make switchgrass an ideal species as a dedicated biomass producer for lignocellulosic ethanol production. Genetic transformation is an important tool for studying gene function and for germplasm improvement in switchgrass, the genome of which has been sequenced recently. This paper intends to provide a comprehensive review on plant regeneration and genetic transformation in switchgrass. We first reviewed the effect of explants, basal medium and plant growth regulators on plant regeneration in switchgrass, which is a prerequisite for genetic transformation. We then reviewed the progresses on genetic transformation with either the biolistic or Agrobacterium-mediated method in switchgrass, and discussed various techniques employed to improve the transformation efficiency. Finally we reviewed the recent progresses on the use of genetic transformation in improving biomass quality such as the reduction of lignin, and in increasing biomass yield in switchgrass. We also provided a future perspective on the use of new genome editing technologies in switchgrass and its potential impact on regulatory processes.

Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of (-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected with Agrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of (-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.

Plant Regeneration from Immature Inflorescence Culture and Genetic Transformation of Wheatgrass(Agropyron cristatum×A. desertorum cv. Hycrest-Mengnong)

The plants of hybrid wheatgrass （A. cristatum×A. desertorum cv. Hycrest-Mengnong） were directly induced from embryogenic callus regenerated from immature inflorescence. Immature inflorescence was cultured on improved MS medium containing 2.0-3.0 mg L^-1 2,4-D to regenerate callus. The calli were then transferred to hormone-free MS medium for differentiation and 1/2 MS medium for rooting. Results showed that callus initiation frequency was 83.4% and plant regeneration frequency was 59.6%. Phosphinothricin acetyltransferase （bar） gene was transformed into the hybrid wheatgrass by particle bombardment. Resistant callus was obtained using selecting agent, herbicide glufosinate of 0.5 mg L^-1, and some transgenic plants were recovered in vitro. The transgenic plants were identified by PCR and Southern blot analysis and these plants developed normally in the glufosinate medium, whereas the nontransgenic plants did not. The results demonstrated that bar cDNA integrated into the genomic DNA of the transgenic plants. The transgenic frequencies of bar gene were 1.1%.