In Vitro Propagation, Genetic Assessment, and Medium-Term Conservation of the Coastal Endangered Species Tetraclinis Articulata (Vahl) Masters (Cupressaceae) from Adult Trees

Tetraclinis articulata (Vahl) Masters is an endangered tree growing in coastal and arid environments that is widely exploited by the timber and resin industry, among other applications. In this context, the use of in vitro techniques is highly encouraged for its propagation. We present a protocol for micropropagation using twigs from adult trees as a source of explants. The Schenk and Hildebrandt basal medium (SH) supplemented with 30 g L−1 sucrose, 6.5 g L−1 plant agar, 4.0 mg L−1 6-benzyladenine (BA), and 0.05 mg L−1 1-naphthaleneacetic acid (NAA) provided the optimum multiplication rate (90.48 ± 9.52 explants with basal shoots and 2.58 ± 0.29 basal shoots per explant). Application of activated charcoal (AC) or ½ Knop solution in a liquid overlay produced significantly longer shoots. Supplementation of solid media with indole-3-butyric acid (IBA) or NAA gave low rooting percentages (<17%). Addition of 0.9 g L−1 AC improved rooting (40%) but rooting performance was optimal (66.7%) after a pulse treatment consisting of 4 h immersion in liquid SH medium without growth regulators, followed by 8 weeks of cultivation. Rooted microplants were successfully acclimatized (93.33%) in a peat moss and vermiculite mixture (1:1 v/v ratio). The genetic stability of the in vitro regenerated plantlets was confirmed using the randomly amplified polymorphic DNA (RAPD) technique. Explant survival and growth remained higher than 90% after 28 weeks of cold storage at both 4 °C and 10 °C. The protocol presented here allows for largescale T. articulata production and could be applied for both ex situ conservation strategies and industrial purposes.

Effect of activated charcoal and ascorbic acid on in vitro morphogenesis and o-dihydroxyphenols content in Paphiopedilum insigne

Phenolic compounds limit micropropagation of many orchids in vitro. The aim of the study was to estimate the effect of activated charcoal (AC);1, 2 or 4 g/L) or ascorbic acid (AA; 10, 20 or 30 mg/L) added to the half strength MS medium on the growth and o-dihydroxyphenols content in Paphiopedilum insigne in vitro. A positive effect of AC on the shoot and root formation has been found. The highest multiplication rate (5.6 shoots/explant) and rooting frequency were obtained on medium containing 2 g/L of AC. However, AC reduced the leaf number as compared to the control. The lowest content of o-dihydroxyphenols was marked in Paphiopedilum insigne leaves when the shoots were grown on medium with 10 mg/L AA, followed by AC at 1 or 2 g/L.

The Effect of TIBA and NPA on Shoot Regeneration of Cannabis sativa L. Epicotyl Explants

Industrial hemp (Cannabis sativa L., family Cannabaceae) is a multi-purpose crop, used in the production of food, nutraceuticals, cosmetics and medicines. Therefore, development of new varieties with specific chemical profiles is necessary. In vitro culture methods could be complementary to conventional breeding and a useful tool for large-scale propagation. Strong apical dominance is considered as one of the factors contributing to the recalcitrance of industrial hemp in shoot proliferation. In this study, we tested the polar transport inhibitors N-1-naphtylphtalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA) to enhance shoot regeneration as the result of suppression of apical dominance and to develop in vitro protocols for Diana, Finola and Fedora 17 cultivars. Shoot tips derived from epicotyls were cultivated on Murashige and Skoog medium (MS) supplemented with meta-topolin (mT) and NPA, and also thidiazuron (TDZ) with a combination of TIBA and NPA. The results showed that the combination of TDZ with NPA (1–5 mg L−1) and TDZ with TIBA (0.5–2.5 mg L−1) increased the response of explants and the multiplication rate, but the effect was genotype-dependent and malformations were observed. To optimize the developed protocol, a two-step procedure with shortened time of exposure to inhibitors and reduced concentrations of them was applied. Shoots were rooted on media containing indole-3-butyric acid (IBA) and then successfully acclimatized. The obtained results will be useful in micropropagation of recalcitrant industrial hemp varieties.

Pengaruh Jenis Eksplan dan Asam Amino pada Inisiasi dan Proliferasi Kalus Embriogenik Phalaenopsis Var. ‘Raiza Agrihorti’

<p><strong>(<em>The Effect of Explant Types and Amino Acids on Embryogenic Callus Initiation and Proliferation of Phalaenopsis Var. ‘Raiza Agrihorti’</em>)</strong></p><p>Penyiapan kalus embriogenik (KE) yang optimal memiliki peranan penting dalam menghasilkan benih bermutu Phalaenopsis skala komersial. Kendala utama yang dihadapi ialah inisiasi dan proliferasi KE yang masih rendah, serta akumulasi fenolik yang tinggi. Penelitian dilakukan di Laboratorium Kultur Jaringan Balithi dari Agustus 2019 hingga Juli 2020. Penelitian menggunakan Rancangan Acak Kelompok (RAK) pola split plot dan faktorial dengan lima ulangan. Percobaan-1: jenis eksplan (pucuk, pangkal, dan daun plantlet) sebagai petak utama dan perlakuan asam amino (tanpa asam amino, L-Proline, L-Glutamine, L-Cysteine, dan Casein-Hydrolisate) dengan konsentrasi 150 mg/l pada medium PC1 (1/2 MS + 1,0 mg/l TDZ + 0,5 mg/l BAP + 20 g/l sukrosa) sebagai anak petak. Percobaan-2: faktor-1 ialah jenis asam amino (L-Proline, L-Cysteine; L-Glutamine, dan Casein-Hydrolisate) dan faktor-2 ialah konsentrasi asam amino (0, 75, 150, 225, dan 300 mg/l). Hasil penelitian menunjukkan bahwa inisiasi KE Phalaenopsis var. ‘Raiza Agrihorti’ terbaik didapatkan dari pangkal plantlet dan 150 mg/l L-Glutamine dengan waktu inisiasi 18,3-24,0 hari, 80-100% pembentukan KE, dan ukuran KE 0,4-0,5 cm3. Proliferasi KE terbaik ditemukan pada L-Glutamine dengan konsentrasi 150 mg/l. Proliferasi KE mencapai 100% dengan penambahan berat segar sebesar 0,39 g, tingkat multiplikasi (MR) 4,55 kali dan pencokelatan 4,0%. Hasil penelitian ini berpotensi tinggi untuk diterapkan pada kultur starter Phalaenopsis hibrida lain.</p><p><strong>Keywords</strong></p><p>Phalaenopsis hibrid; Asam amino; Inisiasi; Kalus embriogenik; Proliferasi</p><p><strong>Abstract</strong></p><p>Setup of the optimum Phalaenopsis embryogenic callus (EC) is an important role in producing qualified-seedlings of Phalaenopsis in commercial scale. The main constraints that are still being faced are the low rate of culture proliferation and high phenolic accumulations. The research was carried out at the Tissue Culture Laboratory-Indonesian Ornamental Plants Research Institite, from August 2019 through July 2020. The split plot and factorial designs were arranged using a Randomized Completely Block Design (RCBD) with five replications. Experiment-1: explants type (shoot tip, basal part, and leaf of plantlet) was used as main plot and amino acids (amino acids free, L-Proline, L-Glutamine, L-Cysteine, and Casein-Hydrolisate) with 150 mg/l concentration on medium PC1 (1/2 MS + 1,0 mg/l TDZ + 0,5 mg/l BAP + 20 g/l sukrosa) as subplot. Experiment-2: the first factor was amino acids type (L-Proline, L-Cysteine; L-Glutamine, and Casein-Hydrolisate) and the second factor was amino acids concentration (0, 75, 150, 225, and 300 mg/l). Results of the studies revealed that the best EC initiation of Phalaenopsis var. ‘Raiza Agrihorti’ was produced by basal part of plantlet and PC1 medium containing 150 mg/l L-Glutamine with EC Initiation time was 18.3-24.0 days, 80-100% of EC formation and size of 0.4-0.5 cm3. The best proliferation of EC was found in L-Glutamine with 150 mg/l concentration. EC proliferation reached 100% with 4.55 EC multiplication rate, 0.39 g EC fresh weight added, and EC browning as low as 4.0%. The established method is high possibly applied for other Phalaenopsis hybrids.</p>

Asynchrony of Apical Polarization, Luminogenesis, and Functional Differentiation in the Developing Thyroid Gland

Follicular thyroid tissue originates from progenitors derived from a midline endodermal primordium. Current understanding infers that folliculogenesis in the embryonic thyroid designates the latest morphogenetic event taking place after the final anatomical shape and position of the gland is established. However, this concept does not consider the fact that the thyroid isthmus develops chronologically before the lobes and also contains all progenitors required for lobulation. To elucidate whether cells committed to a thyroid fate might be triggered to differentiate asynchronously related to maturation and developmental stage, mouse embryonic thyroid tissues from E12.5-17.5 were subjected to immunofluorescent labeling of biomarkers (progenitors: NKX2-1; differentiation: thyroglobulin/TG); folliculogenesis: E-cadherin/CDH1; luminogenesis: mucin 1/MUC1; apical polarity: pericentrin/PCNT; basement membrane: laminin; growth: Ki67), quantitative RT-PCR analysis (Nkx2.1, Tg, Muc1) and transmission electron microscopy. Tg expression was detectable as early as E12.5 and gradually increased &gt;1000-fold until E17.5. Muc1 and Nkx2.1 transcript levels increased in the same time interval. Prior to lobulation (E12.5-13.5), MUC1 and TG distinguished pre-follicular from progenitor cells in the developing isthmus characterized by intense cell proliferation. Luminogenesis comprised redistribution of MUC1+ vesicles or vacuoles, transiently associated with PCNT, to the apical cytoplasm and the subsequent formation of MUC1+ nascent lumens. Apical polarization of pre-follicular cells and lumen initiation involved submembraneous vesicular traffic, reorganization of adherens junctions and ciliogenesis. MUC1 did not co-localize with TG until a lumen with a MUC1+ apical membrane was established. MUC1 delineated the lumen of all newly formed follicles encountered in the developing lobes at E15.5-17.5. Folliculogenesis started before establishment of a complete follicular basal lamina. These observations indicate that embryonic thyroid differentiation is an asynchronous process consistent with the idea that progenitors attaining a stationary position in the connecting isthmus portion undergo apical polarization and generate follicles already at a primordial stage of thyroid development, i.e. foregoing growth of the lobes. Although the thyroid isthmus eventually comprises minute amounts of the total thyroid volume and contributes little to the overall hormone production, it is of principal interest that local cues related to the residence status of cells – independently of a prevailing high multiplication rate – govern the thyroid differentiation program.

IN VITRO PROPAGATION OF PLUM ROOTSTOCK DOCERA 6

This paper describes research on the application of tissue culture techniques to the micropropagation of interspecific rootstock ʹDocera 6ʹ. The experimental work was carried out in the period 2017-2018, in the in vitro propagation laboratory of the Fruit Growing Institute Plovdiv. Аxillary buds were employed as initial explantеs in two different seasons (March-May; September-October). The action of the mineral medium was studied in the multiplication stage. The best result was obtained on LS medium included BAP 0.5 mg/l and IAA 0.05 mg/l. Тhe obtained average multiplication rate is 3.08. The concentration of auxin applied to the basal medium influence the quality of the root system Treatment with high concentrations of IBA added to the rooting medium gives the best results (V5). Тhe influence of the season on growth and development of micropropagated ʹDocera 6ʹ rootstock during ex vitro acclimatization is also part of our research. The spring acclimatization gives better results than the autumn.

Micropropagation of Cassava (Manihot esculenta Crantz): Review

Cassava is a vital crop to the food security of millions of people worldwide, particularly in Sub-Saharan Africa. Because the crop produced a reasonable yield on marginal soils, it could help relieve global hunger. As a result, increasing cassava output and its quality attributes are significant. However, the low multiplication rate of this main crop has resulted in the delayed dissemination of improved varieties among farmers. As a result, tissue culture techniques may be a feasible solution for overcoming these challenges. Cassava in vitro propagation had done using either the shoots multiplication technique or somatic embryogenesis. However, the shoot multiplication approach is preferable since it retains clonal fidelity. Plant regeneration via somatic embryogenesis or organogenesis entailed the use of numerous basal media containing various plant growth hormones. Several studies found that each type of cassava clone required a unique protocol to achieve optimal shoot initiation, shoot multiplication, root induction, and elongation. This review describes recent research on cassava micropropagation that makes use of a variety of experimental systems. While each of these systems focuses on a different aspect of technique, they can be significant in understanding the in vitro production of cassava planting material.

Role of Organic Growth Supplement In vitro Multiplication of Orchid Species- A Review

The main purpose of this article is to review role of several organic growth additives such Apple juice, coconut water (CW), maize extract, banana homogenate (BH), peptone and protocorms etc which stimulate the multiplication rate of various orchid species in in vitro multiplication. These organic growth supplements help to increase the number of shoots, root and leaf in culture medium. In many orchid tissue culture, organic growth supplements, which are the most essential medium aspect to stimulate tissue growth, production and facilitate the regeneration of shoot. The banana homogenate (BH) had the highest rate of regeneration and root developments. The use of organic growth supplements resulted in increased regeneration, the creation of more shoots and the development of fresh plantlets. Amino acids, proteins, vitamins, carbohydrates and various types of organic compounds are present in these growth supplements. These components have the potential to play a significant role in the development and creation of culture. Now more research is needed to figure out which factors are responsible for the organic additives’ promoter effect.

Yam seed system characteristics in Nigeria: Local practices, preferences, and the implications for seed system interventions

Yam is an important food and cash crop in Nigeria. The seed system is mostly farmer-based and its challenges and opportunities are not well documented. This study reports results of surveys, interviews, focus group discussions and participant observations that describe local practices characterizing yam cultivation and the farmer-based seed system in five states in Nigeria. Our findings show that ware and seed yam are dynamic and highly commercialized goods in Nigeria. There is a high demand for, and a high turn-over of, seed yam. The reasons for the high demand are the low multiplication rate and degeneration of seed yam which lead farmers to frequently acquire fresh seeds and replace varieties, investing significantly in the seed of ware yam. The study found some farmers specializing in seed yam production but less than expected for such a highly commercial crop. The market is the major source for off-farm sourced seed yam, and although completely informal, the seed yam sector is vibrant and well organized. The identification of strategically positioned farmers and traders can offer opportunities and entry points for introduction of new varieties and improved seed production techniques. Building on the existing farmer-based system is a more logical strategy than replacing it with formal structures and legislative regulation.

Protocol Optimization for Rapid Multiplication of Cassava (Manihot esculenta Crantz) using the Nodal Segment

Background: Cassava is a vital food security crop for millions of people, especially in sub-Saharan Africa. Since the crop produces a reasonable yield on marginal soils, it could help alleviate world hunger. Consequently, the increase in cassava production and its quality characteristics are significant. However, the low multiplication rate of this main crop has delayed the spread of improved varieties among farmers. As a result, tissue culture techniques can be a viable solution to overcome these challenges. Methods: The study used a nodal segment as an explant to evaluate different concentrations of BAP and NAA for an efficient, cost-effective in vitro mass multiplication of the AWC-1 cassava variety. CuSO4, commercial bleach and ethanol had used to sterilize nodal explants taken from greenhouse-grown plants at various time intervals. Result: The best medium for micro shoots induction had found to be medium without growth regulators. Among different treatments used for shoot multiplication purposes, the maximum shoot number has been recorded on M.S. medium supplemented with 0.75 mg/l BAP and 0.2 mg/l NAA. Medium with 0.5 mg/l NAA concentration was the best for rooting induction. A survival rate of 86% has obtained in the greenhouse and the plantlets appeared to be morphologically normal.