Successful <Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Genetic Transformation of Maize Elite Inbred lines

An efficient transformation system was developed for maize (Zea mays L.) elite inbred lines using Agrobacterium-mediated gene transfer by identifying important factors that affected transformation efficiency. The hypervirulent Agrobacterium tumefaciens strain EHA105 proved to be better than octopine LBA4404 and nopaline GV3101. Improved transformation efficiencies were obtained when immature embryos were inocubated with Agrobacterium suspension cells (A₆₀₀ = 0.8) for 20 min in the presence of 0.1% (v/v) of a surfactant (Tween20) in the infection medium. Optimized cocultivation was performed in the acidic medium (pH5.4) at 22 °C in the dark for 3 days. Using the optimized system, we obtained 42 morphologically normal, independent transgenic plants in four maize elite inbred lines representing different genetic backgrounds. Most of them (about 85%) are fertile. The transformation frequency (the number of independent, PCR-positive transgenic plants per 100 embryos infected) ranged from 2.35 to 5.26%. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to three copies of the transgene were integrated into the maize nuclear genome. About 70% of the transgenic plants received a single insertion of the transgenes based on Southern analysis of 10 transformed events. T₁ plants were analyzed and transmission of transgenes to the T₁ generation in a Mendelian fashion was verified. This system should facilitate the introduction of agronomically important genes into commercial genotypes.     

根癌农杆菌介导真菌遗传转化的研究及应用

根癌农杆菌介导转化法（Agrobacterium tumefaciens-mediated transformation,ATMT）具有转化效率高、遗传稳定、适用范围广等诸多优点,已成为真菌遗传转化研究中的强有力手段,在真菌基因资源开发、真菌性疾病研究和外源蛋白表达研究中发挥巨大作用。本文概述了根癌农杆菌转化法在真菌转化中的研究进展、技术优缺点、转化机制、实验方法和应用现状,着重介绍影响其转化效率的因素并对优化方法进行探讨,展望了该技术在真菌基因资源发掘、基因编辑等方面的应用前景,为今后真菌的遗传转化研究提供参考。     

Effect of age and type of tissue on genetic transformation of Lotus corniculatus by Agrobacterium tumefaciens

Various experiments of Lotus corniculatus cv. Leo were infected with Agrobacterium tumefaciens strains C58 (wild-type) and GV3101 (control). A maximum of 83 per cent of cotyledons excised from 7 day old seedlings and 63 per cent of leaves excised from seedlings grown in vitro formed galls in culture. The genotype of the seedling had an effect on the response.     

Transformation of vitis tissue by different strains of Agrobacterium tumefaciens containing the T-6b gene

Summary Stem pieces and leaf disks of Vitis spp. were cocultured with Agrobacterium tumefaciens strains carrying the UidA (ß-glucuronidase = GUS) gene. The transformation efficiency was highly increased by using a modified T-6b gene (a gene from pTiTm4) which interferes with normal growth and allows regeneration of normal Nicotiana rustica plants (Tinland 1990). The strains first tested on stem segments were subsequently tested in a leaf explant system. On leaves the transformation efficiency of the strains was much lower than with stems. Both the T-6b gene and the hsp 70-T-6b gene (a modified T-6b gene under the control of a heat shock promoter) allowed the initiation of GUS-positive buds.Abbreviations GUS ß-glucuronidase - BAP benzylaminopurine - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide     

T-DNA transfer to maize plants

Agrobacterium-mediated transformation is the method of choice to engineer desirable genes into plants. Here we describe a protocol for demonstrating T-DNA transfer from Agrobacterium into the economically important graminaceous plant maize. Expression of the T-DNA-located GUS gene was observed with high efficiency on shoots of young maize seedlings after cocultivation with Agrobacterium.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> is a useful transporter for introducing T-DNA of the binary plasmid into the chrysanthemum, <Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Kitamura,genome

Agrobacterium rhizogenes was used for efficient transformation of chrysanthemum. Two types of Agrobacterium, A. rhizogenes (A-13) and A. tumefaciens (LBA4404), which harbor pIG121-Hm, were employed for infection. In the A. rhizogenes-infected explants, hairy roots were not observed on any tested medium or culture condition. When explants were cultured on shoot induction medium, calli were formed at the cutting edge within 4–6 weeks of culture, and shoot primordia were observed on the callus surface after 2 weeks of callus formation. Consequently, with gus introduction, a significantly higher transformation rate was observed for A. rhizogenes (6.0%) compared with A. tumefaciens (3.3%). However, only 0.6% of the frequency of rol insertion was exhibited in A. rhizogenes mediation. These results indicate that A. rhizogenes effectively introduces T-DNA of the binary plasmid into the chrysanthemum genome by introducing Ri T-DNA at a low frequency. It also indicates that the system is a useful alternative for the transformation of chrysanthemum.     

Transformation of cauliflower (Brassica oleracea L. var. botrytis) — an experimental survey

The paper compares different approaches for the genetic transformation of cauliflower (Agrobacterium-mediated, PEG-mediated and/or electroporation). Transient expression of the neomycin phosphotransferase II (NPTII) gene could be detected after direct gene transfer. Stable transformation was achieved using both Agrobacterium-mediated and direct gene transfer. Expression as well as incorporation of the NPTII sequence could be demonstrated.     

Transgenic regal pelargoniums that express the rolC gene from Agrobacterium rhizogenes exhibit a dwarf floral and vegetative phenotype

Summary The regal pelargonium, ev. Dubonnet, was transformed using the disarmed Agrobacterium tumefaciens strains LBA4404 or EHA105 containing the binary vector pLN70. This plasmid carries on its T-DNA the rolC gene from Agrobacterium rhizogenes under control of the CaMV 35S promoter and the npt II selectable marker gene under a NOS promoter. Six independent transformants were produced and grouped according to their phenotypic characteristics. Two transformants showed the same phenotype as the untransformed control plants. Three transformants exhibited a dwarf phenotype and one displayed a super-dwarf phenotype. Southern hybridization analyses of the T-DNA left border region using a npt II probe showed that the six transformants all arose from independent transformation events. Northern hybridization analyses showed that the rolC gene was expressed only in the four transformants that exhibited a dwarf phenotype. Our data show that the phenotypic effects of rolC expression in regal pelargoniums include reductions in plant height, leaf area, petal area, and corolla length. Earlier flowering of the rolC transgenics by up to 22d was also observed.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.     

Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R₁ and R₂ generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.     

Deviating T-DNA transfer fromAgrobacterium tumefaciens to plants

We analyzed 29 T-DNA inserts in transgenicArabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA. DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data. Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences. Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T-region. On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat.     

Analysis of conditions forAgrobacterium-mediated transformation of tobacco cells in suspension

We have developed anAgrobacterium-mediated transformation system, using tobacco cell suspensions, that permits evaluation of factors affecting transformation within seven days of co-cultivation. Tobacco cell transformation was determined by monitoring -glucuronidase (GUS) activity detected in plant cell extracts. The use of a chimeric gene construct, 35S-GUS/INT, containing a portable intron in theuidA reading frame, assured only plant-specific GUS expression. During the co-cultivation period, induction of the bacterialvir-region was monitored using a heterologous gene construct composed of avirB promoter fragment from pTiC58 fused to the chloramphenicol acetyltranferase (CAT) gene ofTn9. Tobacco cell transformants were confirmed by antibiotic selection of transformed plant cells and by X-Gluc staining. Maximum transformation was obtained when plant suspension cultures were growing rapidly which also was coincidental with elevated levels of bacterialvir-region expression. One week after co-cultivation, the transformed cultures exhibited a stable pattern of GUS activity which remained constant without antibiotic selection. The system was used to compare the virulence of a number ofAgrobacterium strains. GUS activity of plant cells co-cultivated with a strain containing a cointegrate plasmid was 3-fold higher than that of one with a binary configuration of the T-DNA. When the co-cultivatingAgrobacterium strain also carried the plasmid used to monitorvir induction, the frequency of transformation was reduced by as much, as 97%.     

Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds

A protocol for the transformation of castor embryo axes using the pCAMBIA vector 1304 in disarmed Agrobacterium tumefaciens strain EHA105 is presented. Co-cultivated explants were initially subjected to expansion and proliferation on MS medium with 0.5 mg l^–1 TDZ followed by three cycles of selection on medium with 0.5 mg l^–1 BA and increasing concentrations of hygromycin (20–40–60 mg l^–1). Selected shoot clusters were transferred to medium with 0.5 mg l^–1 BA for proliferation and 0.2 mg l^–1 BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium with 2.0 mg l^–1 NAA. The presence and stable integration of the hpt gene was confirmed through PCR, RT-PCR, PCR-Southern blot, sequence analysis, Southern blot analysis and PCR analysis of progeny. Southern blot analysis of the primary transformants showed single copy integration and progeny analysis revealed monogenic inheritance of the introduced gene. This paper reports the first successful attempt at producing transgenic castor.     

Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium

A chimaeric gene has been constructed that expresses -D-glucuronidase (GUS) in transformed plant tissues, but not in bacterial cells. This gene has proved extremely useful for monitoring transformation during the period immediately following gene transfer from Agrobacterium tumefaciens. GUS expression was detectable 2 days after inoculation, peaked at 3–4 days and then declined; if selection was imposed expression increased again after 10–14 days. The extent of transient expression after 4 days correlated well with stable integration as measured by kanamycin resistance, hormone independence, and gall formation. Histochemical staining of inoculated leaf discs confirmed the transient peak of GUS expression 3–4 days after inoculation. The most surprising result was that the blue staining was concentrated in localized zones on the circumference of the disc; within these zones, essentially all the cells appeared to be expressing GUS. We suggest that the frequency of gene transfer from Agrobacterium is extremely high within localized regions of leaf explants, but that the frequency of stable integration is several orders of magnitude lower.     

Multiple copies of virG enhance the transient transformation of celery,carrot and rice tissues by Agrobacterium tumefaciens

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical -glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.Current address: Department of Agronomy, Purdue University     

农杆菌介导的携带egfp基因载体转化寡雄腐霉

【目的】寡雄腐霉(Pythium oligandrum Drechsler)是一种对动、植物和环境无害,兼具杀菌和增产效果的生防真菌。通过研究建立农杆菌介导的寡雄腐霉遗传转化体系。【方法】选用EHA105、AGL-1、LBA4404三种农杆菌菌株对寡雄腐霉进行遗传转化研究,通过对影响遗传转化效果的条件参数试验优化,确立适宜寡雄腐霉遗传转化的农杆菌菌株及转化条件,建立农杆菌介导的寡雄腐霉遗传转化体系。【结果】经研究发现,所选3种农杆菌菌株中EHA105菌株对寡雄腐霉的遗传转化效果最好,其次是AGL-1菌株,LBA4404菌株转化效果不好。EHA105菌株经IM(含300μmol/L AS)诱导培养至OD_(600)=0.6时,与浓度为10~6–10~7个/m L的寡雄腐霉孢子悬浮液以1–10:1的比例混合,在25–26°C以液体振荡的方式避光共培养72 h(pH 5.0,含300μmol/L AS),寡雄腐霉菌体液体振荡恢复培养24 h,涂布抗性选择平板筛选寡雄腐霉转化子,即可得到寡雄腐霉基因工程菌株,其转化率可达到130个转化子/106个孢子。【结论】本研究首次构建了农杆菌介导的寡雄腐霉遗传转化体系,研究结果可为寡雄腐霉的生防机制及分子育种研究提供技术支撑。     

Genetic transformation and regeneration of transgenic plants in grapevine (Vitis rupestris S.)

Isolated somatic embryos from petiole-derived callus cultures ofVitis rupestris Scheele have been employed in experiments on genetic transformation. Co-cultivation of somatic embryos during embryogenesis induction withAgrobacterium tumefaciens strain LBA4404, which contains the plasmid pBI121 carrying the neomycin phosphotranspherase and the-glucuronidase genes, produced transformed cellular lines capable of recurrent somatic embryogenesis. Precocious selection for high levels of kanamycin (100 mgl^-1) was an important part of our transformation protocol. Transformed lines still have strong-glucuronidase expression as well as stable insertion of the marker genes after 3 years of in-vitro culture, during which they have maintained their capacity to organize secondary embryos and to regenerate transgenic plants with an agreeable efficiency (13%).