Neftalí Ochoa-Alejo

Chilli peppers--a review on tissue culture and transgenesis.

Biotechnology techniques involving plant tissue culture and recombinant DNA technologies are powerful tools that can complement conventional breeding and expedite Capsicum improvement. The rate of progress in Capsicum is relatively slower than other members of Solanaceae because of its high genotypic dependence and recalcitrant nature. Capsicum is a recalcitrant plant in terms of in vitro cell, tissue and organ differentiation, plant regeneration and genetic transformation which makes it difficult to apply recombinant DNA technologies aimed at genetic improvement against pests, diseases and abiotic stress. Despite this, application of tissue culture and genetic transformation have led to significant development in chilli pepper plants, and studies are underway to achieve the targets of pre-harvest improvement and post-harvest characterization for value addition to this crop. This review presents a consolidated account of in vitro propagation and focuses upon contemporary information on biotechnological advances made in Capsicum.

In vitro chili pepper biotechnology

SummaryChili 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.

Melon Fruits: Genetic Diversity, Physiology, and Biotechnology Features

Among Cucurbitaceae, Cucumis melo is one of the most important cultivated cucurbits. They are grown primarily for their fruit, which generally have a sweet aromatic flavor, with great diversity and size (50 g to 15 kg), flesh color (orange, green, white, and pink), rind color (green, yellow, white, orange, red, and gray), form (round, flat, and elongated), and dimension (4 to 200 cm). C. melo can be broken down into seven distinct types based on the previously discussed variations in the species. The melon fruits can be either climacteric or nonclimacteric, and as such, fruit can adhere to the stem or have an abscission layer where they will fall from the plant naturally at maturity. Traditional plant breeding of melons has been done for 100 years wherein plants were primarily developed as open-pollinated cultivars. More recently, in the past 30 years, melon improvement has been done by more traditional hybridization techniques. An improvement in germplasm is relatively slow and is limited by a restricted gene pool. Strong sexual incompatibility at the interspecific and intergeneric levels has restricted rapid development of new cultivars with high levels of disease resistance, insect resistance, flavor, and sweetness. In order to increase the rate and diversity of new traits in melon it would be advantageous to introduce new genes needed to enhance both melon productivity and melon fruit quality. This requires plant tissue and plant transformation techniques to introduce new or foreign genes into C. melo germplasm. In order to achieve a successful commercial application from biotechnology, a competent plant regeneration system of in vitro cultures for melon is required. More than 40 in vitro melon regeneration programs have been reported; however, regeneration of the various melon types has been highly variable and in some cases impossible. The reasons for this are still unknown, but this plays a heavy negative role on trying to use plant transformation technology to improve melon germplasm. In vitro manipulation of melon is difficult; genotypic responses to the culture method (i.e., organogenesis, somatic embryogenesis, etc.) as well as conditions for environmental and hormonal requirements for plant growth and regeneration continue to be poorly understood for developing simple in vitro procedures to culture and transform all C. melo genotypes. In many cases, this has to be done on an individual line basis. The present paper describes the various research findings related to successful approaches to plant regeneration and transgenic transformation of C. melo. It also describes potential improvement of melon to improve fruit quality characteristics and postharvest handling. Despite more than 140 transgenic melon field trials in the United States in 1996, there are still no commercial transgenic melon cultivars on the market. This may be a combination of technical or performance factors, intellectual property rights concerns, and, most likely, a lack of public acceptance. Regardless, the future for improvement of melon germplasm is bright when considering the knowledge base for both techniques and gene pools potentially useable for melon improvement.

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.

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

Abstract 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.

Cultivar differences in shoot-forming capacity of hypocotyl tissues of chilli pepper (Capsicum annuum L.) cultured in vitro.

Abstract Cultured hypocotyl segments of 16 cultivars of chilli pepper (Capsicum annuum L.) were tested for their ability to form adventitious shoots. Explants were grown on six culture media prepared with the basal medium formulated by Murashige and Skoog, supplemented with four combinations of indoleacetic acid (IAA)/benzyladenine (BA) or with three combinations of IAA/2-isopentenyladenine (2iP). Cultivar differences were observed with regard to their capacity for in vitro differentiation of shoots and approximately one-third ( 5 16 ) of the cultivars tested exhibited relatively high differentiation capabilities (determined by the number of shoots per explant and by the frequency of shoot formation). Optimal shoot regeneration medium varied with cultivar. Cultivar ‘Anaheim TMR-23’ displayed the highest shoot-regeneration response.

Virus-induced silencing of Comt, pAmt and Kas genes results in a reduction of capsaicinoid accumulation in chili pepper fruits

Capsaicinoids are responsible for the pungent taste of chili pepper fruits of Capsicum species. Capsaicinoids are biosynthesized through both the phenylpropanoid and the branched-fatty acids pathways. Fragments of Comt (encoding a caffeic acid O-methyltransferase), pAmt (a putative aminotransferase), and Kas (a β-keto-acyl-[acyl-carrier-protein] synthase) genes, that are differentially expressed in placenta tissue of pungent chili pepper, were individually inserted into a Pepper huasteco yellow veins virus (PHYVV)-derived vector to determine, by virus-induced gene silencing, irrespective of whether these genes are involved in the biosynthesis of capsaicinoids. Reduction of the respective mRNA levels as well as the presence of related siRNAs confirmed the silencing of these three genes. Morphological alterations were evident in plants inoculated with PHYVV::Comt and PHYVV::Kas constructs; however, plants inoculated with PHYVV::pAmt showed no evident alterations. On the other hand, fruit setting was normal in all cases. Biochemical analysis of placenta tissues showed that, indeed, independent silencing of all three genes led to a dramatic reduction in capsaicinoid content in the fruits demonstrating the participation of these genes in capsaicinoid biosynthesis. Using this approach it was possible to generate non-pungent chili peppers at high efficiency.

An improved and reliable chili pepper (Capsicum annuum L.) plant regeneration method.

In this work we report a new method forin vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 ± 2°C in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 ± 2°C under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 μmol m-2 s--1) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation (≥ 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10–14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L−1 for 3 h and then subculturing on MS medium without growth regulators.

Biochemistry and Molecular Biology of Carotenoid Biosynthesis in Chili Peppers (Capsicum spp.)

Capsicum species produce fruits that synthesize and accumulate carotenoid pigments, which are responsible for the fruits’ yellow, orange and red colors. Chili peppers have been used as an experimental model for studying the biochemical and molecular aspects of carotenoid biosynthesis. Most reports refer to the characterization of carotenoids and content determination in chili pepper fruits from different species, cultivars, varieties or genotypes. The types and levels of carotenoids differ between different chili pepper fruits, and they are also influenced by environmental conditions. Yellow-orange colors of chili pepper fruits are mainly due to the accumulation of α- and β-carotene, zeaxanthin, lutein and β-cryptoxanthin. Carotenoids such as capsanthin, capsorubin and capsanthin-5,6-epoxide confer the red colors. Chromoplasts are the sites of carotenoid pigment synthesis and storage. According to the most accepted theory, the synthesis of carotenoids in chili peppers is controlled by three loci: c1, c2 and y. Several enzymes participating in carotenoid biosynthesis in chili pepper fruits have been isolated and characterized, and the corresponding gene sequences have been reported. However, there is currently limited information on the molecular mechanisms that regulate this biosynthetic pathway. Approaches to gain more knowledge of the regulation of carotenoid biosynthesis are discussed.

PEG-tolerant cell clones of chili pepper: Growth, osmotic potentials and solute accumulation

Cell cultures of chili pepper (Capsicum annuum L.) were established from callus tissue inoculated in MS liquid medium supplemented with 6.25 μM 2,4-d and 0.44 μM BA. Cell clones were isolated by plating the cell suspension on filter paper discs supported by polyurethane foam that were bathed with culture medium containing 15% PEG. The cell clones T6 and T7 were chosen based on their characteristics of growth and friability. These cell clones were established as cell suspensions in the presence of 15% PEG and subsequently subcultured in increasing concentrations of osmoticum. By this approach the cell clones T7 and T6 were capable of growing in the presence of 20 and 25% PEG, respectively. The cell clone T7 was found to grow better in the presence of 5–10% PEG after a period of subculturing in the absence of osmoticum indicating that the tolerance trait was stable. The tolerant cell clones exhibited a 3 to 3.5-fold decrease in the osmotic potentials in comparison with the nonselected cells suggesting that osmotic adjustment occurred. K+ was the major contributing solute to the osmotic potential in all the cell cultures among those tested and was found to be higher in concentration in the PEG-tolerant clones (1.3–3 times higher than nonselected cells). Proline and glycine betaine levels showed a positive correlation with the degree of tolerance to water deficit in the PEG-tolerant cell clones. The levels of proline in the cell clone T7 subcultured in the absence of PEG in the culture medium decreased to values similar to those of nonselected cells, whereas the contents of glycine betaine in the same conditions were maintained at high levels.