Polyamines- and growth inducers-mediated enhanced mono-phasic in vitro regeneration of sugar leaf plant (Stevia rebaudiana Bert.) in liquid medium

• Published in: South African journal of botany Vol. 173; pp. 34 - 45
• Main Authors: Subrahmanyeswari, Tsama, Gantait, Saikat, Sarkar, Rittika, Kamble, Suchita N., Singh, Sudhir, Bhattacharyya, Somnath
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01-10-2024
• Subjects: meta-Topolin; Putrescine; Salicylic acid; Silver nitrate; Spermidine; Spermine; Salicylic acid; SPD; MSL; mT; IBA; Putrescine; Silver nitrate; PUT; SA; PCA; KIN; SPM; Spermine; meta-Topolin; Spermidine; SN; MJ; PGRs; BA
• ISSN: 0254-6299
• DOI: 10.1016/j.sajb.2024.08.003

•Stevia is an industrially and medicinally valued herb from Asteraceae family.•First report on polyamine- and growth inducer-mediated mono-phasic regeneration in liquid medium.•meta-Topolin-putrescine combination enhanced in vitro growth and proliferation.•Cumulative impact on growth and development was assessed via multiple statistical analyses.•Successfully developed mono-phasic in vitro regenerationn protocol with reduced production time.
In the present study, a high-frequency simultaneous muliple shoot-root regeneration protocol for stevia using a liquid medium was developed successfully. Shoot tip explants were grown in Murashige and Skoog liquid (MSL) medium supplemented with different cytokinins i.e., 6-benzyladenine (BA), kinetin (KIN), and meta-Topolin (mT) in three varied concentrations in combination with 4.9 µM indole-3-butyric acid (IBA) to evaluate their performance on mono-phasic growth and development traits. The mT (6.2 µM) + IBA (4.9 µM) combination induced the highest number of de novo shoots, leaves, and roots (6.3, 30.7, and 5.3, respectively) compared to other treatments during the four-week culture. To assess the effect of polyamines and growth inducers on multiple shoot proliferation, shoot tips from optimized multiple shoot cultures were grown individually in putrescine (PUT), spermidine, spermine, silver nitrate, and salicylic acid (at 100–500 µM) supplemented medium for four weeks and compared against control (without polyamines or growth inducers). The cumulative and individual impact of these polyamines and growth inducers on in vitro growth parameters were comprehensively assessed by employing combined jitter plot and box plot, correlation, network plot, principal component analysis, and unweighted pair-group method with arithmetic mean clustering based on the Euclidean distance matrix. Among different treatments, 200 µM PUT exhibited the most promising results in terms of earliest shoot-root formation (4.7 days, 7.0 days), maximum shoot-root proliferation (counting 12.0, 11.0), and elongation (97.3 mm, 50.0 mm), having maximum leaf count (87). This protocol should be useful to meet the ever-increasing consumers’ demand for stevia plantlets, a source of commercially valuable natural sweetener.