Meristem Culture Technique Research Articles

Effects of thermotherapy and meristem culture techniques on macro and micronutrients content in elephant grass cultivars

Elephant grass is a tropical forage crop highly used in dairy cattle production, in Brazil. It has been getting special attention, because of its bioenergy potential, medicinal properties, and bioremediation profile. The aim of the present study is to investigate the effects of thermotherapy based on clonal cleaning methods and meristem culture on the mineral content of elephant grass (Cenchrus purpureus (Schumach.) Morrone). Cultivars “Mineiro”, “Taiwan A-147” and “Pioneiro” were subjected to the following methods: thermotherapy (T) combined to meristem culture (MC), meristem culture and mature stems (control). The experiment assessed the mineral contents of phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulfur (S) at three cuttings, which were performed every 60 days, for 180 days. There was lack of effects from these methods on the mineral content of approximately 66% of the carried out assessments, standing out unanimity for all cultivars, methods, and most of the cuts. T+MC was the only method showing positive effect on P and Ca content, in all cuts, in the cultivars Taiwan A-147 and Pioneiro, respectively. There was clear negative effect of cleaning methods on P and Ca content, in all cuts, for cultivar Mineiro, and on Mg, for cultivar Taiwan A-147. These results, along with the positive effects observed in vegetative and nutritional parameters shown in other articles published in this Journal, show that the clonal cleaning methods are strongly recommended for cultivars with more than 15 years of ripe stem propagation.

Thermotherapy of cloves for in-vitro mericlone production for healthy and sustainable management of garlic germplasm

Garlic is second most important crop of the genus Allium, known for its traditional medicinal uses. In spite of having medicinal prosperities, it gets infected by several viruses called as garlic viral complex. The garlic viral complex severely affects bulb yield of garlic, due to accumulation of virus load year after year from asexual propagule. Hence we attempted experiment to eliminated garlic viral complex through thermotherapy in combination with meristem culture. The viruses were eliminated from infected garlic bulb with the treatment at 37 OC for 90 day could eliminate the Garlic Common Latent Virus, Onion Yellow Dwarf Virus and Allexiviruses. In this study we confounded that the combination of thermotherapy and meristem culture technique protocal have wider prospects for virus elimination in garlic.

HTS-Based Monitoring of the Efficiency of Somatic Embryogenesis and Meristem Cultures Used for Virus Elimination in Grapevine.

Meristem culture and somatic embryogenesis are effective tools for virus elimination of vegetatively propagated crops including grapevine (Vitis vinifera L.). While both have been shown to be useful to eliminate the main grapevine viruses, their efficiency differs depending on the virus and grapevine variety. In our work, we investigated the efficiency of these two virus elimination methods using small RNA high-throughput sequencing (HTS) and RT-PCR as virus diagnostics. Field grown mother plants of four clones representing three cultivars, infected with different viruses and viroids, were selected for elimination via somatic embryogenesis (SE) and meristem culture (ME). Our results show for the first time that using SE, elimination in mother plants was effective for all viruses, i.e., grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus 1 (GSyV-1), Grapevine virus T (GVT) and grapevine Pinot gris virus (GPGV). This study also confirms previous studies showing that SE is a possible strategy for the elimination of GFkV, GRSPaV, HSVd, and GYSVd-1. Our results demonstrate that the efficacy of virus elimination via SE is relatively high while the purging of viroids is lower. Our work provides evidence that the efficiency of SE is comparable to that of the technically difficult ME technique, and that SE will offer a more effective strategy for the production of virus-free grapevine in the future.

English

Taro leaf blight disease has recently been reported in Cameroon to cause between 50 and 100% yields loss of taro in most of the agro-ecological crop growing regions. This has led to a significant reduction in disease-free planting materials, edible crop and increased. The Meristem culture technique has been used to produce crop plants free of viruses and fungi especially in vegetative propagated colocassia plants. This aimed at applying in-vitro micro-propagation technique for sustainable production of four local taro cultivars in Cameroon. This study was conducted at the Root and Tuber Tissue Culture Laboratory, of the Institute of Agricultural Research for Development (IRAD), Bambui from April 2015 to November 2016. Micro-plants from four local taro cultivars were produced in vitro from apical meristem tips. The tip meristems were excised from corms of the four local taro cultivars. The excised explants were surface sterilized with alcohol and sodium hypochlorite in sequence steps at different concentrations. Meristems were cultured at establishment stage on Murashige and Skoog (MS) medium with 30 g of sugar, 1.1 ml of 6-benzylaminopurine and 7 g of agar. Shoots proliferation was induced in MS with 2.2 ml of 6-benzylaminopurine (BAP). Result shows a significant difference at p≤0.5 in number of shoots, petiole length, open leaf and corm diameter among the cultivars and no significant variation in mean number of senescence leaf with respect to all the cultivars, at 60 days of shoot tip culture. At rooting stage, taro shoots were cultured on MS media supplemented with 10 ml of 0.1 mg/ml naphthalene acetic-acid (NAA). Roots were produced on all the cultivars with excellent mean growth rate of 14.7 ± 0.69 recorded in cultivar with dark green petiole and small leaves.   Key words: Taro cultivars, in-vitro micro-propagation, benzylaminopurine, naphthalene acetic-acid, Murashige and Skoog.

Mass in-vitro micro propagation of banana (Musa sp.)

Production of Banana is constrained by many biotic as well as abiotic factors. The production of disease free planting material of banana by meristem culture technique is an urgent need.An experiment has been designed to develop a suitable and efficient protocol for plant regeneration in banana through micro propagation.The surface sterilization of banana plant is the most difficult practice.So it is very important to optimize the concentration and duration of different sterilants for explants surface sterilization. The best sterilants for the surface sterilization of the explant of banana were 0.1 per cent HgCl 2 for 5 min duration followed by NaOCl (40 %, commercially available) solution for 8 min duration and 70 per cent ethyl alcohol for 2 min. And the best medium for establishment of banana explant was supplemented with 3mg/l BAP, 0.2mg/l IAA with 30 mg ascorbic acid followed by Proliferation with 5mg/l BAP,0.5mg/ l IAA with 30 mg ascorbic acid and 10 mg/l BAP,0.5 mg/l IAA with 30 mg ascorbic acid.Significantly highest number of roots and greater root length were produced by 0.5 mg/l IAA + 0.5 mg/l IBA. How to view point the article : Kumari, Namrata and Misra, Pragati (2016). Mass in-vitro micro propagation of banana (Musa sp.). Internat. J. Plant Protec., 9(1) : 204-210.

Use of Tissue Culture Techniques for Producing Virus‐Free Plant in Garlic and Their Identification through Real‐Time PCR

This study was performed for comparison of meristem culture technique with shoot tip culture technique for obtaining virus-free plant, comparison of micropropagation success of two different nutrient media, and determination of effectiveness of real-time PCR assay for the detection of viruses. Two different garlic species (Allium sativum and Allium tuncelianum) and two different nutrient media were used in this experiment. Results showed that Medium 2 was more successful compared to Medium 1 for both A. tuncelianum and A. sativum (Kastamonu garlic clone). In vitro plants obtained via meristem and shoot tip cultures were tested for determination of onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV) through real-time PCR assay. In garlic plants propagated via meristem culture, we could not detect any virus. OYDV and LYSV viruses were detected in plants obtained via shoot tip culture. OYDV virus was observed in amount of 80% and 73% of tested plants for A. tuncelianum and A. sativum, respectively. LYSV virus was found in amount of 67% of tested plants of A. tuncelianum and in amount of 87% of tested plants of A. sativum in this study.

Effects of growth regulators on plant to meristem culture and set up to complete plant in physic nut plant (Jatropha curcas L.)

Physic nut plant (Jatropha curcas L.) is considered as a potential source for a non-edible biofuel-producing energy crop throughout the world. This optimized meristem culture technique would be useful for developing uniform and virus-free clones of physic nut plant. Physic nut plant shoot apical meristems with 2 leaf primordia were aseptically isolated to sterilize and culture on Murashige and Skoog (MS) medium supplemented with kinetin (0; 0.1; 0.5; 1.0; 1.5; 2.0 mg.l-1), BA (0; 0.1; 0.5; 1.0; 1.5; 2.0 mg.l-1), TDZ (0; 0.01; 0.05; 0.10; 0.50; 1.0 mg.l-1) individually. Primary shoot induction was most effectively promoted by MS medium supplemented with 0.1 mg.l-1 TDZ. In this concentration, more than 95% of the shoots were regenerated without intervening any callus formation. The use of TDZ in combination with GA3 did not have good effects on the formation of shoots from meristem, in that only long meristems resulted. Also, when surveying the state of culture medium, that semi-liquid MS medium supplemented with 0.1 mg.l-1 TDZ will be suitable for the development of meristems. Rooting of adventitious shoots was achieved on MS medium supplemented with 1.0 mg.l-1 IBA and 3.0 mg.l-1 thiamin.

Detection and elimination of Begomovirus infecting Trichosanthes dioica (pointed gourd) plants in Uttar Pradesh, India

Begomovirus is widely spread on cultivated crops in India. In a survey, infected plants of Trichosanthes dioica (Pointed gourd) with the symptoms of mosaic and leaf curling were collected and checked for Begomovirus infection through PCR analysis. Application of meristem culture technique for regeneration of virus free plants of T. dioica was assessed. Plantlets were regenerated from infected plants of T. dioica through meristem culture. Regenerated plants were found Begomovirus free as evident by PCR analysis suggesting the suitability of meristem culture technique for elimination of Begomovirus from infected plants of T. dioica. This is the first report on elimination of Begomovirus in T. dioica in India.

Elimination of sugarcane grassy shoot disease through apical meristem culture

Elimination of sugarcane grassy shoot disease (SGSD) through apical meristem culture technique for producing clean planting material of sugarcane has been attempted in the present study. The results showed that meristems length of 2 and 3 mm were free from the SGSD pathogen at higher frequency than larger meristem length of 4 mm. However, the frequency of survival of explants during initiation of shoot cultures was higher in larger meristems (60%) in comparison to smaller ones (40%). The micropropagated plantlets raised from meristem culture were confirmed for disease-free by nested polymerase chain reaction (PCR) analysis at monthly interval up to 6 months. This is the first report on the elimination of SGSD phytoplasma through meristem culture in India.

Use of Sodium Hypochlorite as Media Sterilant in Sugarcane Micropropagation at Commercial Scale

Sodium hypochlorite has been successfully utilized as media sterilant (at total active chlorine concentration 0.002% in the medium) in production of sugarcane plantlets by applying apical meristem culture technique. The process has been scaled up to produce 2.5 million sugarcane plantlets per year. Use of sodium hypochlorite in place of autoclaving has resulted in reducing considerable amount of electricity cost and ultimately reducing the cost of production of sugarcane plantlets.

Eliminasi Sweet Potato Feathery Mottle Virus (SPFMV) pada Empat Kultivar Ubijalar Unggul Lokal Asal Papua melalui Teknik Kultur Meristem

The objective of this research was to eliminate Sweet Potato Feathery Mottle Virus (SPFMV) infection from local superior sweet potato varieties (PN-II, Maria, Seri, and Numfor) by using meristem culture technique. Shoot tips of sweet potato were grown on solid MS modified medium enriched with naphthalene acetic acid (NAA) and benzyl adenine (BA) with various concentrations. Growth and development of explants were recorded for three months. The results showed that elimination of SPFMV infection of four varieties by using shoot tip explants were successfully achieved. Shoot tips were the best explants used to eliminate SPFMV infection. Murashige and Skoog (MS) modification called Ub I medium, which enriched with NAA (0.02 mg/l) and BA (0.2 mg/l) is the best medium for developing meristem segment of sweet potato. Nitrocellulose membrane-enzyme linked immunosorbent assay (NCM- ELISA) analysis showed that plantlets produced from shoot tip explants of four varieties, tested were free from SPFMV infection. The plantlets of four varieties were maintained in in vitro culture in Biotechnology Laboratory, Faculty of Agriculture, State University of Papua (UNIPA), Manokwari. Key words: Sweet potato, SPFMV; Meristem culture technique

Untersuchungen zur Therapie virusinfizierten Hopfens (Humulus lupulus L) durch Wärmebehandlung von Fechsern und Triebspitzen

Summary Hot water treatment of dormant hop cuttings at temperatures ranging from 35°C to 65°C in intervals of 5 min. to 4 h. proved to be inadequate for the inactivation of hop viruses. In comparable research works different types of heat chambers have been tested for use in hot air treatments of hop plants. Relatively most satisfying was a stationary chamber in which plants after a period of heat adaptation grew under artificial light conditions in temperatures between 37°C to 40 °C, in relative air humidities between 60 to 80 per cent and in soil temperature of 28°C sufficiently survived a treatment of at least 25 days. From more than 1000 about 2 to 3 cm. long shoot tips of 25 to 118 days heat treated hop plants belonging to 15 varieties, 3 clones and 2 breeding lines, 7401 young hop plants could be developed. RMV (rose mosaic virus), NRSV ( Prunus necrotic ringspot virus), AMV ( arabis mosaic virus), and hop mosaic virus in an extend of 34.0, 27.3, 16.7, and 24.3 per cent have been eliminated. However, elimination of the latent rod shaped hop virus (656 nm) only in 2 varieties extreme rarely succeeded even if the longest heat periods have been used. In order to increase the curing rate the combination of hot air treatment and tip meristem culture techniques is recommended. Healthy or virus-poor hop material has been selected on the basis of 2 to 8 times repeated indexings on test plants, partly repeated agar gel double diffusion tests, using antiseras against RMV, NRSV, and AMV and by the reaction of indicators and electron-microscopical tests in course of several years.