Dynamic Hormone Gradients Regulate Wound-Induced de novo Organ Formation in Tomato Hypocotyl Explants

Plants have a remarkable regenerative capacity, which allows them to survive tissue damage after biotic and abiotic stresses. In this study, we use Solanum lycopersicum ‘Micro-Tom’ explants as a model to investigate wound-induced de novo organ formation, as these explants can regenerate the missing structures without the exogenous application of plant hormones. Here, we performed simultaneous targeted profiling of 22 phytohormone-related metabolites during de novo organ formation and found that endogenous hormone levels dynamically changed after root and shoot excision, according to region-specific patterns. Our results indicate that a defined temporal window of high auxin-to-cytokinin accumulation in the basal region of the explants was required for adventitious root formation and that was dependent on a concerted regulation of polar auxin transport through the hypocotyl, of local induction of auxin biosynthesis, and of local inhibition of auxin degradation. In the apical region, though, a minimum of auxin-to-cytokinin ratio is established shortly after wounding both by decreasing active auxin levels and by draining auxin via its basipetal transport and internalization. Cross-validation with transcriptomic data highlighted the main hormonal gradients involved in wound-induced de novo organ formation in tomato hypocotyl explants.

Development of an efficient in vitro callus proliferation protocol for edible wild rhubarb (Rheum ribes L.)

Rheum ribes L. is a perennial wild species. Young shoots and flower bunches are freshly consumed, and root and rhizomes are generally used for medicinal purposes. The aim of the present study was to improve the callus proliferation protocol for R. ribes L. under in vitro conditions. For callus induction, hypocotyl explants taken from 14-day old plantlets germinated in Murashige and Skoog (MS) media were cultured in MS media with 9 plant growth regulator (PGR) combinations containing 6-benzylaminopurine (BAP) (2, 3, and 4 mg/L) + naphthylacetic acid (NAA) (0.1, 0.5, and 1 mg/L). Then, for callus proliferation, 4 PGR combinations containing NAA (0.2 mg/L) + thidiazuron (TDZ) (0.5, 1, 2, and 3 mg) were used in the first set of experiments, and 36 PGR combinations containing BAP (1, 2, 3, and 4 mg/L) + indole-3-butyric acid (IBA) (0.2, 0.5, and 1 mg/L), BAP (1, 2, 3, and 4 mg/L) + NAA (0.2, 0.5, and 1 mg/L), and TDZ (1, 2, 3, and 4 mg/L) + NAA (0.2, 0.5, and 1 mg/L) were used in the second set of experiments. At the end of the second set of experiments, the greatest callus regeneration ratios were obtained due to the combinations including BAP and IBA as well as the low-dose TDZ- (especially 1 mg/L) and NAA- (0.2, 0.5, 1 mg/L) combinations. Regarding callus fresh weights, TDZ + NAA combinations were found to be more successful. The greatest callus fresh weight (12.7 ±0.4 g) was obtained from MS medium supplemented with 2 mg/L TDZ and 0.2 mg/L NAA.

Tissue-Specific Metabolic Reprogramming during Wound-Induced Organ Formation in Tomato Hypocotyl Explants

Plants have remarkable regenerative capacity, which allows them to survive tissue damage after exposure to biotic and abiotic stresses. Some of the key transcription factors and hormone crosstalk mechanisms involved in wound-induced organ regeneration have been extensively studied in the model plant Arabidopsis thaliana. However, little is known about the role of metabolism in wound-induced organ formation. Here, we performed detailed transcriptome analysis and used a targeted metabolomics approach to study de novo organ formation in tomato hypocotyl explants and found tissue-specific metabolic differences and divergent developmental pathways. Our results indicate that successful regeneration in the apical region of the hypocotyl depends on a specific metabolic switch involving the upregulation of photorespiratory pathway components and the differential regulation of photosynthesis-related gene expression and gluconeogenesis pathway activation. These findings provide a useful resource for further investigation of the molecular mechanisms involved in wound-induced organ formation in crop species such as tomato.

Alteration of Flower Color in Viola cornuta cv. “Lutea Splendens” through Metabolic Engineering of Capsanthin/Capsorubin Synthesis

Flower color is an important characteristic that determines the commercial value of ornamental plants. The development of modern biotechnology methods such as genetic engineering enables the creation of new flower colors that cannot be achieved with classical methods of hybridization or mutational breeding. This is the first report on the successful Agrobacterium-mediated genetic transformation of Viola cornuta L. The hypocotyl explants of cv. “Lutea Splendens” variety with yellow flowers were transformed with A. tumefaciens carrying empty pWBVec10a vector (Llccs−) or pWBVec10a/CaMV 35S::Llccs::TNos vector (Llccs+) for capsanthin/capsorubin synthase gene (Llccs) from tiger lily (Lilium lancifolium). A comparative study of shoot multiplication, rooting ability during culture in vitro, as well as phenotypic characteristics of untransformed (control) and transgenic Llccs− and Llccs+ plants during ex vitro growth and flowering is presented. Successful integration of Llccs transgene allows the synthesis of red pigment capsanthin in petal cells that gives flowers different shades of an orange/reddish color. We demonstrate that the ectopic expression of Llccs gene in ornamental plants, such as V. cornuta “Lutea Splendens” could successfully be used to change flower color from yellow to different shades of orange.

Screening of Suitable Plant Regeneration Protocols for Several Capsicum spp. through Direct Organogenesis

Peppers (Capsicum spp.) are recalcitrant to in vitro culture regeneration, making the application of in vitro-based breeding strategies difficult. We evaluated the impact of different combinations of auxins, cytokinins and micronutrients on the induction of direct organogenesis in cotyledon and hypocotyl explants of C. annuum, C. baccatum and C. chinense. We found variation in the regeneration response among species and type of explant. In this way, the average numbers of shoots per cotyledon and hypocotyl explant were, respectively, 1.44 and 0.28 for C. annuum, 4.17 and 3.20 for C. baccatum and 0.08 and 0.00 for C. chinense. Out of the six media, the best overall results were obtained with the medium Pep1, which contained 5 mg/L BAP (6-benzylaminopurine), 0.5 mg/L IAA (indole-3-acetic acid) and 0.47 mg/L CuSO4, followed by a subculture in the same medium supplemented with 10 mg/L AgNO3 (medium Pep1.2). The best result for the Pep1 + Pep1.2 medium was obtained for C. baccatum using cotyledon explants, with 8.87 shoots per explant. The explants grown in medium Pep1 + Pep1.2 were the ones with greener tissue, while overall the hypocotyl explants were greener than the cotyledon explants. Our results indicate that there is wide variation among Capsicum species in terms of regeneration. Our results suggest that the synergistic effect of copper and silver resulted in a higher regeneration rate of Capsicum explants. Explants with shoots were transferred to different media for elongation, rooting and acclimatization. Although acclimatized plantlets were obtained for C. baccatum and C. chinense, an improvement in these latter stages would be desirable for a high throughput regeneration pipeline. This work contributes to the improvement of Capsicum regeneration protocols using specific combinations of medium, explant and genotype, reaching the levels of efficiency required for genetic transformation and of gene editing technologies for other crops.

Tissue-specific metabolic reprogramming during wound induced de novo organ formation in tomato hypocotyl explants

SUMMARYPlants have remarkable regenerative capacity, which allows them to survive tissue damaging after biotic and abiotic stress. Some of the key transcription factors and the hormone crosstalk involved in wound-induced organ regeneration have been extensively studied in the model plant Arabidopsis thaliana. However, little is known about the role of metabolism in wound-induced organ regeneration.Here, we performed detailed transcriptome analysis and targeted metabolomics approach during de novo organ formation in tomato hypocotyl explants and found tissue-specific metabolic differences and divergent developmental pathways after wounding.Our results indicate that callus growth in the apical region of the hypocotyl depends on a specific metabolic switch involving the upregulation of the photorespiratory pathway and the differential regulation of photosynthesis-related genes and of the gluconeogenesis pathway.The endogenous pattern of ROS accumulation in the apical and basal region of the hypocotyl during the time-course were dynamically regulated, and contributed to tissue-specific wound-induced regeneration.Our findings provide a useful resource for further investigation on the molecular mechanisms involved in wound-induced organ formation in a crop species such as tomato.One-sentence SummaryMetabolic switch during wound-induced regeneration

The induction and development of somatic embryos from the in vitro culture of Catharanthus roseus (L.) G. Don

Catharanthus roseus is containing anticancer alkaloids vincristine and vinblastine as is an important medicinal plant. Several studies have conducted on in-vitro culture of this plant. To optimize the somatic embryogenesis, a factorial based on CRD experiment with 10 replications was conducted. Root, hypocotyl and leaf explants grown in-vitro were transferred and cultured on MS media containing different combinations of 2,4-D, NAA and 2,4-D×BAP. The results revealed that in callogenesis, the interaction effects of root and hypocotyl explants×2,4-D and NAA as well as hypocotyl×(1 mg l-1 NAA+1 mg l-1 BAP) was superior than other treatments (p≤0.01). For calli fresh weight, hypocotyl×NAA and hypocotyl×(1 mg l-1 NAA+1 mg l-1 BAP) was the treatment of choice (p≤0.01). The calli produced were sub-cultured to attain the pre-embryos and somatic embryos. For the number of pre-embryos and somatic embryos; the interaction of hypocotyl×2,4-D was the best and most efficient treatment. Seemingly, the production of somatic embryos is accessible in this plant by the logical management of growth regulator combinations. Furthermore, the production and genetic engineering of the somatic embryos could be a promising trend in the subsequent production of high-valued metabolites from this plant.