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Grapevine fanleaf virus (GFLV) is the causal agent of a widespread disease that affects vineyards. Since it is difficult to culture viruses, the availability of an easy and efficient method of virus maintenance in the laboratory would be of interest to virologists. The objective of this research was to determine an adequate culture medium that promotes callus growth and permits the preservation of GFLV on Vitis vinifera tissue. Fragments of in vitro cultured leaves (25 mm 2 ), originated from Cabernet Sauvignon positive for GFLV, were cultivated on a Murashige and Skoog (1962) medium, and the callus was monitored for the presence of GFLV every two weeks using ELISA. Higher 2,4-D concentration induced a higher growth, particularly when combined with a low BA concentration. The medium enriched with 1.0 ppm of 2,4-D combined with 0.5 ppm of BA showed the best result, with the callus area reaching more than 250 mm 2 after 8 weeks in culture. ELISA absorbance observed on callus tissues during the whole period was, at least, three times higher than that observed on leaves positive for GFLV kept either in vivo or in vitro and more than 18 times higher than that of the negative control. Any remarkable difference in absorbance was recorded during the period of callus cultivation. It was concluded that the viral load on the callus was not affected during this time, suggesting that this kind of in vitro culture is an efficient method to preserve GFLV.
365 372 Brown, P. T. H., Gobel, E., Lorz, H. (1991) RFLP analysis of Zea mays callus cultures and their regenerated plants. Theor. Appl. Genet. 81 , 227
263 265 Bajji, M., Lutts, S., Kinet, J. M. (2000): Physiological changes after exposure to and recovery from polyethylene glycol-induced water deficit in callus cultures issued
A simple and efficient protocol has been developed for high frequency plant regeneration through callus cultures derived from leaf bases of abiotic stress sensitive Asian indica rice variety IR 64. Leaf base segments (4–5 mm diameter) were obtained from 6-day-old dark grown seedlings germinated on halfstrength Murashige and Skoog medium and cultured on MS medium supplemented with different concentrations of 2,4-Dichlorophenoxyacetic acid (2.2–18 μM) and Kinetin (0.2–1.7 μM). Among the various combinations, 13.5 μM 2,4-D and 1.3 μM Kn resulted in high callus induction frequency (87.5%) with a maximum fresh weight of 0.22 g per segment. The regeneration frequency was 75.5% with multiple shoots within 3 weeks of transfer on MS medium supplemented with 13.3 μM 6-benzylamino purine and 8 μM Naphthaleneacetic acid. The shoots readily rooted on half-strength MS medium without any hormonal supplements. In vitro regenerated plantlets with multiple shoots and roots were transferred to sterile soil and vermiculite mix and maintained in shade house for 30 days. Complete plantlets were then transferred to nursery and acclimatized to the external environment until seed set. RAPD profile reveals monomorphism and thus confirming the genetic stability of the regenerated plants. This method has the potential for both direct as well as indirect method of transformation for the production of genetically modified plants.
54 Dahleen LS (1995) Improved plant regeneration from barley callus cultures by improved copper levels. Plant Cell Tiss Organ Cult 43
229 236 György, Z., Tolonen, A., Neubauer P. & Hohtola, A. (2005): Enhanced biotransformation capacity of Rhodiola rosea callus cultures for glycosid production
Endogenous carbohydrate (fructose, glucose and sucrose) fractions were measured in calli of potato genotypes with different field tolerance to drought. Under in vitro stress conditions induced by 0.8 M mannitol, sucrose level of calli increased extremely in medium-tolerant (by 424.5%) and sensitive (by 302.7%) genotypes whilst the rate of increase was 12–18-times lower in the drought tolerant variety. Results suggest the applicability of sucrose as biochemical marker for distinguish drought tolerant genotypes from great population in callus culture.
Densitometric HPTLC has been used for determination of catechins in shoot and callus cultures of two species from the genus Phyllanthus, P. juglandifolius and P. grandifolius . The catechin composition of intact P. juglandifolius (leaves and stems) was also compared with that of biomass obtained by biotechnological methods. A mixture of the standards (−)-epicatechin, (+)-catechin, (−)-epigallocatechin, (−)-gallocatechin, (−)-catechin gallate, and (−)-epicatechin gallate was separated on C 18 plates, with methanol-water-formic acid 30:70:6 ( v/v ) as mobile phase, in an automated development chamber at 75% humidity. The catechins were detected at 440 nm after derivatization with diazotized sulfanilic acid. The total catechin content of the plant material was calculated on the basis of the amount of (+)-catechin. The method was validated for precision and repeatability. (−)-Epicatechin, (+)-catechin, (−)-epigallocatechin, and (−)-gallocatechin were shown to be present in all shoot and callus cultures of the species mentioned above and in the leaves of intact P. juglandifolius plants. Amounts of catechins were similar in all the samples — from 2.39 mg g −1 in the leaves of P. juglandifolius to 1.94–1.68 mg g −1 in the shoot cultures of both species.
In vitro plant regeneration was optimized for Iranian purple coneflower via organogenesis from callus cultures derived from cotyledon and hypocotyl tissues by placing them on MS medium supplemented with different concentrations and combinations of BAP and NAA. The experiment was laid out as a completely randomized design in a factorial arrangement with three replications. The results indicated that the mean callus induction was influenced by explant type, with a significant difference between cotyledon (77.81%) and hypocotyl (65.33%) explants at the 0.01 probability level. In relation with the regeneration rate, no significant differences were observed between the two types of explants. For both cotyledons and hypocotyls the optimum shoot regeneration frequency (31.5% and 32.5%, respectively) and number of shoots per explant (5.2 and 5.3, respectively) were achieved using medium supplemented with 0.4 mg l−1 BAP. Proliferated shoots were elongated in hormone-free MS medium and well-developed shoots were rooted on MS medium, both with and without the addition of 2 mg l−1 IBA. All the plantlets survived acclimatization, producing normal plants under controlled conditions. This study revealed that cotyledon and hypocotyl explants of E. purpurea have relatively good potential for callus induction and shoot formation. Furthermore, a beneficial method has been established for the micropropagation of this valuable medicinal species.
Summary
Gymnemic acid (GA), a well known anti-diabetic compound has been detected in methanol extracts of intact leaves and in vitro callus cultures derived from leaf explants of Gymnema sylvestre. Callus biomass was developed in MS medium with optimum plant growth regulators (OPGRs) of 2,4-D (1.5 mg L−1) + KN (0.5 mg L−1) under abiotic stress conditions at 45 days determined by growth curve analysis. GA detection and quantification were carried out using thin-layer chromatography (TLC), highperformance thin-layer chromatography (HPTLC), high-performance liquid chromatography (HPLC), and gravimetric techniques. GA detection peak area and their absorption spectra were evaluated through HPTLC and HPLC with the standard GA. Quantification of GA had showed the linearity, accuracy, robustness and precision by HPLC. GA content was significantly higher in gravimetric method than HPLC. All these methods were found to be simple, accurate, selective and rapid and could be successfully applied for the determination of GA. It could have potential as a pharmaceutical drug for Type 1 diabetes mellitus (IDDM) and obesity.
