Transformation of blueberry without antibiotic selection

Transformation of the blueberry cultivar North Country (Vaccinium corymbosum × V. angustifolium) was achieved using the disarmed Agrobacterium tumefaciens isolate LBA4404, containing a binary vector with an intron-containing β-glucuronidase (GUS) marker gene. Plant regeneration was carried out in the absence of antibiotics due to their toxicity to blueberry explants. Agrobacterium contamination was controlled by dipping the explants in antibiotics and rinsing in sterile distilled water. Once regeneration had been achieved, the plantlets were placed on to medium containing the antibiotic ticaricillin at 250 mg litre^-1 to control and try to eliminate any remaining Agrobacterium. Selection of regenerating explants expressing GUS was achieved by growing the plant material for 2 days on a medium containing 4-methyl umbelliferyl glucuronide (MUG), and examining the medium under UV light to detect fluorescent activity. From the 19 explants showing signs of regeneration, seven produced fluorescent patches on MUG medium. From the selected explants, five plantlets were found to express the GUS gene as detected by fluorometric and histochemical analysis. PCR was used to confirm the presence of the GUS and/or NPTII marker genes after 2 years in culture. Bacterial contamination isolated from plant material (which appeared free of contamination) was examined for GUS activity and analysed using PCR with GUS and NPTII specific primers, but no positive results were obtained.     

Comparison of Biolistic and Agrobacterium -mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice

Transferring foreign DNA into plant cells by biolistic and Agrobacterium -mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG-CAM, into rice (BX)Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium -mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium -mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two-cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium -mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium -mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium -mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.     

Virulence of different Agrobacterium strains on hairy root formation of Hyoscyamus muticus

Hyoscyamus muticus accession was evaluated for its response to inoculation with Agrobacterium rhizogenes strains LBA9402, A4, 15834, and Agrobacterium tumefaciens strain C58 CI pRT GUS104. The Agrobacterium strain used for the transformation has a significant influence on the phenotype of the clone as well as on the growth rate and hyoscyamine production of these root culture clones. The most virulent strains were C58 CI pRT GUS104 and LBA9402. More roots were obtained on LSO medium than on B50 medium. Acetosyringone addition and the time from wounding affected root formation. The alkaloid content was highest in clones C58 and A4 (90mg·l^-1). There are great differences between individual hairy root clones, and hence they are not as uniform as has often been speculated. The Agrobacterium strain used for the transformation has a great influence in this respect.     

Stable transformation of lettuce cultivar South Bay from cotyledon explants

Transgenic plants of lettuce cultivar (cv.) South Bay were produced by using Agrobacterium tumefaciens vectors containing the -glucuronidase (GUS) reporter gene and the NPT II gene for kanamycin resistance as a selectable marker. High frequency of transformation, based on kanamycin resistance and assays for GUS expression, was obtained with 24 to 72-h-old cotyledon explants cocultivated for 48 h with Agrobacterium tumefaciens. After the cocultivation period, the explants were placed in selection medium containing 50 or 100 mg l^–1 of kanamycin, 100 mg l^–1 cefotaxime and 500 mg l^–1 carbenicillin for 10 days. Surviving explants were transferred every 14 days on shoot elongation medium. Progenies of R₀ plants demonstrated linked monogenic segregation for kanamycin resistance and GUS activity.Florida Agricultural Experiment Station Journal Series R-02231. This research was partially supported by CNPq/RHAE (Brazil).     

Transient Expression of β-Glucuronidase in Embryo Axes of Cotton by Agrobacterium and Particle Bombardment Methods

Transient expression of -glucuronidase (GUS) in zygotic embryo axes of two cotton (Gossypium hirsutum L.) cultivars NHH-44 and DCH-32 was induced by Agrobacterium mediated transformation or by particle bombardment. For Agrobacterium transformation, disarmed A. tumefaciens strain GV 2260/p35SGUSINT was used. In cv. NHH-44, the maximum frequency of transient expression (14.28 %) was achieved on spotting Agrobacterium paste on the apical regions of the split embryo axes. The method resulted in a transformed callus line, which showed strong GUS activity. Integration of NPTII gene was confirmed by Southern analysis. Transgene expression by particle bombardment was achieved with p35SGUSINT and pIBGUS plasmids independently. The maximum frequency of GUS expression in 29.16 % explants was observed in cultivar NHH-44 with gold microcarriers (1.1 µm) when bombarded once with rupture disc of 7586 kPa at target cell distance of 6 cm. A transformed callus line was obtained when explants were bombarded with p35SGUSINT and cultured on Murashige and Skoog's medium supplemented with B₅ vitamins, 0.1 mg dm^–3 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, 0.01 mg dm^–3 -naphthaleneacetic acid, 3 % glucose + 50 mg dm^–3 kanamycin. High GUS activity was observed in callus tissue as well as in somatic embryo like structures achieved in liquid shake cultures.     

农杆菌介导的苜蓿次级体细胞胚的遗传转化

采用农杆菌菌株GV3101感染子叶期苜蓿体细胞胚来研究苜蓿次级体细胞胚的遗传转化方法。农杆菌菌株GV3101双相载体pCAMBIA2301,此双相载体具有gus报告基因和nptⅡ抗卡那霉素筛选基因。感染的子叶期苜蓿体细胞在75 mg/L卡那霉素筛选压下,经过一系列诱导培养,最终获得转基因植株。然后,通过GUS组织化学定位分析来检测转基因植株不同器官中的GUS表达,并进一步通过PCR和Southern杂交确定转基因的稳定整合和转化率。结果表明转基因植株不同器官均有GUS表达,整合的nptⅡ基因的拷贝数是1~4,获得的转基因植株的转化率是65.82%。     

Agrobacterium mediated transformation of Vigna sesquipedalis Koern (asparagus bean)

Agrobacterium mediated transformation of Vigna sesquipedalis was achieved using cotyledonary node explants prepared from 5 days old seedlings germinated on B5 basal medium, and transformed using Agrobacterium tumefaciens strain EHA101, carrying the phosphinothricin-N-acetyltransferase gene and neomycin-3-phosphotransferase-II gene as selectable markers and GUS gene as a screenable marker. Gene transfer was achieved by inoculation of cotyledonary node explants with a bacterial suspension and a further cocultivation with Agrobacterium suspension for 3 days on B5 basal medium. Only 10% of the explants were transformed with EHA101 and exhibited transient expression of GUS genes, while 2% of shoots exhibited stable integration of genes and developed into plants. Transgenic character of tissues was confirmed by GUS assay and Southern analysis. Histological analysis of GUS gene expression directly after cocultivation revealed a high competence of subepidermal cell layers of cotyledonary node and associated cotyledons for transformation with Agrobacterium.     

Transformation of the limonene synthase gene into peppermint (Mentha piperita L.) and preliminary studies on the essential oil profiles of single transgenic plants

Agrobacterium-mediated and direct gene transfer into protoplasts using PEG were both successfully used to produce stable, transformed peppermint plants (Mentha×piperita L. cultivar Black Mitcham) with the limonene synthase gene. Stem internode explants found to possess a high level of organogenesis through adventitious shoot formation were subjected to Agrobacterium tumefaciens disarmed strain GV3101 (pMP90). Following the development of an efficient protoplast-to-plant cycle from stem-isolated protoplasts, they were used in direct gene transformations. In both cases the binary vector pGA643 carrying the nptII/GUS genes, both driven by the CaMV35S promoter, was used in preliminary plant-transformation studies. Later, GUS was replaced with the limonene synthase gene. Kanamycin was used as a selective agent in all transformation experiments to obtain both transformed protoplast-derived calli as well as putative transgenic shoots regenerated from internode explants. Both types of transformation resulted in transgenic plants which were detected using PCR and confirmed by Southern-blot hybridizations. Southern analysis revealed that the method of Agrobacterium-mediated transformation is superior to the direct DNA uptake into protoplasts with regard to the stability of the insert during the transformation event. Single transgenic plants were grown to 10% flowering in a greenhouse and the plants derived both by the Agrobacterium and the protoplast-derived methods were generally observed to have essential oil profiles characterized by a high-menthone, low-menthol, high-menthofuran and –pulegone content in comparison to a typical mid-west peppermint. Limonene varied only slightly, around 1.2%, in transgenic plants produced by both methods. Received: 22 November 1998 / Accepted: 4 Januar 1999     

农杆菌介导红江橙遗传转化的研究初报

用红江橙实生苗的上胚轴为材料 ,初步研究以根癌农杆菌介导的 GUS基因转化。结果表明 :以卡那霉素作为选择试剂进行选择培养时 ,Km浓度为 5 0 mg/L;外植体以平放为好 ;抑菌剂选择头孢霉素较好。GUS基因瞬时表达检测 ,70 .4%的外植体呈阳性反应 ;GUS基因稳定表达检测 ,在获得的 1 2株抗性植株中 ,GUS反应呈阳性所占比例为 1 6.7%。     

根癌农杆菌介导的金发草遗传转化条件的优化

金发草(Pogonatherum paniceum)是一种多年生岩生草本植物,在生态恢复和景观建设中起着重要的作用。利用根癌农杆菌介导转化金发草胚性愈伤组织,通过GUS(β-葡萄糖苷酸酶)瞬时表达率研究菌液浓度、浸染时间、乙酰丁香酮(AS)浓度、葡萄糖浓度、共培养时间等因素对金发草转化的影响,并利用确定的最佳条件将GUS基因转入金发草,获得稳定表达转化植株。结果表明:菌液浓度(OD₆₀₀)为0.6,浸染时间为10min,添加20mg/L的乙酰丁香酮(AS)和10g/L的葡萄糖,共培养时间5d为最佳条件,GUS瞬时表达率最高。经过抗性筛选后最终获得阳性转基因植株频率为57%。再生植株经GUS染色和PCR检测证明,GUS基因已成功整合到金发草基因组中。此转化体系的建立为金发草的遗传改良及相关功能基因的研究奠定了基础。     

The use of the two T-DNA binary system to derive marker-free transgenic soybeans

Summary A binary vector, pPTN133, was assembled that harbored two separate T-DNAs. T-DNA one contained a bar cassette, while T-DNA two carried a GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strain EHA101. Mature soybean cotyledonary node explants were inoculated and regenerated on medium amended with glufosinate. Transgenic soybeans were grown to maturity in the greenhouse. Fifteen primary transformants (T₀) representing 10 independent events were characterized. Seven of the 10 independent T₀ events co-expressed GUS. Progeny analysis was conducted by sowing the T₁ seeds and monitoring the expression of the GUS gene after 21 d. Individual T₁ plants were subsequently scored for herbicide tolerance by leaf painting a unifoliate leaf with a 100 mgl⁻¹ solution of glufosinate and scoring the leaf 5 d post application. Herbicide-sensitive and GUS-positive individuals were observed in four of the 10 independent events. Southern blot analysis confirmed the absence of the bar gene in the GUS positive/herbicide-sensitive individuals. These results demonstrate that simultaneous integration of two T-DNAs followed by their independent segregation in progeny is a viable means to obtain soybeans that lack a selectable marker.     

Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants

An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l^–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis, further confirming the integration and expression of T-DNA in these plants. Received: 1 August 1997 / Revision received: 11 December 1997 / Accepted: 24 January 1998     

Agrobacterium-Mediated Transformation of Switchgrass and Inheritance of the Transgenes

Switchgrass (Panicum virgatum L.) has been developed into an important biofuel crop. Embryogenic calli induced from caryopses or inflorescences of the lowland switchgrass cultivar Alamo were used for Agrobacterium-mediated transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin as the selection agent. Embryogenic calli were infected with Agrobacterium tumefaciens strain EHA105. Calli resistant to hygromycin were obtained after 5 to 8 weeks of selection. Soil-grown transgenic switchgrass plants were obtained 4 to 5 months after Agrobacterium infection. The transgenic nature of the regenerated plants was demonstrated by PCR, Southern blot hybridization analysis, and GUS staining. T1 progeny were obtained after reciprocal crosses between transgenic and untransformed control plants. Molecular analyses of the T1 progeny revealed various patterns of segregation. Transgene silencing was observed in the progeny with multiple inserts. Interestingly, reversal of the expression of the silenced transgene was found in segregating progeny with a single insert.     

Exogenous application of penconazole regulates plant growth and antioxidative responses in salt-stressed Mentha pulegium L.

The mechanism of growth amelioration in salt-stressed pennyroyal (Mentha pulegium L.) was investigated by exogenous application of penconazole (PEN). Seven weeks after sowing, seedlings were treated with increasing NaCl concentrations (0, 25, 50, and 75 mM) with or without PEN (15 mg l^?1) and were harvested randomly at different times. Results showed that some growth parameters and the relative water content (RWC) decreased under salt stress, while lipid peroxidation, H₂O₂ content, activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POX; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.1), catalase (CAT; EC 1.11.1.6), and ascorbate peroxidase (APX; EC 1.11.1.1) remarkably increased. Exogenous application of PEN increased some growth parameters, RWC, antioxidant enzyme activities, and H₂O₂ content, but the effects of PEN were more significant under salt stress conditions. PEN treatment also decreased lipid peroxidation. These results suggest that PEN-induced tolerance to salt stress in M. pulegium plants may be related to regulation of antioxidative responses and H₂O₂ level.     

金鱼草基因转化和转基因植株再生

本实验采用根癌农杆菌ＬＢＡ４４０４（ｐ３５ＳＧＵＳＩＮＴ与金鱼草下胚轴切段共培养,将ＧＵＳ基因导入金鱼草细胞,通过不定芽发生途径获得抗Ｇ４１８再生植株．经ＤＮＡ／ＤＮＡ斑点杂交及ＧＵＳ活性原位组织检测初步证实外源基因ＧＵＳ已整合进金鱼草基因组并得到表达．     

抗氧化剂对农杆菌介导的大豆下胚轴GUS基因瞬时表达的影响

大豆(Glycine max)下胚轴作为大豆遗传转化的外植体材料,能快速高频再生不定芽。然而,在遗传转化过程中褐化影响基因转化效率。在该研究中,我们用含有GUS染色基因和hpt II(Hygromycin phosphotransferase II)筛选基因的农杆菌(Agrobacterium tumefaciens) LBA4404侵染大豆下胚轴,并用组织化学定位法测定了GUS基因的瞬时表达,以确定大豆的优化基因转化条件。结果显示,在共培养基中加入硫代硫酸钠、L_半胱氨酸以及二硫苏糖醇等抗氧化剂,可以有效地抑制大豆下胚轴在组培过程中褐化的发生,并大幅度提高农杆菌在下胚轴的瞬时表达率。这些结果说明抗氧化剂可以降低这种影响并有效提高基因转化效率。     

In vitro regeneration and transformation of pigeonpea [Cajanus cajan (L.) Millsp]

A requirement for generating transgenic pigeonpea [Cajanuscajan (L.) Millsp] plants is the development of a highly efficientin vitro regeneration procedure. This goal was achieved byusing germinated seedlings grown on B5 medium supplemented with 10 mgl^–1 6-benzylaminopurine, which induced differentiatingcallus formation in the cotyledonary node region. The calli were transferred onB5 medium with 0.2 mg l^–1 6-benzylaminopurine toobtain shoot induction. Elongated shoots were then further cultured on a B5hormone-free medium for rooting. Using this regeneration system transgenicpigeonpea plants were obtained both by particle bombardment andAgrobacterium tumefaciens-mediated gene transfer. Thepresence of the transgenes in the pigeonpea genome was confirmed by GUS assays,PCR and Southern hybridisation. The transgenic rooted plants were successfullytransferred to soil in the greenhouse. GUS and PCR assays of T1 progeniesconfirmed that the transgenes were stably transmitted to the next generation.This is the first report of successful use ofAgrobacteriumas well as particle bombardment for production of transgenic pigeonpea plants.     

Transgenic plants of coffee Coffea canephora from embryogenic callus via Agrobacterium tumefaciens-mediated transformation

 Embryogenic calli were induced from leaf explants of coffee (Coffea canephora) on McCown's woody plant medium (WPM) supplemented with 5 μM N⁶–(2-isopentenyl)-adenosine (2-iP). These calli were co-cultured with Agrobacterium tumefaciens EHA101 harboring pIG121-Hm, containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase II genes. Selection of putative transgenic callus was performed by gradual increase in hygromycin concentration (5, 50, 100 mg/l). The embryogenic calli surviving on medium containing 100 mg/l hygromycin showed a strong GUS-positive reaction with X-Gluc solution. Somatic embryos were formed from these putative transgenic calli and germinated on WPM medium with 5 μM 2-iP. Regenerated small plantlets with shoots and roots were transferred to medium containing both 100 mg/l hygromycin and 100 mg/l kanamycin for final selection of transgenic plants. The selected plantlets exhibited strong GUS activity in leaves and roots as indicated by a deep blue color. GUS and HPT genes were confirmed to be stably integrated into the genome of the coffee plants by the polymerase chain reaction. Received: 14 December 1998 / Revision received: 12 March 1999 / Accepted: 24 March 1999     

Factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis> mediated genetic transformation of kenaf

As a first step in the development of a successful Agrobacterium tumefaciens mediated transformation method for kenaf, factors influencing the successful T-DNA integration and expression (as measured by the GUS expression) were investigated. Transformation was carried out using two kenaf cultivars and Agrobacterium strain EHA 105 carrying different vectors, plasmid pIG 121-Hm or pEC:gus. Pre-culturing the explants for 2days in benzyl adenine containing medium, and wounding the explant before inoculation were found to enhance the transient GUS expression. Increasing the duration of pre-culture and co-culture period enhanced the transient GUS expression up to a threshold level. Increased transient GUS expression did not correlate with an increase in stable expression. Gene integration was confirmed by PCR analysis.     

Gene delivery using microinjection of agrobacterium to embryonic shoot apical meristem of elite indica rice cultivars

A method of gene transfer to elite indica rice cultivars (BPT5204, IW-Ponni and CR1009) by microinjecting the embryonic shoot apical meristem (ESAM) of seeds with Agrobacterium is described. The ESAM microinjected with Agrobacterium cells containing binary plasmid was incubated in co-cultivation media at 28°C for 3?days in the dark and subsequently transferred to selection medium with appropriate supplements and antibiotic. The frequency of gene transfer under various concentrations of Agrobacterium and acetosyringone used were analyzed by staining for the presence of the marker enzyme ??-glucuronidase (GUS) carried as part of the binary plasmid and growth in the presence of antibiotic hygromycin. Results of this investigation show that the maximum efficiency of gene transfer took place when Agrobacterium was used at an OD₆₀₀ of 0.3 and grown in the presence of 20?mg?1^?1 acetosyringone.