Random-amplified-polymorphic DNA markers in sorghum

Conditions have been identified that allow reproducible amplification of RAPD markers in sorghum. High resolution of RAPD markers was accomplished by radiolabeling PCR-amplified DNAs followed by separation on denaturing 5% polyacrylamide gels. Reaction parameters including MgCl₂ concentration and temperature significantly influenced yield and the type of amplification products synthesized. Unexplained amplified DNAs increased when more than 35 cycles of PCR amplification were used. Under standard conditions, approximately 80% of the primers tested amplified DNA, and most revealed 1–5 polymorphisms between BTx 623 and IS 3620C. Primers were used to amplify RAPDs in 32 genotypes of sorghum. In addition, 8 primers detected RAPDs in a population previously used to create an RFLP map for sorghum. These RAPDs were mapped successfully using a population of 50 F₂ plants.     

Antioxidative response in different sorghum species under short-term salinity stress

Seedlings of sorghum varieties (M35-1, a drought tolerant species; SPV-839, a drought sensitive one) differing in their drought tolerance were subjected to 150 mM NaCl stress for a short duration of time (up to 72 h). Both the varieties failed to exhibit efficient ion exclusion mechanism like that of salt tolerant species, but in turn resulted in higher accumulation of Na⁺ and Cl⁻ ions over a period of 72 h salt stress. In addition, accumulation of calcium, potassium and proline in seedlings of sorghum varieties was moderate to short-term NaCl stress. The modulation of antioxidant components significantly diverged between the two varieties during seed germination, further the efficiency of antioxidant scavenging system is maintained during short-term NaCl treatments. In comparison to tolerant variety, the sensitive variety depicted higher SOD activity under control and salinity treatments but specific activity of catalase was significantly reduced. In contrast, drought tolerant variety exhibited efficient hydrogen peroxide scavenging mechanisms with higher catalase and GST activities under control and salt stress conditions, but not in the sensitive one. In conclusion, our comparative studies indicate that drought tolerant and susceptible varieties of sorghum induce efficient differential oxidative components of enzymatic machinery for scavenging ROS thereby alleviating the oxidative stress generated by salt stress during seedling growth.     

Rapid evaluation of sorghum for aluminum tolerance

Two screening methods tested in this study were based on the observation that roots of freshly germinated seeds exhibit their relative tolerance to Al during the first 48 hours of growth. Sorghum (Sorghum bicolor L. Moench) varieties were evaluated using toxic/nontoxic soil pairs in petri dishes and toxic/nontoxic solution pairs in a flowing aqueous system. The soils had 0, 61, and 72% Al saturation and the solutions had 0, 1.85 and 3.70 M l^–1 Al from AlCl₃ and 0.25 mM l^–1 Ca from Ca(NO₃)₂. Relative root lengths in both systems correctly differentiated 13 genotypes of known Al tolerance into tolerant and intolerant groups. Twenty six other sorghums genotypes were also screened using genotypes of known Al reactions as checks. The soil with 72% Al saturation and the 1.85 M l^–1 Al solution gave the highest correlation between the two systems and both effectively arrayed sorghums of known and unknown tolerance. For routine screening the authors prefer the soil system for its simplicity, efficiency, and use of a natural growth medium.Journal paper 11637. Purdue University Agricultural Experiment Station, Lafayette, Indiana. Contribution from the Agronomy Department.     

Waterlogging injury in sorghum seedlings and their kinetin effected rapid recovery

Short term waterlogging affected the growth of sorghum seedlings as indicated by a high mortality rate of the seedlings and a decrease in the shoot and root biomass, net assimilation rate (NAR), leaf area ratio (LAR), relative growth rate (RGR), total photosynthetic area and rate of photosynthesis. Plant resistance to the stress was exhibited by decreased stomatal index (SI), rate of transpiration and rate of respiration. When the stressed plants were exposed to normal conditions, the recovery of the seedlings was rapid and was enhanced by foliar application of kinetin at 5 mgl^-1.     

Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus)

Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T₁ plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1–8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt -endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer.     

Relationship between contents of leucoanthocyanidin and dhurrin in sorghum leaves

Summary Flag leaves of Colman forage sorghum (Sorghum bicolor) contain at least 25 times as much leucoanthocyanidin (LAC) and approximately half as much of the cyanogenic glucoside, dhurrin, as do flag leaves of White Collier forage sorghum. Assays of flag leaves from 119 F₂ plants and 11 F₅ lines from crosses between these two cultivars revealed a statistically significant negative association between levels of LAC and dhurrin. Both LAC and dhurrin are aromatic compounds, and the negative association between the two may be the result of competition for intermediates or products of the aromatic biosynthetic pathway. This rationale appears to be quite different from that for the negative association reported for levels of tannin and cyanide in Lotus corniculatus. Although the negative relationship between LAC and dhurrin in sorghum was statistically significant, the association was not consistent enough to suggest that either trait could be used reliably in selecting or breeding to modify the other trait.Contribution from USDA-ARS and the University of Nebraska Agric. Res. Div. Published as Paper No. 8003, Journal Series, Agric. Res. Div. The work reported was done under Agric. Res. Div. Project 12–114     

Nutrition of sorghum plants fertilized with nitrogen or inoculated withAzospirillum brasilense

Summary Sorghum plants were inoculated withAzospirillum brasilense or received an N-amended nutrient solution. Azospirillum inoculation increased plant dry weight and nitrogen assimilation by 25%. Most plant growth responses to Azospirillum were comparable to application of 2.0 mM N. Increased scavenging of nutrients, altered root permeability or nitrogen fixation are possible explanations for these effects.This work was supported by the United States Department of Agriculture, Agricultural Research Service (CRIS No. 5102-20170-001) in collaboration with the University of California, Berkeley. Requests for reqrints to G. J. Bethlenfalvay.     

Mechanism of whorl feeding resistance to fall armyworm among converted sorghum accessions

Field studies were conducted in 1989 to evaluate selected converted sorghum (Sorghum bicolor [L.] Moench) accessions for resistance to whorl-stage feeding by larvae of the fall armyworm (Spodoptera frugiperda J. E. Smith) and to determine the mechanism of resistance. The sorghum was infested in the whorl 26 days after planting (DAP) in experiment 1 and 33 DAP in experiment 2. In experiment 1, the plant accessions IS7273C, IS7444C, IS12573C, IS12678C, and IS12679C were more resistant (rating <3) to damage by S. frugiperda larvae than the resistant check CIMMYT (CM) 1821 (rating 6.2) at 14 days after infestation (DAI). These genotypes were also more resistant (ratings 4 at 7 DAI and <3 at 14 DAI) than the resistant check CM1821 (ratings 5.6 at 7 DAI and 8 at 14 DAI) in experiment 2. The number of larvae that established/plant on IS7273C, IS7444C, IS12573C, or IS12679C was significantly less compared with establishment on the resistant check CM1821 at 14 DAI in experiment 1 and at 7 and 14 DAI in experiment 2. Resistance in IS7273C, IS7444C, IS12573C, and IS12679C was mainly due to their rapid rate of growth which induced a quick change in the plant morphology from the whorl- to the panicle-stage and did not permit a sustained colonization of larvae. This new type of resistance could be referred to as morphological non-preference as apposed to chemical non-preference where non-preference is due to plant chemical factors. These genotypes had a significantly shorter cycle than the other sorghum genotypes. Host evasion, a type of pseudoresistance, was the basis for resistance in IS7794C and IS7947C. Tolerance was the major mechanism of resistance in the resistant check CM1821.     

RAPD cluster analysis and chlorate sensitivity of some Indian isolates of Macrophomina phaseolina from sorghum and their relationships with pathogenicity

Charcoal rot caused by Macrophomina phaseolina is an economically important disease in sorghum grown during the post rainy season in India. Variations in random amplified polymorphic DNA (RAPD) polymorphisms, chlorate sensitivity and pathogenicity were studied among sorghum isolates of M. phaseolina collected from different parts of India. RAPD data based on 14 random primers of Kit A and C (OPA and OPC) on 20 isolates showed a high degree of polymorphism (98.1%) in different isolates. UPGMA dendrogram on RAPD data produced 7 clusters at the level of 37% similarity. Isolates from the same locations showed a tendency to group closer, substantiating closer genetic relatedness. Sorghum infecting Macrophomina isolates showed a mixed response for sensitivity to potassium chlorate (120 mM). Chlorate-resistant isolates were predominant (>65% of the isolates) over sensitive isolates. Chlorate-sensitive isolates were found to be genetically closer among them than the resistant ones. For the first time it was shown that chlorate sensitivity in Macrophomina had some relations with charcoal rot severity in sorghum.     

Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences

Summary Transgenic sorghum plants (Sorghum bicolor L. Moench, cv. SRN39) were obtained by microprojectile-mediated DNA delivery (Bio-Rad PDS 1000/He Biolistic Delivery System) to explants derived from immature inflorescences. Explants were precultured on medium supplemented with 2.5 mg/l (11.31 μM) 2,4-D, 0.5 mg/l (2.32 μM) kinetin, and 60 g/l sucrose for 1 to 2 wk prior to bombardment. Bialaphos selectron pressure was imposed 2 wk after bombardment and maintained throughout all the culture stages leading to plant regeneration. More than 2500 explants from 1.5 to 3.0 cm inflorescences were bombarded and subjected to bialaphos selection. Out of more than 190 regenerated plants, 5 were determined to be Ignite resistant. Southern analyses confirmed the likelihood that the 5 herbicide resistant plants derived from two independent transformation events. The phosphinothricin acetyltransferase gene (bar) was inherited by and functionally expressed in T₁ progeny. However, no β-glucuronidase (GUS) activity could be detected in T₁ plants that contained uidA restriction fragments. Histological analyses indicated that in the absence of bialaphos morphogenesis was primarily via embryogenesis while organogenesis was more predominant in callus maintained with herbicide selection.     

Aluminum stress on sorghum growth and nutrient relationships

Summary Sorghum plants were grown in the greenhouse in modified Steinberg nutrient solution containing ten Al rates (0 to 297 μM) and harvested 28 days after transplanting. Top and root dry weight were not affected by added Al up to 74 μM; but decreased sharply at concentration of 148 μM and greater. Aluminum concentrations in blade 1 (recently matured blade) and plants remained constant from 0 to 297 μM added Al. Root Al concentration increased as added Al increased. No correlation existed between top dry weight and Al concentration in blade 1 or in plant. Root Al concentration was related to top dry weight and root dry weight to estimate the Al critical toxicity level. The Al critical toxicity levle in the root was 54 mmol kg⁻¹ root dry weight basis for either top or root dry weight. In blade 1 Cu concentration negatively correlated with Al while Fe and P were positively correlated. In roots Ca, Mg, Mn and Fe concentrations were negatively correlated with Al while Zn, Cu, P, and K were positively correlated with Al concentration.     

Effect of moisture stress, plant population density and pathogen inoculation on charcoal stalk rot of sorghum

The effects of moisture deficit stress, plant population density and pathogen inoculation technique on charcoal stalk rot in the sorghum hybrid CSH 6 were studied in the 1980–81 and 1981–82 post-rainy seasons at three locations in India. Incidence and severity of charcoal rot caused by Macrophomina phaseolina were compared in three plant population densities, subjected to different moisture stress regimes created by withholding irrigation at various plant growth stages. Natural infections were compared to artificial inoculation with M. phaseolina. Combinations of moisture stress, plant population and inoculation treatments were compared to identify the combination most likely to develop maximum disease. Lodging, the first external symptom of charcoal rot, was significantly correlated with other disease symptoms used to measure charcoal rot, such as soft stalk, number of nodes crossed by M. phaseolina infection, root damage and plant senescence. In both seasons the highest incidence of lodging occurred when moisture stress was induced at the 'flag leaf visible in the whorl' growth stage. The greatest incidence of the disease was recorded in the highest plant population (266 700 plant ha^-) at all three locations. No significant differences were found between artificially and naturally inoculated treatments. The maximum number of lodged plants was found at a density of 266 700 plants ha^-1 when moisture stress was induced at the 'flag leaf visible in the whorl' growth stage.     

Control of charcoal root rot in <Emphasis Type="Italic">Pinus radiata</Emphasis> nurseries with antagonistic bacteria

The objective of this study was to determine the potential of antagonistic bacteria to control charcoal root rot of coniferous seedlings caused by Macrophomina phaseolina (Tassi) Goid. in forest nurseries. Bacterial isolates were collected from nurseries located between Region Metropolitana and the VIII Region of Chile. Antagonists were initially evaluated in in vitro assays based on the ability to inhibit mycelial growth of M. phaseolina, and subsequently in two trials in a Pinus radiata nursery with a natural infestation of the pathogen. For nursery trials, the isolates were selected according to in vitro and field trial pathogen controls. The bacteria were applied as seed treatments and via water irrigation. The trials were conduced in a completely randomized block design. Among 568 bacterial isolates tested in vitro, 19.8% displayed some capacity to inhibit the mycelial growth of M. phaseolina, with inhibition between 1.7% and 67.6%. In the first nursery trial, Bacillus amyloliquefaciens VII 015, Bacillus pumilus IX 030, Bacillus stearothermophilus TM 008 and other two Bacillus sp. (VI 009 and IX 049) strains, significantly reduced the total, pre- and post-emergency mortality of seedlings, but no isolate reduced the incidence of M. phaseolina in seedlings. In the second trial, Bacillus sp. IX 049, VI 099, B. subtilis (IX 007) and a non-identified isolate V 005, decreased the incidence of charcoal root rot. It is concluded that the best of these bacterial antagonists have the potential to control M. phaseolina in P. radiata nurseries.     

Cloning and expression of a sorghum gene with homology to maize vp1. Its potential involvement in pre-harvest sprouting resistance

Pre-harvest sprouting (PHS) in sorghum is related to the lack of a normal dormancy level during seed development and maturation. Based on previous evidence that seed dormancy in maize is controlled by the vp1 gene, we used a PCR-based approach to isolate two Sorghum bicolor genomic and cDNA clones from two genotypes exhibiting different PHS behaviour and sensitivity to abscisic acid (ABA). The two 699 amino acid predicted protein sequences differ in two residues at positions 341 (Gly or Cys within the repression domain) and 448 (Pro or Ser) and show over 80, 70 and 60% homology to maize, rice and oat VP1 proteins respectively.Expression analysis of the sorghum vp1 gene in the two lines shows a slightly higher level of vp1 mRNA in the embryos susceptible to PHS than in those resistant to PHS during embryogenesis. However, timing of expression was different between these genotypes during this developmental process. Whereas for the former the main peak of expression was observed at 20 days after pollination (DAP), the peak in the latter was found at later developmental stages when seed maturation was almost complete.Under favourable germination conditions and in the presence of fluridone (an inhibitor of ABA biosynthesis), sorghum vp1 mRNA showed to be consistently correlated with sensitivity to ABA but not with ABA content and dormancy.     

Identification of genomic regions affecting plant height in sorghum and maize

The objective of this study was to use restriction fragment length polymorphisms (RFLPs) to determine the genetic location and effects of genomic regions controlling plant height in sorghum. F₂ plants (152) from the cross CK60 x PI229828 were used. Genomic and cDNA clones (106) identified 111 loci distributed among ten linkage groups covering 1299 cM. Interval mapping identified four regions, each in a separate linkage group. These regions may correspond to loci (dw) previously identified by alleles with qualitative effects. Also, these regions identified in sorghum may be orthologous to those previously reported for plant height in maize. Gene effects and gene action varied among genomic regions. In each region, PI229828 alleles resulted in increased plant height. Each region accounted for 9.2–28.7% of the phenotypic variation. Positive, additive effects ranged from 15 to 32cm. Tallness was dominant or overdominant and conferred by alleles from PI229828 for three quantitative trait loci (QTL). At the fourth QTL, PI229828 contributed to increased plant height, but short stature was partially dominant. One digenic interaction was significant. The presence of a PI229828 allele at one region diminished the effects of the other region. A multiple model indicated that these four regions collectively accounted for 63.4% of the total phenotypic variation. The utility of this information for germplasm conversion through backcross breeding is discussed.Journal Paper No. J. 15649 of the Iowa Agriculture and Home Economic Experiment Station, Ames, Iowa. Project No. 3134     

Heterosis and the metabolism of gibberellin A20 in sorghum

The correlation between gibberellin (GA) metabolism and growth rate was investigated using two Sorghum bicolor inbred lines, Hegari and AT×623, and their heterotic F₁ hybrid. Previous studies have demonstrated that this hybrid is taller and has substantially greater shoot dry weights and leaf areas than either parental inbred. [³H]GA₂₀ was applied to the leaf whorl of seedlings and after 24 hours, plants were harvested and separated into roots, shoot cylinders containing the apical meristems, and leaf blades. Chromatographic analyses of metabolites indicated the conversions of [³H]GA₂₀ to [³H]GA_{1,8 and 29}. The conversion of [²H]GA₂₀ to [²H]GA₁ was demonstrated by gas chromatography-selected ion monitoring (GC-SIM). Putative glucosyl conjugates of all of the [³H]GAs were also produced and GA₈ was identified by GC-SIM following enzymic cleavage of the putative [³H]GA₈ glucosyl conjugate fraction. Comparing the genotypes, [³H]GA₂₀ metabolism was more rapid in the shoot cylinders of the hybrid than in the shoot cylinders from inbreds. In the hybrid samples, there was a three-fold increase in the putative conjugate(s) of [³H]GA₁ which was the principal metabolite, and increased production of [³H]GA₈ and the putative conjugates of [³H]GA₂₉ and [³H]GA₈. Conversely, levels of the remaining precursor, [³H]GA₂₀, and its putative conjugate(s) were reduced in the hybrid. The rate of GA₂₀ metabolism was thus positively correlated with growth rate across these sorghum genotypes. This correlation supports a promotive role of GA in the regulation of shoot growth and in the expression of heterosis (hybrid vigor) in sorghum.     

Genetic variability among sorghum accessions from the Sahelian agroecological region of Burkina Faso

The authors collected Sahelian sorghum landraces of Burkina Faso in 1984 and 74 of these accessions were characterized in 1985–1986 at Gampela in Burkina Faso (West Africa). The five races of cultivated sorghum were represented in this zone but 63.5% of the accessions were Guinea type. Great intra-and inter-accession variability was found. Plants were tall and had long panicles and small to intermediate seeds. There was a strong association between days-to-flowering, number of internodes, panicle length and height. The 100-kernel weight showed an antagonism with days to flowering and tillering. Multivariate analyses were made which enabled the accessions to be classified into four groups. The group most adapted to the sahelian zone, sahelian group, was semi-late, developed intermediate size of vegetative organs, had moderate tillering and had the best yield per plant.     

Screening sorghum for tolerance to excess manganese in solution culture

This study was conducted to define traits to screen sorghum (Sorghum bicolor L. Moench) genotypes for tolerance to excess Mn. Visual Mn toxicity symptoms, net and total root lengths, shoot and root dry matter yields, and shoot and root Mn concentrations were determined for plants grown in nutrient solutions (pH 4.5) at different levels of Mn (0, 3, 6, 9 and 12 mM above the initial 18 M) to assess plant responses to excess Mn. Dry matter yields showed greatest variability among genotypes, and was an effective trait to evaluate sorghum for tolerance to excess Mn. Reductions in dry matter yields did not occur until Mn levels were above 3 mM. Levels of Mn between 3 and 6 mM could effectively be used to screen sorghum for genotypic differences to excess Mn. Manganese levels above 6 mM were too severe to allow good genotypic differentiation. Of genotypes tested, NB9040 and Wheatland showed good tolerance and SC283 and ICA-Nataima were sensitive to excess Mn.     

Screening for grain polyphenol variants from high-tannin sorghum somaclones

Several hundred somaclones established from plants regenerated from embryogenic callus cultures of six high tannin sorghum lines were screened for variants with altered levels of polyphenols in the grain. Grain from over 6000 plants including the R ₁ (primary), R₂, and R₃ generations were analyzed for total phenols, flavan-4-ols, and proanthocyanidins (condensed tannins). Although many variants which had lost the ability to synthesize chlorophyll were found, none of the somaclones tested had lost or greatly reduced the ability to synthesize any of the polyphenols assayed. However, we did observe statistically significant differences in polyphenol concentration between tissue culture-derived R₁ plants and the parental controls. In the R₂ generation the proportion of somaclones which differed significantly from the parents varied from 47% to 68% depending upon genotype. The average somaclonal variation rate and somaclonal variant frequency estimated in the tested population for the three polyphenol characteristics ranged from 37.3% to 40.7% and 5.3% to 7.8%, respectively. Variants with decreased levels of polyphenols were usually epigenetic and reverted back to normal levels in subsequent generations, but those with increased levels usually persisted after two meiotic cycles, indicating they are heritable. Variants with polyphenol levels increased up to 80% or decreased by 30% were selected for in the R₃ generation.