Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants

A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation.     

Differential organ infection studies, potyvirus elimination, and field performance of virus-free garlic plants produced by tissue culture

Presence of potyvirus in single garlic (Allium sativum L.) cloves from the same bulb, and in five single leaves excised from commercial field-grown individual plants was studied using ELISA. It was found that the viruses were not present in all organs of the same plant, since some cloves of the same bulb were infected with potyvirus but some others were potyvirus-free. Analyzed leaves from a given plant also exhibited irregular distribution of potyvirus. This study also aimed to obtain potyvirus-free plants from two commercial garlic cultivars (Taiwan and Chileno) using cloves subjected to thermotherapy, chemotherapy or meristematic dissection followed by in vitro culture. Thermotherapy (sequential treatment at 32°C for a week, 36°C for 2 weeks, and 38°C for 3 weeks) was found to affect survival of explants and 36.5% cloves from Taiwan and 26.8% from Chileno cultivars were recovered after the treatment. ELISA tests showed that 63% of the cloves of Taiwan that survived the treatment and 70.9% of Chileno explants were potyvirus-negative. Regarding chemotherapy (205 μM Ribavirin solution), the explants (cloves) survived, but only an average of 27.0–34.8% were negative for the presence of potyvirus. When meristematic dissection was applied, an average of 41.7% explants of Taiwan and 34.2% of Chileno survived the treatment, and approximately 64% of these explants from both cultivars were potyvirus-negative. Potyvirus-free garlic plants grown in field conditions showed longer stems with a major fresh and dry weight per bulb, and also exhibited a higher yield than non-treated plants.     

In vitro propagation of three <Emphasis Type="Italic">Agave</Emphasis> species used for liquor distillation and three for landscape

In vitro bud clusters of Calathea orbifolia (Linden) Kennedy were obtained and subcultured in semi-solid (agar) medium and temporary immersion system (TIS) for 12 weeks. Uniform young plants were selected and transferred to soilless mix in a growth chamber for ex vitro acclimatization during 35 days, followed by growing in a shaded greenhouse for 65 days. Comparison of in vitro leaf anatomy, ex vitro photosynthetic behaviors and growth was made between two cultural systems. Plants in TIS produced thicker leaf chlorenchyma and aquiferous parenchyma, lower stomatal frequency and more epicuticular wax than did those in semi-solid medium. Plants from semi-solid medium had consistently lower leaf Fv/Fm values than plants from TIS. Leaf Fv/Fm value in plants from TIS decreased to 0.65 at day 7 after transfer and increased soon up to 0.76 thereafter. In contrast, leaf Fv/Fm value in plants from semi-solid medium reduced to 0.27 at day 7 after transfer and increased slowly up to 0.68 at day 35. During ex vitro acclimatization, plants in TIS had substantial higher photosynthetic rates than plants in semi-solid medium. Plants from TIS had subsequent higher leaf area, fresh and dry weights than plants from semi-solid medium.     

Molecular biology of capsaicinoid biosynthesis in chili pepper (Capsicum spp.)

Capsicum species produce fruits that synthesize and accumulate unique hot compounds known as capsaicinoids in placental tissues. The capsaicinoid biosynthetic pathway has been established, but the enzymes and genes participating in this process have not been extensively studied or characterized. Capsaicinoids are synthesized through the convergence of two biosynthetic pathways: the phenylpropanoid and the branched-chain fatty acid pathways, which provide the precursors phenylalanine, and valine or leucine, respectively. Capsaicinoid biosynthesis and accumulation is a genetically determined trait in chili pepper fruits as different cultivars or genotypes exhibit differences in pungency; furthermore, this characteristic is also developmentally and environmentally regulated. The establishment of cDNA libraries and comparative gene expression studies in pungent and non-pungent chili pepper fruits has identified candidate genes possibly involved in capsaicinoid biosynthesis. Genetic and molecular approaches have also contributed to the knowledge of this biosynthetic pathway; however, more studies are necessary for a better understanding of the regulatory process that accounts for different accumulation levels of capsaicinoids in chili pepper fruits.     

In vitro embryo formation and plant regeneration from anther culture of different cultivars of Mexican husk tomato (<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> Brot.)

Eight cultivars and two accessions of Physalis ixocarpa Brot. were tested for their capacity to regenerate embryos and plants from anther cultures. Anthers were pretreated at 4°C for 2 days and then at 35°C for 8 days in the dark while cultured on MS medium supplemented with 0.045 μM 2,4-D + 0.03 mg l⁻¹ vitamin B₁₂ (MS1) or with 2.26 μM 2,4-D + 0.1 mg l⁻¹ vitamin B₁₂ (MS3). Anther incubation proceeded under a 16 h photoperiod at 25 ± 2°C. Embryo formation occurred after 6 weeks of incubation in these conditions. Androgenetic responses were cultivar- and culture medium-dependent, with the greatest embryo yields recorded for cv. Chapingo (36.3%) on MS1 medium, and with wild-type 2 (21.8%) on MS3. Further development of regenerated embryos was promoted on MS medium supplemented with 0.54 μM NAA, 8.88 μM BA and 50 mg l⁻¹ casein hydrolysate. The regenerated plants were cultured on half-strength mineral salts MS medium with 2.85 μM IAA to enhance root formation. Rooted plantlets were transferred to pots and acclimatized to the greenhouse. Ploidy analysis of regenerated plants using flow cytometry revealed 72% diploids, 15% haploids and 7% triploids. AFLP analysis of regenerated plants from anthers of a single parental plant showed different polymorphic patterns indicating their gametophytic origin.     

Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes-transformed tissues

Transgenic Mexican lime [Citrus aurantifolia (Christm.) Swing] plants were regenerated from tissues transformed by Agrobacterium rhizogenes strain A4, containing the wild-type plasmid pRiA4 and the binary vector pESC4 with nos-npt II and cab-gus genes. Transgenic shoots were generated by two different approaches. The first approach used internodal stem segments cocultured with A. rhizogenes. These were placed onto regeneration medium containing Murashige and Skoog salts and B5 organic compounds supplemented with 8 g ⋅ l^–1 agar, 7.5 mg ⋅ l^–1 6-benzylaminopurine, 1.0 mg ⋅ l^–1 -naphthaleneacetic acid, 300 mg ⋅ l^–1 cefotaxime and 80 mg ⋅ l^–1 kanamycin as a selective agent, and incubated under continuous light at 25 °C. Under these conditions, 76% of the explants produced shoots directly with no hairy root phase, with a mean of 1.3 shoots per explant, and 88% of these shoots were genetically transformed as determined by β-glucuronidase (GUS) assays. In the second approach, segments of transformed roots (15 mm long) obtained from internodal stem segments cocultured with A. rhizogenes were cultured on the above regeneration medium under similar conditions. Forty-one percent of these transformed root segments produced adventitious shoots, with a mean of 2.2 shoots per explant and with 90% of shoots transformed. GUS activity was evident in the transformed roots and in all parts of both transformed shoots and regenerated plants. The presence of the npt II and rolB genes in the regenerated plants was confirmed by PCR analysis. The presence of the npt II gene in the regenerated plants was also confirmed by Southern blot. Using these transformation systems, more than 300 Mexican lime transgenic plants were obtained, 60 of which were adapted to growing in soil. Received: 15 March 1997 / Revision received: 30 December 1997 / Accepted: 19 January 1998     

Phenylalanine ammonia-lyase activity and capsaicin-precursor compounds in p-fluorophenylalanine-resistant and -sensitive variant cells of chili pepper (Capsicum annuum)

Cell suspension cultures of chili pepper ( Capsicum annuum L. cv. Tampiqueño 74) displaying differences in their resistance to p -fluorophenylalanine (PFP) and in their contents of capsaicin (the compound which is responsible for the hot taste of chili pepper fruits) were characterized in relation to the activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the levels of free l -phenylalanine, phenolics and the phenylpropanoid acids involved in capsaicin biosynthesis. A nonselected cell line, a sensitive line (CA-02), a moderately resistant cell line (CA-29) and two resistant cell lines (CA-04 and CA-16) were studied. Higher PAL activities and higher levels of phenylalanine and phenolics were found in the PFP-resistant cells even after a minimum of 9 subcultures (15 days each) in the absence of the analog, indicating that the selected trait was stable. PFP-resistant chili pepper cells accumulated higher amounts of capsaicin precursors (cinnamic, caffeic and ferulic acids) than either the nonselected cells or the sensitive cell line. p -Coumaric acid was not detected at significant levels in any of the cell cultures. Overall, accumulation of free phenyl-alanine correlated well with PAL activity, phenolics, phenylpropanoids and capsaicin levels, suggesting an active flow through the phenylpropanoid pathway in PFP-resistant cells of chili pepper.     

In vitro chili pepper biotechnology

Summary Chili pepper is an important horticultural crop that can surely benefit from plant biotechnology. However, although it is a Solanaceous member, developments in plant cell, tissue, and organ culture, as well as on plant genetic transformation, have lagged far behind those achieved for other members of the same family, such as tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and potato (Solanum tuberosum), species frequently used as model systems because of their facility to regenerate organs and eventually whole plants in vitro, and also for their ability to be genetically engineered by the currently available transformation methods. Capsicum members have been shown to be recalcitrant to differentiation and plant regeneration under in vitro conditions, which in turn makes it very difficult or inefficient to apply recombinant DNA technologies via genetic transformation aimed at genetic improvement against pests and diseases. Some approaches, however, have made possible the regeneration of chili pepper plants from in vitro-cultured cells, tissues, and organs through organogenesis or embryogenesis. Anther culture has been successfully applied to obtain haploid and doubledhaploid plants. Organogenic systems have been used for in vitro micropropagation as well as for genetic transformation. Application of both tissue culture and genetic transformation techniques have led to the development of chili pepper plants more resistant to at least one type of virus. Cell and tissue cultures have been applied successfully to the selection of variant cells exhibiting increased resistance to abiotic stresses, but no plants exhibiting the selected traits have been regenerated. Production of capsaicinoids, the hot principle of chili pepper fruits, by cells and callus tissues has been another area of intense research. The advances, limitations, and applications of chili pepper biotechnology are discussed.