In vitro propagation of Indonesian stevia (Stevia rebaudiana) genotype using axenic nodal segments.

OBJECTIVE: The high industrial demand for Stevia cultivation (Stevia rebaudiana) has increased due to its high stevioside content derived from the leaves. However, the low germination rate makes the cultivation of the plant become the main obstacle. Therefore, an efficient cultivation technique is required. This present work aims to analyze the effect of five combinations of Kinetin (Kin) and benzyladenine (BA) on stevia micropropagation using nodal segment explants. RESULTS: The micropropagation of stevia was performed using Murashige and Skoog (MS) medium supplemented with BA and Kin. We analyzed different organogenesis and callogenesis responses. In addition, the number of shoots and root formed during in vitro culture were also observed. Our results demonstrated that all treatments with Kin, both alone and in combination with BA, resulted in the development of callus on all nodal segment explants. Explants treated in MS with 1 mg L-1 BA exhibited the best average of shoot number (36.27). In contrast, the treatment without PGR resulted in the best root formation (2.6). The overall results suggested that different combination of BA and Kin resulted in distinct organogenesis responses, where 1 mg L-1 of BA was potentially used for boosting the number of shoots in micropropagation of stevia accession Mini.

Plastid DNA Barcoding and RtActin cDNA Fragment Isolation of Reutealis Trisperma: A Promising Bioresource for Biodiesel Production.

Reutealis trisperma belonging to the family Euphorbiaceae is currently used for biodiesel production, and rapid development in plant-based biofuel production has led to its increasing demand. However, massive utilization of bio-industrial plants has led to conservation issues. Moreover, genetic information on R trisperma is still limited, which is crucial for developmental, physiological, and molecular studies. Studying gene expression is essential to explain plant physiological processes. Nonetheless, this technique requires sensitive and precise measurement of messenger RNA (mRNA). In addition, the presence of internal control genes is important to avoid bias. Therefore, collecting and preserving genetic data for R trisperma is indispensable. In this study, we aimed to evaluate the application of plastid loci, rbcL, and matK, to the DNA barcode of R trisperma for use in conservation programs. In addition, we isolated and cloned the RtActin (RtACT) gene fragment for use in gene expression studies. Sequence information was analyzed in silico by comparison with other Euphorbiaceae plants. For actin fragment isolation, reverse-transcription polymerase chain reaction was used. Molecular cloning of RtActin was performed using the pTA2 plasmid before sequencing. We successfully isolated and cloned 592 and 840 bp of RtrbcL and RtmatK fragment genes, respectively. The RtrbcL barcoding marker, rather than the RtmatK plastidial marker, provided discriminative molecular phylogenetic data for R Trisperma. We also isolated 986 bp of RtACT gene fragments. Our phylogenetic analysis demonstrated that R trisperma is closely related to the Vernicia fordii Actin gene (97% identity). Our results suggest that RtrbcL could be further developed and used as a barcoding marker for R trisperma. Moreover, the RtACT gene could be further investigated for use in gene expression studies of plant.