Development and maturation of surghum seeds on detached panicles grown in vitro

Summary A procedure for culturing detached panicles of sorghum, Sorghum bicolor (L.) Moench, was developed to achieve flowering, fertilization, and subsequent seed development and maturation in vitro. Sixteen sorghum genotypes (five high and eleven low in tannin) were tested for their ability to develop normally in culture. Panicles collected one to two days before the initiation of anthesis were cultured in flasks containing liquid medium. Contamination and medium darkening were the major obstacles encountered. Up to 55% of the panicles cultured reached physiological maturity in vitro. The frequency of seed set ranged from 30 to 97% depending upon genotype and medium. Seed and glume color were normal. Seed produced in vitro resembled those grown in vivo and germinated well, but were smaller than normal (100 kernel weight reached 50 to 70% of the control). Grain polyphenols were synthesized in the cultured panicles. Seed of high tannin genotypes produced in vitro were lower in total phenols and tannins and higher in flavan-4-ols and the 3-deoxyanthocyanidin pigments than control seed. This technique can be used for harvesting late-maturing stocks and for various sorghum studies.     

Treatment Outcome of Tuberculosis Patients under Directly Observed Treatment Short Course and Factors Affecting Outcome in Southern Ethiopia: A Five-Year Retrospective Study

Tuberculosis (TB) is one of the major public health and socio-economic issues in the 21^st century globally. Assessment of TB treatment outcomes, and monitoring and evaluation of its risk factors in Directly Observed Treatment Short Course (DOTS) are among the major indicators of the performance of a national TB control program. Hence, this institution-based retrospective study was conducted to determine the treatment outcome of TB patients and investigate factors associated with unsuccessful outcome at Dilla University Referral Hospital, southern Ethiopia. Five years (2008 to 2013) TB record of TB clinic of the hospital was reviewed. A total 1537 registered TB patients with complete information were included. Of these, 942 (61.3%) were male, 1015 (66%) were from rural areas, 544 (35.4%) were smear positive pulmonary TB (PTB+), 816 (53.1%) were smear negative pulmonary TB (PTB-) and 177(11.5%) were extra pulmonary TB (EPTB) patients. Records of the 1537 TB patients showed that 181 (11.8%) were cured, 1129(73.5%) completed treatment, 171 (11.1%) defaulted, 52 (3.4%) died and 4 (0.3%) had treatment failure. The overall mean treatment success rate of the TB patients was 85.2%. The treatment success rate of the TB patients increased from 80.5% in September 2008-August 2009 to 84.8% in September 2012–May 2013. Tuberculosis type, age, residence and year of treatment were significantly associated with unsuccessful treatment outcome. The risk of unsuccessful outcome was significantly higher among TB patients from rural areas (AOR = 1.63, 95% CI: 1.21–2.20) compared to their urban counterparts. Unsuccessful treatment outcome was also observed in PTB- patients (AOR = 1.77, 95% CI: 1.26–2.50) and EPTB (AOR = 2.07, 95% CI: 1.28–3.37) compared to the PTB+ patients. In conclusion, it appears that DOTS have improved treatment success in the hospital during five years. Regular follow-up of patients with poor treatment outcome and provision of health information on TB treatment to patients from rural area is recommended.     

QTL analysis of early-season cold tolerance in sorghum

Cool temperatures during the early-growing season are a major limitation to growing sorghum [Sorghum bicolor (L.) Moench] in temperate areas. Several landraces from China have been found to exhibit higher emergence and greater seedling vigor under cool conditions than most breeding lines currently available, but tend to lack desirable agronomic characteristics. The introgression of desirable genes from Chinese landraces into elite lines could be expedited by marker-assisted selection. Using a population of 153 RI lines, developed from a cross between Chinese landrace ‘Shan Qui Red,’ (SQR, cold-tolerant) and SRN39 (cold-sensitive), QTL associated with early-season performance under both cold and optimal conditions were identified by single marker analysis, simple interval mapping (SIM), and composite interval mapping (CIM). Germination was observed under controlled conditions, and other traits were measured in field plantings. Two QTL for germination were identified: one on linkage group SBI-03a, derived from SRN39, was significant under cold and optimal temperatures. The other, on group SBI-07b, showed greater significance under cold temperatures and was contributed by SQR. A region of group SBI-01a, derived from SQR, showed strong associations with seedling emergence and seedling vigor scores under early and late field plantings. A QTL for both early and late emergence was identified by CIM on SBI-02 which favored the SRN39 allele. SIM identified a QTL for early vigor on SBI-04 favoring the SQR genotype. Further studies are needed to validate the effects of these QTL, but they represent the first step in development of a marker-assisted breeding effort to improve early-season performance in sorghum.     

Heterogeneous inbred family (HIF) analysis: a method for developing near-isogenic lines that differ at quantitative trait loci

Abtract  Analysis of near-isogenic lines (NILs) that differ at quantitative trait loci (QTL) can be an effective approach for the detailed mapping and characterization of individual loci. Although NILs are useful for genetic and physiological studies, the time and effort required to develop these lines have limited their use. Here we describe a procedure to identify NILs for any region of the genome that can be analyzed with molecular or other genetic markers. The procedure utilizes molecular markers to identify heterogeneous inbred families (HIFs) that segregate for a genomic region of interest. Each HIF is isogenic at the majority of loci in the genome, but NILs differing for markers linked to QTL of interest can be extracted from segregating families. The application of this procedure is described for two QTL associated with seed weight in sorghum. A population of 98 HIFs was screened with two RAPD markers from different linkage groups that were associated with seed weight. Three segregating families were identified for each marker. The progeny of these HIFs were characterized for the segregation of seed weight and other yield components and for markers flanking each QTL. NILs derived from each HIF had significantly different seed weights confirming the presence of at least two loci that influence seed weight in sorghum. Received: 16 September 1996 / Accepted: 25 April 1997     

Towards effective resistance to Striga in African maize

The fascinating biology of Striga parasitism is manifest through a series of signal exchanges between the parasite and its host. As an obligate root hemi-parasite, Striga development is cued to exudates and solutes of host roots but with negative ramifications on host plant health. Striga control in crops, via a variety of biotechnological approaches, needs to be based on increased understanding of this intricate biology. Maize has become the major cereal crop of Africa. However, this New World transplant has shown a paucity of Striga resistance characters relative to native sorghum. In this paper, we review growing evidence for maize genetic defenses against early pre-emergent phases of the Striga life cycle, when the tolls of parasitism are first manifest. Resistance characters first described in maize wild relatives have now been captured in Zea mays. The possible stacking of new and complementary sources of resistance in improved maize varieties targeted for Striga prone areas is discussed. An integrated approach combining genetic with other control measures is advocated with a more realistic view of the resource challenges prevalent in African agriculture.Key words: Striga, parasitic weed, maize, sorghum, resistance, integrated control     

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.     

Somaclonal variation in high tannin sorghums

Summary 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.     

A Genomewide Analysis of the Cinnamyl Alcohol Dehydrogenase Family in Sorghum [Sorghum bicolor (L.) Moench] Identifies SbCAD2 as the Brown midrib6 Gene

The content and composition of the plant cell wall polymer lignin affect plant fitness, carbon sequestration potential, and agro-industrial processing. These characteristics, are heavily influenced by the supply of hydroxycinnamyl alcohol precursors synthesized by the enzyme cinnamyl alcohol dehydrogenase (CAD). In angiosperms, CAD is encoded by a multigene family consisting of members thought to have distinct roles in different stages of plant development. Due to the high sequence similarity among CAD genes, it has been challenging to identify and study the role of the individual genes without a genome sequence. Analysis of the recently released sorghum genome revealed the existence of 14 CAD-like genes at seven genomic locations. Comparisons with maize and rice revealed subtle differences in gene number, arrangement, and expression patterns. Sorghum CAD2 is the predominant CAD involved in lignification based on the phylogenetic relationship with CADs from other species and genetic evidence showing that a set of three allelic brown midrib (bmr) lignin mutants contained mutations in this gene. The impact of the mutations on the structure of the protein was assessed using molecular modeling based on X-ray crystallography data of the closely related Arabidopsis CAD5. The modeling revealed unique changes in structure consistent with the observed phenotypes of the mutants.     

Molecular tagging and validation of microsatellite markers linked to the low germination stimulant gene (<Emphasis Type="Italic">lgs</Emphasis>) for <Emphasis Type="Italic">Striga</Emphasis> resistance in sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench]

Striga is a devastating parasitic weed in Africa and parts of Asia. Low Striga germination stimulant activity, a well-known resistance mechanism in sorghum, is controlled by a single recessive gene (lgs). Molecular markers linked to the lgs gene can accelerate development of Striga-resistant cultivars. Using a high density linkage map constructed with 367 markers (DArT and SSRs) and an in vitro assay for germination stimulant activity towards Striga asiatica in 354 recombinant inbred lines derived from SRN39 (low stimulant) × Shanqui Red (high stimulant), we precisely tagged and mapped the lgs gene on SBI-05 between two tightly linked microsatellite markers SB3344 and SB3352 at a distance of 0.5 and 1.5 cM, respectively. The fine-mapped lgs region was delimited to a 5.8 cM interval with the closest three markers SB3344, SB3346 and SB3343 positioned at 0.5, 0.7 and 0.9 cM, respectively. We validated tightly linked markers in a set of 23 diverse sorghum accessions, most of which were known to be Striga resistant, by genotyping and phenotyping for germination stimulant activity towards both S. asiatica and S. hermonthica. The markers co-segregated with Striga germination stimulant activity in 21 of the 23 tested lines. The lgs locus similarly affected germination stimulant activity for both Striga species. The identified markers would be useful in marker-assisted selection for introgressing this trait into susceptible sorghum cultivars. Examination of the sorghum genome sequence and comparative analysis with the rice genome suggests some candidate genes in the fine-mapped region (400 kb) that may affect strigolactone biosynthesis or exudation. This work should form a foundation for map-based cloning of the lgs gene and aid in elucidation of an exact mechanism for resistance based on low Striga germination stimulant activity.     

Allelic Association, Chemical Characterization and Saccharification Properties of brown midrib Mutants of Sorghum (Sorghum bicolor (L.) Moench)

Genetic improvement of biomass crops can significantly reduce the overall cost of biomass-to-ethanol conversion. The conversion of cellulose to monomeric sugar units is affected by lignin content and composition. Sorghum has attracted the attention of the scientific and industrial community as a promising source of biomass for bioenergy due to its great yield potential and tolerance to stresses. The brown midrib (bmr) mutants of sorghum are characterized by brown vascular tissue associated with altered lignin content. Twenty-eight bmr mutants have been identified since the late 1970s, but the allelic relationships have not been fully established, and the function of only one of the Bmr loci has been unequivocally established. In this study, we combined genetic and chemical approaches to establish that there are mutations at least four independent bmr loci, represented by the bmr2, bmr6, bmr12 and bmr19 groups. Since each allelic group presents unique staining characteristics, rapid classification of emerging bmr lines into the existing groups can be achieved using phloroglucinol-HCl as a histochemical stain. In addition, pyrolysis-gas chromatography-mass spectrometry, enabled the characterization of changes in subunit lignin composition in each of the allelic groups, to help predict the genes underlying the mutations. Enzymatic saccharification of stover from plants representing each allelic bmr group demonstrated that lignin changes in lines belonging to the bmr2, bmr6 and bmr12 groups can increase glucose yields, up to 25% compared to wild-type isolines. In order to expedite the selection of the bmr mutant alleles in breeding populations, we have developed molecular markers specific for bmr7 and bmr25, two novel mutant alleles of the gene encoding caffeic acid O-methyl transferase. Based on the results from this study, we propose to rename the bmr mutants in a manner that reflects the number of independent loci.