Regeneration of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.) from root explants

Summary Eggplant (Solanum melongena L.) was efficiently regenerated from cultured roots of 15-d-old seedlings on Murashige and Skoog (MS) medium containing 0.45 μM thidiazuron and 13.3 μM 6-benzyladenine. Within 28d of culture initiation, induction of organogenic calluses and subsequent differentiation into shoot buds were observed. Shoot buds upon subculture to MS basal medium elongated into healthy shoots. Excised shoots (2–4 cm) were rooted on Soilrite^? irrigated with water either in vitro or in vivo. Plants with well-developed root systems were established under field conditions after hardening in the glasshouse, where they developed into flowering plants and produced mature fruits with viable seeds.     

Plastid transformation in eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

We have developed a method for plastid transformation in eggplant (Solanum melongena L.), a solanaceous plant species. Plastid transformation in eggplant was achieved by bombardment of green stem segments with pPRV111A plastid expression vector carrying the aadA gene encoding aminoglycoside 3′′-adenylyltransferase. Biolistic delivery of the pPRV111A plasmid yielded transplastomic plants at a frequency of two per 21 bombarded plates containing 25 stem explants each. Integration of the aadA gene in the plastome was verified by PCR analysis and also by Southern blotting using 16S rDNA (targeting sequence) and the aadA gene as a probe. Transplastomic expression of the aadA gene was verified by RT-PCR. The development of transplastomic technology in eggplant may open up exciting possibilities for novel gene introduction and expression in the engineered plastome for agronomic or pharmaceutical 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.     

<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.     

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.     

Efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) from stem explants using a two-step kanamycin-hygromycin selection method

Summary To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants, leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt (p35S) gene for hygromycin (Hyg) resistance, and the gusA (p35S) reporter gene for β-glucuronidase (GUS). After 3 d co-cultivation, selection of calluses from the three explant types began first with culture on 50 mg l⁻¹ of Km for 6 wk and then transfer to 30 mg l⁻¹ of Hyg for 6–16 wk in Linsmaier and Skoog (1965) medium (LS) also containing 6.49 μM 4-fluorophenoxyacetic acid and 250 mgl⁻¹ cefotaxime in the dark. The selected friable calluses regenerated shoots in 4 wk on LS containing 15.13 μM abscisic acid and 2.89 μM gibberellic acid under a 16h photoperiod of 30 μmol m⁻²s⁻¹. The two-step selection method led to successful recovery of transgenic shoots from stem explants at 30.8%, leaf dises 11.2%, and petioles 10.7% stable transformation efficiencies. PCR analyses of 122 GUS-positive lines revealed the expected fragment for hpt. Southern hybridization of genomic DNA from 18 independent transgenic lines detected the presence of the gusA gene. The number of integrated T-DNA copies varied from one to four.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Festuca arundinacea</Emphasis> (Schreb.) and <Emphasis Type="Italic">Lolium multiflorum</Emphasis> (Lam.)

Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and beta-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and surviving colonies plated on embryogenesis media. Eight Lolium (six independent lines) and two Festuca plants (independent lines) were regenerated and established in soil. All plants were hygromycin-resistant, but histochemical determination of GUS activity showed that only one Festuca plant and one Lolium plant expressed GUS. Three GUS-negative transgenic L. multiflorum and the two F. arundinacea plants were vernalised and allowed to flower. All three Lolium plants were male- and female-fertile, but the Festuca plants failed to produce seed. Progeny analysis of L. multiflorum showed a 24-68% inheritance of the hph and uidA genes in the three lines with no significant difference between paternal and maternal gene transmission. However, significant differences were noted between the paternal and maternal expression of hygromycin resistance.     

<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.     

Highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.) through somatic embryogenesis

A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166 and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin (Km) were then recovered on CIM + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM α-naphthaleneacetic acid (NAA) + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and 18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production of transgenic celery plants.     

Efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation using embryogenic suspension cultures in sweetpotato, <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.

Efficient Agrobacterium tumefaciens-mediated transformation was achieved using embryogenic suspension cultures of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Lizixiang. Cell aggregates from embryogenic suspension cultures were cocultivated with the A. tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hygromycin phosphotransferase II gene (hpt II) genes. Selection culture was conducted using 25 mg l⁻¹ hygromycin. A total of 2,218 plants were regenerated from the inoculated 1,776 cell aggregates via somatic embryogenesis. β-glucuronidase (GUS) assay and PCR, dot blot and Southern blot analyses of the regenerated plants randomly sampled showed that 90.37% of the regenerated plants were transgenic plants. The number of integrated T-DNA copies varied from 1 to 4. Transgenic plants, when transferred to soil in a greenhouse and a field, showed 100% survival. No morphological variations were observed in the ex vitro transgenic plants. These results exceed all transformation experiments reported so far in the literature in quantity of independent events per transformation experiment in sweetpotato.     

The influence of flask sealing on <Emphasis Type="Italic">in vitro</Emphasis> morphogenesis of eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

The influence of flask sealing on eggplant morphogenic responses and morpho-anatomical characteristics was evaluated. Eggplant seeds from the cultivar Embu were disinfected and inoculated in MS medium supplemented with B₅ vitamins, 0.55 mM myo-inositol, 2% (w/v) sucrose, and 0.65% (w/v) agar. NAA (53.7 μM) and IAA (0.57 μM) were added to the medium to elicit morphogenic responses from cotyledon and hypocotyl explants via somatic embryogenesis and organogenesis, respectively. The plates were sealed with Micropore® 3M, Parafilm®, or polyvinyl chloride (PVC) film. The effect of glass vessel capping on morphogenesis was also evaluated for shoot apexes inoculated on medium containing half-strength MS where the capping consisted of polypropylene lids with or without two vents (0.45-μm MilliSeal® air vent) and PVC film. Leaf histological analysis and leaf bleaching from each treatment were performed. No significant differences were observed in the number of embryos and root primordia in media containing either 53.7 μM NAA or 0.57 μM IAA. However, embryogenic calli fresh weight was higher for PVC and Parafilm®. Morphogenesis from the shoot apex was influenced, except the plant height. Plants maintained in glass flasks capped with vented lids showed more vigorous growth and differentiated anatomical structures compared to plants under other treatments. This treatment resulted in more expanded leaves, wider stems, and higher dry and fresh weights. In all treatments, the number of stomata was higher in the abaxial surfaces of leaves. Our results indicate that the flasks with vents provided air exchange beneficial for plant morphogenesis.     

<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.     

<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.     

The transformation of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): applicable methods of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated gene transfer

Three methods of transformation of pea (Pisum sativum ssp. sativum L. var. medullare) were tested. The most efficient Agrobacterium tumefaciens-mediated T-DNA transfer was obtained using embryonic segments from mature pea seeds as initial explants. The transformation procedure was based on the transfer of the T-DNA region with the reporter gene uidA and selection gene bar. The expression of β-glucuronidase (GUS) in the regenerated shoots was tested using the histochemical method and the shoots were selected on a medium containing phosphinothricin (PPT). The shoots of putative transformants were rooted and transferred to non-sterile conditions. Transient expression of the uidA gene in the tissues after co-cultivation and in the course of short-term shoot cultivation (confirmed by histochemical analysis of GUS and by RT-PCR of mRNA) was achieved; however, we have not yet succeeded in proving stable incorporation of the transgene in the analysed plants.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): gene integration,expression and inheritance

A reproducible method of Agrobacterium-mediated transformation was developed for Cicer arietinum (chickpea). Initial explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. The plasmid contained a bi-functional fusion gene conferring both -glucuronidase and neomycin phosphotransferase activities, under the control of a 35S35SAMV promoter. Using a series of tissue culture media for co-cultivation, shoot initiation and rooting, we recovered transgenic plants from approximately 1.3% of the sliced embryo axes. The addition of a shoot elongation medium to the protocol improved the success rate to 3.1% but increased the time in tissue culture. Inheritance of the gus gene was followed through four generations, both through expression and Southern hybridization assays, and showed the expected Mendelian inheritance pattern.NRCC Grant No. 46589.     

Functional characterization of the powdery mildew susceptibility gene <Emphasis Type="Italic">SmMLO1</Emphasis> in eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Eggplant (Solanum melongena L.) is one of the most important vegetables among the Solanaceae and can be a host to fungal species causing powdery mildew (PM) disease. Specific homologs of the plant Mildew Locus O (MLO) gene family are PM susceptibility factors, as their loss of function results in a recessive form of resistance known as mlo resistance. In a previous work, we isolated the eggplant MLO homolog SmMLO1. SmMLO1 is closely related to MLO susceptibility genes characterized in other plant species. However, it displays a peculiar non-synonymous substitution that leads to a T → M amino acid change at protein position 422, in correspondence of the MLO calmodulin-binding domain. In this study, we performed the functional characterization of SmMLO1. Transgenic overexpression of SmMLO1 in a tomato mlo mutant compromised resistance to the tomato PM pathogen Oidium neolycopersici, thus indicating that SmMLO1 is a PM susceptibility factor in eggplant. PM susceptibility was also restored by the transgenic expression of a synthetic gene, named s-SmMLO1, encoding a protein identical to SmMLO1, except for the presence of T at position 422. This indicates that the T → M polymorphism does not affect the protein role as PM susceptibility factor. Overall, the results of this work are of interest for the functional characterization of MLO proteins and the introduction of PM resistance in eggplant using reverse genetics.     

<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.