Physical state of the culture medium triggers shift in morphogenetic pattern from shoot bud formation to somatic embryo in Solanum khasianum.

Solanum khasianum is a rich source of steroidal alkaloids that are important secondary metabolites with enormous pharmaceutical uses. Development of plantlets from somatic tissues, under in vitro conditions, takes place both through adventitious shoot bud differentiation or somatic embryogenesis (SE) pathway. We observed that the physical state of medium, solid or liquid, determined the regenerant differentiation patterns from root segment explants in S. khasianum. In the solidified medium, the root segments developed adventitious shoot buds whereas somatic embryos were regenerated in the liquid medium. Varying gradients from liquid to solid medium were further used to confirm the effect of solidified condition on regeneration pathway. Histological analysis of developing shoot buds and somatic embryos was also performed to confirm their development and differentiation patterns. In order to further confirm the developmental pathways, qRT-PCR analysis of the marker genes of SE and shoot regeneration was also performed. While SOMATIC EMBRYOGENESIS RECEPTOR KINASE1 (SkSERK1) expression was significantly up-regulated during the early embryogenic stage, the LATE EMBRYOGENESIS ABUNDANT (SkLEA) protein was found to be highly expressed in the mature embryos. Expression of the HISTONE DEACETYLASE (HDA6), a repressor of SE related genes, was highly decreased during embryogenesis in the liquid culture. Furthermore, expression of the ENHANCER OF SHOOT REGENERATION  (ESR) gene was comparatively increased during shoot regeneration in the culture using solid medium. Our results point out that the physical state of the medium in S. khasianum plays a decisive role in differentiation pattern which was independent of hormonal supplements.

Growth and alkaloid production along with expression profiles of biosynthetic pathway genes in two contrasting morphotypes of prickly and prickleless Solanum viarum Dunal.

Growth and production kinetics of three important glycoalkaloids viz. α-solanine, solanidine, and solasodine in two contrasting prickly and prickleless plants of Solanum viarum Dunal were evaluated under in vitro conditions. The prickleless plants showed improved accumulation of total glycoalkaloid content [7.11 and 6.85 mg g-1 dry weight (DW)] and growth (GI = 11.08 and 19.26) after 45 and 50 days of culture cycle, respectively. For higher biomass (91.18 g l-1) as well as glycoalkaloid (52.56 mg l-1) recovery, the prickleless plants served as highly profitable platform. All the three studied glycoalkaloids were identified and quantified by mass spectrometry and HPLC. All the three studied glycoalkaloids accumulated in age-dependent manner. The presence of two constituents, i.e., solasodine and solanidine mainly contributed for higher accumulation of total glycoalkaloid content in the prickleless plants. However, the synthesis of α-solanine was highly age specific and could be detected after 4 to 5 weeks of culture cycle in both prickle containing as well as prickleless plants of S. viarum. The higher accumulation of glycoalkaloids in prickleless plants was also supported with the expression analysis of six key pathway enzymes viz. mevalonate kinase (MVK), 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), farnesyl diphosphate synthase (FPS), UDP-galactose/solanidine galactosyltransferase (SGT1), UDP-glucose/solanidine glucosyltransferase (SGT2), and cytochrome P450 monooxygenase (CYP). The results indicated that the plants harvested after 45 and 50 days of culture cycle accumulated maximum bioactive in-demand glycoalkaloids in the prickly and prickleless plants of S. viarum Dunal, respectively.

Transcriptome analysis provides insight into prickle development and its link to defense and secondary metabolism in Solanum viarum Dunal.

Prickles are epidermal outgrowth found on the aerial surface of several terrestrial plants. Microscopic studies on prickles of S. viarum Dunal indicated a crucial role of glandular trichomes (GTs) in their development. A spontaneously obtained prickleless mutant showed normal epidermal GTs, but its downstream developmental process to prickle was perturbed. Thus, prickleless mutant offers an ideal opportunity to unveil molecular regulators working downstream to GTs in the prickle formation. Differential transcriptome analysis of epidermis of prickly and prickleless mutant revealed that expression of several defense regulators like ethylene, salicylic acid, PR-proteins, etc. were significantly down-regulated in prickleless mutant, provide an important link between defense and prickle development. It was also noteworthy that the expression of few essential development related TFs like MADS-box, R2R3-MYB, REM, DRL1, were also down-regulated in the stem, petioles, and leaves of prickleless mutant indicating their potential role in prickle development. Interestingly, the gene expression of terpenoid, steroid, flavonoid, glucosinolate, and lignin biosynthesis pathways were up-regulated in prickleless mutant. The biochemical and qRT-PCR analysis also confirmed metabolite elevation. These results indicated that the loss of prickle was compensated by elevated secondary metabolism in the prickleless mutant which played important role in the biotic and abiotic stress management.