Effect of Cytokinins on Micropropagation of Moringa Oleifera Lam. and Genomic Stability Assessment Using Flow-Cytometry

Moringa oleifera Lam. is a multipurpose medicinal plant of the family Moringaceae which has been widely utilized as a pharmaceutical remedy to treat a wide range of diseases. In addition, the tree has several applications in human nutrition as well as livestock feeding. M. oleifera is easily multiplied through epigeal germination (recalcitrant) but seed propagated plants are heterogeneous and take longer to reach fruit-bearing age. As an alternative, branch cuttings have been used but their establishment is erratic and often leads to reduced growth of the mother plant. Thus, to produce superior planting materials, in-vitro propagation has become paramount. As a result, several studies using a limited range of cytokinin have been undertaken to multiply M. oleifera through tissue culture. Otherwise, a study was conducted to examine the effect of five different cytokinins on in-vitro regeneration of this tree species. Results showed that nodal explants cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 6-Benzylaminopurine (BAP) and subsequently rooted on basic MS media was the most optimal treatment. Furthermore, acclimatization of plantlets in sterile soil substrate and perlite (1:3;v/v) under transparent polythene sheet for 7 days resulted in survival rate of 100%. Assessment of genetic fidelity using flow cytometry revealed that surface sterilization alongside cytokinin treatments produced plantlets that were genetically stable regardless of the growth regulator used. Thus, the in-vitro protocol developed in this study can be utilized for in-vitro studies and mass propagation of this imperative plant species.

The Effect of Polyamines and Silver Thiosulphate on Micropropagation of Date Palm Followed by Genetic Stability Assessment

There are some limitations in the practical applications of in vitro date palm tissue culture, such as low multiplication efficiency, low rooting rate, and high mortality experienced by in vitro raised plantlets during laboratory to soil transfer. This study’s objective was to investigate the effect of the two types of polyamines (putrescine "PUT" and spermidine" SPD") in combination with silver thiosulfate (STS) on the growth and development and genetic stability of cultures of Quntar cultivar. Media supplemented with 75 mg L−1 SPD in combination with 10 mgL−1 STS gave the highest percentage of callus producing buds (83.34%) and average bud formation (16.3) per jar. The addition of PUT and STS to the medium was most effective in root formation and the number of roots per shoot, where the best result 91.67% and 6.37 roots per shoot, respectively, were obtained using 75 mgL−1 PUT and 10 mgL−1 STS, resulting in fast-growing plantlets during acclimatization phase, reaching 90% of plant survival. The genetic fidelity assessment of plants derived from micropropagation was confirmed by RAPD analysis. Four operon primers were used, and all of them showed amplified unambiguous (OPA02, OPC-04, OPD-07, and OPE-15). All generated bands were monomorphic and had no variation among the tissue culture-derived plants tested. Accordingly, these results indicate that adding polyamines and silver thiosulfate to the nutrient medium of date palm cv. Quntar is beneficial in improving shoot organogenesis, rooting, and production of genetically stable date palm plants.

Rapid in-vitro micropropagation of Bamboo (Dendrocalamus strictus) and its genetic fidelity testing using ISSR markers

Bamboo is a versatile, arborescent, perennial and non-wood forest tree with tremendous commercial importance. For mass scale propagation of bamboo, the micropropagation is an effective way for producing elite, infection free and true-to-type planting material. Here, the nodal explants of Dendrocalamus strictus used to develop an effective protocol for micropropagation based on tissue culture technique. In this study, the sterilization treatment of 70% ethanol + Tween 20 + Bavistin + Hgcl2 + PPM was successfully controlled the contamination up to 90 % as compared to other treatments. The shoots were initiated from nodal segments in MS medium supplemented with BAP (4 mg/l) and PPM (500µl/l). Shoot multiplication was found best with BAP (4 mg/l) and kinetin (2 mg/l) by using liquid MS medium. Whereas, rooting in solid MS medium has shown good results when supplemented with NAA (4mg/l). Healthy and disease-free plants were obtained after hardening under greenhouse conditions. Genetic fidelity testing by using ISSR markers reported that there was no variation in plantlets developed through micropropagation.

Effects of Polyamines And Silver Thiosulphate On Phoenix Dactylifera L. Cv. Quntar Micropropagation And Molecular Analysis.

There are some limitations in the practical applications of in vitro date palm tissue culture, such as low multiplication efficiency, low rooting rate, and high mortality experienced by in vitro raised plantlets during laboratory to soil transfer. The objective of the present study is to determine the effect of polyamines (putrescine "PUT" and spermidine" SPD") and silver thiosulfate (STS) on enhancing propagation of date palm cv Quntar in vitro. Media supplemented with 75 mg L-1 SPD in combination with 10 mgL-1 STS gave the highest percentage of callus producing buds (83.34%) and average bud formation (16.3) per jar. The addition of PUT and STS to the medium was most effective in root regeneration and the number of roots per shoot, where the best result 91.67% and 6.37 roots per shoot, respectively, were obtained using 75 mgL-1 PUT and 10 mgL-1 STS, resulting in fast-growing plantlets during acclimatization phase, reaching 90% of plant survival. The genetic fidelity assessment of plants derived from micropropagation was confirmed by RAPD analysis. Four operon primers were used, and all of them showed amplified unambiguous (OPA02, OPC-04, OPD-07, and OPE-15). All generated bands were monomorphic and had no variation among the tissue culture-derived plants tested. Accordingly, these results indicate that adding polyamines and silver thiosulfate to the nutrient medium of date palm cv. Quntar is beneficial in improving shoot organogenesis, rooting, and production of genetically stable date palm plants.

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.

Breeding Aspects of Selected Ornamental Bulbous Crops

This article provides an overview of the origin, genetic diversity and methods and trends in breeding of selected ornamental geophytes (Lilium, Tulipa, Narcissus and Hippeastrum). The role of interspecific hybridisation and polyploidisation in assortment development is reviewed. A great variety of cultivars with traits of interest have been generated over the last century by using classical breeding. Geophyte breeders have been interested in a diversity of traits, including resistance to diseases, flower colour and shape, long lasting flowering and a long vase life. Shortening the long breeding process of many geophytes by reducing the juvenile phase and using in vitro techniques are reviewed. Currently, the breeding process has been enhanced by using modern molecular cytogenetic techniques. Genomic in situ hybridisation is frequently used, among other techniques, for genome differentiation in interspecific hybrids, and for assessment of the extent of intergenomic recombination in backcross progenies. Furthermore, several molecular marker techniques are used for verification of hybrid status, identification of genetic diversity, confirmation of the genetic fidelity of in vitro propagated plants and construction of high-density linkage maps. Recently, a myriad of new plant breeding technologies, such as cisgenetics and genome editing technologies have been used to improve the traits of ornamental geophytes, an endeavour that is discussed here. Breeding trends, cultivar novelties as well a new cultivars registered by international authorities during the last five years are presented in detail.

Metabolic profiling, in vitro propagation, and genetic assessment of the endangered rare plant Anarrhinum pubescens

Background Anarrhinum pubescens Fresen. (Plantaginaceae) is a rare plant, endemic to the Saint Catherine area, of South Sinai, Egypt. Earlier studies have reported the isolation of cytotoxic and anti-cholinesterase iridoid glucosides from the aerial parts of the plant. The present study aimed to investigate the chemical profiling of the wild plant shoots as well as establish efficient protocols for in vitro plant regeneration and proliferation with further assessment of the genetic stability of the in vitro regenerated plants. Results Twenty-seven metabolites have been identified in wild plant shoots using the Nuclear Magnetic Resonance (NMR) spectroscopy. The metabolites include alkaloids, amino acids, carbohydrates, organic acids, vitamins, and a phenol. In vitro propagation of the plant was carried out through nodal cutting-micropropagation and leaf segment-direct organogenesis. The best results were obtained when nodal cutting explants were cultured on Murashige and Skoog medium with Gamborg B5 vitamins supplemented with 6-benzylaminopurine (BAP) (1.0 mg/L) and naphthaleneacetic acid (NAA) (0.05 mg/L), which gave a shoot formation capacity of 100% and a mean number of shoots of 27.67 ± 1.4/explant. These shoots were successfully rooted and transferred to the greenhouse and the survival rate was 75%. Genetic fidelity evaluation of the micropropagated clones was carried out using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) molecular markers. Jaccard’s similarity coefficient indicated a similarity as high as 98% and 95% from RAPD and ISSR markers, respectively. Conclusions This study provides the chemical profiling of the aerial part of Anarrhinum pubescens. Moreover, in vitro regeneration through different tissue culture techniques has been established for mass propagation of the plant, and the genetic fidelity of the in vitro regenerated plants was confirmed as well. Our work on the in vitro propagation of A. pubescens will be helpful in ex situ conservation and identification of bioactive metabolites.