In vitro regeneration of Pinus brutia Ten. var. eldarica (Medw.) through organogenesis

This paper describes two in vitro regeneration systems through direct and indirect organogenesis in Pinus brutia using fascicles aseptic cultures as explants. Mechanical scarification and gibberellic acid (GA3) were evaluated on in vitro seed germination. Scarification was the treatment that allowed for in vitro seed germination. The highest direct organogenic response was obtained in Murashige and Skoog (MS) medium containing 4.5 µM thidiazuron, whereas the highest indirect organogenesis was obtained with 9.8 µM thidiazuron and 3.4 µM paclobutrazol. The isolated shoots were rooted on MS medium supplemented with 1.70 µM indoleacetic acid. A large variation in root ability was observed among plantlets. These results suggest that both regeneration systems can be applied to the micropropagation or genetic transformation of P. brutia.Key words: Pinus brutia, organogenesis, fascicles, paclobutrazol, thidiazuron

Changes in the protein profile of Habanero pepper (Capsicum chinense J.) somatic embryos during development

Protein profile was studied during the development of Capsicum chinense somatic embryos. The total protein content and profile of polypeptides (by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of somatic embryos at different developmental stages (globular, heart-shaped, torpedo and cotyledonary stages) were analyzed. The protein profile of zygotic embryos included nine exclusive bands with molecular weights of 4.0, 5.2, 8.1, 13.7, 20.9, 23.7, 41, 50 and 69.3 kDa; these bands were not observed in the protein profile of somatic embryos. Coincidently, five of these bands possessed similar molecular weights to those reported for storage proteins in other plant species. Protein content showed a clear decreasing tendency with increasing somatic embryo development. The lowest protein content was detected in somatic embryos at the cotyledonary stage (0.436 µg/mg fresh weight), and the highest content was found in somatic embryos at the globular stage (2.98 µg/mg fresh weight). Total proteins two-dimensional electrophoresis (2-DE) analysis of mature zygotic embryo (prior to the desiccation) and cotyledonal somatic embryo, showed significant differences in the protein profile of both types of embryos. Zygotic embryo showed the proteins expression of isoelectric point between 4 to 7 and 7 to 10, and molecular weights between 25 to 36 KDa, which were not expressed in the cotyledonal somatic embryo. The low protein content during the development of the somatic embryos, particularly at the cotyledonary stage, is a factor that could be related with the low rate of conversion to plantlets and the high frequency of deformed somatic embryos of C. chinense.Key words: Recalcitrance, maturation, germination, conversion

In vitro regeneration of Pinus brutia Ten. var. eldarica (Medw.) through organogenesis

This paper describes two in vitro regeneration systems through direct and indirect organogenesis in Pinus brutia using fascicles aseptic cultures as explants. Mechanical scarification and gibberellic acid (GA3) were evaluated on in vitro seed germination. Scarification was the treatment that allowed for in vitro seed germination. The highest direct organogenic response was obtained in Murashige and Skoog (MS) medium containing 4.5 μΜ thidiazuron, whereas the highest indirect organogenesis was obtained with 9.8 μΜ thidiazuron and 3.4 μΜ paclobutrazol. The isolated shoots were rooted on MS medium supplemented with 1.70 μM indoleacetic acid. A large variation in root ability was observed among plantlets. These results suggest that both regeneration systems can be applied to the micropropagation or genetic transformation of P. brutia

Floral Biology Studies in Habanero pepper (Capsicum chinense Jacq.) to Implement in a Cross-Breeding Program

Knowledge of the reproductive biology of a species is fundamental in order to develop an efficient program of genetic improvement by hybridization. The viability of the pollen, anther dehiscence, receptivity of the stigma and the anthesis of 12 improved lines of Habanero pepper were studied to develop a cross-breeding program. Among the results, the greatest number of flowers in anthesis was quantified at 8:00 a.m. for most genotypes. The dehiscence of the anther differed significantly in stages evaluated, observing in flower buds 100% of the closed anthers. The receptivity was positive in all the stages evaluated (before, during and after anthesis) and in all the genotypes, the most outstanding being the genotype AKN-08, which presented 100% of receptivity in the three stages evaluated. The viability of the pollen varied among the different conservation times evaluated (0, 24 and 48 h) while the highest percentage of viability (80%) and the largest number of seeds per fruit (56) were obtained when recently collected pollen was used (0 time). These results will have an important repercussion on the improvement of the Habanero pepper by increasing the efficiency of the programs to obtain hybrids and/or improved varieties

Encapsulación de embriones somáticos de Laelia Anceps Ssp. Sawsonii para la producción de semilla sintética

Los embriones somáticos de Laelia anceps ssp. dawsonii, obtenidos a partir de semillas germinadas en un medio MS suplementado con ácido naftalen-acético (ANA), 6, bencilaminopurina (BAP) y ácido indol-3-acético (AIA), 2 mg·litro-1 de cada uno fueron encapsulados en matriz de alginato de sodio en complejo con cloruro de calcio (CaCl2·2H2O) 75 mM, para producir semillas sintéticas, como una estrategia de rescate y conservación. Para determinar el efecto de factores físicos y químicos tales como temperatura y tiempo de almacenamiento, consistencia de la matriz de alginato, concentración de nutrimentos minerales y reguladores del crecimiento vegetal, sobre el porcentaje y tiempo de germinación, así como viabilidad de las semillas sintéticas, se probaron concentraciones de alginato de sodio al 2.0, 3.0 y 4.0 %, de sales de MS (37.5, 50 y 100 %) y BAP (0.5, 1.0 y 2.0 mg·litro-1) adicionados a la matriz de encapsulación, además de diferentes temperaturas (4, 20 y 25 °C) y tiempos de almacenamiento (15 y 30 días). Se observó el 100 % de germinación cuando las semillas fueron encapsuladas con 3 % de alginato de sodio en complejo con CaCl2·2H2O a 75 mM; con las sales MS al 100 y 50 % en la matriz de encapsulación, suplementando 2.0 mg·litro-1 de BAP, se logró un porcentaje de germinación del 64.54 y 62.77 %, respectivamente, sin existir diferencia significativa entre ellas

Regeneración in vitro de Laelia anceps ssp. dawsonii

Se germinaron in vitro semillas de Laelia anceps ssp. dawsonii, una orquídea silvestre amenazada, originaria de México y Mesoamérica, con alto potencial ornamental, utilizando el medio Murashige & Skoog (1962) suplementado con ácido 1-naftalén-acético (ANA), 6-benzyl-amino-purina (BAP), Kinetina (Kin) y ácido indol-3-acético (AIA), 2 mg L-1 de cada uno, el cual resultó óptimo para la inducción de callo bajo fotoperiodo de 16/8 h (20.2 µmol·m-2·s-1). El callo fue subcultivado a intervalos de 45 días en el mismo medio de cultivo, produciendo en promedio 524 embriones somáticos en el tercer subcultivo. Los embriones somáticos producidos se convirtieron en plantas completas con brotes y raíces en el mismo medio, y fueron transferidas al medio VW suplementado con BAP 2 mg L-1, AIA 1 mg L-1 y carbón activado 0.2 % para su desarrollo. Después de aproximadamente tres meses, las plántulas fueron aclimatizadas en el invernadero con un 100 % de tasa de sobrevivencia

Regeneración in vitro de Laelia Anceps ssp. Dawsonii

Seeds of Laelia anceps ssp. dawsonii were germinated in vitro, this is a wild endangered orchid, originated in México and Mesoamerica, with a high ornamental potential. Murashige & Skoog (1962) culture media, supplemented with 1-naftalen-acetic acid (ANA), 6-benzyl-amino-purine (BAP), Kinetin (Kin) and indol-3-acético (AIA) acid, 2 mg L-1 each one, was optimum for callus induction under 16/8 h photoperiod (20.2 μmol·m-2·s-1). Callus was subcultured every 45 days in the same culture medium producing 524 somatic embryoids in average, at the end of the third subculture. Somatic embryoids germinated in plants with shoots and roots in the same culture medium, and were transferred to VW supplemented with 2 mg L-1 BAP, 1 mg L-1 AIA and 0.2 % active charcoal to induce their develop. Three months later, plantlets were acclimatized in a greenhouse, with 100 % survivence.Se germinaron in vitro semillas de Laelia anceps ssp. dawsonii, una orquídea silvestre amenazada, originaria de México y Mesoamérica, con alto potencial ornamental, utilizando el medio Murashige & Skoog (1962) suplementado con ácido 1-naftalén-acético (ANA), 6-benzyl-amino-purina (BAP), Kinetina (Kin) y ácido indol-3-acético (AIA), 2 mg L-1 de cada uno, el cual resultó óptimo para la inducción de callo bajo fotoperiodo de 16/8 h (20.2 μmol·m-2·s-1). El callo fue subcultivado a intervalos de 45 días en el mismo medio de cultivo, produciendo en promedio 524 embriones somáticos en el tercer subcultivo. Los embriones somáticos producidos se convirtieron en plantas completas con brotes y raíces en el mismo medio, y fueron transferidas al medio VW suplementado con BAP 2 mg L-1, AIA 1 mg L-1 y carbón activado 0.2 % para su desarrollo. Después de aproximadamente tres meses, las plántulas fueron aclimatizadas en el invernadero con un 100 % de tasa de sobrevivencia

Development of the ovule and seed of Habanero chili pepper (Capsicum chinense Jacq.): Anatomical characterization and immunocytochemical patterns of pectin methyl-esterification

32 p.-7 fig.-1 fig.supl.Ovule and seed development in plants has long fascinated the scientific community given the complex cell coordination implicated in these processes. These cell events are highly conserved but are not necessarily representative of all plants. In this study, with the aim of obtaining information regarding the cellular patterns that follow the usual development of the ovule and the zygotic embryo, we carried out an integral anatomical study of the Capsicum chinense Jacq., floral buds and seeds at various days during maturation. This study allowed us to identify the main histo-morphological stages accompanying the transition of somatic cells into the macrospore, female gamete, and the zygotic embryogenesis. This knowledge is fundamental for future biotechnological research focused on solving the morphological recalcitrance observed during the in vitro induction of somatic or microspore embryogenesis in Capsicum. For the first time in C. chinense, we have described the hypostases, a putative source of plant growth regulators, and "the corrosion cavity", a space around the embryo. Additionally, the cell wall pectin-esterification status was investigated by immunohistology. At early stages of morphogenesis, the pectin is highly methyl-esterified; however, methyl-esterification decreases gradually throughout the process. A comparison of the results obtained here, together with the histo- and immunological changes occurring during the somatic and microspore embryogenesis, should help to elucidate the biochemical mechanisms that trigger the morphogenic events in Capsicum spp.This work was supportedby Consejo Nacional de Ciencia y Tecnología (CONACYT-México) [grant number 219651] and the Spanish MINECO and European Regional Development Fund (ERDF/FEDER) [grant numbers AGL2014-52028-R and AGL2017-82447-R]Peer reviewe

Endogenous auxin accumulation/localization during zygotic and somatic embryogenesis of Capsicum chinense Jacq

37 p.-9 fig.Zygotic and somatic embryogenesis in plants is a fascinating event that is finely regulated through the expression of a specific group of genes and dynamic levels of plant hormones whose concerted action determines the fate that specific cells follow towards zygotic or somatic embryo development. This work studied different stages of Capsicum chinense Jacq. zygotic and somatic embryogenesis. HPLC quantification determined that the levels of indole-3-acetic acid (IAA) increase as the zygotic or somatic embryogenesis progresses, being higher at maturity, thus supporting a positive correlation between embryo cell differentiation and IAA increase. A monoclonal anti-IAA-antibody was used to detect IAA levels. Findings revealed a dynamic pattern of auxin distribution along the different embryogenic embryonic stages. In the early stages of zygotic embryos, the IAA gradient was observed in the basal cells of the suspensor and the hypostases, suggesting that they are the initial source of the IAA hormone. As embryogenesis proceeds, the dynamic of the IAA gradient is displaced to the embryo and endosperm cells. In the case of induced somatic embryogenesis, the IAA gradient was detected in the dividing cells of the endodermis, from where pre-embryogenic cells emerge. However, the analysis of somatic embryos revealed that IAA was homogeneously distributed. This study shows evidence supporting a correlation between IAA levels during zygotic or somatic embryogenesis in Capsicum chinense species.This manuscript received financing from CONACYT-México through the project with reference number PDCPN2016 3953. This study was funded by Project grant AGL2017-82447-R, funded by Spanish AEI and European ERDF/FEDER too.Peer reviewe

Indirect Somatic Embryogenesis: An Efficient and Genetically Reliable Clonal Propagation System for <i>Ananas comosus</i> L. Merr. Hybrid “MD2”

The objective of this study was to establish an efficient—direct or indirect—regeneration system for pineapple (Ananas comosus L.) plants, with a high rate of multiplication and that would preserve the genetic identity of the donor genotype (Hybrid ‘MD2’) in the regenerated plants. Ten treatments, with different concentrations of 2,4-Dichlorophenoxyacetic (2,4-D) and Picloram (P), in the absence or presence of 6-Benzylaminopurine (BAP), were used for in vitro morphogenesis induction, as well as histological and molecular techniques, in order to characterize the morphogenic responses induced. Significant differences between treatments tested, to induce callus and buds, were assessed by the Kruskal Wallis method and the Mann–Whitney U-tests. Different pineapple regeneration routes were identified, showing the high regeneration potential of this species. The medium containing 2 mg L−1 2,4-D and 2 mg L−1 BAP, where indirect somatic embryogenesis occurred, was selected as the most efficient treatment, with an average of 120 somatic embryos per explant, differing significantly from the rest of the treatments. It was also demonstrated that the pineapple plants regenerated in vitro preserved the genetic identity of the donor genotype, which represents a high degree of confidence for the application of indirect somatic embryogenesis for A. comusus clonal propagation