Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L.).

Anthers from a doubled-haploid line of spring wheat (Triticum aestivum L.) cv. Pavon 76 were plated in liquid P-4 medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at four concentrations (0.5, 1.0, 2.0, 4.0 mg/l) for 5, 10, 15, and 25 days before being transferred to another medium with the same or reduced 2,4-D concentrations for the remainder of the induction phase for a total of 45 days. Incubation with 0.5 mg/l 2,4-D for 45 days produced lower callus yield and plant regeneration, indicative of insufficient auxin for callus induction. Callus yield and regeneration frequencies were higher with 1.0 mg/l 2,4-D. With 2.0 or 4.0 mg/l 2,4-D, an induction period of 10 or 15 days was sufficient for initiation of callus development. The extended presence of 2-4 mg/l 2,4-D in the medium beyond the initiation phase was detrimental to plant regeneration. Thus optimal callus induction and plant regeneration could be obtained through manipulating the 2,4-D concentration and the duration of its presence in the induction medium.

Stably transformed callus of wheat by electroporation-induced direct gene transfer.

Fertile plants of wheat have been regenerated from protoplasts in several laboratories. The objective of this study was to develop a transformation system using protoplasts as target cells. Protoplasts were isolated from cell suspensions initiated from an anther-derived callus. The protoplasts were transformed by electroporation using pBARGUS or pBAS, both carrying the Basta resistance (BAR) gene. A total of 2,761 calli were produced from electroporation transformed protoplasts in 3 independent experiments. Six calli survived selective culture on 10 mg/l phosphinothricin (PPT), a concentration that completely inhibited the growth of non-transformed wheat callus. Five PPT resistant calli showed phosphinothricin acetyltransferase (PAT) activity, whereas the sixth probably was a mutant. The transformed wheat calli could tolerate PPT concentrations up to 2,560 mg/l. Southern blot analyses confirmed the integration of the BAR gene in wheat genomes. The integrated DNA sequence may have partially methylated and tandemly repeated at least once. These results demonstrate the production of stably transformed wheat calli by electroporation-mediated direct gene transfer into protoplasts.

Osmotic potential of media affecting green plant percentage in wheat anther culture.

The percentage of green plants in anther culture is known to be controlled by the genetics of anther donor materials. The objective of this study was to determine whether components in the culture media also would have a significant influence on the percentage of green plants from wheat anther culture. Anthers of a spring wheat cultivar, "Pavon 76", were cultured on potato 4 (P4) induction media with various modifications. Addition of 200 g/l ficoll to the liquid P4 medium significantly increased the percentage of green plants even though the final yield of green plants per 100 anthers was lower than the liquid medium. A higher concentration of maltose (135 g/l) produced significantly higher percentage of green plants than the medium containing 90 g/l maltose or sucrose. These results demonstrate culture medium effects on albinism, indicating that the percentage of green plants in wheat anther culture can be increased by optimizing medium osmotic potential.

Quantitative variation in the kernel proteins among 841 accessions of Triticum dicoccoides estimated by SDS-PAGE.

The relative proportion and amount of proteins in five defined molecular weight (MW) regions (A1=above 71,000=71K, A2=71K-49K, A3=49K-31K, A4=31K-20K, A5=20K and less) were estimated by densitometric analyses of the amount of dye bound by kernel proteins (Fullington et al. 1980) of Triticum dicoccoides SDS-PAGE gels. These MW regions roughly correspond to the wheat protein solubility classes (Cole et al. 1981; Fullington et al. 1983). One purpose of the study was to select accessions whose seed proteins bind relatively high amounts of dye in the glutenin and albumin globulin regions. These accessions will be used for further in-depth studies as possible candidate donors of genes to improve the baking and nutritional quality of wheat. Marked differences in the quantitative relationships were found among the proteins in the five MW regions. Coefficients of variation (CV's) for the highest peak (i.e., most abundant protein) MW in different protein MW regions were similar for A1, A2 and A3, at 11.4, 11.7, and 11.1%, respectively, but only 4.1 for A4, and 10.6% for region A5. The CV for the highest peak MW overall was 29.8. Accession BP0649, for example, had over 44% of its protein in region A5, whereas BP0566 (lowest among the top 10%) had only 21.4% of its protein in that region. Over 37% of the proteins of accessions BP0649 and 0001 to 0005 was in region A5. At least 84 accessions with the highest amount of protein in region A5, and 13 accessions with more protein in region A1 than Chinese Spring may merit further evaluation as possible protein gene donors. High amounts of protein in A1 may be of importance in bread-baking quality, and in A4 and A5 for high lysine wheat. Accessions in both extremes were selected to test these hypotheses. All accessions are now or will be available in the USDA Wheat Collection.

A computer-assisted examination of the storage protein genetic variation in 841 accessions of Triticum dicoccoides.

Triticum turgidum L. var. dicoccoides (wild emmer) is an important genetic resource for increasing the protein content of common wheat (Triticum aestivum L.). Many studies have shown that the presence or absence of bands in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) electrophoregrams of wheat storage proteins to be of a purely genetic character. A total protein extraction and SDS-PAGE technique was used to estimate the storage protein genetic variability among 841 accessions of wild emmer collected from various ecological regions in the Middle East. In addition, a computer data bank was developed, recording the onedimension electrophoregram bands for each accession by molecular weight (MW) and relative Coomassie Blue staining intensity as determined from densitometer scans. Analyses of this information are being used to identify specific accessions for further study by two dimension electrofocusing-electrophoresis and breeding and genetic analyses. The computer-assisted analyses indicated that the greatest genetic variability occurs for proteins in the high MW region (above 70,000 MW) followed by those in the medium range (70,000 to 33,300 MW). Comparatively little variability was revealed for protein subunits of below 33,300 MW.

Artificial mutagenesis as an aid in overcoming genetic vulnerability of crop plants.

Artificially induced genetic variation is being used effectively to supplement or complement sources of natural origin for practical plant breeding. Thus, creating genetic variation uill become increasingly important as crop genetic resources become more difficult to obtain via plant exploration. The aritificial induction of useful genetic variation offers important elements that can be used for overcoming genetic vulnerability: (1) new, previously unknown alleles can be induced in crop plant species to broaden the base of variation; (2) useful genetic variation can be induced in modern cultivars helping to shorten breeding time or to extend production "life"; (3) characteristics of existing genetic resource stocks can be improved to make them more useful in breeding; and (4) recombination in crosses may be enhanced. The performance of induced mutant crop cultivars and the successful uses of induced genetic variation in cross breeding indicate that artificial mutagenesis will play an increasingly greater role in plant breeding.

Radiobiological damage: a new class identified in barley seeds stored after irradiation.

A new class of radiobiological damage has been identified in irradiated barley seeds. Consisting of both physiological and genetic damage, it appears to be independent of oxygen and long-lived free radicals, and develops very slowly during storage after irradiation. Increasing the storage temperature accelerates its development.