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- Author or Editor: Lourdes G. Iglesias-Andreu x
To determine the effect of different nitrogen sources and osmotic regulators on minimal growth of Habanero pepper (Capsicum chinense Jacq.) germplasm for in vitro conservation, different concentrations of nitrate, sucrose, mannitol, and sorbitol were evaluated. The micropropagation system based on culture medium was modified in its nitrate concentrations: reduced to 50% and increased to 150%, and osmoregulators were added to the basal culture media: sucrose (6% and 8%), mannitol (2%, 4%, and 8%), or sorbitol (2%, 4%, and 8%). The apical meristems of germinated plants were cultivated in the different treatments for 35 weeks without subculture. Results have demonstrated that mannitol at 2% had the better effect on minimal growth of the plantlets and did not affect the plant physiology and quality. The plantlets remained small in size, turgent, with green leaves and stems and looked like normal plants until to the end of the evaluation period. Changes in nitrogen media concentration did not prove to be adequate for conserving because they affected the plantlet quality (they became chlorotic). The presence of sorbitol and high osmolite concentrations induced minimal growth but reduced the plant quality. Sucrose at mid or low concentrations did not induce minimal growth.
Vanilla planifolia Jacks. is a species of great economic importance, since vanillin, a compound highly valued in the food and pharmaceutical industry, is extracted from its pods. This species is in the category of special protection, so it is important to take actions for its conservation and to maintain the genetic stability of the conserved germplasm. An adequate way to achieve this is through the minimal growth in vitro conservation techniques. The present work aimed to establish an in vitro conservation protocol for vanilla germplasm that allows the genetic stability of the conserved material. For the establishment of the minimal growth in vitro conservation protocol: two concentrations of basal Murashige and Skoog (MS) medium (50% and 100%), two incubation temperatures (4 and 22 °C) and two concentrations of abscisic acid (ABA) (3 and 5 mg⋅L−1) were evaluated. To evaluate the genetic stability of the germplasms used in this study (cultivated, wild, and V. insignis morphotypes) by analyzing the profiles of molecular markers SSR (simple sequence repeats) and ISSR (inter simple sequence repeats). The MS medium (100%) supplemented with 3 mg⋅L−1 of ABA and incubated at 22 °C, was the best treatment for the in vitro conservation of Vanilla spp. Compared with the control treatment, it allowed us to obtain smaller shoots (1.17 × 0.17 cm), which showed high genetic stability, given by the low percentages of polymorphism detected in morphotypes cultivated and wild (SSR 0%, ISSR 2%) and V. insignis (SSR 0%, ISSR 0%). We conclude the usefulness of the established protocol to conserve the genetic variation of the evaluated Vanilla germplasm.
This article describes the performance of nodal segments from Habanero pepper (Capsicum. chinense) during shoot induction and elongation under different semisolid and liquid culture conditions with various degrees of ventilation in which they were exposed to different levels of immersion and growth regulators. The ethylene content in non-ventilated containers, the age of the explant donor plants as well as the effect of thidiazuron and paclobutrazol on shoot induction and of gibberellic acid and AgNO3 on shoot elongation were also evaluated. A temporary immersion bioreactor (BioMINT™) was used for the multiplication and elongation of isolated shoots with very good results. We report an efficient protocol for the in vitro propagation of Habanero pepper that produces plants with a high survival rate when transplanted to soil.
The in vitro production of ethylene and its effects on the development of Habanero pepper (Capsicum chinense Jacq.) plantlets were evaluated using nonventilated containers (NVCs) and ventilated containers (VCs). Shoots of Habanero pepper between 0.5 and 1.0 cm of height were cultivated in Magenta culture boxes and samples of the headspace atmosphere were taken every four days during the previously established culturing time of 40 days. The presence of ethylene was detected in the NVCs and produced a negative effect on the development of plantlets. In a second phase of this work, the effect of silver nitrate (AgNO3) and cobalt chloride (CoCl2) on ethylene production was evaluated during in vitro development of Habanero pepper plantlets. Concentrations of 50, 300, and 500 μm of each ethylene inhibitor were used in the culture medium. Although cobalt chloride partially inhibited the production of ethylene during in vitro culture of this species, at low concentrations the plantlets presented some degree of vitrification and the highest concentration proved to be toxic for the plantlets. Silver nitrate added to the culture medium did not inhibit ethylene production, however, it did inhibit the effect of this hormone on the plantlets. In fact, when high concentrations of silver nitrate were used (300 μm), high amounts of ethylene were detected in the headspace of the vessels and plantlets were actually healthier.
Intersimple sequence repeat (ISSR) markers were used to evaluate the effects of in vitro culture on genetic variation in Habanero pepper (Capsicum chinense Jacq.) regeneration protocols. A total of 219 ISSR clear and reproducible fragments were generated with 13 ISSR primers in direct organogenesis, direct and indirect somatic embryos, and the embryogenic callus system. A cluster analysis was performed to express in the form of dendrogram the relationships among different regeneration systems and the genetic variability detected. Genetic distance analysis indicated that our regeneration protocols are inappropriate for micropropagation, conservation, or genetic transformation; however, they may be applicable to breeding. This is the first report on the use of molecular analysis to evaluate genetic variation of in vitro-regenerated plants of Habanero pepper using ISSR markers.
A complete and efficient regeneration protocol was developed for Vanilla planifolia ‘Andrews’, an endangered orchid species that represents an important crop in several tropical countries. Axillary buds excised from the first to the eighth node, considering the first to fourth nodes as “young” (zone 1) and the fifth to eighth as “mature” (zone 2), were cultured on Murashige and Skoog (MS) medium supplemented with 5.73, 7.64, 9.55, or 11.46 μm 6-benzylaminopurine (BA) for shoot induction and in combination with 4.45 μm naphthalene acetic acid (NAA) to induce multiple shoot proliferation. Cytokinin concentration and bud position in the stem had a significant effect on the number of shoots regenerated. The greatest shoot formation per explant, for the two tested zones, was obtained with 9.55 μm BA on MS medium supplemented with 100 mg·L−1 myoinositol, 150 mg·L−1 citric acid, 100 mg·L−1 ascorbic acid, and 20 g·L−1 sucrose. Young buds from zone 1 were able to form an average of 18.5 ± 2.4 shoots per explant, whereas buds from zone 2 induced a maximum of 11.0 ± 1.0 shoots per explant. Plants of 2 to 3 cm height developed a root system in half-strength MS medium supplemented with 0.44 μm NAA and, once they reached 5 cm height on average, were transferred to greenhouse conditions for their acclimatization where a 100% rate survival was achieved. The optimal use of both young and mature buds from each mother plant to induce adventitious shoots permitted a marked increase in the number of shoots per explant. By using all buds from the upper stem part (zone 1 + zone 2) and subculturing every 90 d, the multiplication rate was 1.1 to 1.86 × 105 shoots per bud per year.