Regeneration of Chinese cabbage transgenic plants expressing antibacterial peptide gene and cowpea trypsin inhibitor gene

Summary An efficient transformation system for Chinese cabbage cotyledon explants was developed using Agrobacterium tumefaciens strains LBA4404 harbouring the plasmid pMOG 411 and the plasmid pBinΩSCK respectively. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings, growth conditions and status of Agrobacterium suspension, preculture of explants, cocultivation time, ratio between Agrobacterium and explants, acetosyringone and concentration of kanamycin had a significant influence on transformation frequency and plant regeneration. The presences of the antibacterial peptide gene and the cowpea trypsin inhibitor gene in those selected shoots in kanamycin medium were confirmed by PCR, Southern blotting and Northern blotting, The frequency of regeneration from cotyledons was analyzed in eight parental lines of Chinese cabbage and five lines were suitable for obtaining transformed plants, among which Qingmaye C and Qingmaye D were more responsive than other lines.     

High-efficiency transformation of Gossypium hirsutum embryogenic calli mediated by Agrobacterium tumefaciens and regeneration of insect-resistant plants

A simple protocol of transformation of cotton (Gossypium hirsutum L.) at a high frequency has been developed via Agrobacterium mediation, coupled with the use of embryogenic calli as explants. Embryogenic calli derived from only one to two somatic embryogenic calli lines of two Chinese cotton cultivars, the cvs. Ekang 9 and Jihe 321 which have low embryogenic potency were first inoculated with the A. tumefaciens strain LBA4404 harbouring binary vector pBin438 carrying a synthetic Bacillus thuringiensis‐active Cry1Ac and API‐B chimeric gene. Infected embryogenic calli were co‐cultivated for 48 h and were then moved on to the selection medium with kanamycin (100 mg/l) for 7‐8 weeks. Then, the kanamycin‐resistant calli (Km¹) subcultured in proliferation medium would re‐differentiate to form somatic embryos in 30 days. Cotyledon embryos were transferred to 100‐ml Erlenmeyer flasks for germination and regeneration. Putative transformants were confirmed by polymerase chain reaction and Southern blot analysis. Forty‐five regenerated plants were successfully transferred to soil, of which 12 proved to have the active Cry1Ac and API‐B chimeric gene. Insect resistance was tested by bioassay. The transgenic plants were highly resistant to cotton bollworm (Heliothis armigera) larvae, with mortality (insect resistance) ranging from 95.8 to 100%. In comparison with the methods used in Agrobacterium‐mediated transformation of cotton hypocotyls or cotyledons, about 6 months are saved by using the method presented in this paper to obtain a large number of transgenic plants.     

A melon genotype with superior competence for regeneration and transformation

Transformation efficiency of melon is low and is still regarded as a challenge. In this paper, the regeneration and transformation response of ‘BU‐21/3′, a newly characterized melon breeding line, is described. The line seems to be superior in this regard to previously evaluated genotypes. Agrobacterium‐mediated delivery of the GUS or GFP reporter genes into cotyledon explants was used to evaluate efficiency of transient and stable transformation. Good transient expression was observed, and stable transformation frequencies of 0.4‐1.5 transgenic shoots per explant were obtained. Transgenic plantlets were transferred to a contained greenhouse as early as 8‐10 weeks after transformation. Transgenic plants are fertile and exhibit a true‐to‐type phenotype. The ‘BU‐21/3’ line may become a useful tool for the facilitation of transgenic breeding in melon.     

Factors involved in Agrobacterium tumefaciens-mediated gene transfer into Pinus nigra Arn. ssp. salzmannii (Dunal) Franco

Cotyledons from dissected sterile embryos of salgareño pine (Pinus nigra Arn. ssp. salzmannii (Dunal) Franco) were inoculated with different disarmed Agrobacterium tumefaciens strains harbouring the binary vector p35SGUSint. The transient expression of a β-glucuronidase gene (uidA) was studied, using a histochemical staining procedure. Nineteen days after inoculation, the activity of β-glucuronidase was detected in epidermal and subepidermal layers of cotyledonary explants. The EHA105 strain harbouring a disarmed agropine-type Ti-plasmid (pTiBO542) was the most effective for gene transfer of the uidA gene. The effects of exudates and extracts from 0-day-old embryos on induction of vir gene expression in A. tumefaciens were also examined. The results of this study showed that salgarño pine embryo exudates contain a substance(s) that induce vir gene expression, in similar way to that observed with 100 μM acetosyringone (AS).All these findings suggest that T-DNA processing and transfer might take place when Agrobacterium infects suitable tissues of salgareño pine.     

Transgenic fertile soybean plants derived from somatic embryos transformed via the combined DNA-free particle bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis> system

An Agrobacterium-mediated transformation procedure for soybean [Glycine max L. Merrill] proliferating somatic embryos is here described. The Agrobacterium tumefaciens LBA4404 strain harboring pTOK233, pCAMBIA1390-olp or pH7WG2Dwrky plasmids was used to mediate gene transfer into the plant genome. Prior to Agrobacterium inoculation, proliferative soybean embryogenic clusters were microwounded by DNA-free tungsten particle bombardment. Three independent transformation experiments were performed. In Experiment I, 26 transgenic plants were obtained from a unique clone of cv Bragg, while 580 plants were recovered from 105 clones of cv IAS5. In Experiment II, a single hygromycin-resistant clone of cv BRSMG68 Vencedora was recovered and gave rise to five plants. In Experiment III, 19 plants of cv Bragg and 48 plants of IAS5 were recovered, representing five and 14 independent transformation events, respectively. PCR and Southern analyses confirmed the transgenes’ integration into plant genomes. Transgenic plants were fertile. They flowered, set pods and seeds. Transgene segregation in two T₁ progenies fits the Mendelian pattern (3:1 transgenic:non-transgenic plants). This is the first report of transgenic fertile soybean plants obtained from somatic embryogenic tissues transformed by the system that combines DNA-free particle bombardment and Agrobacterium.     

Coat protein-mediated protection against plum pox virus in herbaceous model plants and transformation of apricot and plum

Summary The expression of the viral coat protein gene in transgenic plants has been shown to induce tolerance against virus infection (Beachy et al., 1990). Transgenic plants ofNicotiana clevelandii andNicotiana benthamiana- herbaceous host plants for PPV - transformed withAgrobacterium strain LBA 4404 containing the plasmid pBinPPVm, regenerated on selection media containing kanamycin were tested for the expression of the PPV coat protein gene by ELISA and immuno western blot. After rooting and acclimatisation plants were tested for the protection against PPV Following the inoculation plants were investigated for symptom development and virus accumulation. Different lines were identified, according to the different reaction to the mechanical inoculation, ranging from a complete absence to a strong reduction of symptoms. There have not been many reports on transformation of trees in general, and in fruit trees particularly. It is obvious that the major obstacle is the regeneration of transformed plantlets. Attempts to improve crop plants by genetic engineering techniques will always depend very strongly on the availability of reliable protocols for transformation, selection and regeneration (Laimer et al., 1989, 1990). Different systems involving juvenile and adult plant material have been developed allowing the transfer of foreign genes into apricot and plum cultivars. We report the transformation and regeneration ofPrunus armeniaca andPrunus domestica plants withAgrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker geneβ-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV), the causal agent of Sharka disease. The marker geneGUS was used for the optical evaluation of the efficiency of different transformation systems involving cotyledons of immature embryos as well as somatic embryos and leaf discs. The coat protein gene of PPV was used to introduce the coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area.     

Manipulation of discriminatory T-DNA delivery by Agrobacterium into cells of immature embryos of barley and wheat

Immature embryos (IEs) of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) were co-cultivated with Agrobacterium tumefaciens and the transfer of T-DNA to the cereals was assessed by analysis of the expression of auidA reporter gene. This approach revealed that T-DNA transfer is preferentially targeted to distinct anatomical regions of the cereal embryos that have a low capacity for regenerative growth. This preference may limit the efficiency of generating genetically transformed plants. With the aims of maximizing the frequency of T-DNA delivery and altering the preference of delivery in favour of regeneration-competent areas of IEs, various parameters of the inoculation and co-cultivation procedure were evaluated. It was found that controlling the density of the Agrobacteriuminoculum and/ or the concentration of the Murashige and Skoog (MS) basal salts in the medium produced changes both in the total number and relative distribution of T-DNA transfer events, and these changes may have positive implications for the production of stable transformants. The use of pre-cultured embryos were also important for T-DNA targeting. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transgenic apples display stable gene expression in the fruit and Mendelian segregation of the transgenes in the R1 progeny

Summary The transfer of genes via Agrobacterium to a perennial tree crop such as apple, requires monitoring of the stability of the genes in the target tissues such as the fruit and leaves. If the same genes are required for introgression into a conventional breeding programme, their expression also needs to be stable and their inheritance should follow a normal Mendelian pattern. In the following report we show, for the first time, the stable expression and Mendelian segregation of transgenes in a tree species. We have evidence for a 1:1 segregation of the nos and nptII genes among R1 progeny from a transgenic apple parent. In addition, we present evidence for stable gene expression of both nos and the co-transferred gene nptII in the flesh of apple fruit 7 years after the initial transformation.Abbreviations nos nopaline synthase - nptII neomycin phosphotransferase - ACC 1-aminocyclopropane carboxylic acid - PVP polyvinylpyrrolidone - PCR polymerase chain reaction - EFE ethylene forming enzyme - B.t. Bacillus thuringiensis     

Genetic transformation of lipid transfer protein encoding gene in <Emphasis Type="Italic">Phalaenopsis amabilis</Emphasis> to enhance cold resistance

Embryogenic callus of Phalaenopsis amabilis derived from leaf tissue was cocultivated with Agrobacterium tumefaciens strain LBA4404 harboring a plant cloning vector. The vector carried the lipid transfer protein (LTP) encoding gene cloned from cold tolerant Brazilian upland rice cv. IAPAR 9. The highest transformation efficiency (12.16%) was obtained when 1–2 mm calli were infected and cocultivated with 0.4 (OD₆₀₀) A. tumefaciens for 20 min. Transgene integration of kan-resistant plants was confirmed through polymerase chain reaction analysis and Southern hybridization. Four hundred seventy transgenic plants, each derived from an independent protocorm-like body, were obtained. The expression of rice cold-inducible LTP gene in transgenic P. amabilis improved its adaptive responses to cold stress. The examination of transgenic plants revealed that enhanced cold tolerance was most likely due to the increased accumulation of several compatible solutes such as total soluble sugars, proline, antioxidant superoxide dismutase, decreased accumulation of malondialdehyde, and maintained electrolytes within the membrane compared with controls.     

Amenability of castor to an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation strategy using a <Emphasis Type="Italic">cry1AcF</Emphasis> gene for insect tolerance

Agrobacterium tumefaciens mediated in planta transformation protocol was developed for castor, Ricinus communis. Two-day-old seedlings were infected with Agrobacterium strain EHA105/pBinBt8 harboring cry1AcF and established in the greenhouse. Screening the T₁ generation seedlings on 300 mg L⁻¹ kanamycin identified the putative transformants. Molecular and expression analysis confirmed the transgenic nature and identified high-expressing plants. Western blot analysis confirmed the co-integration of the nptII gene in the selected transgenic plants. Bioassay against Spodoptera litura corroborated with high expression and identified five promising effective lines. Analysis of the T₂ generation plants proved the stability of the transgene indicating the feasibility of the method.     

Efficient In Vitro Shoot-regeneration Systems in Cassava (Manihot esculenta Crantz)

Two different protocols for in vitro regeneration of cassava using zygotic embryos and nodal axillary meristems have been developed. In both cases, buds were regenerated directly from excised explants without an intervening callus phase after a two-step culture procedure. In cotyledonary explants derived from zygotic embryos, prolific shoot formation occurred within 2—3 weeks on MS medium supplemented with 0.5—5 mg/1 BAP alone or in combination with 0.1 mg/1 NAA. Nodal explants with axillary meristems derived from aseptically grown seedlings or stem cuttings were used to initiate a round compact bulb-like structure on MS medium containing 10 mg/1 BAP. These latter structures, when cultured on MS medium supplemented with 0.1 mg/1 NAA, 1 mg/1 BAP and 0.1 mg/1 GA₃, produced multiple shoots. Somatic embryos isolated at the globular/torpedo stage from zygotic embryo explants were also capable of multiple shoot production on medium with 1 mg/1 BAP. Rooting of regenerated shoots exceeded 95 % in phytohormone-free MS medium. No change in their ploidy levels was observed. Therefore, the protocols developed should be of use in the particle gun and Agrobacterium-mediated genetic transformation of cassava.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of faba bean <Emphasis Type="Italic">(Vicia faba</Emphasis> L.) using embryo axes

A system for the production of transgenic faba bean by Agrobacterium-mediated transformation was developed. This system is based upon direct shoot organogenesis after transformation of meristematic cells derived from embryo axes. Explants were co-cultivated with A. tumefaciens strain EHA105/pGlsfa, which harbored a binary vector containing a gene encoding a sulphur rich sunflower albumin (SFA8) linked to the bar gene. Strain EHA 101/pAN109 carrying the binary plasmid containing the coding sequence of a mutant aspartate kinase gene (lysC) from E. coli in combination with neomycinphosphotransferase II gene (nptII) was used as well. The coding sequences of SFA8 and LysC genes were fused to seed specific promoters, either Vicia faba legumin B4 promoter (LeB4) or phaseolin promoter, respectively. Seven phosphinothricin (PPT) resistant clones from Mythos and Albatross cultivars were recovered. Integration, inheritance and expression of the transgenes were confirmed by Southern blot, PCR, enzyme activity assay and Western blot.     

Genetic engineering of cereal crop plants: a review

Summary Many aspects of basic and applied problems in plant biology can be investigated by transformation techniques. In dicotyledonous species, the ability to generate transgenic plants provides the tools for an understanding of plant gene function and regulation as well as for the directed transfer of genes of agronomic interest.For many dicotyledonous plants Agrobacterium tumefaciens can be routinely used to introduce foreign DNA into their genome. However, cereals seem to be recalcitrant to Agrobacterium-mediated transformation.In cereals, many efforts have been made in recent years to establish reliable transformation techniques. Several transformation techniques have been developed but to date only three methods have been found to be suitable for obtaining transgenic cereals: transformation of totipotent protoplasts, particle bombardment of regenerable tissues and, more recently, tissue electroporation. The current state of transformation methods used for cereals will be reviewed.     

Phenotypic expression of quantitative and qualitative components of partial resistance to powdery mildew (Sphaerotheca fuliginea race 1) in melon (Cucumis melo) germplasm

A study was conducted to investigate the expression of four components of partial resistance to Sphaerotheca fuliginea race 1 in selected melon (Cucumis melo L.) lines viz. infection frequency, latent period, spore production, and disease-severity score. Those components were evaluated at two developmental stages of the host: the cotyledon stage and the stage of the first two true leaves. Detached plant parts (disks of cotyledons and true leaves) were inoculated using a vacuum-operated settling tower. All four components showed significant variation among genotypes, and correlations between components at both developmental stages were large and significant. The line ‘CNPH 83–095’ (without any major resistance gene to powdery mildew) presented the highest level of partial resistance in both vegetative stages for almost all components evaluated. The lines ‘W-6’ (Pm1Pm1, Pm2Pm2), ‘Cinco’ (Pm1Pm1, Pm2Pm2), and CNPH ‘84–147’ (Pm1Pm1), even though carrying the major gene Pm1 for complete resistance to race 1 of the fungus, showed slight but significant differences for quantitative components of partial resistance at the cotyledonal stage. Different levels of partial resistance may be expressed, even in lines with a major race-specific resistance gene to powdery mildew, in specific developmental stages of the melon plants.     

A non-ACC pathway for ethylene biosynthesis in Botrytis cinerea

Premature softening and tissue senescence occur in kiwifruit infected with Botrytis cinerea. While ethylene production is enhanced in infected fruit and B. cinerea produces ethylene on defined media in vitro the source of ethylene in this pathosystem is unclear. Ethylene production by B. cinerea was enhanced when methionine or ∝-keto-methylthiobutyric acid (KMBA) was added to a defined (modified Pratts) medium. Although 1-aminocyclopropane-1-carboxylic acid (ACC) did not stimulate ethylene production, ∝-aminooxyacetic acid (AOA) was inhibitory suggesting a role for a pyridoxal phosphate mediated enzyme reaction down stream from the methionine/KMBA stimulated ethylene biosynthetic pathway. Cobalt chloride (Co²⁺) was inhibitory, but after a 4-d lag period ethylene production from B. cinerea cultures containing methionine and Co²⁺ reached the same level as those without Co²⁺. [U ¹⁴C] methionine was converted to ¹⁴C-ethylene with high efficiency indicating that it is a direct precursor, while [2,3 ¹⁴C]-ACC did not yield radioactively labelled ethylene. These results suggest that the ethylene biosynthetic pathway in B. cinerea does not involve ACC as a precursor and that the enzyme responsible for synthesising ethylene is similar to, but different from, ACC oxidase from higher plants. The ethylene biosynthetic pathway in B. cinerea is yet to be determined.     

Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene

Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes. Plants showing vigorous PPT resistance were obtained by a series concentration selection for PPT resistance and subsequent regeneration of leaf explants dissected from the putative chimera. Transgenic plants were confirmed by PCR and genomic Southern blotting, which showed that the bar and pinII genes were integrated into the plant genome. Double haploid homozygous transgenic plants were obtained by microspore culture. The pinII expression was detected using quantitative real time polymerase chain reaction (qRT-PCR) and detection of PINII protein content in the transgenic homozygous lines. Insect-feeding trials using the larvae of cabbage worm (Pieris rapae) and the larvae of the diamondback moth (Plutella xylostella) showed higher larval mortality, stunted larval development, and lower pupal weights, pupation rates, and eclosion rates in most of the transgenic lines in comparison with the corresponding values in the non-transformed wild-type line.     

Medium, Explant and Genotype Factors Influencing Shoot Regeneration in Oilseed Brassica spp.

The effects of culture media, explants and genotypes on shoot regeneration in oilseed Brassica species were examined in this study. The maximum shoot regeneration frequency was obtained in Murashige and Skoog medium supplemented with 3 mg l^?1 6‐benzylaminopurine and 0.15 mg l^?1 1‐naphthaleneacetic acid. The addition of 2.5 mg l^?1 AgNO₃ was very beneficial to shoot regeneration in B. napus and Ag₂S₂O₃ (10 mg l^?1) was even superior to AgNO₃ (2.5 mg l^?1). Explant age, explant type and carbon source also significantly affected shoot regeneration. Four‐day‐old seedlings of cotyledonary explants showed the maximum shoot regeneration frequency and number of shoots per explant. Of the four explants – peduncles, hypocotyls, cotyledons and leaf petioles – cotyledons produced the highest shoot regeneration frequency (56.67 %). Four carbon sources – glucose, maltose, starch and sucrose – were compared for their respective effects on shoot regeneration from cotyledonary explants. Sucrose appeared to be the best carbon source for shoot regeneration with the highest shoot regeneration frequency (76.00 %). Considerable variation in shoot regeneration from cotyledonary explants was observed both between and within Brassica species. The shoot regeneration frequency ranged from 10.00 % for cv. R5 (B. rapa) to 83.61 % for cv. N1 (B. napus). Two B. napus, one B. carinata and one B. juncea cultivars exhibited shoot regeneration frequency higher than 70 %. In terms of the number of shoots produced per explant, B. rapa showed the highest variation, ranging from 5.64 for cv. R3 to 1.33 for cv. R5. Normal plantlets were regenerated from all induced shoots and developed normally. The regenerated plants were fertile and identical with the source plants.     

Rapid induction of a novel ACC synthase gene in deepwater rice seedlings upon complete submergence

Elongation growth is a typical feature of deepwater rice plants in response to submergence. The growth phenomenon is known to be induced by hypoxia, which results in the expression of genes implicated in ethylene biosynthesis. Ethylene is considered to trigger the growth response as it accumulates in the submerged tissues and submergence enhances the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase. However, ACC concentration in rice plants increases much faster after submergence than the activity of the ACC synthase genes studied previously. Here, we studied the expression characteristics of the fifth member of this gene family, OS-ACS5, and show that submergence induces the messenger concentration of this gene before the accumulation of ACC could be observed. OS-ACS5 may play a fundamental role in the growth-promoting increase in ethylene biosynthesis during the first hours of submergence in deepwater rice. This revised version was published online in August 2006 with corrections to the Cover Date.