The ectopic expression of apple <Emphasis Type="Italic">MYB1</Emphasis> and <Emphasis Type="Italic">bHLH3</Emphasis> differentially activates anthocyanin biosynthesis in tobacco

Two direct DNA transfer methods, biolistic transformation and a protoplast transformation approach using the INRA-clone 717 1B4 (Populus tremula?×?P. alba), are applied to poplars and compared. Both the in vitro culture and the transformation parameters were optimized to receive a maximum quantity of transformed cells to achieve a stable transformation. For the first time, the stable integration of gfp and dsred in the poplar genome and their expression as visual reporter genes in regenerated plantlets can be shown. For biolistic transformation, stem segments cut lengthwise and incubated for 10 days on a callus induction medium revealed the highest number of transient Gfp- and dsRed signals. After optimization of the in vitro culture parameter, Gfp and dsRed-expressing transgenic poplars were regenerated, proven by PCR and Southern blot analysis. For protoplast transformation, the focus was initially on the development of a highly efficient protoplast isolation and plant regeneration system. Using an enzyme solution consisting of 1.0% cellulase R10 and 0.24% macerozyme, 1?×?10⁷ protoplasts were obtained from 1 g fresh weight leaves. Following incubation of the protoplasts in 600 mOsm culture medium, a high number of microcalli were obtained, from which plantlets were regenerated. The parameters for isolation and regeneration were then complemented by an efficient protoplast transformation protocol with 40% PEG₁₅₀₀. The results of this study confirm that both the biolistic and the protoplast transformation methods can be considered suitable for transferring cisgenes directly into poplar.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation and agronomic performance of regenerated chili pepper (C<Emphasis Type="Italic">apsicum</Emphasis> spp.) plants from commercially important genotypes

The application of modern biotechnology for improvement of chili pepper productivity requires an efficient in vitro plant regeneration protocol. In this study, a reliable protocol was developed for the in vitro regeneration of four types of chili, Capsicum annuum var. annuum (Jalapeño and Serrano), C. annuum var. glabriusculum/aviculare (Piquin), and C. chinense (Habanero) by direct organogenesis using three different explants (cotyledon, hypocotyls, and embryo) and three induction media. All evaluated culture media promoted the formation of adventitious shoots. When embryos or hypocotyls were used as explants, morphologically normal adventitious shoots developed, while culturing cotyledons resulted in nonelongating rosette-shaped shoots. The highest in vitro regeneration efficiency (14.6 shoots per explant) was achieved when Habanero chili hypocotyls were grown on Murashige and Skoog medium containing 1.7 μM indole-3-acetic acid and 22.2 μM N⁶-benzyladenine. This regeneration rate is higher than that obtained in previous reports. Regenerated plants were ready to be transferred to the greenhouse 13 wk after the explant culture. An evaluation carried out under greenhouse conditions showed differences in agronomic performance between in vitro regenerated plants and plants developed from seeds with the magnitude of the differences depending on the genotype being studied.     

Protoplast preparation and polyethylene glycol (PEG)-mediated transformation of <Emphasis Type="Italic">Candida glycerinogenes</Emphasis>

The regeneration of Candida glycerinogenes protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. An investigation of protoplast formation and cytological examination was used to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency (98.6% protoplasts/mL) were isolated, using lysozyme dissolved in 1M sorbitol osmoticum. The commercial enzyme preparations, osmotic stabilisers, and growth phase were effective in raising the protoplast yield. Sodium chloride was effective for protoplast preparation; however, sugars and sugar alcohols were better for protoplast regeneration. Sorbitol at a concentration of 1 M was used in regeneration agar for further studies. Regeneration of colonies from protoplasts was maximal (11 ~ 15%) when protoplasts were incorporated in cooled agar containing 0.5% glucose, supplemented with 1M sorbitol as osmotic stabilizer. C. glycerinogenes strain was highly sensitive to zeocin, so transformation of protoplasts and PEG-mediated was achieved with an improved transformation system, using plasmid pURGAP-gfp containing zeocin gene driven by a PCgGAP promoter from C. glycerinogenes to express gfp gene and be transformed into the 5.8S rDNA site of C. glycerinogenes in order to test the system for studying the yeast osmoregulation. We developed an efficient method for transformation of C. glycerinogenes, and parameters involved in transformation efficiency were optimized. Expressions of gfp at different levels were conducted under osmotic stress containing NaCl, KCl, sorbitol or glycerol for the recombinant strains. These improved procedures for protoplast isolation, regeneration and transformation proved to be useful applications in genetic studies for other Candida species and industrial yeast.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and shoot regeneration in elite breeding lines of western shipper cantaloupe and honeydew melons (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

A reliable Agrobacterium-mediated transformation and shoot regeneration protocol was developed for breeding lines of commercially important western-shipper cantaloupe and honeydew melons, ‘F39’ and ‘150’, respectively. Different media were tested to select a shoot regeneration system for each of three elite breeding lines ‘F39’, ‘141’ and ‘TMS’. Murashige &; Skoog (MS) basal medium supplemented with 1 mg l^?1 benzyladenine (BA), 0.26 mg l^?1 abscisic acid (ABA) and 0.8 mg l^?1 indole-3-acetic acid (IAA) was used for shoot regeneration from cotyledonary explants in ‘F39’ and ‘150’. Kanamycin sensitivity as well as Timentin^? and Clavamox^® were evaluated using wild-type ‘F39’ and ‘150’ cotyledons. Kanamycin concentrations of 200 and 150 mg l^?1 were chosen as the threshold levels for ‘F39’ and ‘150’, respectively. No significant differences were found between Timentin^? and Clavamox^® in ‘F39’; however, Clavamox^® reduced the incidence of vitrification and increased the frequency of shoot elongation in ‘150’. A. tumefaciens strain EHA105, harboring pCNL56 carrying neomycin phosphotransferase II (nptII) and gusA reporter genes, was selected to establish a transformation protocol for ‘F39’ and ‘150’. Putative transformants were evaluated using β-glucuronidase (GUS) histochemical assay, polymerase chain reaction (PCR) and Southern blot analyses. Based on these parameters, the transformation efficiency for cantaloupe ‘F39’ was 0.3% and that for honeydew ‘150’ was 0.5%.     

Improved <Emphasis Type="Italic">Agrobacterium tumefaciens-</Emphasis>mediated transformation of soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.] following optimization of culture conditions and mechanical techniques

In the present study, Agrobacterium tumefaciens-mediated transformation of Glycine max (L.) Merr. (soybean) cv. DS-9712 using half-seed explants was optimized for eight different parameters, including seed imbibition, medium pH, infection mode (sonication and vacuum infiltration), co-cultivation conditions, concentrations of supplementary compounds, and selection. Using this improved protocol, maximum transformation of 14% and regeneration efficiencies of 45% were achieved by using explants prepared from mature seeds imbibed for 36 h, infected with A. tumefaciens strain EHA105 at an optical density (OD₆₀₀) of 0.8, suspended in pH 5.4 medium containing 0.2 mM acetosyringone and 450 mg L^?1 L-cysteine, followed by sonication for 10 s, vacuum infiltration for 2 min, and co-cultivated for 3 d on 35 mg L^?1 kanamycin-containing medium. Independent transgenic lines were confirmed to be transgenic after ß-glucuronidase histochemical assays, polymerase chain reaction, and southern hybridization analysis. The protocol developed in the present study showed high regeneration efficiency within a relatively short time of 76 d. This rapid and efficient protocol might overcome some hurdles associated with the genetic manipulation of soybean.     

Regeneration of protoplasts after somatic hybridisation of <Emphasis Type="Italic">Hydrangea</Emphasis>

Somatic hybridisation of Hydrangea is a promising tool to obtain new basic material for breeding. Viable mesophyll protoplasts were isolated from 21 cultivars and accessions of H. macrophylla, H. paniculata, H. arborescens, H. quercifolia and H. febrifuga. Induction of cell divisions was observed after electromanipulation and fusion by polyethylene glycol. Multi-cellular structures developed in liquid media. Plated microcalli grew on different solid regeneration media. Several phytohormones, their concentration and combination influenced the development of callus and roots. The regular appearance of endophytes challenged successful plant regeneration. As a result of endophytes, the development of microcolonies stopped and they died in liquid protoplast media. Plated microcalli or growing calli turned brown on regeneration medium. Antibiotic Timentin^® inhibited expansion of endophytes for a short time. The addition of ascorbic and citric acid to the regeneration media had inhibiting effect on endophyte growth. Calli showed more vitality and grew faster. The supplement of the regeneration media with karrikinolide, a recently discovered new plant growth regulator, brought contradictory results. After addition of karrikinolide microcolonies looked healthier by their shining green colour in liquid media followed by a severe browning of callus soon afterwards. In the further course, karrikinolide promoted the development of endophytes. Shoot induction and plant regeneration succeeded only once from callus that was a result from H. macrophylla ‘Schneeball’ and H. macrophylla ‘Nachtigall’ fusion.     

Construction of high efficiency regeneration and transformation systems of <Emphasis Type="Italic">Pyrus ussuriensis</Emphasis> Maxim

The purpose of the present study was to establish a simple and efficient protocol for pear regeneration and transformation. Major factors that influence multiplication, rooting, regeneration, and transformation (concentrations of plant growth regulators, pre-culture or co-culture times, and kanamycin concentration) were examined in wild Pyrus ussuriensis Maxim ‘Shanli’. The results showed that the best multiplication index (5.1) was obtained on Murashige and Skoog (MS) medium containing 1.5 mg L^?1 N6-benzyladenine (BA) and 0.2 mg L^?1 indole-3-butyric acid (IBA) with healthy and strong growth. On 1/2 MS medium supplemented with 1.0 mg L^?1 IBA or 0.2 mg L^?1 indole-3-acetic acid (IAA), the rooting percentage was up to 94.4%. The highest regeneration rate (100%) and number of adventitious shoots per explant (6.30) were obtained on Nitsch and Nitsch 1969 (NN69) medium containing 2.0 or 3.0 mg L^?1 thidiazuron (TDZ) and 0.2 mg L^?1 IAA with leaves as explants. Transformation was successfully achieved using the following protocol: cut-wounding leaves pre-cultured for 5 days were co-cultivated with the Agrobacterium tumefaciens strain EHA105 harboring 35S::DHN3 plasmid for 2 days, then were transferred to regeneration medium containing 15 mg L^?1 kanamycin to select the resistant plants, and followed by being cultured on rooting medium supplemented with 5 mg L^?1 kanamycin. PCR analysis revealed that 27 independent transgenic lines were obtained, with the transformation rate up to 11.72%. We postulate that the regeneration and transformation system in this study is an alternative method for pear breeding.     

Effect of over-expression of <Emphasis Type="Italic">Linum usitatissimum</Emphasis> PINORESINOL LARICIRESINOL REDUCTASE (<Emphasis Type="Italic">LuPLR</Emphasis>) gene in transgenic <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l^?1, kan 50 mg l^?1 and cephotaxime 62.5 mg l^?1. The optimum medium for regeneration of multiple shoots was MS supplemented with TDZ 1.54 mg l^?1, kan 50 mg l^?1. Efficient and effective rooting of plantlets was achieved by culturing the in vitro regenerated shoots on liquid ½ MS medium containing 0.7 mg l^?1 indole 3-butyric acid (IBA) and 5 mg l^?1 kan. Rooted plants were acclimatized in the mixtures of vermiculite and soil. The transformation of kan-resistant plantlets regenerated from shoot-tip explants was confirmed by GUS and polymerase chain reaction (PCR) analysis. Southern blot and reverse transcribed PCR (RT-PCR) analysis confirmed successful integration and expression of Lu-PLR gene. Quantitative analysis of phyllanthin performed on transgenic and wild plants using high-performance liquid chromatography (HPLC) revealed that transgenic lines contained higher phyllanthin content (0.3–0.81% w/w) than wild plants (0.09% w/w). The highest yield of phyllanthin was detected in transgenic lines was up to 1.16, 1.22 and 1.23 folds higher than that of wild plant. This report highlights the transgenic approach to enhance the contents of phyllanthin and hypophyllanthin.     

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. ‘W. Murcott’ mandarin (a hybrid of ‘Murcott’ and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of ‘W. Murcott’ mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of ‘W. Murcott’ mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3–5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic ‘W. Murcott’ mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.     

In vitro regeneration and optimization of factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis> mediated transformation in <Emphasis Type="Italic">Artemisia Pallens</Emphasis>, an important medicinal plant

Artemisia pallens is an important medicinal plant. In-vitro regeneration and multiplication of A. pallens have been established using attached cotyledons. Different growth regulators were considered for regeneration of multiple shoots. An average of 36 shoots per explants were obtained by culturing attached cotyledons on Murashige and Skoog’s medium containing 2 mg/L BAP and 0.1 mg/L NAA, after 45 days. The shoots were rooted best on half Murashige and Skoog’s medium with respect to media containing 1 mg/L IBA or 1 mg/L NAA. Different parameters such as type of bacterial strains, OD₆₀₀ of bacterial culture, co-cultivation duration, concentration of acetosyringone and explants type were optimized for transient expression of the reporter gene. Agrobacterium tumefaciens harbouring pCambia1301 plasmid carrying β-glucuronidase as a reporter gene and hygromycin phosphotransferase as plant selectable marker genes were used for genetic transformation of A. pallens. Hygromycin lethality test showed concentration of 15 mg/L were sufficient to inhibit the growth of attached cotyledons and multiple shoot buds of nontransgenics in selection media. Up to 83 % transient transformation was found when attached cotyledons were co-cultivated with Agrobacterium strain AGL1 for 2 days at 22 °C on shoot induction medium. The bacterial growth was eliminated by addition of cefotaxime (200 mg/L) in selection media. T₀ transgenic plants were confirmed by GUS histochemical assay and further by polymerase chain reaction (PCR) using uidA and hpt gene specific primers. The study is useful in establishing technological improvement in A. pallens by genetic engineering.     

Enhanced production of fructosyltransferase in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> by genome shuffling

Objective

To breed Aspergillus oryzae strains with high fructosyltransferase (FTase) activity using intraspecific protoplast fusion via genome-shuffling.

Results

A candidate library was developed using UV/LiCl of the conidia of A. oryzae SBB201. By screening for enzyme activity and cell biomass, two mutants (UV-11 and UV-76) were chosen for protoplast fusion and subsequent genome shuffling. After three rounds of genome recombination, a fusion mutant RIII-7 was obtained. Its FTase activity was 180 U g^?1, approximately double that of the original strain, and RIII-7 was genetically stable. In fermentation culture, FTase activity of the genome-shuffled strain reached a maximum of 353 U g^?1 using substrate-feeding method, and this value was approximately 3.4-times higher than that of the original strain A. oryzae SBB201.

Conclusions

Intraspecific protoplast fusion of A. oryzae significantly enhanced FTase activity and generated a potentially useful strain for industrial production.

     

Glandular trichome-specific expression of alcohol dehydrogenase 1 (<Emphasis Type="Italic">ADH1</Emphasis>) using a promoter-GUS fusion in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Petunia, a commercially important ornamental plant worldwide, has been subjected to breeding programs that have yielded a high number of varieties. One of the key factors in the commercial value of these varieties is plant compactness. Currently, compact petunias are obtained through the application of expensive, harmful and short-lasting chemicals. To avoid the use of these chemicals, transgenic plants that over-express dwarf-inducing genes have been recently proposed as an alternative, but the current legislation regarding transgenic plants restricts their commercialization. In this work, we studied the effect of polyploidization in the plant architecture of Petunia axillaris, an Argentine native petunia. We developed a new polyploidization protocol that consisted in culturing petunia leaves in RL medium (MS medium supplemented with 30 g l^?1 sucrose, 1 mg l^?1 BA and 0.2 mg l^?1 IAA) supplemented with 0.2 g l^?1 colchicine for 15 days. This protocol allowed the regeneration of stable autotetraploid petunias (polyploidization rate: 29.0?±?8,2%), which were 54% more compact than the diploid ones. Furthermore, they exhibited no variations in agronomical traits compared to the initial genotypes, except for a short delay in blooming. These autotetraploid plants can be used in different breeding programs and the polyploidization method developed can be tested in others cultivars of the genus Petunia for the same purpose.     

High responsiveness in <Emphasis Type="Italic">de novo</Emphasis> shoot organogenesis induction of <Emphasis Type="Italic">Passiflora cristalina</Emphasis> (Passifloraceae), a wild Amazonian passion fruit species

The aim of the present study was to establish a regeneration system via de novo organogenesis from different types of non-meristematic explants of Passiflora cristalina. Leaf, hypocotyl, root segments, cotyledons, and endosperm of P. cristalina seeds were inoculated in Murashige and Skoog (MS)-basal medium, supplemented with different concentrations of 6-Benzyladenine (BA), Thidiazuron (TDZ), or Kinetin (KIN). BA was found to be the most efficient cytokinin in induction of de novo organogenesis from most the explants used in the study. The highest frequencies of adventitious bud formation in the hypocotyl and cotyledon explants were observed in medium supplemented with 1.0 mg L^?1 BA. For leaf and endosperm segments, the best concentration was 2.0 mg L^?1 BA; while for root segments, the highest mean values were observed with 1.0 mg L^?1 KIN. The different morphogenetic responses obtained from each explant source were characterized using light microscopy. P. cristalina revealed a remarkable organogenic potential, with superior production of adventitious shoots compared with the other Passiflora species evaluated elsewhere. These results will be helpful to establish a reproducible and reliable micropropagation protocol, as well as to implement conservationist and biotechnological-based genetic breeding strategies for this wild Passiflora species.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

Improvement of in vitro embryo maturation,plantlet regeneration and transformation efficiency from alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.) somatic embryos using <Emphasis Type="Italic">Cuscuta campestris</Emphasis> extract

Developmental deficiency of somatic embryos and regeneration to plantlets, especially in the case of transformation, are major problems of somatic embryo regeneration in alfalfa. One of the ways to overcome these problems is the use of natural plant regulators and nutrients in the culture medium of somatic embryos. For investigating the influence of Cuscuta campestris extract on the efficiency of plant regeneration and transformation, chimeric tissue type plasminogen activator was transferred to explants using Agrobacterium tumefaciens, and transgenic plants were recovered using medium supplemented with different concentration of the extract. Transgenic plants were analyzed by PCR and RT-PCR. Somatic embryos of Medicago sativa L. developed into plantlets at high frequency level (52 %) in the maturation medium supplemented with 50 mg 1^?1 C. campestris extract as compared to the medium without extract (26 %). Transformation efficiency was 29.3 and 15.2 % for medium supplemented with dodder extract and without the extract, respectively. HPLC and GC/MS analysis of the extract indicated high level of ABA and some compounds such as Phytol, which can affect the somatic embryo maturation. The antibacterial assay showed that the extract was effective against some strains of A. tumefaciens. These results have provided a scientific basis for using of C. campestris extract as a good natural source of antimicrobial agents and plant growth regulator as well, that can be used in tissue culture of transgenic plants.     

Complete chloroplast genome sequences of <Emphasis Type="Italic">Solanum commersonii</Emphasis> and its application to chloroplast genotype in somatic hybrids with <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

Key message

Chloroplast genome of Solanum commersonii and S olanum tuberosum were completely sequenced, and Indel markers were successfully applied to distinguish chlorotypes demonstrating the chloroplast genome was randomly distributed during protoplast fusion.

Abstract

Somatic hybridization has been widely employed for the introgression of resistance to several diseases from wild Solanum species to overcome sexual barriers in potato breeding. Solanum commersonii is a major resource used as a parent line in somatic hybridization to improve bacterial wilt resistance in interspecies transfer to cultivated potato (S. tuberosum). Here, we sequenced the complete chloroplast genomes of Lz3.2 (S. commersonii) and S. tuberosum (PT56), which were used to develop fusion products, then compared them with those of five members of the Solanaceae family, S. tuberosum, Capsicum annum, S. lycopersicum, S. bulbocastanum and S. nigrum and Coffea arabica as an out-group. We then developed Indel markers for application in chloroplast genotyping. The complete chloroplast genome of Lz3.2 is composed of 155,525 bp, which is larger than the PT56 genome with 155,296 bp. Gene content, order and orientation of the S. commersonii chloroplast genome were highly conserved with those of other Solanaceae species, and the phylogenetic tree revealed that S. commersonii is located within the same node of S. tuberosum. However, sequence alignment revealed nine Indels between S. commersonii and S. tuberosum in their chloroplast genomes, allowing two Indel markers to be developed. The markers could distinguish the two species and were successfully applied to chloroplast genotyping (chlorotype) in somatic hybrids and their progenies. The results obtained in this study confirmed the random distribution of the chloroplast genome during protoplast fusion and its maternal inheritance and can be applied to select proper plastid genotypes in potato breeding program.

     

An efficient <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation protocol of <Emphasis Type="Italic">Withania somnifera</Emphasis>

This is the first report on Agrobacterium rhizogenes-mediated transformation of Withania somnifera for expression of a foreign gene in hairy roots. We transformed leaf and shoot tip explants using binary vector having gusA as a reporter gene and nptII as a selectable marker gene. To improve the transformation efficiency, acetosyringone (AS) was added in three stages, Agrobacterium liquid culture, Agrobacterium infection and co-culture of explants with Agrobacterium. The addition of 75 μM AS to Agrobacterium liquid culture was found to be optimum for induction of vir genes. Moreover, the gusA gene expression in hairy roots was found to be best when the leaves and shoot tips were sonicated for 10 and 20s, respectively. Based on transformation efficiency, the Agrobacterium infection for 60 and 120 min was found to be suitable for leaves and shoot tips, respectively. Amongst the various culture media tested, MS basal medium was found to be best in hairy roots. The transformation efficiency of the improved protocol was recorded 66.5 and 59.5?% in the case of leaf and shoot tip explants, respectively. When compared with other protocols the transformation efficiency of this improved protocol was found to be 2.5 fold higher for leaves and 3.7 fold more for shoot tips. Southern blot analyses confirmed 1–2 copies of the gusA transgene in the lines W1-W4, while 1–4 transgene copies were detected in the line W5 generated by the improved protocol. Thus, we have established a robust and efficient A. rhizogenes mediated expression of transgene (s) in hairy roots of W. somnifera.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) somatic embryos with fluorescent marker genes and optimization of transgenic plant recovery

Avocado globular somatic embryos were transformed with three binary vectors, pK7FNF2, pK7RNR2 and pK7S*NF2, harboring the marker genes gfp, DsRed and a gfp-gus fusion gene, respectively. GFP and DsRed fluorescence was detected in embryogenic lines growing in selection medium 2 months after Agrobacterium inoculation. The fluorescence signal was maintained thereafter in transgenic calli, as well as in mature somatic embryos. Red fluorescence in pK7RNR2 transgenic lines was higher and more easily observable than GFP fluorescence. Furthermore, calli transformed with pK7S*NF2, harboring gfp-gus, showed higher level of fluorescence than those transformed with pK7FNF2, containing two gfp. To improve plant recovery, maturated transgenic embryos that failed to germinate or showed an underdeveloped shoot were cultured for 4 weeks in a medium with 1 mg l^?1 TDZ and 1 mg l^?1 BA after partial removal of cotyledons. A 50% of embryos developed one or several shoots on the cut surface. These embryos were cultured for 4 additional weeks in a medium with 1 mg l^?1 BA for shoot elongation and then, shoots were grafted in vitro onto seedling rootstocks. Culture of micrografts in solid MS medium supplemented with 1 mg l^?1 BA allowed a 60–80% success rate. Young leaves from transgenic plants showed GFP or DsRed fluorescence located in the nucleus. The results obtained indicate that fluorescent marker genes, especially DsRed, could be useful for early selection of transgenic material and optimization of the transformation parameters in avocado. Furthermore, the protocol established allowed the successful recovery of transgenic plants, one of the main limiting steps in avocado transformation.     

Efficient protoplast isolation and transient gene expression system for <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrid cultivar ‘Ruili Beauty’

With the release of the Phalaenopsis equestris (Schauer) Rchb.f. genome database, more in-depth studies of Phalaenopsis spp. will be carried out in the future. Transient gene expression in protoplasts is a useful system for gene function analysis, which is especially true for Phalaenopsis, whose stable genetic transformation is difficult and extremely time-consuming. In this study, juvenile leaves from aseptic Phalaenopsis seedlings were used as the starting material for protoplast isolation. After protocol refinement, the highest yield of viable protoplasts [5.94 × 10⁶ protoplasts g^?1 fresh weight (FW)] was achieved with 1.0% (w/v) Cellulase Onozuka R-10, 0.7% (w/v) Macerozyme R-10, and 0.4 M D-mannitol, with an enzymolysis duration of 6 h. As indicated by transient expression of green fluorescent protein (GFP), a transformation efficiency of 41.7% was achieved with 20% (w/v) polyethylene glycol (PEG-4000), 20 μg plasmid DNA, 2 × 10⁵ mL^?1 protoplasts, and a transfection duration of 30 min. The protocol established here will be valuable for functional studies of Phalaenopsis genes.     

Efficient callus-mediated regeneration and <Emphasis Type="Italic">in vitro</Emphasis> root tuberization in <Emphasis Type="Italic">Trichosanthes kirilowii</Emphasis> Maxim., a medicinal plant

Trichosanthes kirilowii Maxim. is a climbing herb with considerable medicinal value. In this study, efficient protocols for callus-mediated regeneration and in vitro tuberization of this plant were developed. Sterilized stem and leaf tissues were cultured on Murashige and Skoog (MS) medium with plant growth regulators (PGRs), and additives that promoted callus induction and regeneration. Both stem and leaf tissues showed the best response (100%) for callus initiation on MS medium supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D). Efficient shoot organogenesis was obtained by exposing the callus tissue to 4.6-μM kinetin, 2.2-μM 6-benzylaminopurine, and 2.7-μM 1-naphthylacetic acid (NAA) along with 12.6-μM copper sulfate, which yielded a shoot regeneration rate of 85.5% and 28 shoots derived from each callus. In vitro shoots were best rooted on half-strength (1/2) MS medium with 2.7-μM NAA. Tuberous roots were efficiently induced on rooting medium with 5% (w/v) sucrose under short illumination conditions (8 h photoperiod). Rooted plantlets were successfully acclimatized in pots with a >?90% survival rate. This protocol provides an effective method for callus-mediated regeneration and in vitro root tuberization.