Cytokinin activities in cells of wheat inflorescence in depependence of its developmental stage.

We demonstrate that differences in the endogenous biologically active levels and patterns of cytokinins depend on the organ differentiation, i. e. immature inflorescence before meiosis and immature embryo 14 days after anthesis, respectively, in winter wheat of two genotypes: Grana and Almari. Two cell lines were subjected to analysis of cytokinin contents. Higher levels of endogenous cytokinin were found in cells derived from immature embryo (E) in comparison to cells obtained from immature inflorescence (I). The levels of zeatin ryboside ([9R]Z) and benzyladenosine ([9R]BAP) were predominant and isopentenyladenine (iP) was very low for the cells E of both genotypes. The cytokinin amounts in cells E and I of the zeatin group were smaller than those of other groups. We suggest that the concentration of cytokinins in tissue capable of generative development in winter wheat should be on a level that occurred in inflorescence cell lines, in comparison to immature embryo cell lines. These results indicate that the effect of the developmental stage of ears of wheat on the capability to flower in winter wheat plants regenerated in in vitro culture may be induced by the hormone level.

Factors affecting transient expression of vector constructs in wheat protoplasts.

Direct uptake of reporter gene constructs with the bacterial beta-glucuronidase (GUS) gene fused to various promoters was achieved to embryogenic cell suspension culture-derived protoplasts of GK Ságvári winter wheat (Triticum aestivum L.) with polyethylene glycol (PEG) treatment. Based on GUS specific activity values, it was found that Mg2+ with PEG at 20% final concentration can significantly increase the transient expression in wheat protoplasts in comparison to the Ca(2+)-containing medium. The optimum incubation time in transformation mixture was 5-10 min at 25 degrees C. Transient GUS expression as detected by spectrofluorimetry was positively correlated with the time elapsed after DNA uptake (with maximum activity at 48 h), and the incubation time in GUS reaction mixture. It was also found that the protoplast culture medium plays an important role in the efficiency, and the treated wheat protoplasts cultured in KM medium showed a higher GUS activity than those kept in GM medium. Among the five plasmid constructs 6-16-fold higher promoter activity has been achieved with pKM794 driven by CaMV 35S promoter plus two enhancer elements than with the other constructs tested.

High-efficiency plant regeneration from an embryogenic cell suspension culture of winter wheat (Triticum aestivum L.).

Highly embryogenic cell suspension cultures were established from immature embryo-derived embryogenic calli of winter wheat (Triticum aestivum L., cv. GK Ságvári). Weekly subcultures were made in liquid MS medium supplemented with 2 mg 1(-1) 2,4-D. An average of twenty-two compact, organized calli were obtained from each 1 ml suspension cells when plated on solid MS medium containing IAA and zeatin under a 16/8 h light/dark cycle, while only 9 calli were produced in the dark. Variation in the callus inducing ability was correlated to the time elapsed after subculture. Plated cells responded best 9 days after the subculture and 59% of the calli were regenerable, retaining their embryogenic capacity over 6 months. Several hundred green shoots and plants were regenerated on differentiation media supplemented with IAA and BAP, and 300 plants were transferred to the greenhouse. The majority of plants had an abnormal chromosome number and a low viability.

Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.).

Regenerable embryogenic cell suspensions initiated from immature embryo-derived friable, fast growing, embryogenic calli of GK Ságvári winter wheat (Triticum aestivum L.) served as sources of protoplasts, which were cultured in different liquid or agarose-solidified media. Protocallus formation was best on KM8p (Kao and Michayluk 1975) and GM (Li and Murai 1990) media, and protocallus growth on MS (Murashige and Skoog 1962) callus growing medium. Green shoot/plant regeneration occurred on MS regenerating medium, and rooting on MS or N6M (Mórocz et al. 1990) hormone-free media. Protocalli maintained their morphogenic capacity over 4 months, and with multiple subcultures on half-strength MS regenerating medium, the total number of regenerants could be increased. Approximately 1000 shoots/plants were regenerated and over 500 plants were transplanted in the greenhouse. The majority of them had an abnormal chromosome number and low viability, however, one plant grew to maturity and set seed.