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.

Differential Expression of lncRNAs and miRNAs Between Self-Rooting Juvenile and Donor Clones Unveils Novel Insight Into the Molecular Regulation of Rubber Biosynthesis in Hevea brasiliensis

The rubber tree (Hevea brasiliensis Muell. Arg.) is a tropical tree species that produce natural rubber. Self-rooted juvenile clones (SRJCs) are novel rubber tree planting materials developed through primary somatic embryogenesis. SRJCs have a higher rubber yield compared with donor clones (DCs). The molecular basis underlying increased rubber yield in SRJCs remains largely unknown. Here, the latex from SRJCs and DCs were collected for strand-specific and small RNA-seq methods. A total of 196 differentially expressed long noncoding RNAs (DELs), and 11 differentially expressed microRNAs were identified in latex between SRJCs and DCs. Targeted genes of DELs were markedly enriched for various biological pathways related to plant hormone signal transduction, photosynthesis, glutathione metabolism, and amino acids biosynthesis. DELs probably acted as cis-acting regulation was calculated, and these DELs relevant to potentially regulate rubber biosynthesis, reactive oxygen species metabolism, and epigenetic modification. Furthermore, the DELs acting as microRNA targets were studied. The interaction of microRNA and DELs might involve in the regulation of natural rubber biosynthesis.

Micropropagation of Feverfew (Tanacetum parthenium) and Quantification of Parthenolide Content in Its Micropropagated and Conventionally Grown Plants

Feverfew (Tanacetum parthenium) is a well-known multi-functional plant with anti-inflammatory, cardiotonic, antiangiogenic, and anticancer effects. The therapeutic value of this plant is due to its phytochemical constitutes, especially parthenolide. Tissue culture techniques have been applied to improve the bioactive components of many herbal plants. Hence, this study, was carried out to establish a protocol for micropropagation of the feverfew plant and to quantify parthenolide content in its micropropagated and conventionally grown plants. To establish an aseptic culture, different concentrations of sodium hypochlorite (NaOCl) were investigated for seed surface sterilization. Besides, the effects of plant growth regulators (PGRs) on the callus induction, shoot organogenesis from callus and in vitro rooting were evaluated. Additionally, the parthenolide yield of the micropropagated and conventionally grown plants was determined by using high-performance liquid chromatography (HPLC). The results showed that surface sterilization of feverfew seeds with 6% NaOCl for 15 min obtained 65.00 ± 2.69% aseptic seeds. Murashige and Skoog (MS) medium supplemented with 0.4 mg/L thidiazuron (TDZ) and 2 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) resulted in 86.00 ± 1.72% callus induction. The highest number of shoots (5.00 ± 0.15) per explant was obtained in the treatment of MS medium supplemented with 5 mg/L zeatin. MS medium fortified with 3 mg/L indole-3-butyric acid (IBA) produced the maximum number of roots per plantlet (8.90 ± 0.35). A total of 90% of the micropropagated plantlets survived when planted in perlite + peat moss (1:1 v/v); the micropropagated plantlets were successfully established in the ex vitro conditions. According to parthenolide analysis, its level was significantly higher in the micropropagated plants than conventionally grown plants. Among different solvents, ethanolic extraction obtained the highest parthenolide content of the feverfew plant. Hence, it can be concluded that micropropagation of feverfew could be applied to produce disease-free planting materials and to improve the parthenolide content of the feverfew plant.

Geographical Distribution and Genetic Diversity of the Banana Fusarium Wilt Fungus in Laos and Vietnam

Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), poses a major threat to global banana production. The tropical race 4 (TR4) variant of Foc is a highly virulent form with a large host range, and severely affects Cavendish bananas. Foc TR4 was recently observed within the Greater Mekong Subregion, after Chinese private companies expanded Cavendish production to the region. In this study, extensive surveys conducted across Laos and Vietnam show that Foc TR4 is still mainly constricted to the northern regions of these countries and is limited to Cavendish cultivation settings. In Laos, Foc TR4 is associated with large-scale Cavendish plantations owned by or involved with Chinese companies through which infected planting material could have been imported. In Vietnam, mostly small-holder Cavendish farmers and backyard gardens were affected by Foc TR4. In Vietnam, no direct link is found with Chinese growers, and it is expected the pathogen mainly spreads through local and regional movement of infected planting materials. Foc TR4 was not recorded on banana cultivars other than Cavendish. The extensively cultivated ‘Pisang Awak’ cultivar was solely infected by VCGs belonging to Foc race 1 and 2, with a high occurrence of VCG 0123 across Laos, and of VCG 0124/5 in Vietnam. Substantial diversity of Foc VCGs was recorded (VCGs 0123, 0124/5, 01218 and 01221) from northern to southern regions in both countries, suggesting that Fusarium wilt is well established in the region. Interviews with farmers indicated that the local knowledge of Fusarium wilt epidemiology and options for disease management was limited. Clear communication efforts on disease epidemiology and management with emphasis on biosecurity practices need to be improved in order to prevent further spread of Foc TR4 to mixed variety smallholder settings.

In vitro Microrhizome Induction and Essential Oil Production from Aromatic Ginger Kaempferia galanga : An Economically Important Medicinal Herb

Kaempferia galanga L. or ‘aromatic ginger’ is a stem-less herb in Zingiberaceae having different pharmacological properties like antioxidant, antimicrobial, nemeticidal, vasorelaxant and wound healing activity. The plant is generally a vegetatively propagated annual herb; its conservation using conventional methods takes more time to get sufficient amount of planting materials for commercial cultivation. Micropropagation by in vitro methods helps to overcome the present demand for this high sought medicinal and aromatic species. At present the concern on in vitro propagation is directed to rhizome or storage organ induction for productive acclimatization and to reduce the injury during transportation. Microrhizomes are the small rhizomes developed in in vitro conditions and its induction is an effective biotechnological tool for the production of quality planting materials as they are genetically stable and disease free. The present study is discussing the role of silver nitrate (AgNO3) along with sucrose in in vitro microrhizome induction in K. galanga for the first time. MS medium fortified with 2.0 mgl-1 AgNO3 along with 6% (w/v) sucrose produced maximum amount of microrhizomes i.e., 4.52±0.11 g after 3 months that increased to 5.70±0.20 g in six months of harvesting. Here we also reports the comparative analysis of chemical constituents in the essential oil of in vivo rhizomes and in vitro microrhizome through GC-MS analysis that further reveals the superior characteristics of the microrhizomes in terms of the bioactive components ethyl p-methoxy cinnamate and ethyl cinnamate, the esters that contribute the nematicidal, antituberculosis, anti-inflammatory, antifungal and larvicidal properties to the oil. This protocol for in vitro microrhizome induction can be used for the commercial production of rhizomes and essential oil in K. galanga and the outcome of this study can be further used for mass production of pathogen-free microrhizomes and conservation for its sustainable utilization of the species.

The Response of Vegetable Sweet Potato (Ipomoea batatas Lam) Nodes to Different Concentrations of Encapsulation Agent and MS Salts

As an emerging technology, shoot encapsulation has been employed in germplasm conservation, distribution, and micropropagation of elite plant species. However, the production of synthetic seeds of sweet potato via non-zygotic embryogenesis requires a large number of embryos per cultured callus suspension and is labour-intensive. Here, we reported a simple method of encapsulating in vitro derived vegetable sweet potato nodal segments with sodium alginate, calcium chloride (CaCl2), and Murashige and Skoog (MS) salts. The nodes encapsulated with 4% sodium alginate (w/v) and 100 mM CaCl2 were the most suitable for propagation. They had uniform spherical beads and took the least number of days to shoot and root emergence. These plantlets produced more leaves, roots, and long shoots. Further evaluation of the MS salts concentration revealed that the plantlets encapsulated and grown with ½ MS salts had the least days to shoot and root emergence. They also had a longer shoot, the highest conversion rate (99%), and the least leaf abscission (17%). Thus, the sweet potato nodal segments encapsulated with 4% sodium alginate, 100 mM CaCl2, and ½ MS salts could be used as excellent material for micropropagation, germplasm conservation, and exchange of sweet potato planting materials.

Farmers' Sources and Varieties of Cassava Planting Materials in Coastal Kenya

Cassava (Manihot esculenta Crantz) grows in diverse agro-ecological zones. In Kenya, it is widely cultivated in Western and Coastal regions. It is mainly grown for food and nutrition security and excess roots are sold to generate income for the farm households. Its productivity per unit of the land area is high compared to maize and wheat which are staple crops in the country. However, scarcity of cassava planting materials and pests and diseases limit production in these regions. This study aimed at revealing the sources and varieties of cassava planting materials used by farmers and other farmers' practices in coastal Kenya. Four focus group discussions (FGD) and a survey were conducted in 2018, using a semi-structured questionnaire targeting 250 farmers. The data collected on the sources of planting materials, preferred varieties and the practices employed by the farmers in cassava production, was analyzed using descriptive statistics. Majority of farmers (83%) interviewed indicated that they recycled planting materials from the previous crop while some 67% respondents obtained the planting material from their neighbors. Kenya Agricultural and Livestock Research Organization's (KALRO) and the local markets were reported as sources of planting materials by 11 and 5% farmer respondents, respectively. The only formal seed source reported was KALRO. The rest, own seed, neighbors, and the local markets, were informal seed sources. Piecemeal harvesting practiced by 98% of the farmers favored planting material recycling. Farmers dedicated a small proportion of their land (mean 0.2 ha) to cassava production as reported by 60 and 72.8% of farmers from Taita Taveta and Kilifi, respectively. Slightly above half (56%) of the farmers in Kilifi dedicated slightly more land to cassava, planting between 100 and 4,000 cuttings (2.5% of an acre up to a full acre) compared to 87% farmers from Taita Taveta who planted 100 cuttings or less (which is about 2.5% of an acre or less). A majority (81.1%) of farmers in Taita Taveta planted local cassava varieties compared to Kilifi's 57.8%. Slightly above half of the farmer respondents reported Kibandameno as the preferred variety followed by Tajirika as the second preferred variety as reported by 18% farmers. Kibandameno was preferred for its sweet taste by 75.6% farmers while Tajirika was preferred by 52.4% farmers because of the high yielding capacity. Nearly all farmer respondents, in Taita Taveta County obtained the planting material from informal seed sources, except a negligible number, who reported buying their planting material from KALRO, a formal seed source, far from their locality. Farmers sourcing cuttings from a formal seed source such as those from Kilifi County were more likely to use a tractor for land preparation compared to those who sourced planting materials informally who more likely had scarce knowledge on cassava production and the value of cassava. Therefore, interventions to establish a sustainable healthy cassava planting materials seed system are needed to address the systemic constraint and help develop a viable cassava value chain.

Multi-layer Budding of Oversized Rubber (Hevea brasiliences) Seedlings: Success and Growth in Different Soil Media

The study on Multi-layer budding of oversized rubber seedling aimed at evaluating the success of multiple budding operation on single seedling of rubber tree species comprising of 4 to 7 budding layers per seedling. The experiment used randomized complete blocked design layout with 3 treatments such as 4, 5 and 7 layer budding in a single seedling replicated 4 times. Results revealed that the highest number of budding layers succeeded significantly higher as compared to 4 and 5 layers. The main survival percentage is about 79.17% for the 7 layers, 76.52% for the 5 layers and only about 55.36% for the 4 layers. Pure garden soil reported to have significant results on sprouting rate, sprout length, stalk diameter and number of leaves. This finding emphasized that budding of rejected oversized rubber seedlings grown in an abandoned nurseries can be reutilized for mass production of budded cuttings through multiple budding of 7 layers per seedlings to maximize planting materials derived from single seedlings.

Characterizing cassava farmer typologies and their seed sourcing practices to explore opportunities for economically sustainable seed business models in Rwanda

The overdependency on local cassava varieties and informal seed sources by farmers in Rwanda has contributed to the spread of cassava viral diseases. The use of improved planting materials made available through formal seed sources, that assure seed quality, is one way to prevent future disease outbreaks. In order to increase the availability of, and farmers access to, such materials there is increasing interest to develop seed business models. This study aims to understand seed sourcing practices of different farm typologies to inform the development of tailored seed business models. A total of 390 farmers were interviewed and the collected data was analyzed into clusters, resulting in seven farm typologies. Seed sourcing strategies, seed replacement dynamics and purchasing behavior of these typologies were explored via a seed tracing study. We find that more commercial oriented farmers have better access to formal seed sources. Nevertheless, the majority of farmers in all typologies accessed new varieties and quality cassava seed via informal channels. At both formal and informal sources, cash investments in seed were mainly made by the categories of better-off farmers, and were one-time investments to acquire a new variety. Based on farmers current seed sourcing practices, clarifications on the differences between farmers and their willingness-to-pay, the roles of seed degeneration, cost-benefit analysis, value propositions and profit formulas seem important requirements for the further development of viable cassava seed business models. We conclude that tailoring seed business models can have a high potential as it acknowledges differences among farmers, but that careful coordination is needed to ensure that one approach or intervention does not contrast with and/or undermine the others.

In Vitro Propagation And Rejuvenation of Senescent Maternal Plant of Ardisia Crenata Var. Bicolor (Primulaceae)

Ardisia crenata var. bicolor is an ornamental shrub, owing to its declined wild population, recalcitrant seeds and few high-quality cuttings, the main objective of this study was to optimize an in vitro propagation protocol by using tip shoot and nodal segment as explants from senescent plant. Explants were sterilized and cultured on Muraghige and Skoog medium contained 1.0 mg·L-1 benzylaminopurine and 0.05 mg·L-1 1-naphthaleneacetic acid for shoot initiation. For shoot proliferation, explants were cultured on MS medium with 1.0 mg·L-1 BAP, 0.1 mg·L-1 NAA, and 0.5 mg·L-1 kinetin, and the proliferation coefficient were 3.1 and 2.5. Rooting was achieved by two explants in half-strength MS medium containing 0.5 mg·L-1 indole-3-butyric acid + 0.1 mg·L-1 or 0.2 mg·L-1 NAA, and 0.5 g·L-1 activated charcoal. The highest rooting rate were 72.7% and 65.1% with the highest mean number of roots (4.2 and 2.8, respectively). After acclimatization, 83.3% and 81.2% of plants were survived in the greenhouse. The plant can be rejuvenated via in vitro propagation and provide a reference for supplying the planting materials quickly with an uniform genotype.