Micropropagation and In Vitro Inflorescence of Pentas lanceolata

In this study, different concentrations of 6-benzyladenine (BA) on in vitro shoot and inflorescence inductions of P. lanceolata were investigated. The in vivo and in vitro floral characteristics of this plant were also compared. Nodal explants of P. lanceolata were cultured vertically with the cut ends inserted into semi-solid Murashige and Skoog (MS) medium supplemented with 0, 0.5, 1, 2, 4, and 8 mg L–1 BA. The results showed that the explants formed the highest numbers of shoots even when cultured in MS basal medium without any addition of BA, while the shoots formed in the explants cultured in MS medium supplemented with 1 mg L–1 BA were the longest. No inflorescence was found in the shoots cultured in MS medium supplemented with 8 mg L–1 BA, while the highest percentage of inflorescence induction was found in the shoots cultured in the medium supplemented with 0.5 mg L–1 BA. The apperances of in vivo and in vitro flowers of P. lanceolata were the same in many aspects except that the number of flower/inflorescence formed was different. In addition, water accumulation was observed only inside the in vitro flowers. Water deposit in the long tubular structure of P. lanceolata flower could cause anther injury, suggesting that flowers developed in vitro may not always produce pollen.

In Vitro Shoot Culture of Sesuvium portulacastrum: An Important Plant for Phytoremediation

Sesuvium portulacastrum L., a member of the family Aizoaceae, is an important coastal halophyte. Due to its adaptability to salinity and heavy metals, S. portulacastrum has now been widely used for the phytoremediation of saline soils and wastewater and the protection of the coast from erosion. The increasing use of this plant requires a large number of propagules. Stem cutting propagation and seed germination cannot meet this demand, and such propagations can initiate and spread diseases. A recent occurrence of Bipolaris sesuvii J.Z. Zhang and Gibbago trianthemae E.G. Simmons in S. portulacastrum resulted in the substantial loss of the plants during the remediation of aquaculture wastewater. Thus, there is an urgent need for establishing efficient methods of propagating disease-free starting materials. In the present study, we evaluated different growth regulators in the induction of axillary shoots from nodal explants cultured on Murashige and Skoog medium and identified that zeatin (ZT) and α-naphthaleneacetic acid (NAA) was an appropriate combination for inducing high numbers of axillary shoots. The nodal explants were then cultured on MS medium supplemented with different concentrations of ZT and NAA, and the combination of ZT at 1.0 mg L−1 and NAA at 0.3 mg L−1 induced more than 12 axillary shoots per explant. The axillary shoots were excised to produce microcuttings or microshoots, which were rooted on half-strength MS medium supplemented with different concentrations of indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). The results showed that IBA at 0.6 mg L−1 induced 91.7% of the microcuttings to root with root numbers of over 36 per cutting. The rooted plantlets were healthy and true-to-type and grew vigorously in plug trays or plastic containers with a 100% survey rate in a greenhouse. Thus, this established protocol could be used for the rapid propagation of genetically identical and disease-free plants of S. portulacastrum for phytoremediation and the protection of shoreline soils from erosion.

Multiple shoot induction and regeneration of Vanilla borneensis Rolfe - a critically endangered orchid of Assam, India

In the present investigation, a micropropagation protocol has been developed for Vanilla borneensis Rolfe – a critically endangered orchid through multiple shoot regeneration. Through in vitro multiple shoot regeneration from both nodal and shoot tip explants, maximum (100%) shoot induction was observed. The minimum time required for shoot bud induction was observed from the shoot tip (5–7 days) on medium supplemented with BAP (4.44 mM) + KIN (2.32 mM) as compared to the nodal explants. Maximum multiple shoot regeneration was observed from nodal explants on the medium supplemented with BAP (4.44 mM) + TDZ (6.82 mM). However, maximum shoot length was observed on the medium supplemented with BAP (4.44 mM) + 15% CW and the number of nodes (5.27±0.33) per shoot after 90 days. Maximum (80-100%) of root initiation was observed in almost all the concentrations of NAA. The shortest time of root initiation was found on the medium supplemented with NAA (5.37 mM). Further, acclimatization period was found to be 15 days with 70% acclimatization while 60% of survivability was observed in the field condition. This efficient micropropagation method of V. borneensis could be successfully used for mass propagation as well as conservation of the critically endangered wild orchid.

Is Endogenous Ethylene Production Associated with Increased Shoot Number in Gentian Cultured In Vitro? Developing a Method for Ethylene Measurement at Nano Level within a Gas-exchangeable In Vitro System

We investigated the possibility of either exogenous ethylene or endogenous ethylene production having an association with the increase in shoot number when nodal explants of Gentiana spp. ‘Little Pinkie’ were cultured in an in vitro medium supplemented with ethephon (10 mg⋅L–1). For the first time within an in vitro system, we report the application of laser ethylene detector technology, and optimization of the methodology to quantify concentrations of ethylene (in the part-per-billion range) released from ethephon decomposition within the atmosphere of gas-exchangeable culture vessels including nodal explants. Compared with continuous (continuous measurements on the same replicate of vessels) and repeated (sampling same replicate of vessels every 48 hours) sampling methodologies, the nonrepeated (sampling fresh replicate of vessels every 48 hours) method of measurement of ethylene concentration was more representative of the actual condition within vessels. Although no prior published data exist showing the positive or negative effect of gaseous ethylene in the headspace of culture vessels on bud outgrowth in gentian, our study shows gaseous ethylene in the headspace of culture vessels was not effective in increasing shoot formation in gentian explants cultured in vitro, whereas ethephon supplementation in agar was effective. Plant material in culture vessels did not have a significant effect on ethylene production regardless of the presence or absence of ethephon. Therefore, although ethephon supplementation in the medium produced gaseous ethylene in the headspace, it was unlikely to cause endogenous ethylene production in explants, but it did trigger shoot formation in ‘Little Pinkie’, perhaps through decomposition to ethylene within the explant tissue, enhancing the internal ethylene level possibly at a locally high concentration.

Variables Affecting Shoot Growth and Plantlet Recovery in Tissue Cultures of Drug-Type Cannabis sativa L.

Tissue culture approaches are widely used in crop plants for the purposes of micropropagation, regeneration of plants through organogenesis, obtaining pathogen-free plantlets from meristem culture, and developing genetically modified plants. In this research, we evaluated variables that can influence the success of shoot growth and plantlet production in tissue cultures of drug-type Cannabis sativa L. (marijuana). Various sterilization methods were tested to ensure shoot development from nodal explants by limiting the frequency of contaminating endophytes, which otherwise caused the death of explants. Seven commercially grown tetrahydrocannabinol (THC)-containing cannabis genotypes (strains) showed significant differences in response to shoot growth from meristems and nodal explants on Murashige and Skoog (MS) medium containing thidiazuron (1 μM) and naphthaleneacetic acid (0.5 μM) plus 1% activated charcoal. The effect of Driver and Kuniyuki Walnut (DKW) or MS basal salts in media on shoot length and leaf numbers from nodal explants was compared and showed genotype dependency with regard to the growth response. To obtain rooted plantlets, shoots from meristems and nodal explants of genotype Moby Dick were evaluated for rooting, following the addition of sodium metasilicate, silver nitrate, indole-3-butyric acid (IBA), kinetin, or 2,4-D. Sodium metasilicate improved the visual appearance of the foliage and improved the rate of rooting. Silver nitrate also promoted rooting. Following acclimatization, plantlet survival in hydroponic culture, peat plugs, and rockwool substrate was 57, 76, and 83%, respectively. The development of plantlets from meristems is described for the first time in C. sativa and has potential for obtaining pathogen-free plants. The callogenesis response of leaf explants of 11 genotypes on MS medium without activated charcoal was 35% to 100%, depending on the genotype; organogenesis was not observed. The success in recovery of plantlets from meristems and nodal explants is influenced by cannabis genotype, degree of endophytic contamination of the explants, and frequency of rooting. The procedures described here have potential applications for research and commercial utility to obtain plantlets in stage 1 tissue cultures of C. sativa.

Cryopreservation of 13 Commercial Cannabis sativa Genotypes Using In Vitro Nodal Explants

Cannabis has developed into a multi-billion-dollar industry that relies on clonal propagation of elite genetics with desirable agronomic and chemical phenotypes. While the goal of clonal propagation is to produce genetically uniform plants, somatic mutations can accumulate during growth and compromise long-term genetic fidelity. Cryopreservation is a process in which tissues are stored at cryogenic temperatures, halting cell division and metabolic processes to facilitate high fidelity germplasm preservation. In this study, a series of experiments were conducted to optimize various stages of cryopreservation and develop a protocol for long-term germplasm storage of Cannabis sativa. The resulting protocol uses a standard vitrification procedure to cryopreserve nodal explants from in vitro shoots as follows: nodes were cultured for 17 h in a pre-culture solution (PCS), followed by a 20-min treatment in a loading solution (LS), and a 60 min incubation in plant vitrification solution 2 (PVS2). The nodes were then flash frozen in liquid nitrogen, re-warmed in an unloading solution at 40 °C, and cultured on basal MS culture medium in the dark for 5 days followed by transfer to standard culture conditions. This protocol was tested across 13 genotypes to assess the genotypic variability. The protocol was successful across all 13 genotypes, but significant variation was observed in tissue survival (43.3–80%) and regrowth of shoots (26.7–66.7%). Plants grown from cryopreserved samples were morphologically and chemically similar to control plants for most major traits, but some differences were observed in the minor cannabinoid and terpene profiles. While further improvements are likely possible, this study provides a functional cryopreservation system that works across multiple commercial genotypes for long-term germplasm preservation.

An efficient protocol for in vitro regeneration from the nodal explants of Withania coagulans (Stocks) Dunal: a valuable medicinal herb

<p>In order to develop a protocol for the effective micropropagation of the important medicinal plant Withania coagulans (Stocks) Dunal, the effects of different concentrations and combinations of growth regulators on the nodal explants in two independent experiments were investigated. For shooting, a MS medium fortified with different concentrations and combinations of IBA (0.01, 0.1 and 0.5 mg l-1), BA (0.5, 1 and 2 mg l-1), Kin (0.5 and 1 mg l-1), PG (0.5 mg l-1) and GA (0.5 mg l-1) was used and the highest shooting response, shoot number and shoot length were obtained in the MS + IBA (0.01 mg l-1) + BA (0.5 mg l-1) + PG (0.5 mg l-1) + GA (0.5 mg l-1) treatment. In the second experiment, the effect of MS supplemented with different combinations and concentrations of IBA (0.1, 0.5, 1 and 2 mg l-1), NAA (0.1 and 1 mg l-1) and PG (1 mg l-1) on rooting of the nodal explants was investigated, which showed that the highest rooting response (%) was observed in the MS fortified with NAA (0.1 mg l-1), NAA (1 mg l-1), NAA (0.1 mg l-1) + PG (1 mg l-1), and NAA (1 mg l-1) + PG (1 mg l-1) treatments, as well as the highest number of roots at NAA (0.1 mg l-1) and the highest root length at IBA (1 mg l-1). Our findings highlight a complete micropropagation method for W. coagulans from the nodal explant that can make a significant contribution to the development of W. coagulans material for medical applications.</p>