Search results for: subculture

Authors: Emma K. Sales, Nilda G. Butardo

Abstract:

The project was undertaken to determine the effects of modified tissue culture protocols e.g. age of culture and hormone levels (2,4-D) in generating somaclonal variation. Moreover, the utility of molecular markers (SSR and MSAP) in sorting off types/somaclones were investigated.

Results show that somaclonal variation is in effect due to prolonged subculture and high 2,4-D concentration. The resultant variation was observed to be due to high level of methylation events specifically cytosine methylation either at the internal or external cytosine and was identified by methylation sensitive amplification polymorphism (MSAP).Simple sequence repeats (SSR) on the other hand, was able to associate a marker to a trait of interest.

These therefore, show that molecular markers can be an important tool in sorting out variation/mutants at an early stage.

Keywords: Methylation, MSAP, somaclones, SSR, subculture, 2, 4-D.

Authors: Valbona Sota, Efigjeni Kongjika

Abstract:

Wild almond is a woody species, which is difficult to propagate either generatively by seed or by vegetative methods (grafting or cuttings) and also considered as Endangered (EN) in Albania based on IUCN criteria. As a wild relative of cultivated fruit trees, this species represents a source of genetic variability and can be very important in breeding programs and cultivation. For this reason, it would be of interest to use an effective method of in vitro mid-term conservation, which involves strategies to slow plant growth through physicochemical alterations of in vitro growth conditions. Multiplication of wild almond was carried out using zygotic embryos, as primary explants, with the purpose to develop a successful propagation protocol. Results showed that zygotic embryos can proliferate through direct or indirect organogenesis. During subculture, stage was obtained a great number of new plantlets identical to mother plants derived from the zygotic embryos. All in vitro plantlets obtained from subcultures underwent in vitro conservation by minimal growth in low temperature (4ºC) and darkness. The efficiency of this technique was evaluated for 3, 6, and 10 months of conservation period. Maintenance in these conditions reduced micro cuttings growth. Survival and regeneration rates for each period were evaluated and resulted that the maximal time of conservation without subculture on 4ºC was 10 months, but survival and regeneration rates were significantly reduced, specifically 15.6% and 7.6%. An optimal period of conservation in these conditions can be considered the 5-6 months storage, which can lead to 60-50% of survival and regeneration rates. This protocol may be beneficial for mass propagation, mid-term conservation, and for genetic manipulation of wild almond.

Keywords: Micropropagation, minimal growth, storage, wild almond.

Authors: J. Linjikao, P. Inthima, A. Kongbangkerd

Abstract:

In vitro conservation of orchid germplasm provides an effective technique for ex situ conservation of orchid diversity. In this study, an efficient protocol for in vitro conservation of Vandopsis lissochiloides (Gaudich.) Pfitz. plantlet under slow growth conditions was investigated. Plantlets were cultured on different strength of Vacin and Went medium (½VW and ¼VW) supplemented with different concentrations of mannitol (0, 2, 4, 6 and 8%), sucrose (0 and 3%) and 50 g/L potato extract, 150 mL/L coconut water. The cultures were incubated at 25±2 °C and maintained under 20 µmol/m²s light intensity for 24 weeks without subculture. At the end of preservation period, the plantlets were subcultured to fresh medium for growth recovery. The results found that the highest leaf number per plantlet could be observed on ¼VW medium without adding sucrose and mannitol while the highest root number per plantlet was found on ½VW added with 3% sucrose without adding mannitol after 24 weeks of in vitro storage. The results showed that the maximum number of leaves (5.8 leaves) and roots (5.0 roots) of preserved plantlets were produced on ¼VW medium without adding sucrose and mannitol. Therefore, ¼VW medium without adding sucrose and mannitol was the best minimum growth conditions for medium-term storage of V. lissochiloides plantlets.

Keywords: Preservation, Vandopsis, germplasm, in vitro.

Authors: A. Pourjabar, S.A. Mohammadi, R. Ghahramanzadeh, Gh. Salimi

Abstract:

The extract of milk thistle contains a mix of flavonolignans termed silymarine.. In order to analysis influence of growth regulators, genotype, explant and subculture on the accumulation of flavonolignans, a study was carried out by using two genotype (Budakalszi and Noor abad moghan cultivars), cotyledon and hypocotyle explants, solid media of MS supplemented by different combinations of two growth regulators; Kinetin (0.1, 1 mg/l) and 2,4-D (1, 2 mg/l). Seeds of the plant were germinated in MS media whitout growth regulators in growth chamber at 26°C and darkness condition. In order to callus induction, the culture media was supplemented whit different concentrations of 2,4-D and kinetin. Calli obtained from explants were sub-cultured four times into the fresh media of the first experiment. flavonoides was extracted from calli in four subcultures. The flavonoid components were determined by high- performance liquid choromatography (HPLC) and separated into Taxifolin, Silydianin+Silychristin, Silybin A+B and Isosilybin A+B. Results showed that with increasing callus age, increased accumulation of silybin A+B, but reduced Isosilybin A+B content. Highest accumulation of Taxifolin was observed at first calli. Calli produced from cotyledon explant of Budakalszi cultivar were superior for Silybin A+B, where calli from hypocotyl explant produced higher amount of Taxifolin and Silydianin+Silychristin. The best cultivar for Silymarin production in this study was Budakalszi cultivar. High amount of SBN A+B and TXF were obtained from hypocotil explant.

Keywords: Callus culture, Flavonolignans, Silimarine