Single medium-based efficient protocols for large-scale multiplication of the rare woody aromatic medicinal plant Rotula aquatica Lour. by means of axillary bud multiplication and indirect organogenesis were established using Murashige and Skoog (MS) medium. There were no significant differences with respect to the induction of shoots per node or callus and roots per shoot on media prepared either with tap water and commercial sugar or those prepared with double distilled water and tissue culture-grade sucrose. The most effective medium for axillary bud proliferation was MS medium fortified with 1.0 mg l–1 N6-benzylaminopurine (BAP) and 0.5 mg l–1 indole-3-butyric acid (IBA), on which shoots were induced at the rate of 15 per node. The excision of node segments from the in vitro-derived shoots and their subsequent culture on medium supplemented with same concentrations of BAP and IBA facilitated enhanced axillary bud proliferation. Callus that developed from the lower cut end of the node explants induced shoots during subculture on half-strength MS medium with 1.0 mg l–1 BAP and 0.5 mg l–1 kinetin. The shoots developed rooted best on half-strength MS medium supplemented with 0.5 mg l–1 naphthaleneacetic acid (NAA). Rooted shoots, following acclimation in the greenhouse, were successfully transferred to field conditions, and 80% of the plantlets survived. When the basal ends of shoots harvested from multiplication medium were dipped in an NAA (0.5 mg l–1) solution for 25 days, a mean of 5.6 roots per shoot developed; the transfer to small pots facilitated the survival of 75% of the rooted shoots. Ex vitro rooting by direct transfer of the shoots from the multiplication medium to the greenhouse resulted in a 65% survival. Commercial sugar and tap water and ex vitro rooting make the protocol economically advantageous. About 750 plantlets were procured in a 3-month period starting from a single node explant.

Rapid in vitro multiplication and ex vitro rooting of Rotula aquatica Lour., a rare rhoeophytic woody medicinal plant.

The genus Catharanthus consists of eight species of which seven are native to Madagascar and one, C. pusillus, to India. Catharanthus roseus, Madagascar periwinkle is one of the few pharmacological plants that have a long history of therapeutic voyage from Mesopotamian folklore of 2600 BCE till today playing a considerable role as herbal and traditional medicine of various diseases. As previously published reviews on Madagascar periwinkle mostly concentrate on the pharmaceutical and chemical compounds of the herb, lack of information about the other features of the species is extremely tangible. This organised review provided insights into agro technological, biological, ecological, and medicinal aspects (particularly anticancer compounds) of Madagascar periwinkle along with possible pathways and mode of action. The diverse medicinal and therapeutic potentials of Catharanthus roseus have been revealed that this magical herb may be utilized to its full potential and future research orientation may be directed towards it.

https://www.phytojournal.com/archives/2017/vol6issue5/PartY/6-5-182-590.pdf

Madagascar periwinkle (Catharanthus roseus L.): Diverse medicinal and therapeutic benefits to humankind

Development of Agrobacterium-mediated transient transformation in persimmon (Diospyros kaki Thunb.)

Somatic embryo (bipolar) or shoot (monopolar) morphogenesis in mesophyll cells of Euphorbia nivulia Buch.-Ham in vitro was dependent on the type of auxin supplementing Murashige and Skoog (MS) medium containing benzyladenine. Direct in vitro morphogenesis, i.e., organogenesis, and somatic embryogenesis were significantly influenced by seasonal growth of the donor plant, explant position (proximal, mid, and distal), and light. Explants collected in march/April were superior to July/August material. Proximal explants underwent morphogenesis more readily than mid- and tip-derived explants. Incubation in the light favored morphogenesis while darkness was inhibitory. Kinetin (Kn) was also inhibitory to morphogenesis. MS medium enriched with different levels of N6-benzyladenine (BA) alone, or in combination with α-naphthaleneacetic acid (NAA) or indole-3-acetic acid (IAA), induced adventitious shoots directly. Explants collected in March/April cultured on medium with 13.3 μM BA and 2.69 μM NAA developed the highest number of shoots, a mean of 15.2 shoots per proximal explant. Developed shoots rooted the best on half-strength MS medium with 2.46 μM indole-3-butyric acid, which developed a mean of 5.2 roots per shoot. Rooted healthy shoots could be transplanted to small pots, with an 80% survival rate. Addition of 2,4-dichlorophenoxyacetic acid (2.4-D) to BA-supplemented medium was obligatory to develop somatic embryos. MS medium containing 2.26 μM 2,4-D and 4.44 μM BA induced a mean of 44.8 somatic embryos per proximal explant. The embryos passed through distinct stages of embryogenesis, namely globular, heart, torpedo, and early cotyledonary. The embryos (88%) underwent maturation on half-strength MS medium with 2.89 μM gibberellic acid (GA3), and its subsequent transfer on half-strength MS basal medium in light conditions facilitated 80% conversion of embryos to plantlets. Direct shoots or embryos were originated from the mesophyll cells. Somatic embryo development was concurrent with the independent origin of vasculature in the bulbous basal portion. The survival rate of embryo-derived plants was 90%.

Influence of auxins in direct in vitro morphogenesis of euphorbia nivulia, a lectinaceous medicinal plant

An efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation using cotyledon explants of oriental melon (Cucumis melo L. var. makuwa) has been developed. All six oriental melon cultivars evaluated in the study showed a >90 % shoot regeneration frequency and produced 1.8–3.6 shoots per cotyledon explant when cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg L−1 benzyladenine and 0.01 mg L−1 indoleacetic acid. Kanamycin (Km) and geneticin (Gt) in the shoot induction medium (SIM) were compared both qualitatively and quantitatively for their efficiency as a selection agent for the selection and regeneration of transgenic plants after Agrobacterium-mediated transformation. Shoot formation was completely inhibited at 50 mg L−1 Km and 10 mg L−1 Gt. Relatively high concentrations of both Gt and Km (>100 mg L−1 Km and >25 mg L−1 Gt) were necessary because large numbers of non-transgenic shoots survived during the selection process. The incorporation of a selectable marker (neomycin phosphotransferase II) into the genome of transgenic plants was confirmed using β-glucuronidase (GUS), PCR and Southern blot analysis. Shoot regeneration frequencies were 41.2 % at 100 mg L−1 Km and 15.2 % at 30 mg L−1 Gt 8 weeks after transformation, whereas the transformation frequencies based on the PCR were 2.9 and 7.1 %, respectively, 16 weeks after transformation. These results demonstrate that a large portion of the regenerated shoots on SIM supplemented with 100 mg L−1 Km consisted of non-transformed or escaped shoots, indicating that 30 mg L−1 Gt is the more suitable for the selection and regeneration of transgenic plants in oriental melon.

An efficient selection and regeneration protocol for Agrobacterium -mediated transformation of oriental melon ( Cucumis melo L. var. makuwa )

An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki Thunb.) cultivars ‘Fuyu’ and ‘Nishimurawase’ has been developed. The regeneration capacity was influenced by the culture vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were cultured on a modified Murashige and Skoog medium (MS1/2N), for 16 wk without transfer to fresh medium. Adventious shoots appeared after 4 and 8 wk in culture of ‘Nishimurawase’ and ‘Fuyu’ tissues, respectively. The culture of leaf explants in Erlenmeyer flasks with medium containing 4 g l−1 agar enhanced shoot formation in comparison to media with increased agar concentrations. Optimal shoot regeneration was obtained with 5 mg l−1 (22.8 μM) zeatin and 0.1 mg l−1 (0.05 μM) indole-3-butyric acid (IBA) for ‘Nishimurawase’, and 10 mg l−1 (45.6 μM) zeatin and 0.1 mg l−1 (0.05 μM) IBA for ‘Fuyn’. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to 9.2 for ‘Nishimurawase’ and 2.2 for ‘Fuyu’. Dark incubation during the first 4–5 wk was the most effective condition to successfully influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot formation by ‘Fuyu’, it was only slightly beneficial to ‘Nishimurawase’. More than 80% of the regenerated shoots rooted within 4 wk on hormone-free MS1/2N demium after having been dipped for 30 s in 250 mg l−1 (1.1. mM) IBA solution.

C. H. Lee

Papers

Efficient and simple plant regeneration via organogenesis from leaf segment cultures of persimmon (Diospyros kaki thunb.)