Cytokinin-Based Tissue Cultures for Stable Medicinal Plant Production: Regeneration and Phytochemical Profiling of Salvia bulleyana Shoots

Salvia bulleyana is a rare Chinese medicinal plant that due to the presence of polyphenols lowers the risk of some chronic diseases especially those related to the cardiovascular system. The present study examines the organogenic competence of various combinations and concentrations of plant growth regulators to develop an efficient protocol for in vitro regeneration of S. bulleyana via leaf explants, maintaining the high production of active constituents. The purpose of the study was also to assess the possibilities of using a cytokinin-based regeneration to effectively produce therapeutic compounds. The adventitious shoot formation was observed through direct organogenesis on media with purine derivatives (meta-topolin, mT and benzylaminopurine, BAP), and through indirect organogenesis on media with urea derivatives (tidiazuron, TDZ and forchlorfenuron, CPPU). The highest regeneration frequency (95%) with 5.2 shoots per explant was obtained on leaves cultured on Murashige and Skoog (MS) medium containing 0.1 mg/L naphthalene-1-acetic acid (NAA) and 2 mg/L BAP. Following inter simple sequence repeat (ISSR) marker-based profiling, the obtained organogenic shoot lines revealed a similar banding pattern to the mother line, with total variability of 4.2–13.7%, indicating high level of genetic stability. The similar genetic profile of the studied lines translated into similar growth parameters. Moreover, HPLC analysis revealed no qualitative differences in the profile of bioactive metabolites; also, the total polyphenol content was similar for different lines, with the exception of the shoots obtained in the presence of CPPU that produced higher level of bioactive compounds. This is the first report of an effective and rapid in vitro organogenesis protocol for S. bulleyana, which can be efficiently employed for obtaining stable cultures rich in bioactive metabolites.

Development of a Rapid Selection System for Salt-Resistant Mutants of Nicotiana benthamiana through Protoplast Culture after Gamma Irradiation

We aimed to develop a novel technology capable of rapidly selecting mutant plant cell lines. Salt resistance was chosen as a rapid selection trait that is easily applicable to protoplast-derived cell colonies. Mesophyll protoplasts were cultured in a medium supplemented with 0, 50, 100, 150, 200, 250, and 300 mM NaCl. At NaCl concentrations ≥ 100 mM, cell colony formation was strongly inhibited after 4 weeks of culture. Tobacco protoplasts irradiated with 0, 50, 100, 200, and 400 Gy were then cultured to investigate the effects of radiation intensity on cell division. The optimal radiation intensity was 50 Gy. To develop salt-resistant tobacco mutant plants, protoplasts irradiated with 50 Gy were cultured in a medium containing 100 mM NaCl. The efficiency of cell colony formation from these protoplasts was approximately 0.002%. A salt-resistant mutant callus was selected and proliferated in the same medium and then transferred to a shoot inducing medium for adventitious shoot formation. The obtained shoots were then cultured in a medium supplemented with 200 mM NaCl and developed into normal plantlets. This rapid selection technology for generating salt-resistant tobacco mutants will be useful for the development of crop varieties resistant to environmental stresses.

The Effect of Sodium Butyrate on Adventitious Shoot Formation Varies among the Plant Species and the Explant Types

Histone acetylation plays an important role in plant growth and development. Here, we investigated the effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on adventitious shoot formation from protoplast-derived calli and cotyledon explants of tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum). The frequency of adventitious shoot formation from protoplast-derived calli was higher in shoot induction medium (SIM) containing NaB than in the control. However, the frequency of adventitious shoot formation from cotyledon explants of tobacco under the 0.1 mM NaB treatment was similar to that in the control, but it decreased with increasing NaB concentration. Unlike in tobacco, NaB decreased adventitious shoot formation in tomato explants in a concentration-dependent manner, but it did not have any effect on adventitious shoot formation in calli. NaB inhibited or delayed the expression of D-type cyclin (CYCD3-1) and shoot-regeneration regulatory gene WUSCHEL (WUS) in cotyledon explants of tobacco and tomato. However, compared to that in control SIM, the expression of WUS was promoted more rapidly in tobacco calli cultured in NaB-containing SIM, but the expression of CYCD3-1 was inhibited. In conclusion, the effect of NaB on adventitious shoot formation and expression of CYCD3-1 and WUS genes depended on the plant species and whether the effects were tested on explants or protoplast-derived calli.

Temporal and Spatial Expression Analysis of Shoot-Regeneration Regulatory Genes during the Adventitious Shoot Formation in Hypocotyl and Cotyledon Explants of Tomato (CV. Micro-Tom)

Enhancing the competence for plant regeneration in tissue culture studies is an important issue not only for efficient genetic transformation of commercial crops but also for the reproducibility of scientific reports. In this study, we investigated optimization of several tissue culture conditions including plant growth regulators, types and ages of explants, culture densities, and plant position in order to improve the competence of adventitious shoot formation of the tomato (Solanum lycopersicum cv. Micro-Tom). In addition, we examined the differential expression of D-type cyclin (CYCD3-1) and several shoot regeneration regulatory genes from hypocotyl and cotyledon explants of tomato during shoot organogenesis. A treatment of 1 mg L−1 Zeatin and 0.1 mg L−1 Indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium containing 3% sucrose was optimal for adventitious shoot formation from hypocotyl and cotyledon explants. The younger explants exhibited more shoot formation regardless of explant types. Additionally, those closest to the shoot apical meristem produced more shoots compared to the other regions in the hypocotyl and the cotyledon explants. Gene expression of CYCD3-1, SHOOT MERISTEMLESS (STM), and cytokinin dependent WUSCHEL (WUS) was significantly higher in younger explants than in older ones. Furthermore, an increase in CYCD3-1, STM, and WUS expression was evident at the distal part of hypocotyls and the proximal part of cotyledons compared to other regions. These differential gene expression profiles exhibited good agreement with the results of shoot formation obtained from diverse explants of tomato. These results suggest that temporal and spatial gene expression of shoot regeneration regulatory genes plays an important role in enhancing the competence and the reproducibility of adventitious shoot formation from tomato explants.

Comparative Efficiency Analysis of Different Explants and Contribution of Polyamines on in vitro Propagation of Dendrobium Hybrid Sonia

An efficient method of plant regeneration and subsequent growth and development of Dendrobium hybrid Sonia was developed using intact seedling, shoot tip and thin cell layer. Maximum number of PLBs was obtained in MS liquid medium supplemented with 0.5 mg/l NAA and 1mg/l BAP through shoot tip and thin cell layer culture (TCL). Highest number of adventitious shoot formation was recorded in 0.5 mg/l of NAA and 2 mg/l of BAP through shoot tip culture. In shoot tip and TCL cultures, the necrosis was checked in presence of NAA (0.5 mg/l) and BAP (1 mg/l). Maximum callus frequency was recorded in NAA and BAP (0.5 mg/l) through thin cell layer culture. Direct PLB formation was better at 1.0 μM concentration in all the three polyamines tested. Three polyamines tested were effective in direct PLB formation as well as adventitious shoots. Plant Tissue Cult. & Biotech. 30(1): 77-86, 2020 (June)

Micropropagation and in vitro rooting of Robinia pseudoacacia L. recalcitrant genotypes

In forest production, there is an emerging tendency towards the planting of fast-growing trees as attractive, renewable energy sources. Hence, efforts were made to develop a method of micropropagation by organogenesis of seven clones of black locust (Robinia pseudoacacia L.) that are resistant to propagation by traditional vegetative methods, as well as one plus tree (no. 9755) at the age of 60, to see if the age of the mother plant is a limitation in the micropropagation of black locust trees. Overall results suggest that Murashige and Skoog medium supplemented with 30 g l−1 sucrose, 0.6 mg l−1 6-benzylaminopurine (BAP) and 0.1 mg l−1 naphthalene acetic acid (NAA) is better for the propagation of each genotype of R. pseudoacacia than Woody Plant Medium with the same growth regulators, and the age of the donor plant does not affect the organogenic potential. Recalcitrance to adventitious rooting from adventitious shoot formation is a major limitation for the clonal micropropagation of forest trees. Our results showed that although the roots were also formed spontaneously in the growth medium without growth hormones for the tested black locust clones, the application of auxin increased the total root length compared to that in the medium with active carbon and control. A significant effect of the additives of hormone and sucrose on the total root length was found. Increasing the sucrose concentration stimulated the induction of roots in each of the tested concentrations (5, 10, 15 or 20 g l−1). Additionally, the change in sugar dose in the rooting medium caused significant differences in total root length.

Development of an In Vitro Propagation Protocol and a Sequence Characterized Amplified Region (SCAR) Marker of Viola serpens Wall. ex Ging

An efficient protocol of plant regeneration through indirect organogenesis in Viola serpens was developed in the present study. Culture of leaf explants on MS (Murashige and Skoog) medium supplemented with 2.0 mg/L 6-benzyladenine and 0.13 mg/L 2,4-dichloro phenoxy acetic acid. Adventitious shoot formation was observed when calli were transferred on to MS medium containing 0.5 mg/L α-naphthalene acetic acid and 2.25 mg/L kinetin, which showed the maximum 86% shoot regeneration frequency. The highest root frequency (80.92%) with the 5.6 roots per explant and 1.87 cm root length was observed on MS medium supplemented with 2 mg/L indole-3-butyric acid. The plantlets were transferred to the mixture of sand, coffee husk and soil in the ratio of 1:2:1 in a pot, and placed under 80% shade net for one month. It was then transferred to 30% shade net for another one month, prior to transplantation in the field. These plantlets successfully acclimatized under field conditions. A Sequence Characterized Amplified Region (SCAR) marker was also developed using a 1135 bp amplicon that was obtained from RAPD (Random Amplification of Polymorphic DNA) analysis of six accessions of V. serpens. Testing of several market samples of V. serpens using the SCAR marker revealed successful identification of the genuine samples of V. serpens. This study, therefore, provides a proficient in vitro propagation protocol of V. serpens using leaf explants and a SCAR marker for the authentic identification of V. serpens. This study will be helpful for conservation of authentic V. serpens.