Improvement of regeneration in pepper: a recalcitrant species

Abstract

Organogenesis is influenced by factors like genotype, type of explant, culture medium components, and incubation conditions. The influence of ethylene, which can be produced in the culture process, can also be a liming factor in recalcitrant species like pepper. In this work, bud induction was achieved from cotyledons and hypocotyls -from eight pepper cultivars -on Murashige and Skoog (MS) medium supplemented with 22.2 µM 6-benzyladenine (6BA) and 5.7 µM indole-3-acetic acid (IAA), in media with or without silver nitrate (SN) (58.9 µM), a suppressor of ethylene action. In the SN-supplemented medium, the frequencies of explants with buds and with callus formation were lower in both kinds of explant, but higher numbers of developed shoots were isolated from explants cultured on SN. Bud elongation was better in medium with gibberellic acid (GA3) (2.9 µM) than in medium free of growth regulators or supplemented with 1aminocyclopropane-1-carboxylic acid (ACC) at 34.5 µM. However, isolation of shoots was difficult and few plants were recovered. The effect of adding SN following bud induction (at 7 d) and that of dark incubation (the first 7 d of culture) was also assessed in order to improve the previous results. When SN was added after bud induction, similar percentages of bud induction were found for cotyledons (average frequency 89.4% without SN and 94.4% with SN) whereas they doubled in hypocotyls (50 % without SN and 87.7% with SN). In addition, in both kinds of explant, the number of developed plants able to be transferred to soil (developed and rooted) was greatly increased by SN. Dark incubation does not seem to improve organogenesis in pepper, and hypocotyl explants clearly represent a better explant choice -with respect to cotyledonary explants -for the pepper cultivars assayed. REGENERATION IN PEPPER3

Biolistics transformation of callus and cell suspension cultures of Capsicum annuum L. ‘Serrano’ is useful for in vitro studies of the relative contents of secondary metabolites

“…Five samples of 1 g of callus were used per treatment for the cultures in solid medium, and the increase in fresh weight was monitored after 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, and 26 days. For liquid cultures, aliquots of 1 mL were withdrawn from the flasks after 1, 3,5,8,10,12,15,17,19, and 22 days and, after centrifugation at 1,000 g, the accumulated mass was estimated by gravimetric analysis as the fresh weight of the pelleted material. The pellet then was dried to constant weight in a vacuum stove at 60°C and weighed again to record its dry weight.…”

“…Its importance resides in the fruit's high content of pigments, vitamins, and valuable secondary metabolites, including flavonoids and the distinctive capsaicinoids and capsinoids [2,3]. Capsicum annuum L. is currently the chilli pepper of high economic impact, and the genetic manipulation of this plant species has become an important biotechnological goal [1,4,5]. However, major obstacles have limited the obtention of genetically modified chilli peppers with improved agricultural traits [4,6].…”

“…The somatic embryogenesis has been reported but the number of plants recovered is low [5] and the results seem to depend on the genotype and its previous history [8,9]. In fact, the presence of genetic features limiting the rate of somatic embryogenesis in C. annuum was demonstrated by the overexpression of the BABY BOOM transcription factor from Arabidopsis thaliana L. [10].…”

“…Plant regeneration seems to occur more consistently by direct organogenesis [5,12]. In this case, consistent regeneration from shoot segments has been achieved using spermidine and avoiding the formation of callus [13].…”

Isolation of Sperm and Egg Cells from Pepper

“…Nowadays, genetic transformation of the plant has become an important alternative for both basic and commercial plant breeding programs. However, pepper is considered to be a highly recalcitrant species with respect to in vitro regeneration and genetic transformation (Gammoudi et al, 2018). Isolated egg and sperm from different species can create distant hybridization between two species, which has a great potency during crop breeding.…”