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.     

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.     

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     

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.     

Resistance to Striga hermonthica in a maize inbred line derived from Zea diploperennis

Breeding for resistance to Striga in maize (Zea mays), with paucity of donor source and known mechanisms of resistance, has been challenging. Here, post-attachment development of S. hermonthica was monitored on two maize inbreds selected for field resistance and susceptibility reactions to Striga at the International Institute of Tropical Agriculture. Haustorial invasion of the parasite into roots of these inbreds was examined histologically. Morphological differences were observed between roots of the susceptible and the resistant inbreds. The resistant maize had fewer Striga attachments, delayed parasitic development and higher mortality of attached parasites compared with the susceptible inbred. Striga on the susceptible inbred usually penetrated the xylem and showed substantial internal haustorial development. Haustorial ingress on the resistant inbred was often stopped at the endodermis. Parasites able to reach resistant host xylem vessels showed diminished haustorial development relative to those invading susceptible roots. These results suggest that the resistant inbred expresses a developmental barrier and incompatible response against Striga parasitism.     

Marker-assisted selection for early-season cold tolerance in sorghum: QTL validation across populations and environments

Sorghum [Sorghum bicolor (L.) Moench] landraces from China generally exhibit excellent emergence and seedling vigor under cool conditions, and are being used as sources of genes for improvement of seedling cold tolerance in other cultivars. Marker-assisted selection (MAS) could expedite the introgression of genes from landraces into elite lines, however, only a few studies have empirically demonstrated efficacy of MAS for quantitatively inherited agronomic traits. In a preceding study we identified quantitative trait loci (QTL) for early-season performance in a recombinant inbred (RI) population, one parent of which was a cold-tolerant Chinese line, ‘Shan Qui Red’ (SQR). In this study, three SSR markers (Xtxp43, Xtxp51, and Xtxp211), each representing a QTL, were tested in two new populations: (Tx2794 × SQR F₃) and (Wheatland × SQR BC₁F₃). Individual families were genotyped, and early-season field performance was measured for two years. Statistical analyses showed that the SQR allele of Xtxp43 had favorable effects on seedling vigor in both populations, and on emergence in the Tx2794 population. A large positive effect of the SQR allele of Xtxp51 was observed in the Tx2794 population for vigor and emergence. Slight genotype by environment interaction was observed for Xtxp51 in the Wheatland population. Marker Xtxp211 had small but significant effects on seedling vigor and emergence in both populations. Various interactions between loci were also significant. This study validated QTL markers in various genetic backgrounds, and demonstrated the utility of MAS for a quantitative trait, early-season cold tolerance, evaluated in the field.     

Enhancement of ethylene biosynthesis and germination with thidiazuron and some selected auxins in Striga asiatica seeds

Witchweed [ Striga asiatica (L.) Kuntze], an economically important parasitic weed on several poaceous crops, is difficult to control. In nature, germination and subsequent morphogenesis of Striga are cued to specific host-derived chemical signals. Seeds (approximately 2.4 mg) treated with thidiazuron (TDZ) or the auxins 2,4-dichlorophenoxy-acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), or 2-(4-chloro- o -tolyloxy) propionic acid (MCPP) produced little ethylene (66-138 nl l⁻¹). Combinations of TDZ with the auxins increased ethylene production by 4- to 18-fold. Ethylene production was strongly inhibited (86–92%) by aminoethoxyvinylglycine (AVG), inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. Ethylene evolved from seeds treated with TDZ in combination with 2,4-D increased after a lag period and was promoted by a pretreatment in 2,4-D. TDZ or any of the auxins, at the rates tested, effected negligible to low levels of germination (0 to 16%), whereas mixtures of TDZ with the above auxins stimulated 38 to 84% germination. Test solutions containing TDZ and indole-3-acetic acid (IAA) were, however, less effective. TDZ/auxin-induced germination was inhibited by AVG and the ethylene action inhibitor silver thiosulfate (STS). The inhibitory effect of the former was reversed by treatment with ACC. In vitro studies revealed negligible germination (< 1%) on control medium. Seeds germinating on media containing TDZ alone developed into seedlings with distinct shoots and rudimentary roots. Seeds germinating on media containing 2,4-D, irrespective of TDZ concentration, were induced to form calli. The results are consistent with a model in which both germination and subsequent morphogenesis in Striga are associated with exogenous and endogenous phytohormones.     

Genetic analysis of post-flowering drought tolerance and components of grain development in Sorghum bicolor (L.) Moench

Drought is a serious agronomic problem and the single greatest factor contributing to crop yield loss in the world today. This problem may be alleviated by developing crops that are well adapted to dry-land environments. Sorghum (Sorghum bicolor (L.) Moench) is one of the most drought-tolerant grain crops and is an excellent crop model for evaluating mechanisms of drought tolerance. In this study, a set of 98 recombinant inbred (RI) sorghum lines was developed from a cross between two genotypes with contrasting drought reactions, TX7078 (pre-flowering-tolerant, post-flowering susceptible) and B35 (pre-flowering susceptible, post-flowering-tolerant). The RI population was characterized under drought and non-drought conditions for the inheritance of traits associated with post-flowering drought tolerance and for potentially related components of grain development. Quantitative trait loci (QTL) analysis identified 13 regions of the genome associated with one or more measures of post-flowering drought tolerance. Two QTL were identified with major effects on yield and 'staygreen under post-flowering drought. These loci were also associated with yield under fully irrigated conditions suggesting that these tolerance loci have pleiotropic effects on yield under non-drought conditions. Loci associated with rate and/or duration of grain development were also identified. QTL analysis indicated many loci that were associated with both rate and duration of grain development. High rate and short duration of grain development were generally associated with larger seed size, but only two of these loci were associated with differences in stability of performance under drought.