Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice.

The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

Exposure to nitric oxide increases the nitrosyl-iron complexes content in sorghum embryonic axes.

This work was aimed to investigate nitrosyl-Fe complexes formation by reaction of endogenous ligands and Fe, in sorghum embryonic axes exposed to NO-donors. Electron paramagnetic resonance (EPR) was employed to detect the presence of nitrosyl-Fe complexes in plant embryos, as well as changes in labile iron pool (LIP). Nitrosyl-Fe complexes formation was detected in sorghum embryonic axes homogenates incubated in vitro in the presence of 1 mM of NO donors: diethylenetriamine NONOate (DETA NONOate), S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP). In axes isolated from seeds incubated in vivo in the presence of 1 mM SNP for 24 h, the content of NO was increased by 2-fold, and the EPR spectrum from mononitrosyl-Fe complexes (MNIC) was observed with a concomitant increase in the fresh weight of sorghum axes. The simultaneous exposure to deferoxamine and the NO donor precluded the increase in fresh weight observed in the presence of excess NO. While total Fe content in the axes isolated from seeds exposed to 1mM SNP was not significantly affected as compared to control axes, the LIP was increased by over 2-fold.The data reported suggest a critical role for the generation of complexes between Fe and NO when cells faced a situation leading to a significant increase in NO content. Moreover, demonstrate the presence of MNICs as one of the important components of the LIP, which could actively participate in Fe cellular mobilization.

Apospory appears to accelerate onset of meiosis and sexual embryo sac formation in sorghum ovules.

BACKGROUND: Genetically unreduced (2n) embryo sacs (ES) form in ovules of gametophytic apomicts, the 2n eggs of which develop into embryos parthenogenetically. In many apomicts, 2n ES form precociously during ovule development. Whether meiosis and sexual ES formation also occur precociously in facultative apomicts (capable of apomictic and sexual reproduction) has not been studied. We determined onset timing of meiosis and sexual ES formation for 569 Sorghum bicolor genotypes, many of which produced 2n ES facultatively. RESULTS: Genotype differences for onset timing of meiosis and sexual ES formation, relative to ovule development, were highly significant. A major source of variation in timing of sexual germline development was presence or absence of apomictic ES, which formed from nucellar cells (apospory) in some genotypes. Genotypes that produced these aposporous ES underwent meiosis and sexual ES formation precociously. Aposporous ES formation was most prevalent in subsp. verticilliflorum and in breeding lines of subsp. bicolor. It was uncommon in land races. CONCLUSIONS: The present study adds meiosis and sexual ES formation to floral induction, apomictic ES formation, and parthenogenesis as processes observed to occur precociously in apomictic plants. The temporally diverse nature of these events suggests that an epigenetic memory of the plants' apomixis status exists throughout its life cycle, which triggers, during multiple life cycle phases, temporally distinct processes that accelerate reproduction.

Importance of seed-borne fungi of sorghum and pearl millet in Burkina Faso and their control using plant extracts.

Seed-borne fungi of sorghum and pearl millet in Burkina Faso were surveyed. A total of 188 seed samples from various locations, collected in 1989 (42) and 2002 (146), were tested, using the blotter, dry inspection and washing methods. Infection experiments were carried out with the major fungi recorded on each crop by the blotter test. Six essential oils of plants were investigated for their inhibitory activity against eight pathogenic fungi. Thirty four and 27 fungal species were found in seed samples of sorghum and pearl millet, respectively. Phoma sp. and Fusarium moniliforme infected 95 to 100% of the seed samples of both sorghum and pearl millet. Sphacelotheca sorghi and Tolyposporium ehrenbergii were encountered in respectively, 75 and 33% of seed samples of sorghum. T. penicillariae, Sclerospora graminicola and Claviceps fusiformis were present in 88, 41 and 32% of seed samples of pearl millet, respectively. Seeds inoculated with Acremonium strictum, Curvularia oryzae, F. equiseti, F. moniliforme and F. subglutinans and sown in sterilized soil, showed considerable mortality of the seedlings. Three essential oils inhibited in vitro the mycelial growth of all the fungi used by 85 to 100% and reduced significantly sorghum and pearl millet seed infection rates of Phoma sp., Fusarium sp., Curvularia sp., Colletotrichum graminicola and Exserohilum sp. Presence of many pathogenic fungi in considerable number of seed samples indicates the need of field surveys for these and other pathogens. Development of plant extracts for the control of seed-borne pathogens and public awareness on seed-borne diseases management measures for maintaining quality seed should be increased.

Evaluation of dyes decolourisation by the crude enzyme from Pleurotus sajor-caju grown on sorghum seed media.

The extracellular enzymes from Pleurotus sajor-caju were studied for lignin degrading enzyme patterns and dye decolourisation potential. Laccases are major ligninolytic enzymes excreted by the fungus. The results from a native-PAGE revealed that there were at least two isoenzymes. The crude enzyme had a pH and a temperature optimum at 6.0 and 40 degrees C, respectively when 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was used as substrate. The pH and thermal stability were at 5.0 and 30 degrees C. The pH optima for decolourisation of Indigo Carmine and Methyl Red were at 5.0 and 6.0, respectively. Indigo Carmine could be decolorized efficiently above 90% within 180 min, whereas Methyl Red could be decolorized only 3.5%. High efficiency decolourisation of Indigo Carmine makes this fungus to be a promise choice in biological treatment of waste water containing Indigo Carmine.

Expression of cell wall invertase and several other genes of sugar metabolism in relation to seed development in sorghum (Sorghum bicolor).

We report expression profiles of several genes of carbohydrate metabolism, cell wall invertase (CWI) in particular, to better understand sugar transport and its utilization in developing caryopses of grain sorghum [Sorghum bicolor (L.) Moench]. Gene expression analyses for CWI using RNA gel blot and real-time quantitative PCR approaches on developing caryopses, including the glumes (maternal tissue appended to the seeds), showed expression of SbIncw (ZmIncw2 ortholog) primarily in the basal sugar unloading zone of endosperm. The expression of ZmIncw1 ortholog was significantly less abundant and restricted to the glumes. The protein and enzyme activity data corroborated the temporal transcript expression profile that showed maximal CWI protein (INCW) expression preceding the starch-filling phase of endosperm development, i.e. 6-12d-after-pollination (DAP). Protein gel blot analysis using polyclonal maize INCW1 antibodies showed a single polypeptide of 72kDa. The highest level of enzyme activity was unique to the basal part of the endosperm, in particular the basal endosperm transfer cell (BETC) layer and the maternal pedicel region that were highly enriched for the INCW protein, as seen by immunolocalization. High hexose-to-sucrose ratio in 6-12 DAP seeds, and negligible starch deposition in glumes corroborated the CWI activity data. Additionally, we report transcription profiles of several other genes related to sugar-to-starch metabolism in developing sorghum endosperm. As in maize, the INCW-mediated apoplastic cleavage of sucrose in the BETC and pedicel during the early developmental stages of caryopses is essential for the normal development of filial tissues. The unique cell-specificity of the INCW protein to both proximal and distal ends of placental sac shown here for the first time is likely to greatly increase uptakes of both hexose sugars and water through turgor sensing into developing seed. This trait is unique to sorghum among cereals and may facilitate its survival in drought environment.

Endosperm-specific hypomethylation, and meiotic inheritance and variation of DNA methylation level and pattern in sorghum (Sorghum bicolor L.) inter-strain hybrids.

Understanding dynamics and inheritance of DNA methylation represents important facets for elucidating epigenetic paradigms in plant development and evolution. Using four sets of sorghum (Sorghum bicolor L.) inter-strain hybrids and their inbred parents, the developmental stability and inheritance of cytosine methylation in two tissues, leaf and endosperm, by MSAP analysis were investigated. It was found that in all lines (inbred and hybrid) studied, endosperm exhibited a markedly reduced level of full methylation of the external cytosine or both cytosines at the CCGG sites relative to leaf, which caused a variable reduction in the estimated total methylation level in endosperm by 6.89-19.69% (11.47% on average). For both tissues, a great majority of cytosine methylation profiles transmitted to F1 hybrids, however, from 1.69 to 3.22% of the profiles showed altered patterns in hybrids. Both inherited and altered methylation profiles can be divided into distinct groups, and their frequencies are variable among the cross-combinations, and between the two tissues. The variations in methylation level and pattern detected in the hybrids were not caused by parental heterozygosity, and they could be either non-random or stochastic among hybrid individuals. Homology analysis of isolated bands that showed endosperm-specific hypomethylation or variation in hybrids indicated that diverse sequences were involved, including known-function cellular genes and mobile elements. RT-PCR analysis of six genes representing endosperm-specific hypomethylation in MSAP profiles indicated that all showed higher expression in endosperm than in leaf, suggesting involvement of methylation state in regulating tissue-specific or tissue-biased expression in sorghum. Analysis on leaf-RNA from 5-azacytidine-treated plants further corroborated this possibility.

Effects of phosphorus and potassium on growth, yield and fodder quality of IS 23585 forage sorghum cultivar (Sorghum bicolor L. Moench).

This growth analysis experiment was carried out at Khon Kaen University Experimental Farm, Khon Kaen, Northeast Thailand in the 2000, in order to justify the effects of phosphorus (P) and potassium (K) levels added to Yasothon soil series (Oxic Paleustults) on dry matter yield and fodder quality of IS 23585 forage sorghum cultivar. The experiment was laid in a 4x4 factorial arranged in a randomized complete block design (RCBD) with four replications. The P levels used were 0, 37.50, 75.00 and 150.00 kg P2O5 ha(-1) and K levels were 0, 56.25, 112.50 and 225.00 kg K2O ha(-1). The results showed that total dry weight (yield) and other growth parameters in most sampling periods significantly increased with an increase in K levels but the increase was only up to K1. Higher levels of both P and K had no significant effect on total dry weight (yield) and other growth. There were no consistent trends due to both P and K application rates. However, mean Leaf Area Index (LAI) reached a maximum value of 10.21 (P3K3) at 8 weeks after emergence (WAE) and then a decline to 5.37 (P0K3) at 10 WAE. K significantly affected LAI only at 2 and 6 WAE. Crop growth rate (CGR) and leaf area duration (D) significantly increased up to K1, whilst P did not. Both optimum total dry weight of 1,7221 kg ha(-1) (10 WAE) and seed yields of 5,169 kg ha(-1) were obtained from P0K1. Both P and K had no significant effect on 1000-seed weight. P did not affect brix value, whilst K significantly depressed this parameter. K had no significant effect on crude protein (CP), whilst P did. P had no significant effect on neutral detergent fibre (NDF), whilst K significantly did it. Both P and K had no significant effect on both acid detergent fibre (ADF) and ruminal dry matter degradability (DMD) of the sorghum plants.

Rapid and reproducible Agrobacterium-mediated transformation of sorghum.

A rapid and reproducible Agrobacterium-mediated transformation protocol for sorghum has been developed. The protocol uses the nptII selectable marker gene with either of the aminoglycosides geneticin or paromomycin. A screen of various A. tumefaciens strains revealed that a novel C58 nopaline chromosomal background carrying the chrysanthopine disarmed Ti plasmid pTiKPSF(2), designated NTL(4)/Chry5, was most efficient for gene transfer to sorghum immature embryos. A NTL(4)/Chry5 transconjugant harboring the pPTN290 binary plasmid, which carries nptII and GUSPlus expression cassettes, was used in a series of stable transformation experiments with Tx430 and C2-97 sorghum genotypes and approximately 80% of these transformation experiments resulted in the recovery of at least one transgenic event. The transformation frequencies among the successful experiments ranged from 0.3 to 4.5%, with the average transformation frequency being approximately 1% for both genotypes. Over 97% of the transgenic events were successfully established in the greenhouse and were fully fertile. Co-expression of GUSPlus occurred in 89% of the transgenic T(0) events. Seed set for the primary transgenic plants ranged from 145 to 1400 seed/plant. Analysis of T(1) progeny demonstrated transmission of the transgenes in a simple Mendelian fashion in the majority of events.

In vitro culture methods in sorghum with shoot tip as the explant material.

Twenty-four diverse genotypes of sorghum were evaluated for response to callus induction and plant regeneration with two media viz., MS and NBKNB using shoot tips as the start material to identify a model genotype. None of the genotypes tested showed promising results. Therefore, alternative methods of in vitro pathways using shoot meristem isolated from shoot tips were explored. Shoot apical meristems were isolated and were induced to multiple shoots or multiple shoot buds pathway by manipulation of thidiazuron (TDZ), 6-benzyl adenine (BAP) and 2, 4-dichlorophenoxy acetic acid (2, 4-D). Choice of the pathway whether large-scale multiplication of shoots or production of target tissues for transformation can be exercised based on the needs and applications. A simple procedure, for large scale handling of shoot tips is described in detail. Electron microscopic studies revealed that meristems isolated from 7-day-old seedlings are superior because of possessing greater number of transformation competent cells.