Unknown proteins isolated from mutant tissues of rice (Oryza sativa L.) recovered from inhibitor selections were subsequently peptide microsequenced. Database searches putatively identified one peptide as fructose 1,6-bisphosphate aldolase (EC 4.1.2.13). Tissues of mutant rice, PI564784, and wild type (cv Calrose 76) tissues were evaluated for aldolase activity. Total enzyme activities were slightly lower in the mutant than the control but the differences were not significant. Although the mutant phenotype is for enhanced lysine and protein, we ascribe the small aldolase differences to physiological adjustments, rather than to DNA modifications of the aldolase gene(s). Homologies of rice peptides with aldolases from a range of species, as well as rice cell culture expressed sequence tags (ESTs) are presented. Some amino acids sequences are highly conserved. The mutant phenotype expressing stress proteins is not likely to be defined by a change in rice aldolases.

Free amino acids were determined in developing seed of a rice mutant with enhanced grain lysine. This phenotype frequently has enhanced protein. Some free amino acids of developing seed are inversely related to the level of total amino acids in proteins of the mature grain. Amino acids that were enhanced in protein, including aspartic acid, threonine, methionine and lysine, were notably lower in the free amino-acid pool. Our conclusion is that mutant-developing grains process aspartate amino acids more rapidly than the controls. Conversely, arginine, valine and glutamic acid/glutamine accumulate as free amino acids with mutant/control ratios of 1.39, 1.29 and 1.12, respectively. Glutamic acid/glutamine in proteins of mature seeds is lower in the mutant than the control. 3 H-lysine incorporation showed enhanced isotope incorporation into at least four proteins. One mutant protein was less actively labelled than analogous controls. The 3 Hlysine pattern indicates processing modifications in this useful rice mutant.

Seed storage proteins from in vitro-derived rice mutants improved by several backcrosses to ‘Calrose 76’ and BC 2 and BC 3 were characterized for changes in five different solubility classes. Albumins, rsealb (water-soluble globulins), true salt-soluble globulins, prolamins and glutelins were SDS-PAGE separated in a single dimension, and some two-dimensionally, to identify protein modifications. The genetic transmission of the enhanced-lysine mutants in backcrosses and the linkage of lysine with grain chalkiness were confirmed. Advanced lines had altered globulin profiles similar to those of unimproved lines. Chalky/ enhanced-lysine phenotypes had similar prolamin and glutelin profiles in the mutant and controls at the same protein level. Mutants had increased levels of globulins at 50 kDa and 33 kDa but had substantially less protein at 25 kDa than the controls. High protein in the mutant contributed to an increase in prolamins and the major storage proteins in both the globulins and glutelins. A significant decrease in low-molecular-weight, 15- to 18-kDa albumins was associated with the chalky/enhanced-lysine mutant phenotype. Two proteins in the 15- to 18-kDa group were amino acid sequenced, and database comparisons identified these proteins as allergens. Advanced lines downregulated for allergens and with enhanced-lysine/protein but with normal fertility and seed weight should be useful in breeding programs for nutritional quality.

Mutant rice cells (Oryza sativa L.) grown in liquid suspension cultures exported greater quantities of protein and β-glucanases than controls. These mutants were isolated from anther calli resistant to 1 mM lysine plus threonine (LT), regenerated and reestablished as cell suspension cultures from seeds. Cellular protein levels are genetically conditioned, and the levels of extracellular proteins and enzyme activities are inversely related to that of the cellular portions. The rechallenge of cells with 1 mM LT inhibited the expression of both β-1,3-glucanases and β-1,4-glucosidases but had no significant effect upon the levels of chitinase activity. Mutant cells were more sensitive than controls to stress caused by exogenous LT. In general, under exogenous LT stress the mutant/control ratio for extracellular glucanases increased as the assay conditions were changed from a basic to an acidic pH. The specific activity of βglucanases was highest in media and lowest in cells. Both the mutant and control cells exported β-glucanases into the suspension medium, but the level of activity in media was greater in that in which the mutant was suspended. The export was probably modulated by the internal protein levels which were highest in mutant cells without LT. Seedlings from mutants with enhanced lysine also had enhanced acidic β-glucanase activity.

Crop ScienceVolume 34, Issue 5 cropsci1994.0011183X003400050077x p. 1424-1425 Registration of Germplasm Registration of Five Lysine-Enhanced Rice Germplasm Lines: 2K41, 2K539, 2K(C193), 2K497, and 2K601 G. W. Schaeffer, Corresponding Author G. W. Schaeffer n/[email protected] USDA-ARS, Plant Molecular Biology Lab, Beltsville, MD, 20705Corresponding author.Search for more papers by this authorF. T. Sharpe Jr., F. T. Sharpe Jr. USDA-ARS, Plant Molecular Biology Lab, Beltsville, MD, 20705Search for more papers by this authorJ. T. Dudley, J. T. Dudley USDA-ARS, Plant Molecular Biology Lab, Beltsville, MD, 20705Search for more papers by this author G. W. Schaeffer, Corresponding Author G. W. Schaeffer n/[email protected] USDA-ARS, Plant Molecular Biology Lab, Beltsville, MD, 20705Corresponding author.Search for more papers by this authorF. T. Sharpe Jr., F. T. Sharpe Jr. USDA-ARS, Plant Molecular Biology Lab, Beltsville, MD, 20705Search for more papers by this authorJ. T. Dudley, J. T. Dudley USDA-ARS, Plant Molecular Biology Lab, Beltsville, MD, 20705Search for more papers by this author First published: 01 September 1994 https://doi.org/10.2135/cropsci1994.0011183X003400050077xCitations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume34, Issue5September–October 1994Pages 1424-1425 RelatedInformation

Experiments were designed to test whether an enhanced lysine phenotype from in vitro selections was expressed in milled rice, Oryza sativa L. Analysis of the endosperm tissue after removal of the aleurone and outer layers proved the grains maintained enhanced lysine after milling. Mutant endosperm had 15% greater lysine than the control and the enhanced lysine was correlated with reduced glutamic acid in two different mutants. Glutelins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed increases in the protein quantity of the 33-kDa alpha and 21-kDa beta subunits.

A rice mutant with unique protein expression/ transport properties has been established as cells in liquid suspension and partially characterized. Mutants were originally recovered from anther calli grown for three cycles at inhibitory levels of lysine + threonine and one cycle of S-(2-aminoethyl)cysteine. Cell suspension cultures were started from high lysine-containing seeds regenerated from the inhibitor selections. Cultures of the mutant produce 2 times as much protein per unit weight as is produced by the control. Significant portions of the proteins are exported from the cells into the surrounding medium. The mutant also has 20% greater lysine content in the exported protein than the control. This cell suspension line should be particularly useful for biochemical and molecular studies on protein synthesis and processing phenomena in cereals.

When rice (Oryza sativa) cell suspension cultures are grown in the presence of [terminal methylenes-(3)H]spermidine, label is incorporated in a single polypeptide with a molecular mass of 18 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preincubation of cell cultures with polyamine biosynthesis inhibitors difluoromethylarginine and difluoromethylornithine, resulted in increased incorporation of the label into the 18 kilodalton polypeptide. In cells in which protein synthesis was arrested by cycloheximide, no label was detected in the 18 kilodalton polypeptide, suggesting a requirement for de novo protein synthesis.

The interactions of genotype and several variables related to culture environment, including temperature pretreatment, conditioned medium and agar concentrations were examined in a series of experiments for their effects on percent anthers producing callus and number of embryoids produced per 100 anthers scored. Significant genotypic interaction was observed for both traits with all environmental variables except methods of medium conditioning. Such interactions involve both changes of response magnitude and changes of rank order of genotypes. The highest response frequencies observed were in excess of 30% of anthers callusing. Most lines examined responded relatively well to a culture regime utilizing a 4°C treatment for 7–14 d prior to anther excision, followed by float culture, without transfer and without preconditioning of the culture medium. The results indicate, however, that particular genotypes may have specific requirements with respect to various environmental conditions so that culture conditions may need to be adjusted, especially for the least responsive genotypes.

The effects of long-term tissue culture on mitochondrial DNAs were examined using rice (Oryza sativa) cell suspension cultures. Mitochondrial DNAs were isolated from P. I. 353705 (an indica subspecies of rice similar to 'Asam 5'), its anther-culture-derived line BL2 (an 8-year-old cell suspension culture), and five other cell lines (A1, A7, A11, A13, and A23), also derived from BL2 and independently selected for resistance to the lysine analog, S-(2-amino)-ethyl-L-cysteine. Mitochondrial DNAs of the rice lines were digested with ten restriction endonucleases (BamHI, BglII, EcoRI, EcoRV, HindIII, PstI, PvuII, SalI, SmaI, and XhoI), electrophoresed, and transferred to nylon membranes. Southern blots were hybridized with one rice and five maize probes containing mitochondrial genes. The restriction patterns of ten Southern blots and hybridization patterns of 60 endonuclease/probe combinations were analyzed. DNAs from all sources produced unique restriction patterns when digested with HindIII or BglII; with the other endonucleases an array of similarities and differences was observed. Lines BL2 and A11 showed unique patterns with all restriction endonucleases tested. No hybridization pattern differences were observed among the lines when probes containing apt9 and atpA were used. However, extensive hybridization pattern differences were observed with coxI, coxII, rrn18-rrn5, and atp6 probes. Both restriction and hybridization patterns revealed variation due to tissue culture effect. Coxll was most efficient in revealing the uniqueness of BL2. Among the analog selected lines A11 was most divergent, and probes rrn18-rrn5 and atp6 were most efficient in revealing its distinctiveness. Unique mitochondrial genomic organizations were found to be associated with long-term tissue culture.

Endosperm protein mutants in rice may be recovered by biochemical selections with inhibitory levels of lysine and threonine. Among the phenotypes recovered from in vitro selections are lines with increased protein and percent lysine in the protein. This work was designed to identify changes in proteins of rice mutants and to further our understanding of the mechanisms of lysine plus threonine selections in rice. Among the most obvious amino acid changes in mutants was a higher lysine level in all protein solubility fractions and a decrease in tyrosine. Methionine and glutamate are reduced in some protein fractions. However, methionine is significantly higher in the mutant than the control in the glutelin fraction. Several other aspartate pathway amino acids are higher in the mutant than the unselected controls. Separation of proteins in SDS-PAGE gels showed shifts in the protein profiles in the mutants, including a decrease in the major 30 kDa low lysine globulin component, and an increase in several high-molecular-weight components, approximately 60–100 kDa. Increases in the lysine content of proteins of different solubility classes and different proteins within classes are detailed.

Lysine is a limiting amino acid for optimal nutritional quality in rice grain. In vitro selections using inhibitory levels of lysine plus threonine or s-aminoethylcysteine allow the predictable recovery of variants with elevated levels of lysine and protein. These methods may generate useful starting germplasm for plant breeders. This study was conducted to define the genetics of lysine mutants in progeny from crosses of mutants derived from cells cultured in vitro in the presence of inhibitory levels of lysine plus threonine and s-(2-aminoethyl)-cysteine. In vitro selections produce a wide range of mutants, including endosperm mutants with elevated lysine and protein levels as well as mutants for high and low seed weights. Mutants were analyzed for lysine content by the endosperm half-seed method in which the halves without the embryo were ground and acid hydrolyzed for amino acid determinations. The halves with the embryos were preserved for later germination. In two different F2 populations derived from a cross of a selected mutant x M-101, a parental marker, there was an inverse relationship between seed weight and percent lysine in endosperm protein (R2 0.52 and 0.56). The F2 segregation patterns show that elevated lysine is inherited as a recessive gene and that increased lysine is correlated with decreased seed size. F3 and F4 data provide evidence for the transmission of high lysine genes to advanced germplasm in rice. This work supports our earlier conclusions that high lysine phenotypes can be recovered predictably from in vitro selections. The elevated lysine phenotypes are frequently, but not exclusively, associated with opaque seed. Some segregants from crosses produced increased lysine in plants with near normal seed weight and good fertility.

To determine the level of gametoclonal variation among doubled-haploid lines (DHLs) of Triticum aestivum L. developed using anther culture techniques and its effect on agronomic performance, 70 anther culture-derived DHLs of ‘Kitt’ were compared for 7 agronomic traits to 50 single-seed descent-derived lines (SSDLs) of ‘Kitt’ and to the cultivar ‘Kitt’. In a second experiment, 26 DHLs of ‘Chris’ were compared for 7 agronomic traits to 29 SSDLs of ‘Chris’ and to the cultivar ‘Chris’. Each experiment was grown as a randomized complete block design with three replications in three environments. For ‘Kitt’, the DHLs averaged significantly lower grain yields than the comparable SSDLs. For ‘Chris’, the DHLs averaged lower, but not significantly lower, grain yield than the SSDLs. In both ‘Kitt’ and ‘Chris’, the genetic component of variance for yield of the DHLs was significantly larger than that of the SSDLs indicating the presence of gametoclonal variation. The lower average grain yield of the DHLs was explained by a larger group of low-yielding DHLs than was present in the SSDLs. Six ‘Kitt’ DHLs and 3 ‘Chris’ DHLs were lower yielding than the lowest yielding SSDL, respectively. Elite DHLs and SSDLs were similar for mean grain yield performance. Though the DHLs and SSDLs were significantly different for some yield components, the affected yield component changed with the cultivar and there was no consistent effect. Significant genotype × environment interactions were detected for some traits which were caused by changes in the magnitude of differences, rather than reversals in ranking, indicating that low yielding DHLs could be culled on the basis of visual selection or single-environment testing. Hence, gametoclonal variation was induced by the anther culture techniques used in this study, tended to be deleterious for grain yield, and was sensitive to the growing environment. However, as the DHLs and SSDLs had similar expected population means based upon expected gains from selection, this gametoclonal variation should not be a major hindrance to wheat breeding.

Plants were regenerated from anther calli of the winter wheat cultivar 'Centurk' (Triticum aestivum L. em. Thell.). Cells of root tips of young regenerated plants were assayed for ploidy and plants were categorized as polyhaploid, mixoploid, or hexaploid. Tillering and seed set were analyzed in plants that survived to maturity. Less than 1% of the tiller population produced by polyhaploid plants set seed. In contrast, 73% of the tiller population produced by hexaploid plants set seed, with significantly greater seed set per fertile tiller. These data were taken to indicate that the ploidy composition of root tips of young regenerated plants reflected that of the reproductive structures of mature regenerated plants. Common patterns of aneuploidy in hexaploid and hyperploid cells found among roots of individual plants confirmed the idea that doubling of the cell genome occurred before plant regeneration. Polyhaploid and hexaploid cells were found in individual root tips of mixoploid plants regenerated from calli that were known to be cytochimeric. The possibility that regeneration of plants can occur from more than a single cell of an anther callus is discussed.Key words: anther culture, Triticum aestivum, wheat, mixoploidy, aneuploidy, regeneration.

Chloroplast (ct) and mitochondrial (mt) DNAs were isolated from two subspecies of rice (Oryza sativa), japonica (Calrose 76) and indica (PI353705) and compared by restriction endonuclease fragment pattern analysis. Similarly, PI353705 (A5) mtDNA was also compared with the mtDNA of its long term tissue cultured line, BL2. Variation in the ctDNA of the 2 subspecies was detected with two (AvaI and BglI) of the 11 restriction endonucleases tested, whereas their mtDNAs showed considerable variation when restricted by PstI, BamHI, HindIII and XhoI endonucleases. Thus, the chloroplast DNA was more highly conserved than the mtDNA in the subspecies comparisons. Only minor variation was observed between the restriction endonuclease patterns of the mtDNAs of BL2 and A5. Southern blots of mtDNA were hybridized with heterologous probes from maize and spinach organelle genes. Differences were found in the hybridization patterns of the two subspecies for six of the eight (mitochondrial and chloroplast) probes tested. Two of the seven (mitochondrial) probes (coxII and 26S rRNA) detected tissue culture generated variation in mtDNA. The relative values of restriction endonuclease and hybridization patterns for studying phylogenetic and genetic relationships in rice are discussed.

Experiments were designed to test whether variation in percent lysine in seed proteins could be recovered in plants regenerated from callus subjected to inhibitory levels of lysine plus threonine. Anther-derived callus was subjected to 1 millimolar lysine plus threonine for three successive passages and then once to the same concentration of S-(2-aminoethyl)cysteine. Plants were regenerated from the resistant callus. Plants recovered directly from tissue culture were normal in color, size and were 50% or less fertile. Second and third generation plants produced a wide range of variants including albinos, deep green plants both short and tall, and totally fertile as well as partially fertile plants. All regenerated plants produced chalky or opaque seed. One unique second generation line had 14% more lysine in seed storage proteins than the controls. This characteristic was transmitted to the next generation. The high lysine plants had reduced seed size with significantly higher levels of seed storage protein than the controls. The phenotypes recovered provide experimental materials for basic studies in protein synthesis and lysine metabolism and may become a source of material for rice breeding.

Modification of a potato extract medium was undertaken to increase pollen embryogenesis from anthers of a hard red spring wheat (Triticum aestivum L. cv. Chris). Whole potato tubers (Solatium tuberosum L. cv. Kennebec) were separated into peeled tubers and peel from which extracts were prepared. A 3 ✕ 3 latin square design with three replications in time was used to compare media prepared from: (i) extract of whole potato tubers, (ii) extract of the peel only, and (iii) extract of peeled tubers. A 60% increase in anther induction resulted from the peeled tuber extract medium compared to the whole potato medium. Removal of the peel from tubers probably reduces the concentration of compounds that may be inhibitory to wheat embryogenesis during anther culture. Anther source, defined by spike section (basal, middle, and distal), was a second variable in the experiment but did not significantly affect embryogenesis.