Leaf sheath cuticular waxes on bloomless and sparse-bloom mutants of Sorghum bicolor.

Leaf sheath cuticular waxes on wild-type Sorghum bicolor were approximately 96% free fatty acids, with the C28 and C30 acids being 77 and 20% of these acids, respectively. Twelve mutants with markedly reduced wax load were characterized for chemical composition. In all of the 12 mutants, reduction in the amount of C28 and C30 acids accounted for essentially all of the reduction in total wax load relative to wildtype. The bm2 mutation caused a 99% reduction in total waxes. The bm4, bm5, bm6, bm7 and h10 mutations caused more than 91% reduction in total waxes, whereas the remaining six mutants, bm9, bm11, h7, h11, h12 and h13, caused between 35 and 78% reduction in total wax load. Relative to wild-type, bm4 caused a large increase in the absolute amount of C22, C24 and C26 acids, and reduction in the C28 and longer acids, suggesting that bm4 may suppress elongation of C26, acyl-CoA primarily. The h10 mutation increased the absolute amounts of the longest chain length acids, but reduced shorter acids, suggesting that h10 may suppress termination of acyl-CoA elongation. The bm6, bm9, bm11, h7, h11, h12 and h13 mutations increased the relative amounts, but not absolute amounts, of longer chain acids. Based on chemical composition alone, it is still uncertain which genes and their products were altered by these mutations. Nevertheless, these Sorghum cuticular wax mutants should provide a valuable resource for future studies to elucidate gene involvement in the biosynthesis of cuticular waxes, in particular, the very-long-chain fatty acids.

A highly digestible sorghum mutant cultivar exhibits a unique folded structure of endosperm protein bodies.

The endosperm of a sorghum mutant cultivar, with high in vitro uncooked and cooked protein digestibilities, was examined by transmission electron microscopy and alpha-, beta-, and gamma-kafirins (storage proteins) were localized within its protein bodies. Transmission electron microscopy micrographs revealed that these protein bodies had a unique microstructure related to high protein digestibility. They were irregular in shape and had numerous invaginations, often reaching to the central area of the protein body. Protein bodies from normal cultivars, such as P721N studied here, with much lower uncooked and cooked digestibilities are spherical and contain no invaginations. Immunocytochemistry results showed that the relative location of alpha- and beta-kafirins within the protein bodies of the highly digestible genotype were similar to the normal cultivar, P721N. Gamma-kafirin, however, was concentrated in dark-staining regions at the base of the folds instead of at the protein body periphery, as is typical of normal cultivars. The resulting easy accessibility of digestive enzymes to alpha-kafirin, the major storage protein, in addition to the increased surface area of the protein bodies of the highly digestible cultivar appear to account for its high in vitro protein digestibility.

Molecular characterization of a mutable pigmentation phenotype and isolation of the first active transposable element from Sorghum bicolor.

Accumulation of red phlobaphene pigments in sorghum grain pericarp is under the control of the Y gene. A mutable allele of Y, designated as y-cs (y-candystripe), produces a variegated pericarp phenotype. Using probes from the maize p1 gene that cross-hybridize with the sorghum Y gene, we isolated the y-cs allele containing a large insertion element. Our results show that the Y gene is a member of the MYB-transcription factor family. The insertion element, named Candystripe1 (Cs1), is present in the second intron of the Y gene and shares features of the CACTA superfamily of transposons. Cs1 is 23,018 bp in size and is bordered by 20-bp terminal inverted repeat sequences. It generated a 3-bp target site duplication upon insertion within the Y gene and excised from y-cs, leaving a 2-bp footprint in two cases analyzed. Reinsertion of the excised copy of Cs1 was identified by Southern hybridization in the genome of each of seven red pericarp revertant lines tested. Cs1 is the first active transposable element isolated from sorghum. Our analysis suggests that Cs1-homologous sequences are present in low copy number in sorghum and other grasses, including sudangrass, maize, rice, teosinte, and sugarcane. The low copy number and high transposition frequency of Cs1 imply that this transposon could prove to be an efficient gene isolation tool in sorghum.

Chemically Induced Cuticle Mutation Affecting Epidermal Conductance to Water Vapor and Disease Susceptibility in Sorghum bicolor (L.) Moench.

Analysis of Sorghum bicolor bloomless (bm) mutants with altered epicuticular wax (EW) structure uncovered a mutation affecting both EW and cuticle deposition. The cuticle of mutant bm-22 was about 60% thinner and approximately one-fifth the weight of the wild-type parent P954035 (WT-P954035) cuticles. Reduced cuticle deposition was associated with increased epidermal conductance to water vapor. The reduction in EW and cuticle deposition increased susceptibility to the fungal pathogen Exserohilum turcicum. Evidence suggests that this recessive mutation occurs at a single locus with pleiotropic effects. The independently occurring gene mutations of bm-2, bm-6, bm-22, and bm-33 are allelic. These chemically induced mutants had essentially identical EW structure, water loss, and cuticle deposition. Furthermore, 138 F2 plants from a bm-22 x WT-P954035 backcross showed no recombination of these traits. This unique mutation in a near-isogenic background provides a useful biological system to examine plant cuticle biosynthesis, physiology, and function.

Transgenic sorghum plants via microprojectile bombardment.

Transgenic sorghum plants have been obtained after microprojectile bombardment of immature zygotic embryos of a drought-resistant sorghum cultivar, P898012. DNA delivery parameters were optimized based on transient expression of R and C1 maize anthocyanin regulatory elements in scutellar cells. The protocol for obtaining transgenic plants consists of the delivery of the bar gene to immature zygotic embryos and the imposition of bialaphos selection pressure at various stages during culture, from induction of somatic embryogenesis to rooting of regenerated plantlets. One in about every 350 embryos produced embryogenic tissues that survived bialaphos treatment; six transformed callus lines were obtained from three of the eight sorghum cultivars used in this research. Transgenic (T0) plants were obtained from cultivar P898012 (two independent transformation events). The presence of the bar and uidA genes in the T0 plants was confirmed by Southern blot analysis of genomic DNA. Phosphinothricin acetyltransferase activity was detected in extracts of the T0 plants. These plants were resistant to local application of the herbicide Ignite/Basta, and the resistance was inherited in T1 plants as a single dominant locus.

Genetic mapping and characterization of sorghum and related crops by means of maize DNA probes.

Cloned DNA fragments from 14 characterized maize genes and 91 random fragments used for genetic mapping in maize were tested for their ability to hybridize and detect restriction fragment length polymorphisms in sorghum and other related crop species. Most DNA fragments tested hybridized strongly to DNA from sorghum, foxtail millet, Johnsongrass, and sugarcane. Hybridization to pearl millet DNA was generally weaker, and only a few probes hybridized to barley DNA under the conditions used. Patterns of hybridization of low-copy sequences to maize and sorghum DNA indicated that the two genomes are very similar. Most probes detected two loci in maize; these usually detected two loci in sorghum. Probes that detected one locus in maize generally detected a single locus in sorghum. However, maize repetitive DNA sequences present on some of the genomic clones did not hybridize to sorghum DNA. Most of the probes tested detected polymorphisms among a group of seven diverse sorghum lines tested; over one-third of the probes detected polymorphism in a single F2 population from two of these lines. Cosegregation analysis of 55 F2 individuals enabled several linkage groups to be constructed and compared with the linkage relationships of the same loci in maize. The linkage relationships of the polymorphic loci in the two species were usually conserved, but several rearrangements were detected.

Somaclonal variation in high tannin sorghums.

Genetic variants were found among over 6,000 primary plants (R1) regenerated from embryogenic tissue cultures of eight high tannin sorghums [Sorghum bicolor (L.) Moench]. Field assessment of somaclonal variation has progressed to the R2 population, with over 48,000 R2 seedlings (27,000 plants) in 1,126 rows from 1,055 R1 plants. A total of 43 variant phenotypes was recovered, including several types of chlorophyll deficiencies, dwarfism, short culm, sterility, narrow leaf, and several previously unreported variants, such as ragged leaf, multibranched heads, and Hydra, a developmental variant which produces large numbers of panicles. Variation production greatly depends on parent genotype and appears to increase with increasing time in cultures. The toal average somaclonal variation rate (based per 100 R1 plants) and somaclonal variant frequency (based per 100 R2 plants) estimated in the tested population were 11.3 and 1.6, respectively. Chimerism was found in regenerants. The estimated size of the mutated sector carried by mutant regenerants ranged from the whole plant to less than 3% of a single head. The average proportion of mutated R1 heads carrying large (80%-100%), medium (40%-80%), and small (<40%) mutated sectors was 38.7%, 26.0% and 35.3%, respectively. Some sector mutations do not appear until the R3 generation. In order to avoid losing variants, the population for selecting somaclonal variation should be as large as possible. Some of these variants found may be useful for further study or for use in breeding programs.

Improving the in vitro protein digestibility of sorghum with reducing agents.

We have shown in previous reports that cooked sorghum protein is less digestible than other cooked cereal proteins. The pepsin-indigestible proteins in sorghum were found to be mainly prolamin proteins. Cooking sorghum in the presence of 2-mercaptoethanol increased protein digestibility (in vitro with pepsin or trypsin/chymotrypsin) to a level comparable with other cereals. At a concentration of 100 mM, other reducing agents (dithiothreitol, sodium bisulfite, and L-cysteine) were equally effective in improving sorghum digestibility. When maize was cooked in the presence of 2-mercaptoethanol, protein digestibility increased 5% compared to 25% for sorghum. Cooking barley, rice, and wheat with 2-mercaptoethanol had no significant effect on protein digestibility. The addition of reducing agents appears to prevent the formation of protein polymers linked by disulfide bonds.

Digestibility and utilization of protein and energy from Nasha, a traditional Sudanese fermented sorghum weaning food.

Whole grain sorghum flour was fermented into Nasha, a traditional Sudanese food, and freeze-dried or drum-dried. It was cooked and fed to convalescent malnourished infants and small children as 61% of total diet calories and all of 6.4% protein calories, with (Lys+) and without lysine supplementation to 3% of protein. Apparent absorptions of nitrogen were 73 +/- 5 and 74 +/- 6% of intake, significantly (P less than 0.01) less than those from preceding (Cas-1, 86 +/- 3%) and following (Cas-2, 85 +/- 3%) isonitrogenous casein diets. Apparent retentions of nitrogen from Nasha (26 +/- 10%) were significantly lower than those from Lys + (34 +/- 9%, P less than 0.05), Cas-1 (35 +/- 11%, P less than 0.01) or Cas-2 (49 +/- 9%, P less than 0.01). Retentions from Cas-2 were higher than those from Cas-1 or Lys + (P less than 0.01). Fecal wet and dry weights were higher (P less than 0.02) during both Nasha diets and Cas-2 than during Cas-1. Fecal energy and carbohydrate were significantly (P less than 0.01) higher from either Nasha diet than from either casein diet; fecal fat was not different. Two children received drum-dried Nasha without further cooking; digestibilities were not different from those of the cooked product but biological value was much lower. When properly cooked and consumed along with small amounts of a good source of lysine, Nasha is a satisfactory weaning food.

Pepsin digestibility of proteins in sorghum and other major cereals.

We have shown previously that sorghum is highly digestible in the rat. However, other workers have shown that sorghum is much less digestible than wheat, maize, and rice in young children. Because the rat does not show these digestibility differences, we developed an empirical pepsin digestion method, first reported in 1981, which simulates the digestion values found in children. In this report the method has been improved and used to analyze wheat, maize, rice, millet, and sorghum and certain processed samples of millet and sorghum. The pepsin digestion values parallel those found in children for wheat, maize, rice, and sorghum. In addition, a processed sorghum product that gave a high digestion value in children also gave a high value with the in vitro pepsin method.

Digestibility of sorghum proteins.

Published information indicates that rice, maize, and wheat proteins are much more digestible in children than sorghum proteins are (66-81% compared with 46%). However, this digestibility difference cannot be demonstrated with the weanling rat, which gave digestibility values of 80% for cooked and 85% for uncooked sorghum gruels. Therefore, a search was made for a laboratory system sensitive to the digestibility differences between sorghum and other cereals. We found that porcine pepsin in vitro shows these digestibility differences. Using pepsin, we have found that uncooked sorghum proteins have a high digestibility (78-100%), which drops to a range of 45-55% after cooking. Two fermented sheet-baked sorghum products (kisra and abrey) from Sudan gave pepsin digestibility values of 65-86%. In contrast, unfermented cooked gruels made in our laboratory from the same flours used for the kisra and abrey gave pepsin values of only 44-56%. Therefore, fermentation improves pepsin digestibility of sorghum. The digestibility values of other sorghum-based foods prepared in the semiarid tropics need surveying. Those with high pepsin digestibility values hopefully should be more digestible (in children) than are the cooked sorghum gruels studied to date by human nutritionists.