Low variability of internal transcribed spacer rDNA and trnL (UAA) intron sequences of several taxa in the Festuca ovina aggregate (POACEAE).

Identification and classification of numerous Festuca species is still a difficult problem due to the close morphological resemblance. The most difficult fine fescues to identify belong to the Festuca ovina aggregate, which is the largest group in the genus Festuca. Many taxons are considered to be separate species based on quantitative taxonomic characters, differences in ploidy level or the structure of sclerenchyma cells. In order to evaluate the taxonomic value of DNA-based markers, sequence analysis of the internal transcribed spacer (ITS1-5.8S-ITS2) region and the chloroplast trnL (UAA) intron was performed in the ten most problematic fine fescues belonging to the Festuca ovina aggregate. Intraspecific ITS variants were found in a single case while in other cases only intragenomic ITS polymorphisms were detected with 1-2 ambiguous positions. Among the sequences of the trnL (UAA) intron even intragenomic polymorphisms were not detected in any of the Festuca species studied. Thus, the results do not support the species status of these ten taxa.

High efficiency callus induction and plant regeneration in petiole culture of four poplar genotypes.

In order to develop a simple and feasible approach to achieve high frequency plant regeneration for protoplast isolation and transformation experiments, a method was elaborated by using a new type of explant (petiole segments) for the four Populus nigra genotypes. Callus initiation from the petioles took place on N6 medium containing 2,4-D (0.1-1 mg/l). The highest rate of callus initiation (100%) was achieved when the basic medium was supplemented with 0.5 mg/l of 2,4-D, in all tested genotypes. For shoot regeneration, calli were transferred to MS and WPM medium supplemented with BA (1.0-2.5 mg/l) and NAA (0.2 mg/l). Multiple shoot regeneration was observed in each shoot induction medium. The highest rate of shoot regeneration (6.83 shoot/callus) was observed on MS medium containing 2.5 mg/l BA and 0.2 mg/l NAA. The results showed highly significant differences between the media. There was no significant difference between the genotypes and genotype x medium interaction.

Developmental analysis of primary and secondary somatic embryogenesis in soybean tissue culture.

Somatic embryogenesis from immature soybean cotyledons was induced on Murashige & Skoog medium, supplemented with 2,4-dichlorophenoxyacetic acid. The full developmental analyses of primary and secondary somatic embryogenesis was monitored by scanning electron microscopy. The developmental stages of embryogenesis from the first unequal cell division through the three- and four-cell embryos, and multi-cell globular and heart shape embryos, to the fully developed plants were determined. When the globular stage embryos were incubated on the embryo maturation medium for 4 to 6 weeks a secondary embryogenesis was observed and scanned. Chimeric embryos with epidermis zones similar to mature leaf were characterized.

Responses to continuous and discontinuous NaCl stress of long-term cultured rice (Oryza sativa L.) cells.

Rice (Oryza sativa L.) cells, grown under a continuous stress of 1.5% NaCl, produced a homogeneous mass of dry, compact, nodular callus with high regeneration potential, typical of embryogenic cultures. When transferred and subcultured in conditions without salt, the pre-adapted cells underwent several changes, leading to the formation of heterogeneous populations that comprised different cell types. This newly formed callus, in the 6-month period without salt stress, resembled cell populations of the untreated cultures. These cells are characterized by fast growth, high water content, friable texture, high salt sensitivity and low culture response. They also produce a protein pattern in the SDS-PAGE analysis, that differs from that of cells cultured under the continuous salt stress. This observation indicates that different salt stress regimes induced different responses in the cultured cells in rice.

Callus initiation and plant regeneration from inflorescence primordia of the intergeneric hybrid Agropyron repens (L.) Beauv.xBromus inermis Leyss. cv. nanus on a modified nutritive medium.

Plant regeneration from callus of intergeneric hybrid Agropyron repens (L.) Beauv. x Bromus inermis Leyss cv. nanus (AGROMUS) was carried out on a new culture medium designated medium-F. Within 21 days of the plating of inflorescence primordia the initiated callus showed globular structures. From the 21st day of culture, one step plant regeneration occurred on the callus without subculture. The new basal medium reported in this work was effective in callus initiation and plant regeneration of the hybrid AGROMUS by (i) the reduction of the total ion strength (2.6 g/l, 22.5 mM) of macroelements compared to MS (4.5 g/l,45.2 mM), (ii) the use of NH4NO3 as the sole N-source, and (iii) the application of KH2PO4 at an 8 times higher concentration (1160 mg/l,8.5 mM) when compared to the Murashige and Skoog medium composition. This medium provided a 2 to 10 fold reduction in the 2,4-dichlorophenoxyacetic acid supplement needed for the callus initiation and one step plant regeneration after a gibberellic acid (2 mg/l, for 5 days) pretreatment of tillers. The regenerated plantlets were subcultured in multi-shoot culture and potted in soil to grow for further analysis.

Neomorph and leaf differentiation as alternative morphogenetic pathways in soybean tissue culture.

Somatic embryogenesis and organogenesis were induced on immature cotyledons of different soybean cultivars. The anatomical investigation of morphogenesis proved neomorph differentiation instead of somatic embryos, and leaf formation instead of shoot development. While normal embryos were induced in 0-3.1% of the explants, neomorphs developed at a much higher rate i.e. in 10.5-78.9% depending on the genotype. Likewise organogenesis preferably followed the pathway of leaflet development (3.1-26.3%) than that of shoot tip formation (0-2.6%). Low plant regeneration frequency of soybean can partly be explained with these two alternative abortive pathways of morphogenesis probably induced with higher frequency than the normal pathways by the generally used in vitro methods.

Organ-specific and ploidy-dependent somaclonal variation; a new tool in breeding.

The organ-specific somaclonal variation means the differences between the variability of somaclones originated from different somatic tissue of plant. Significant differences in some agronomical characters were achieved among somaclones of seed and plumule meristem origin. The ploidy-dependent somaclonal variation means the differences between the variability of somaclones originated from different ploidy-level tissue. Increased variation among regenerated plants was postulated by origin from cultured cells of reduced ploidy level. The comparison of somaclonal variation in the progenies of diploid plants regenerated from callus of haploid and diploid origin supported the ploidy dependent theory. The pollenhaploid somaclone method (PHS-method) was developed and tested for utilization somaclonal variation in rice breeding. The PHS-method comprises the two well-known and widely applied in vitro methods which are the androgenesis (another culture) and genetic instability of cultured haploid somatic cells (callus cultures). Developmental varieties produced by this breeding sheme are under certification in Hungary.

Effect of different cryoprotectants and transfer temperatures on the survival rate of hemp (Cannabis sativa L.) cell suspension in deep freezing.

Adequate cell dehydration is the precipitating element in the successful cryopreservation of plant cells and organs. This could be achieved by using different cooling rates, transfer temperatures and cryoprotectants. Experiments were performed to determine these critical points in the freeze preservation procedure of Cannabis sativa (L.) suspension cultures. The explants were frozen at a cooling rate of 2 degrees C/min, while the transfer temperatures were -10 degrees C, -20 degrees C, -30 degrees C, -40 degrees C and -50 degrees C. The applied cryoprotectants were the DMSO, glycerol, proline and PEG in different concentration. The highest viability (58%) was obtained by using 10% DMSO and at -10 degrees C transfer temperature. The optimum transfer temperature varied remarkably by different cryoprotectant concentrations indicating the importance of their interactions.

Increase of green plant regeneration efficiency by callus selection in Puccinellia limosa (Schur.) Holmbg.

Three main types of callus have been selected from seeds of salt marsh grass(Puccinellia limosa (Schur.) Holmbg.) subcultured on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and kinetin. Callus type I differentiated only occasionally. Callus type II produced roots but no shoots under all tested culture conditions. Both green (47 %) and albino plants have been obtained from the embryogenic callus type III. Callus type III was divided into two subtypes (greening and non-greening) according to the presence or absence of green spots. Separated greening embryogenic callus gave up to 87 % green plants, whereas non-greening callus produced only 4 %.