<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Botryosphaeria dothidea</Emphasis>

Botryosphaeria dothidea is a severe causal agent of die-back and cankers of many woody plants and causes great losses in many regions. The pathogenic mechanism of this pathogen has not been well explored due to lack of mutants and genetic information. In this study, we developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for B. dothidea protoplasts using vector pBHt2 containing the hph gene as a selection marker under the control of trp C promoter. Using this protocol we successfully generated the B. dothidea transformants with efficiency about 23 transformants per 10⁵ protoplasts. This is the first report of genetic transformation of B. dothidea via ATMT and this protocol provides an effective tool for B. dothidea genome manipulation, gene identification and functional analysis.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of blackgram: An assessment of factors influencing the efficiency of <Emphasis Type="Italic">uidA</Emphasis> gene transfer

Agrobacterium tumefaciens strain EHA105 carrying a binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron, was used for transformation of Vigna mungo cotyledonary node explants. Various factors such as preculture and wounding of explants, manipulations in inoculation and co-cultivation conditions were found to play a significant role in influencing tissue competence, Agrobacterium virulence and compatibility of both, for achieving the maximum transformation frequencies. The stable transformation with 4.31 % efficiency was achieved using the optimized conditions. The transformed green shoots that were selected and rooted on medium containing kanamycin and tested positive for nptII gene by polymerase chain reaction were established in soil to collect seeds. GUS activity was detected in leaves, roots, pollen grains and T₁ seedlings. Southern analysis of T₀ plants showed the integration of nptII into the plant genome.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of oleaginous yeast <Emphasis Type="Italic">Lipomyces</Emphasis> species

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species.     

Frond transformation system mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> for <Emphasis Type="Italic">Lemna minor</Emphasis>

The Lemnaceae, known as duckweed, the smallest flowering aquatic plant, shows promise as a plant bioreactor. For applying this potential plant bioreactor, establishing a stable and efficient genetic transformation system is necessary. The currently favored callus-based method for duckweed transformation is time consuming and genotype limited, as it requires callus culture and regeneration, which is inapplicable to many elite duckweed strains suitable for bioreactor exploitation. In this study, we attempted to establish a simple frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor, one of the most widespread duckweed species in the world. To evaluate the feasibility of the new transformation system, the gene CYP710A11 was overexpressed to improve the yield of stigmasterol, which has multiple medicinal purposes. Three L. minor strains, ZH0055, D0158 and M0165, were transformed by both a conventional callus transformation system (CTS) and the simple frond transformation system (FTS). GUS staining, PCR, quantitative PCR and stigmasterol content detection showed that FTS can produce stable transgenic lines as well as CTS. Moreover, compared to CTS, FTS can avoid the genotype constraints of callus induction, thus saving at least half of the required processing time (CTS took 8–9 months while FTS took approximately 3 months in this study). Therefore, this transformation system is feasible in producing stable transgenic lines for a wide range of L. minor genotypes.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Factors influencing <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn

Agrobacterium-mediated transformation protocol has been developed for Eleusine coracana (var. PR-202) by varying several factors which influence T-DNA delivery. Green nodular regenerative calli with meristematic nodules of seed origin were used as the target tissue for Agrobacterium tumefaciens-mediated gene transfer. The highest frequency of transformation (44.4%) was observed when callus was infected, co-cultivated and incubated at 22°C. Incorporation of higher level of CuSO₄ in the regeneration medium had significantly positive effect on the recovery of transformed plants. PCR analysis of T ₀ and T ₁ generation plants with nptII-specific primers revealed the amplification of nptII gene. Southern blot analysis of six regenerated plants confirmed selectable marker gene integration in three plants. This is a first report on Agrobacterium-mediated genetic transformation of finger millet and will pave the way for further studies in this and other millet crops.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Acacia crassicarpa</Emphasis> via organogenesis

A protocol was developed for Agroacterium-mediated genetic transformation of Acacia crassicarpa via organogenesis by using in vitro phyllode (leaf) as the explant. Phyllode (leaf) explants were co-cultured with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pBI101 (harboring antisense Pt4CL1 with respect to the Pt4CL1P promoter). The selection for transgenic shoots was performed through two consecutive steps on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators and antibiotics in the following order: 0.5 mg/l thidiazuron (TDZ), 0.5 mg/l α-naphthaleneacetic acid (NAA), 300 mg/l carbenicillin (Car) and 20 mg/l kanamycin (Km) for 10 days; 0.1 mg/l TDZ, 200 mg/l Car and 20 mg/l Km for 60 days; 0.5 mg/l indole-3-butyric acid (IBA), 100 mg/l Car and 20 mg/l Km 50 days. 21.7% of nodules produced multiple adventitious shoot buds, of which 27.7% survived in initial selection. The shoot buds were subjected to repeated selection on MS medium supplemented with 0.1 mg/l TDZ, 200 mg/l Car and 20 mg/l Km for 60 days. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.5 mg/l IBA, 100 mg/l Car 20 mg/l Km 50 days. Genomic PCR analysis confirmed the incorporation of the antisense Pt4CL1 with respect to the Pt4CL1P promoter fragment into the host genome.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of a medicinal plant <Emphasis Type="Italic">Taraxacum platycarpum</Emphasis>

Dandelion plants, the genus Taraxacum, are used in herbal medicine owing to their choleretic, diuretic and anti-carcinogenic activities and several medicinal compounds have been isolated from the roots of these plants. Metabolic manipulation of secondary metabolite biosynthesis is a potential strategy to improve the production of high-value secondary metabolites. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is known to control a key regulatory step in the isoprenoid pathway. We report an efficient transformation protocol for stable introduction of HMGR into dandelion plants (Taraxacum platycarpum H. Dablstaed), which is essential for the biotechnological approach. The Agrobacterium tumefaciens strain EHA105 containing the binary vector, pCAMBIA1301, with GUS and HMGR genes, showed high transformation efficiency after 3–5 week hygromycin selection. Southern blotting, GUS staining and RT-PCR analyses demonstrated stable integration of one copy of the HMGR gene into the dandelion genome. Expression of the integrated genes was particularly eminent in root tissues of primary transformant plants. The establishment of an efficient transformation method may facilitate the improvement of medicinal plant in terms of the accumulation levels of secondary metabolites.     

Genetic transformation of <Emphasis Type="Italic">Gentiana dahurica</Emphasis> Fisch by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> using zygotic embryo-derived callus

Gentiana dahurica Fisch is one of four important commercial Radix Gentianae stipulated by the Chinese Pharmacopoeia. We have established a rapid and effective regeneration system using zygotic embryo-derived callus of G. dahurica Fisch. Using this regeneration system, Agrobacterium tumefaciens-mediated transformation of G. dahurica Fisch has been developed. Zygotic embryo-derived callus was infected with an A. tumefaciens strain (GV3130) harboring a pBI121 vector that contained an nptII selective marker gene. In a total of 60 zygotic embryo-derived calli assayed, frequency of calli with nptII gene PCR-positive transgenic plantlets is 5%. Transient glucuronidase (GUS) expression was observed from these transgenic plantlets. Frequency of GUS-positive transgenic plantlets (4/78) was approximately consistent with that of PCR assay (3/60). Our data indicate that we have successfully established a stable G. dahurica Fisch transformation system by A. tumefaciens. In general, this transformation system we have established might be useful for modifying physiological, medicinal and horticultural traits.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.)

Transient expression studies using blueberry leaf explants and monitored by -glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)₃AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 M AS. Explants were then placed on modified WPM supplemented with 1.0 mg l^–1 thidiazuron, 0.5 mg l^–1 -naphthaleneacetic, 10 mg l^–1 kanamycin (Km), and 250 mg l^–1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 E m^–2 s^–1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.     

Genetic transformation of <Emphasis Type="Italic">Agave salmiana</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and particle bombardment

Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive with the GUS assay after 14 months on selective medium while still undergoing regeneration.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.) using root explants

An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed. Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calli were induced in media containing 0.1 mg l(-1) thidiazuron (TDZ), 3.0 mg l(-1) N(6)-benzylaminopurine, 100 mg l(-1) kanamycin and 500 mg l(-1) cefotaxime. The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg l(-1) kanamycin sulphate. Transgenic plants were raised in pots and seeds subsequently collected from mature fruits. Histochemical GUS assay and polymerase chain reaction analysis of field-established transgenic plants and their offsprings confirmed the presence of the GUS and NPTII genes, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both the NPTII and GUS genes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Lotus tenuis</Emphasis> and regeneration of transgenic lines

A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog medium supplemented with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 μg ml⁻¹) and cefotaxime (400 μg ml⁻¹) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten adventitious shoots per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS strain produced 13–15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines were obtained from 160 explants, corresponding to a transformation efficiency of 10 and 18.1%, respectively. More than 90% of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase from stressed ADC- Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter showed strong signals of osmotic stress in the leaves and stem tissues. All of the transgenic plants obtained exhibited the same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the cloned materials was established.     

Developing an <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation for a selenium-hyperaccumulator <Emphasis Type="Italic">Astragalus racemosus</Emphasis>

Agrobacterium tumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The optimal concentration of kanamycin that could effectively inhibit cell growth and division in non-transgenic tissues was 50 mg l⁻¹ and thus all putative transgenic plants were obtained on induction medium containing 50 mg l⁻¹ kanamycin. The verification of transformants was achieved by both histochemical GUS assay and PCR amplification of nptII gene. Southern blot analysis was performed to further confirm that transgene nptII was stably integrated into the A. racemosus genome. A transformation frequency of approximately 10% was achieved using this protocol, but no beneficial effect from the addition of acetosyringone (50 μM) was observed. This transformation system will be a useful tool for future studies of genes responsible for Se-accumulation in A. racemosus.     

Organogenesis and <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Eucalyptus saligna</Emphasis> with <Emphasis Type="Italic">P5CS</Emphasis> gene

The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ¹-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied. The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots was similar in both types of plants.     

An <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation system using callus of <Emphasis Type="Italic">Zoysia tenuifolia</Emphasis> Willd. ex Trin

Zoysia tenuifolia Willd. ex Trin. is one of the most popularly cultivated turfgrass. This is the first report of successful plant regeneration and genetic transformation protocols for Z. tenuifolia using Agrobacterium tumefaciens. Initial calli was induced from stem nodes incubated on a Murashige and Skoog (1962) (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg l⁻¹ 6-benzyladenine (BA), with a frequency of 53%. Compact calli were selected and subcultured monthly on the fresh medium. Sixty-nine percent of the calli could be induced to regenerate plantlets when the calli incubated on a MS medium supplemented with 0.2 mg l⁻¹ BA under darkness. For genetic transformation, calli were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, about 12% of the callus explants produced hygromycin resistant calli on MS medium supplemented with 2 mg l⁻¹ 2,4-D, 1 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, 500 mg l⁻¹ cefotaxime after 8 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 0.2 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 46% of the resistant calli differentiated into shoots. Finally, all the resistant shoots were rooted on 1/2 MS media supplemented with 50 mg l⁻¹ hygromycin, 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. About 5% of the total inoculated callus explants produced transgenic plants after approximately 5 months. The procedure described will be useful for both, the introduction of desired genes into Z. tenuifolia and the molecular analysis of gene function.