Agrobacterium tumefaciens-mediated transformation of cauliflower,Brassica oleracea var. botrytis

We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.     

Optimization of Brassica napus (canola) explant regeneration for genetic transformation

Brassica napus (canola) is the second largest oilseed crop in the world. It is among the first crops to be genetically transformed, and genetically modified cultivars are in commercial production at very significant levels. Despite the early lead with respect to transgenesis, there remain cultivars that are recalcitrant to transformation. To address this, we have conducted an elaborate investigation of the conditions for regenerating shoots from hypocotyl explants from four genetic lines: Invigor 5020, Westar and Topas as well as a microspore culture derived line of Topas (Line 4079). We analyzed the effect of hormonal combinations in regeneration medium, donor plant age and explant type on the regeneration capacity of these plants. The analysis showed that hypocotyls of eight-day-old seedlings grown on media supplemented with 1mg/L dinitrophenylhydrazine (2,4-D) produced the most shoots. Globular somatic embryos emerged following two weeks of 2,4-D treatment. When transferred to the medium containing 5mg/L benzyladenine (BA), approximately 82% of embryos produced shoots within six weeks. Invigor plants were shown to regenerate more efficiently than Topas; the number of plantlets regenerated from Invigor was approximately 40-50% more as compared to Topas or Line 4079. When hypocotyl explants were co-cultivated with the Agrobacterium strain GV3101 harboring a binary vector carrying a firefly luciferase reporter gene (LUC), significant numbers of plantlets were LUC-positive in a luciferase assay. Frequency of such plants were: Invigor 5020 (54.2 ± 2.5%), Westar (53.7 ± 5.3), Topas (16.0 ± 0.24) and Line 4079 (13.4 ± 4).     

根癌农杆菌介导B.t.基因和CpTI基因对花椰菜的转化

用根癌农杆菌介导的方法 ,将B .t.基因和Cp TI基因分别导入花椰菜“杂交 75天”的父本和母本的无菌苗下胚轴切段细胞 ,都获得了转基因植株。经过预培养的下胚轴切段用根癌农杆菌 (LBA44 0 4/ pG BI4A2B ,含B .t .基因 ;LBA44 0 4/pBRLC ,含CpTI基因 )进行感染后 ,共培养 48h ,继续培养 30d后 ,将分化芽转移至筛选培养基上。 10d后 ,大多数分化芽的顶端变成紫色 ,2 0d后紫色芽逐渐变白死亡 ,而转化芽在选择培养基上长成小植株。小植株移至大田能正常生长、开花、结籽。PCR和Southernblot分析表明B .t和CpTI基因已整合在植物基因组中     

The ploidy level of transgenic plants in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of tomato cotyledons (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> L.Mill.) is genotype and procedure dependent

A protocol avoiding the feeder-layer cell system was optimized for Agrobacterium-mediated transformation of tomato cotyledonary explants. Over 500 transgenic plants from five tomato cultivars were regenerated in 15 independent experiments. Depending on both genotype and procedure, transformation frequencies ranged from 1.8% to 11.3%. The optimal transformation rate was obtained by inoculating explants with a bacterial suspension in exponential growth ( D(600) = 10(2)-10(3) cells/ml) and transferring cotyledon explants to fresh selective regeneration medium every 3 weeks. The ploidy level of both tomato genotypes used as explant source and primary transformants, was studied by flow cytometry. The inbred lines and cultivars were diploid but a polysomatic pattern in the cotyledon explant was confirmed. The rate of tetraploid transgenic plants ranged from 24.5% to 80% and depended on both the genotype and the transformation procedure. Surprisingly, the percentages of transformed plants with higher ploidy levels were not related to the proportion of 4C and 8C nuclei in the cotyledonary tissue. For some genotypes the optimisation of the transformation rate resulted in an increase of tetraploid transgenic plants. Results obtained in this work indicate the convenience of checking the ploidy level of the primary transformants before performing basic studies or introducing tomato transgenic material in a breeding program.     

Factors influencing the regeneration capacity of oilseed rape and cauliflower in transformation experiments

The efficiency ofAgrobacterium-based transformation technique in oilseed rape and cauliflower was influenced by cultivar specificity, donor plant age and explant type. Marked differences in demands for plant hormone contents in the regeneration medium were recorded already among different types of nontransformed explants. The highest regeneration capacity was recorded with stem and leaf segments isolated from one-month-old aseptically grown plants. The regeneration was markedly species-dependent. Regeneration of transformed plants from stem segments and thin layers isolated from field-grown oilseed rape plants (at the most 2% of regenerating explants) and from oilseed rape hypocotyls (0.8% of regenerating explants) and cauliflower (1.2% of explant regenerated transformed shoots) was achieved after disarmedAgrobacterium treatment. Hypersensitive reaction of explants could be prevented by using prolongedin vitro precultivation and delayed application of the selective agent.     

Cytokinin vectors mediate marker-free and backbone-free plant transformation

Conventional Agrobacterium-mediated transformation methods rely on complex and genotype-specific tissue culture media for selection, proliferation, and regeneration of genetically modified cells. Resulting transgenic plants may not only contain selectable marker genes but also carry fragments of the vector backbone. Here, we describe a new method for the production of transgenic plants that lack such foreign DNA. This method employs vectors containing the bacterial isopentenyltransferase (ipt) gene as backbone integration marker. Agrobacterium strains carrying the resulting ipt gene-containing "cytokinin" vectors were used to infect explants of various Solanaceous plant species as well as canola (Brassica napus). Upon transfer to hormone-free media, 1.8% to 9.9% of the infected explants produced shoots that contained a marker-free T-DNA while lacking the backbone integration marker. These frequencies often equal or exceed those for backbone-free conventional transformation.     

Regeneration of transgenic vegetable brassicas (Brassica oleracea andB. campestris) via Ri-mediated transformation

A procedure for the production of fertile transgenic brassicas via Ri-mediated transformation is reported in this paper. Transgenic hairy root lines were selected for 12 vegetable brassica cultivars and lines representing six varieties: broccoli, Brussels sprouts, cabbage, cauliflower, rapid-cycling (allBrassica oleracea) and Chinese cabbage (B. campestris). Leaf explants or petioles of intact cotyledons were co-cultivated withAgrobacterium strain A4T harbouring various binary vectors. The T-DNA region of all binary vectors contained a neomycin phosphotransferase II gene for kanamycin resistance, in addition to other genes. Hairy root lines grew prolifically on hormone-free medium containing kanamycin. Transgenic shoots were regenerated from all cultivars either spontaneously or after transfer of hairy roots to a hormone-containing medium. Southern analysis confirmed that the plants were transgenic. Plants from all brassica types were successfully transferred to greenhouse conditions. Plants were fertile and segregation analysis confirmed transmission of traits to progeny.Abbreviations BA 6-Benzylaminopurine - GUS -Glucuronidase - LS Linsmaier and Skoog medium - NAA I-Naphthaleneacetic acid - NPTII Neomycin phosphotransferase II - TDZ thidiazuron     

Increased <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and rooting efficiencies in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) from hypocotyl segment explants

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.     

一种加工型马铃薯品种的农杆菌转化体系研究

大西洋马铃薯是经济价值很高的炸片型加工品种,逆境胁迫下,易产生褐变、空心等问题,影响加工品质。为获取抗逆境胁迫的优质转基因新品种,采用根癌农杆菌介导法,以大西洋马铃薯的茎段为外植体,建立了快速,简便,高效的遗传转化体系。从共培养到转化植株获得只需7-8周,转化频率达80%。结果表明茎段是较好的转化受体,硫代硫酸银可以有效促进不定芽分化并提高再生频率。PCR、Southern杂交分析证明外源基因已经成功整合到马铃薯再生植株的基因组中。该转化体系为大量开发转基因马铃薯植株,进而筛选优质的马铃薯炸片加工型新品种奠定基础。     

Seedling preconditioning in thidiazuron enhances axillary shoot proliferation and recovery of transgenic cowpea plants

Lack of competence of seedling explants for efficient shoot proliferation in recalcitrant grain legume cowpea restricts its genetic manipulation for crop improvement. This study aimed at establishing a protocol to increase the shoot proliferation efficiency during the regeneration of transgenic cowpea plants. Here, we describe how seedling preconditioning in thidiazuron (TDZ) could stimulate the transformation process (by 3.5-fold), shoot proliferation potential of cotyledonary node (by a factor of fourfold) and accelerate the transgenic shoot regeneration. We investigated the effect of TDZ and 6-benzyladenine (BA) at high dose (5?C20???M) in the induction phase of regeneration by preconditioning seedlings for different durations (2?C6?days) with the aim of improving shoot proliferation competence from cultured explants. Cotyledonary node explants from preconditioned seedlings were cultured on MSB₅ medium supplemented with 5???M BA and 0.5???M kinetin for 4?weeks. Best response in terms of maximum shoot proliferation (7.1 shoots per explants), and greatest shoot length (2.6?cm) were obtained with explants derived from seedlings preconditioned in 10???M TDZ for 4?days. This enhanced shoot proliferation ability was maintained through three subsequent 4-week long regeneration passages. On comparison of the transformation rate in absence and presence of seedling preconditioning (in 10???M TDZ for 4?days), a significant enhancement from 0.6 to 2.1% was observed. The promotive effect of seedling preconditioning had a direct beneficial effect on transgenic plant recovery time leading to a reduction of more than 2?weeks. The protocol was found applicable to seven cowpea genotypes.     

In vitro regeneration of plantlets in Brassica alboglabra

Different vegetative parts of Brassica alboglabra seedlings and mature plants were used as explants in culture.A high frequency (60–100%) of shoot regeneration was obtained from hypocotyl explants, nodal stem segments, internodal segments and shoot apices cultured on Murashige-Skoog basal medium. Addition of 6-benzylaminopurine and kinetin increased the average number of shoots per explant. When detached and transferred to basal medium, the shoots readily developed roots. Regenerated plantlets could be successfully transplanted in soil.     

Agrobacterium tumefaciens-mediated transformation of Brassica napus winter cultivars

An efficient protocol for Agrobacterium tumefaciens-mediated transformation of six commercial Brassica napus winter cultivars is described. Two B. napus spring cultivars were analysed for comparison. Five strains of A. tumefaciens with different combinations of nopaline and octopine chromosomal backgrounds and virulence plasmids were used for cocultivation. Selection of putative regenerated transgenic plants was performed on kanamycin- or hygromycin-containing media. The scores of transgenic plants were calculated on the basis of GUS (-glucuronidase) activity, detected by the histochemical X-Gluc test. Target tissue derived from the cut surface of cotyledon petioles resulted in successful transformation with all the winter cultivars tested. Target tissue from hypocotyl segments resulted in a successful transformation with only one winter cultivar. The transformation rates for B. napus winter cultivars in this study were higher than in previous reports. Southern blot analysis revealed that integration of marker genes occurred in single and in multiple copies and at multiple loci in the genome. The transgenic plants all grew normally and developed fertile flowers after a vernalization period. After self-pollination, Southern blot analysis of selected GUS active F1 plants revealed that introduced marker genes were stably inherited to the next generation. These data demonstrate that morphologically normal, fertile transgenic plants of B. napus winter cultivars can be achieved with both nopaline- and octopine-derived A. tumefaciens strains. This protocol should have a broad application in improvement of Brassica napus winter cultivars by introduction of foreign genes     

农杆菌介导的高羊茅高效遗传转化和转基因植株再生

用带有质粒pDBA121(含hpt基因和bar基因)的农杆菌EHA 105转化高羊茅(Festucaarundinacea Schreb.)胚性悬浮细胞,建立了可重复的、高效的农杆菌介导的高羊茅遗传转化系统.商业用的除草剂Basta直接用于转化细胞的筛选.基因型、受体材料的类型、培养基成分和筛选剂影响农杆菌介导的转化频率.悬浮细胞的农杆菌转化效率为每克悬浮细胞再生2.85～10.9株转基因植株,大大高于基因枪法的高羊茅转化效率(2～5株).经PCR分析和Southern杂交检测表明,bar基因已整合进入高羊茅基因组,转基因植株Basta喷洒试验表明bar基因已成功地实现高水平的表达.此转化系统的建立为高效地将外源有用基因导入高羊茅并高效稳定地表达奠定了基础.     

Transgene expression in broccoli (Brassica oleracea var. italica) clones propagated in vitro via leaf explants

We have developed an efficient protocol for the in vitro propagation of transgenic broccoli plants using leaf explants as starting material. A high frequency of shoot formation from leaf explants was obtained on Murashige and Skoog medium containing benzyladenine (BA, 5 mg/l) and naphthaleneacetic acid (0.5 mg/l). Frequent subcultures of existing shoots and shoot clusters to medium containing only BA (2 mg/l) promoted rapid shoot multiplication. The use of a 1:1 mixture of Agargel and Gelrite in the rooting medium increased the number of healthy roots per rooted plant. Applying this protocol, we obtained thousands of clonal rooted plantlets within 6 months from a transgenic broccoli plant carrying the cry1Ac and cry1C genes from Bacillus thuringiensis associated with kanamycin and hygromycin selectable markers, respectively. Thirty randomly selected clones that had been propagated for 1 year on medium containing kanamycin (50 mg/l) all showed resistance to both kanamycin and hygromycin. Genomic DNA and total soluble proteins were isolated from 16 of these clones. Polymerase chain reaction analysis indicated that the cry1Ac and cry1C genes were both maintained. ELISA assays showed that all of the clones produced a high level of Cry1Ac protein similar to the original transgenic plant; however, most clones had significantly lower levels of Cry1C protein than the original plant. This variation indicates that it is important to evaluate transgene expression in transgenic clones propagated long-term in vitro. In vitro propagation starting from leaf explants was also successful with other transgenic and non-transgenic Brassica oleracea materials, including broccoli, cauliflower, and collard.     

Shoot differentiation, regeneration of cauliflower and analysis of somaclonal variation by RAPD

Our major goal in this study was to establish and characterize an efficient callus induction and shoot regeneration protocol for cauliflower (Brassica oleracea var. botrytis). The effects of induction medium, growth regulator combination, organic component, AgNO(3), genotype and explants type on shoot differentiation on the cauliflower regeneration process were taken in account and hereby evaluated. The optimal media for shoot differentiation and rooting were modified MS medium (MS with PG-96 organic components) supplemented with NAA at 0.5 mg l(-1), TDZ at 0.25 mg l(-1), BA at 3.0 mg l(-1), AgNO(3) at 2.0 mg l(-1) and MS supplemented with IBA at 0.4 mg l(-1), respectively. Among the four varieties tested, Saixue gave the best shoot differentiation response (average over 18 shoots per explant) from the upper section of the hypocotyls. We have so far obtained over 500 regenerated plants under this improved protocol. We have further analyzed the somaclonal variation of regenerated plants at the DNA level by using the RAPD molecular markers. By PCR amplification, we were able to amplify 75 scoreable bands from 15 primers out of 40 arbitrary primers screened, where 35 of them were monomorphic and 40 polymorphic bands (53.3%) in four varieties studied. The absence of polymorphism among regenerated plants from the same variety indicated the conformity of the regeneration protocol.     

Agrobacterium-mediated transformation and plant regeneration of Brassica oleracea var. capitata

An efficient protocol for Agrobacterium tumefaciens-mediated transformation of four commercial cultivars of Brassica oleracea var. capitata is described. A strain of A. tumefaciens LBA4404 with the neomycin phosphotransferase gene (nptII) and a CaMV 35S-peroxidase gene cassette were used for co-cultivation. Preliminary selection of regenerated transgenic plants was performed on kanamycin-containing medium. The frequency of transgenic plants was calculated on the basis of GUS (β-glucuronidase) activity detected by the histochemical X-gluc test. Tissue-specific GUS expression driven by the peroxidase gene promoter in transgenic plants was analysed by GUS staining. The transformation rates of the commercial cultivars of B. oleracea was higher than in previous reports. Southern blot analysis revealed that integration of marker genes occurred in single and multiple loci in the genome. All transgenic plants grew normally after a brief vernalization period and showed stable inheritance of the marker gene. The present study demonstrates that morphologically normal, fertile transgenic plants of B. oleracea can be obtained. Received: 24 August 1999 / Revision received: 23 November 1999 / Accepted: 3 December     

Transformation of Recalcitrant Melon (Cucumis melo L.) Cultivars is Facilitated by Wounding with Carborundum

Transformation of the recalcitrant melon (Cucumis melo L.) cultivars Kιrka?aç 637 and Noi Yarok was accomplished by wounding cotyledon explants by vortexing with carborundum prior to inoculation with Agrobacterium tumefaciens. The addition of silver nitrate to the regeneration‐selection medium reduced the transformation efficiency, as the percentage of the explants forming putative transgenic calli and bud‐like protuberances was decreased and no transgenic shoots were produced. Chimeric transgenic plants were obtained after the regeneration of putatively transformed callus, bud‐like protuberances, buds and shoots on selective medium with kanamycin. The treatments producing the most buds or shoots from explants after 30–40 days of cultivation were the most successful for the production of transgenic plants. Only treatments where explants were vortexed with carborundum produced transgenic melon shoots of either cultivar. Subculture every 18–20 days on fresh regeneration‐selection medium containing 50 mg/L kanamycin after either a relatively high (100 mg/L) or low level (50 mg/L) of kanamycin in the first regeneration‐selection medium was necessary for the successful transformation of cultivar Kιrka?aç 637. These techniques are now being used in breeding programs for the production of melon lines bearing resistances to zucchini yellow mosaic virus and cucumber mosaic virus, important viruses limiting agricultural production.     

Conditions of transformation and regeneration of `Induka' and `Elista' strawberry plants

Efficiency of plants' transformation depends on many factors. The genotype, applied techniques and conditions of plant's modification and modified plant regeneration are the most important among them. In our studies regeneration and transformation conditions for two strawberry cultivars were determined and compared. Plants were transformed by Agrobacterium tumefaciens LBA₄₄₀₄ strain containing plasmid pBIN19 with nptII and gus-reporter genes. Experiment was carried out on more than 1300 leaf explants from each cultivar. Generally, `Induka' plants characterized with higher regeneration potential than `Elista'. The highest number of regenerated shoots was obtained on MS medium with 0.4 mg l ^–1 IBA and 1.8 mg l^–1 BA (3.5 and 1.8 shoots/explant for `Induka' and `Elista', respectively). After plant transformation number of regenerated, transgenic shoots was higher for `Elista' (on the average: 8.3 shoots/100 explants). The number of transgenic `Induka' shoots, obtained at the same conditions, was twice lower (4.2). Simultaneously `Induka' plants needed higher kanamycin concentration for transgenic explants selection than `Elista' (25 mg l^–1). Preliminary incubation of A. tumefaciens in LB or MS medium with acetosyringone and IAA resulted in increasing transgenic shoots number (per 100 explants: `Induka' 4.5, `Elista' 8.0–9.5 shoots). After using untreated bacteria for plants' transformation, number of transgenic plants varied (dependently on cultivar) from 3.8 to 7.0/100 explants. Applying LB or MS as basic medium as well as adding tobacco plant extract to these media did not significantly influence transformation efficiency.     

Adventitious shoot regeneration from seven commercial strawberry cultivars (<Emphasis Type="Italic">Fragaria </Emphasis>×<Emphasis Type="Italic"> ananassa</Emphasis> Duch.) using a range of explant types

The parameters for optimal regeneration of seven commercial strawberry cultivars were tested using a range of explants and culture conditions. Efficient levels of regeneration--those needed to carry out transformation experiments--with the cultivars Calypso, Pegasus, Bolero, Tango and Emily were achieved with leaf discs, petioles, roots and stipules. Regeneration from cv. Elsanta proved to be difficult from all explant material, although unpollinated ovaries proved to be a promising explant source, with 12% of the explants regenerating shoots. In cv. Eros, regeneration occurred only from root tissue. A comparison of the genetic background suggests that there is a strong genetic component amongst the different cultivars determining their regeneration capacity. The development of these regeneration systems provides a means to use almost the whole stock plant for the efficient genetic transformation of commercial strawberry varieties.     

甘蓝型油菜的遗传转化

甘蓝型油菜的各种外植体经遗传转化、组织培养后可以再生为转基因植株,但再生频率会因外植体的基因型、年龄、培养基添加成分和农杆菌共培养的不同而发生变化。转化方法包括农杆菌介导转化、基因枪法、花粉介导法、PEG介导法等,其应用前景非常广阔。甘蓝型油菜的遗传转化在其品质改良、抗逆性提高、雄性不育系的获得和一些特殊性状方面都取得了很大成就。简要介绍甘蓝型油菜的再生体系建立、转化方法及所取得的部分成就。