Reliable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of Dianthus spiculifolius

Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus induction,adventitious bud differentiation,and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D),6-benzyl aminopurine(6-BA),and naphthaleneacetic acid(NAA)to Murashige and Skoog medium.The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min,and transgenic regenerated plants were obtained by kanamycin(30mg·L^−1)screening.RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D.spiculifolius.Theβ-glucuronidase(GUS)histochemical staining confirmed GUS gene expression in transgenic calli,adventitious buds,and regenerated plants of D.spiculifolius.The green fluorescent protein(GFP)visual analysis showed GFP gene expression in transgenic calli.Furthermore,subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D.spiculifolius callus cells.These results indicated a successful establishment of a reliable and efficient A.tumefaciens-mediated genetic transformation system,which will contribute to functional gene research and genetic improvement of D.spiculifolius.

Agrobacterium tumefaciens-mediated transformation of modern rose(Rosa hybrida)using leaf-derived embryogenic callus

Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.

Characterization of Avirulent TnphoA Mutants in <i>Agrobacterium tumefaciens</i>to Enhance Transformation Efficiency

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane protein. Agrobacterium strain A6007 was mutagenized with E. coli strain mm294A plasmid pRK609 having TnphoA to obtain mutants defective in virulence. Because alkaline phosphatase activity is only detected when the PhoA gene product from the transposon is secreted out of the protoplasm, the virulence mutants are located in genes that code for transmembrane or periplasmic proteins. Attempts were made to obtain the sequences adjacent to the TnphoA inserts through several different approaches including Inverse PCR, Cloning, and Tail PCR. Transposon-adjacent sequence was obtained from one membrane anchor subunit in Bradyrhizobium japonicum i.e. succinate dehydrogenase which has enhanced transformation efficiency.

Enemies atpeace:Recentprogressin Agrobacterium-mediated cereal transformation

Agrobacterium tumefaciens mediated plant transformation is a versatile tool for plant genetic engineering following its discovery nearly half a century ago.Numerous modifications were made in its application to increase efficiency,especially in the recalcitrant major cereals plants.Recent breakthroughs in transformation efficiency continue its role as a mainstream technique in CRISPR/Cas-based genome editing and gene stacking.These modifications led to higher transformation frequency and lower but more stable transgene copies with the capability to revolutionize modern agriculture.In this review,we provide a brief overview of the history of Agrobacterium-mediated plant transformation and focus on the most recent progress to improve the system in both the Agrobacterium and the host recipient.A promising future for transformation in biotechnology and agriculture is predicted.

Research Progress on Agrobacterium tumefaciens-based Transgenic Technology in Brassica rapa

Brassica rapa L.is cultivated globally and consumed in many areas worldwide.Using the transgenic Agrobacterium-mediated transformation method,which is a reproducible and efficient technique,genes can be transferred into various B.rapa species.This review summarizes the processes involved in Agrobacterium-mediated transformation of B.rapa,including surface seed sterilization,co-cultivation with A.tumefaciens,induction of callus/shoot/root formation,and confirmation of transgenic plants.In addition,factors such as the Agrobacterium strain,plant genotype,explant age,transformation efficiency of the hybrid or inbred line,and the concentrations of N6-benzyl amino purine and naphthalene acetic acid,are discussed.And this review shows clearly how to do it,what to do,and what not to do in the transgenic Agrobacterium-mediated in Brassica rapa.The information presented here lays the foundation for a simple and efficient method that resolves existing problems and improves overall transgenic B.rapa production,thereby benefiting both basic and applied research.

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.

Strain Cultivation Stage and Infectious Concentration on Soybean Cotyledonary Node via Agrobacterium–mediated Transformation System

In soybean cotyledonary transformation,Agrobacterium infection is influenced by many factors.The cultivation stage of Agrobacterium strain and infectious concentration were studied in this research.Results showed that the highest rate of resistant shoots was obtained when the strain cultivation concentration was OD 600 =0.6,and the infectious concentration was OD 600 =0.5.Seven soybean cultivars in North China were also screened for high susceptibility to Agrobacterium and three genotypes were found to be suitable for transformation.This research has built a good foundation for soybean transformation improvement.

Agrobacterium-mediated transient transformation of Pentalinon andrieuxii Müll.Arg.

Sections of hypocotyls, roots and leaves from Pentalinon andrieuxii plantlets were transiently transformed with Agrobacterium tumefaciens LBA4404 bearing the binary plasmid pCAMBIA2301 with an interrupted β-Glucuronidase (GUS) gene. Histochemical GUS assays showed transient gene expression in all infected tissues, being older roots those which displayed the most intense GUS staining. To our knowledge, this is the first report of Pentalinon andrieuxii susceptibility to Agrobacterium tumefaciens-mediated genetic transformation.

等离子体活化水对根癌土壤杆菌的抑制作用及其对樱花根癌病的防治

以樱花幼苗为试材,使用低温等离子体活化水冷杀菌技术(PAW)对樱花幼苗及其病原菌进行处理,研究了PAW对樱花苗木的抗病作用,以期为PAW在樱花根癌病防治中的应用提供参考依据。结果表明:等离子体活化水能有效地抑制樱花根癌病病菌Agrobacterium tumefaciens的生长,其可以对Agrobacterium tumefaciens的细胞结构造成破坏。CK组樱花幼苗的根瘤发生率为100%,根瘤的平均直径为18.8 mm,处理后的幼苗发病率为56.7%,根瘤的平均直径为8.4 mm。此外,PAW可以提高樱花根系多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)及超氧化物歧化酶(SOD)活性的表达,激活樱花植株的抗性。综上,PAW具有一定的生物防治潜力,可以防御樱花根癌病。