Quantitative and qualitative study of endogenous and exogenous growth regulators in eggplant (Solanum melongena) microspore cultures

In eggplant microspore embryogenesis, embryos are produced and then transformed into undifferentiated calli, instead of developing as true embryos. This is the main current bottleneck that precludes this process from being efficient. In this work we aimed to shed light on the factors involved in the successful in vitro development of eggplant haploid embryos by evaluating the role of growth regulators (GRs) in this process. We analyzed the endogenous levels of different GRs, including auxins, cytokinins and gibberelins, as well as salicylic, jasmonic and abscisic acid, in microspores and microspore-derived embryos at different culture stages. We also analyzed the same GR profiles in leaf and anther wall tissues of different eggplant backgrounds. Finally, we assessed the application of different GR combinations to the culture medium. Our results showed that in eggplant there are no genotype-specific endogenous GR profiles that can be associated to a high embryogenic response. Instead, the embryogenic response seems related to different GR accumulation patterns during in vitro culture. The changes observed in the endogenous levels of salicylic and abscisic acid were not related to the embryo transition. There were, however, changes in the levels of indole acetic acid and dihydrozeatin. The best GR combination to promote callus production was 0.5 mg/L 1-naphthaleneacetic acid (NAA) and 0.5 mg/L 6-benzylaminopurine (BAP). A 20% reduction of NAA and BAP reduced embryo production but produced structures more anatomically similar to embryos. These results shed light on the role of GRs during the development of microspore-derived embryos in eggplant microspore cultures.

Somatic Embryogenesis in Vitis for Genome Editing: Optimization of Protocols for Recalcitrant Genotypes

New Plant Breeding Techniques (NPBTs) protocols have been developed to produce new grape varieties with improved quantitative and qualitative characteristics. Reliable transformation protocols for grapes are based on the generation/induction of embryogenic callus cells that are then transformed. Varieties such as Italia have proven to be very recalcitrant to regeneration via somatic embryogenesis. In this work, the development of a protocol for improved production of embryogenic calluses is described. Two sterilization protocols were tested: (a) a lower active chlorine concentration for a longer time (LS); and (b) a higher chlorine concentration for a shorter time (HS), in combination with the absence or presence of citric acid in the growing substrate in the first growth media. The embryogenic calluses formation in Chardonnay, a cv. with a high embryogenic response, was significantly higher in presence of citric acid in the initial growing substrate regardless of the sterilization protocol. In Aglianico, a cv. with a lower embryogenic response, no significant differences were observed. Instead, in a recalcitrant cv. as Italia, we obtained a 13-fold increase in embryogenic calluses formation performing sterilization of flowers with the HS protocol compared to LS.

Effect of Explants and Low Cost Medium on Morphogenic Response of Aerial Stem and Rhizome Bud Explants of Turmeric (Curcuma longa L.) for Plant Regeneration

This study was done to select suitable explants and low cost medium for plant regeneration of turmeric. Therefore, the different explants were excised from the aerial stems and rhizome buds and surface sterilized. The sterilized explants were cultured on MS medium fortified with 2.0 mg/l BAP. From the survived aerial stem explants, 0.5 cm long vertical half of the aerial stem explants exhibited somatic embryogenic response (69.7%). The highest morphogenic response (74%) of shoot bud initiation was observed from the top slice of the surviving rhizome bud explants. Further, Yara Mila complex fertilizer, which is an ideal granular fertilizer mixture, was used as an alternative to MS medium. Three different concentrations of Yara Mila complex fertilizer (1.0, 3.0, and 5.0 g/l ) supplemented with 2.0 mg/l BAP each were tested with the MS medium fortified with 2.0 mg/l BAP (control treatment) for in vitro establishment from aerial stem explants and top slice of the rhizome bud explants. Both explants were surface sterilized and cultured on MS medium and different concentrations of Yara Mila complex fertilizer fortified with 2.0 mg/l BAP. From the survived explants, aerial stem explants exhibited somatic embryogenic response (69.7%) and shoot bud initiation (74%) on normal MS media. The higher performances were observed in 1.0 g/l concentration of complex fertilizer incorporated medium with 51% embryogenic response from the aerial stem explants and 52.3% shoot bud formation response from the top slice of the rhizome bud. The cost of 1 kg complex fertilizer was Rs. 182. It could be concluded that complex fertilizer is a cost effective alternative medium for MS medium for in vitro propagation reducing the cost of the substituted ingredients by 99.87% in the tissue culture of turmeric.

Variability in somatic embryo-forming capacity of spinach

High variability in somatic embryo (SE)-forming capacity has previously been observed in several spinach cultivars. Such variability frequently accounted for more variation in embryogenic response of the explants than the factor being investigated. Hence, the variability in embryogenic capacity was examined in the present study at both the population and the single-seedling level, using seeds of spinach cultivar Matador obtained from nine European seed companies. Seed population obtained from Slovenia (Sl) was superior to others, with the highest regeneration frequency (100%) and the highest mean SE number (14.4). A total of 82% of these seedlings had 80–100% of regenerating explants, while in populations with intermediate embryogenic capacity approximately 40% of seedlings had 20–60% of regenerating explants. The explants from the majority of seedlings (52–100%) in the least responsive populations were irresponsive. Furthermore, the explants from Sl seedlings regenerated from 10–20 (43.5%) up to > 20 (27.6%) SEs on average, while the explants from the majority of seedlings belonging to other populations regenerated 1–10 SEs. The present study strongly indicates that the variability of plant material must not be overlooked, because choosing more responsive individuals for one treatment and less responsive ones for another may lead to misinterpretation of the data.

The miR396–GRF Regulatory Module Controls the Embryogenic Response in Arabidopsis via an Auxin-Related Pathway

In plants, microRNAs have been indicated to control various developmental processes, including somatic embryogenesis (SE), which is triggered in the in vitro cultured somatic cells of plants. Although a transcriptomic analysis has indicated that numerous MIRNAs are differentially expressed in the SE of different plants, the role of specific miRNAs in the embryogenic reprogramming of the somatic cell transcriptome is still poorly understood. In this study, we focused on performing a functional analysis of miR396 in SE given that the transcripts of MIR396 genes and the mature molecules of miR396 were found to be increased during an SE culture of Arabidopsis [1]. In terms of miR396 in embryogenic induction, we observed the SE-associated expression pattern of MIR396b in explants of the β-glucuronidase (GUS) reporter line. In order to gain insight into the miR396-controlled mechanism that is involved in SE induction, the embryogenic response of mir396 mutants and the 35S:MIR396b overexpressor line to media with different 2,4-Dichlorophenoxyacetic acid (2,4-D) concentrations was evaluated. The results suggested that miR396 might contribute to SE induction by controlling the sensitivity of tissues to auxin treatment. Within the targets of miR396 that are associated with SE induction, we identified genes encoding the GROWTH-REGULATING FACTOR (GRF) transcription factors, including GRF1, GRF4, GRF7, GRF8, and GRF9. Moreover, the study suggested a regulatory relationship between miR396, GRF, and the PLETHORA (PLT1 and PLT2) genes during SE induction. A complex regulatory relationship within the miR396–GRF1/4/8/9–PLT1/2 module that involves the negative and positive control of GRFs and PLT (respectively) by miR396 might be assumed.

Gametic Embryogenic Response in Wild Diploid Solanum Species and its Implications for Genome Sequencing Projects and Breeding

The anther culture response in Solanum commersonii (2n = 2x = 24, 1EBN) and S. chacoense (2n = 2x = 24, 2EBN), two wild potato germplasm resources was studied to obtain haploid plants. Three accessions from each of the two species and 3200 anthers from each genotype were cultured. Authors assessed different culture media; ascorbic acid, L?cysteine and silver nitrate (AgNO3) were included to prevent browning of anther cultures. Addition of AgNO3, was effective to induce embryogenesis. The clones from S. commersonii showed different embryogenic response to androgenesis. However, the three accessions from S. chacoense did not induce any embryo in the same conditions. Ploidy level of the regenerated clones was estimated by flow cytometry and confirmed by chromosome counts. This is the first report of haploid plants obtained from anther culture in S. commersonii, with important implications in sequencing efforts and potato breeding.Plant Tissue Cult. & Biotech. 26(2): 159-173, 2016 (December)

EFFECT OF GENOTYPE ON THE in vitro REGENERATION OF Stevia rebaudiana VIA SOMATIC EMBRYOGENESIS

<p class="p1"><strong>ABSTRACT</strong></p><p class="p2"><em>Stevia rebaudiana</em> (Asteraceae) is a plant of economic importance because of its medicinal properties and the presence of sweetener compounds on its leaves. These compounds can be a substitute for sucrose in a wide variety of products used by persons with diabetes and obesity problems. To standardize an efficient and effective propagation method for the different Stevia genotypes grown in Colombia, this study evaluated the effect of different combinations of the plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 6-(gamma, gamma-dimethylallylamino) purine (2iP) and Zeatin on the induction and development of somatic embryos. Adenine and coconut water were also evaluated as supplements in the basal culture medium Murashige and Skoog Basal Salt Mixture (MS) with glutamine. The combination of 2,4-D (18.09 μM) and 2iP (7.38 μM) produced the highest number of somatic embryos per explant, which had well-defined characteristics. The genotype showed a significant effect on the embryogenic response. In the “SRQ-93” genotype, the formation and development of somatic embryos was achieved, whereas the genotypes “Bertoni” and “Morita II” only yielded embryogenic and non-embryogenic calli, respectively. The conversion to seedlings was achieved on the regeneration medium containing gibberellic acid (GA<span class="s1">3</span>) (0.29 μM) and activated charcoal. </p><p class="p1"><strong>RESUMEN</strong></p><p class="p2"><em>Stevia rebaudiana</em> (Asteraceae), es una planta de gran importancia económica debido a sus propiedades medicinales y a la presencia de compuestos endulzantes en sus hojas, los cuales pueden sustituir la sacarosa en gran variedad de productos utilizados por personas con problemas de diabetes y obesidad. Con el propósito de estandarizar un método de propagación eficiente y efectivo para diferentes genotipos de Stevia cultivados en Colombia, en la presente investigación se evaluó el efecto sobre la inducción y desarrollo de embriones somáticos de diferentes combinaciones de los reguladores de crecimiento vegetal 2,4-D, IAA, IBA, 2iP y Zeatina, además de los suplementos adenina y agua de coco en el medio de cultivo basal Murashige y Skoog (1962), adicionado con glutamina. Con la combinación 2,4-D (18.09 μM) y 2iP (7.38 μM) se obtuvo el mayor número embriones somáticos por explante con características bien definidas. El genotipo tuvo un efecto significativo en la repuesta embriogénica, en el genotipo “SRQ-93” se logró la formación y el desarrollo de embriones somáticos, mientras que en los genotipos “Bertoni” y “Morita II”, solo se obtuvo callo embriogénico y no embriogénico respectivamente. La conversión a plántulas se alcanzó en el medio de regeneración conteniendo GA3 (0.29 μM) y carbón activado.</p><p class="p2"> </p>