In vitro shoot regeneration can efficiently contribute to the improvement of recalcitrant Cannabis sativa L. We aimed at developing a highly efficient protocol for in vitro direct regeneration of C. sativa plants from different explants (cotyledon, hypocotyl, and true leaf) from seedlings of monoecious C. sativa short-day varieties Ferimon, Felina32, Fedora17, and USO31, together with dioecious neutral-day variety Finola. Ten regeneration media, including already published protocols, and self-designed combinations of plant growth regulators were tested. The developmental morphology since germination of seeds to the development of rooted plantlets was followed. Additionally, the ploidy level of explants and in vitro regenerants was analyzed. We concluded that hypocotyl is the best explant for in vitro direct regeneration of C. sativa plants with 49.45% of responding explants, while cotyledon and true leaf had a poor response with, respectively, 4.70 and 0.42% of explants developing plantlets. In terms of shoot regeneration, we found significant differences among the culture media evaluated and the varieties studied. Overall, the best regeneration media were ZEA RIB 2.0 (mg/L) and ZEA RIB 1.0 (mg/L) + NAA 0.02 (mg/L) with 66.67% of responding hypocotyls. Amazingly, hypocotyls cultured in medium without plant growth regulators showed an excellent response (61.54% of responding hypocotyls) and spontaneous rooting of regenerants (17.94%). In vitro regenerated plants were acclimatized just 6 weeks after culture initiation. The developmental morphology study suggests that regenerated shoots originate from pericycle cells adjacent to xylem poles. Polysomaty was detected in hypocotyls and cotyledons of all varieties studied, and diploid (>80%) and mixoploid (with diploid and tetraploid cells) plants were regenerated. Our protocol allows a high shoot organogenesis efficiency in different C. sativa varieties. The fact that a significant percentage of plants are mixoploid may provide an alternative way to develop polyploids in C. sativa . Our results show that direct in vitro regeneration may make a significant contribution to the development of improved C. sativa materials for medical applications.

Background: The use of sequencing and genotyping platforms has undergone dramatic improvements, enabling the generation of a wealth of genomic information. Despite this progress, the availability of high-quality genomic DNA (gDNA) in sufficient concentrations is often a main limitation, especially for third-generation sequencing platforms. A variety of DNA extraction methods and commercial kits are available. However, many of these are costly and frequently give either low yield or low-quality DNA, inappropriate for next generation sequencing (NGS) platforms. Here, we describe a fast and inexpensive DNA extraction method (SILEX) applicable to a wide range of plant species and tissues. Results: SILEX is a high-throughput DNA extraction protocol, based on the standard CTAB method with a DNA silica matrix recovery, which allows obtaining NGS-quality high molecular weight genomic plant DNA free of inhibitory compounds. SILEX was compared with a standard CTAB extraction protocol and a common commercial extraction kit in a variety of species, including recalcitrant ones, from different families. In comparison with the other methods, SILEX yielded DNA in higher concentrations and of higher quality. Manual extraction of 48 samples can be done in 96 min by one person at a cost of 0.12 €/sample of reagents and consumables. Hundreds of tomato gDNA samples obtained with either SILEX or the commercial kit were successfully genotyped with Single Primer Enrichment Technology (SPET) with the Illumina HiSeq 2500 platform. Furthermore, DNA extracted from Solanum elaeagnifolium using this protocol was assessed by Pulsed-field gel electrophoresis (PFGE), obtaining a suitable size ranges for most sequencing platforms that required high-molecular-weight DNA such as Nanopore or PacBio. Conclusions: A high-throughput, fast and inexpensive DNA extraction protocol was developed and validated for a wide variety of plants and tissues. SILEX offers an easy, scalable, efficient and inexpensive way to extract DNA for various next-generation sequencing applications including SPET and Nanopore among others.

Background: Efficient organogenesis induction in eggplant (Solanum melongena L.) is required for multiple in vitro culture applications. In this work, we aimed at developing a universal protocol for efficient in vitro regeneration of eggplant mainly based on the use of zeatin riboside (ZR). We evaluated the effect of seven combinations of ZR with indoleacetic acid (IAA) for organogenic regeneration in five genetically diverse S. melongena and one S. insanum L. accessions using two photoperiod conditions. In addition, the effect of six different concentrations of indolebutyric acid (IBA) in order to promote rooting was assessed to facilitate subsequent acclimatization of plants. The ploidy level of regenerated plants was studied. Results: In a first experiment with accessions MEL1 and MEL3, significant (p < 0.05) differences were observed for the four factors evaluated for organogenesis from cotyledon, hypocotyl and leaf explants, with the best results obtained (9 and 11 shoots for MEL1 and MEL3, respectively) using cotyledon tissue, 16 h light / 8 h dark photoperiod conditions, and medium E6 (2 mg/L of ZR and 0 mg/L of IAA). The best combination of conditions was tested in the other four accessions and confirmed its high regeneration efficiency per explant when using both cotyledon and hypocotyl tissues. The best rooting media was R2 (1 mg/L IBA). The analysis of ploidy level revealed that between 25 and 50% of the regenerated plantlets were tetraploid. Conclusions: An efficient protocol for organogenesis of both cultivated and wild accessions of eggplant, based on the use of ZR, is proposed. The universal protocol developed may be useful for fostering in vitro culture applications in eggplant requiring regeneration of plants and, in addition, allows developing tetraploid plants without the need of antimitotic chemicals.

Oxford University PressCorral Martínez, P.; García-Fortea, E.; Bernard, S.; Driouich, A.; Seguí-Simarro, JM. (2016) (1) Growth and development AbstractIn this work, we performed an extensive and detailed analysis of the changes in cell wall composition during Brassica napus anther development. We used immunogold labeling to study the spatial and temporal patterns of composition and distribution of different AGP, pectin, xyloglucan and xylan epitopes in high pressurefrozen/freeze-substituted anthers, quantifying and comparing their relative levels in the different anther tissues and developmental stages. We used the following monoclonal antibodies: JIM13, JIM8, JIM14 and JIM16 for AGPs, LM5, LM6, JIM7, JIM5 and LM7 for pectins, CCRC-M1, CCRC-M89 and LM15 for xyloglucan, and LM11 for xylan. Each cell wall epitope showed a characteristic temporal and spatial labeling pattern.Microspore, pollen and tapetal cells showed similar patterns for each epitope, whereas the outermost anther layers (epidermis, endothecium and middle layers) presented remarkably different patterns. Our results suggested that AGPs, pectins, xyloglucan and xylan have specific roles during anther development. The AGP epitopes studied appeared to belong to AGPs specifically involved in microspore differentiation, and contributed first by the tapetum and then, upon tapetal dismantling, by the endothecium and middle layers. In contrast, the changes in pectin and hemicellulose epitopes suggested a specific role in anther dehiscence, facilitating anther wall weakening and rupture. The distribution of the different cell wall constituents is regulated in a tissue and stage-specific manner, which seems directly related with the role of each tissue at each stage.

Dramatic advances in genomics during the last decades have led to a revolution in the field of vegetable crops breeding. Some vegetables, like tomato, have served as model crops in the application of genomic tools to plant breeding but other important crops, like eggplant (Solanum melongena), lagged behind. The advent of next generation sequencing (NGS) technologies and the continuous decrease of the sequencing costs have allowed to develop genomic tools with a greatly benefit for no-model plants such as eggplant. In this review we present the currently available genomic resources in eggplant and discuss their interest for breeding. The first draft of eggplant genome sequence and the new upcoming improved assembly, as well as the transcriptomes and RNA-based studies represent important genomic tools. The transcriptomes of cultivated eggplant and several wild relatives of eggplant are also available and have provided relevant information for the development of markers and understanding biological processes in eggplant. In addition, a historical overview of the eggplant genetic mapping studies, performed with different types of markers and experimental populations, provides a picture of the increase over time of the precision and resolution in the identification of candidate genes and QTLs for a wide range of stresses, and morphoagronomic and domestication traits. Finally, we discuss how the development of new genetic and genomic tools in eggplant can pave the way for increasing the efficiency of eggplant breeding for developing improved varieties able to cope with the old and new challenges in horticultural production.

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Selection and breeding of eggplant (Solanum melongena) materials with good performance under low nitrogen (N) fertilization inputs is a major breeding objective to reduce environmental degradation, risks for human health, and production costs. Solanum elaeagnifolium, an eggplant wild relative, is a potential source of variation for introgression breeding in eggplant. We evaluated 24 plant, fruit, and composition traits in a set of genotyped advanced backcrosses (BC2 and BC3) of eggplant with S. elaeagnifolium introgressions under low N conditions. Significant differences were found between the two parents for most traits, and a wide phenotypic diversity was observed in the advanced backcrosses, with some individuals with a much higher yield, nitrogen use efficiency (NUE), and phenolics content than the S. melongena parent. In general, the lower the proportion of S. elaeagnifolium genome introgressed in the advanced backcrosses, the higher was the general phenotypic resemblance to S. melongena. Putative QTLs were detected for stem diameter (pd4), presence of prickles in stem (ps6), leaf (pl6) and fruit calyx (pc6), fruit width (fw7), chlorogenic acid content (cg5), total phenolic acid peaks area (ph6), chlorogenic acid peak area (ca1), and phenolic acids pattern (cp1). Our results reveal that introgression breeding of eggplant with S. elaeagnifolium has a great interest for eggplant breeding, particularly for adaptation to low N conditions. These materials can potentially contribute to the development of improved eggplant varieties for a more sustainable agriculture.

Tomato chlorotic dwarf viroid (TCDVd) is a pospiviroid that causes severe disease symptoms in tomato. TCDVd is also naturally found in other crops and plants, in most occasions being asymptomatic. Apart from the natural hosts reported up to now, artificial inoculations have revealed that TCDVd can infect other plants, including eggplant (Solanum melongena). In a screening of seedlings of eggplant from a breeding programme we detected a pospiviroid, which we identified as TCDVd, representing the first report of natural infection of eggplant by TCDVd. The new TCDVd isolate of eggplant was detected by reverse transcription polymerase chain reaction (RT‐PCR) using primers TG21/CT20, initially designed to detect potato spindle tuber viroid. The new isolate sequence is close to a Brugmansia sanguinea isolate of TCDVd from the Netherlands, and most of the nucleotidic changes with respect to this isolate and to the reference genome sequence of TCDVd are found in the TR region. Naturally infected plants of eggplant with this TCDVd isolate did not display any disease symptoms. We demonstrated that in eggplant TCDVd is mechanically transmitted with low to moderate efficiency with cultivation practices, but not by plant‐to‐plant contact. Tomato plants artificially inoculated with the eggplant isolate of TCDVd tested positive for the presence of the viroid at 50 days after inoculation, but did not display any disease symptoms. Seed transmission to germinated seedlings of eggplant was variable among progenies from infected plants, ranging from 7.7% to 100.0%. Disinfection of seeds with chemical treatments with sodium hypochlorite and trisodium phosphate solutions plus thermotherapy at 80°C for 24 hr or 90°C for 6 hr was ineffective in reducing the rate of transmission by seed. We did not find evidence of horizontal transmission of TCDVd by pollen, but vertical transmission was highly efficient when healthy eggplant plants were pollinated with infected pollen. Our results indicate that asymptomatic infection of eggplant by TCDVd and high seed and pollen transmission rates may contribute to the spread of this viroid. The information we obtained is useful in order to implement measures for the prevention, control and eradication of TCDVd in eggplant crops, as well as to avoid their transmission to other hosts.