Lignin reduction in sugarcane by performing CRISPR/Cas9 site-direct mutation of SoLIM transcription factor.

Genetic engineering of plant cell walls is limited for reducing lignocellulose recalcitrance, so mild and/or green-like pretreatment is still required for sequential enzymatic saccharification. Here, we report a method to reduce lignin content in sugarcane stalks using the CRISPR/Cas 9 technique. Three target sequences of SoLIM were designed and fused to pRGEB32. The cassette constructs were introduced into sugarcane calli cv. KK3 through Agrobacterium-mediated transformation. We produced one base substitution and one insertion line for the 1st target site; two insertions, one deletion, and one base substitution for the 2nd target site; and one base substitution and insertion for the 3rd target site. qRT-PCR analysis of SoLIM, SoPAL, SoC4H, and SoCAD showeded that downregulation of SoLIM by single nucleotide insertions or deletions reduced the expression of SoPAL, SoC4H, and SoCAD. Consequently, the edited lines contained 9.74 to 51.46% less lignin content compared to that in the wild-type plants. The syringyl/guaiacyl (S/G) ratio of the edited lines ranged between 0.23 and 0.49, while the wild-type was 0.22. The histochemical evaluation and scanning electron microscopy of the cell walls supported this observation. A low lignin content sugarcane will provide a better feedstock for second-generation bioethanol production.

In vitro and in vivo screening for the identification of salt-tolerant sugarcane (Saccharum officinarum L.) clones: molecular, biochemical, and physiological responses to salt stress.

Sugarcane is a glycophyte whose growth and yield can be negatively affected by salt stress. As the arable lands with potential saline soils expand annually, the increase of salt-tolerance in sugarcane cultivars is highly desired. We, herein, employed in vitro and in vivo conditions in order to screen sugarcane plants for salt tolerance at the cellular and at the whole plant levels. Calli of sugarcane cv. Khon Kaen 3 (KK3) were selected after culturing in selective media containing various NaCl concentrations, and regenerated plants were then reselected after culturing in selective media containing higher NaCl concentrations. The surviving plants were finally selected after an exposure to 254 mM NaCl under greenhouse conditions. A total of 11 sugarcane plants survived the selection process. Four plants that exhibited tolerance to the four different salt concentrations applied during the aforementioned screening process were then selected for the undertaking of further molecular, biochemical, and physiological studies. The construction of a dendrogram has revealed that the most salt-tolerant plant was characterized by the lowest genetic similarity to the original cultivar. The relative expression levels of six genes (i.e., SoDREB, SoNHX1, SoSOS1, SoHKT, SoBADH, and SoMIPS) were found to be significantly higher in the salt-tolerance clones than those measured in the original plant. The measured proline levels, the glycine betaine content, the relative water content, the SPAD unit, the contents of chlorophyll a and b, as well as the K+/Na+ ratios of the salt-tolerant clones were also found to be significantly higher than those of the original plant.When the salt-tolerant clones were grown in a low saline soil, they exhibited a higher Brix percentage than that of the original cultivar.

EMS-induced mutation followed by quizalofop-screening increased lipid productivity in Chlorella sp.

The objective of this study was to enhance biomass and lipid productivity in Chlorella sp. isolate 6-4 by inducing mutagenesis with two growth inhibitors: the herbicide quizalofop-P-ethyl, a known inhibitor of acetyl-CoA carboxylase (ACCase) activity, and chemical mutagen, ethyl methanesulfonate (EMS), at different concentrations and length of times. The induced-mutagenized microalgae were screened on selective medium containing 10-100 µM quizalofop. The biomass yield, biomass productivity, lipid content, and lipid productivity of mutagenized microalgae were determined. The result showed that 100-200 mM EMS concentrations and 30 min incubation time were the most effective. Biomass yield and biomass productivity of the mutagenized microalgae E50-30-40, E100-60-40, and E100-30-60 were statistically significant higher than those of the wild type. The mutagenized microalgae E100-30-60 showed that the highest biomass yield and biomass productivity were 111 and 110% higher than the wild type, respectively (p < 0.01). Lipid content and lipid productivity of the mutagenized microalgae E200-30-40 were 59 and 53% significantly higher than the wild type, respectively. It should be noted that biomass productivity of the mutagenized microalgae E200-30-40 was not significantly different from E100-30-60, meaning that this microalga strain exhibited highest both biomass and lipid productivity. These results indicated that inducing mutagenesis by EMS subsequently screening by herbicide could lead to enhance biomass and lipid accumulation. Therefore, this methodology could be used for improvement microalgae for biofuel production.

New microsatellite markers isolated from mungbean (Vigna radiata (L.) Wilczek).

In this study, we reported the isolation and analysis of new polymorphic microsatellites in mungbean (Vigna radiata (L.) Wilczek). Twelve out of 210 primer pairs screened in 30 mungbean accessions gave polymorphism. The polymorphic markers detected two to three alleles per locus with an average of 2.08. Observed heterozygosity varied from 0 to 0.133, while expected heterozygosity ranged from 0.095 to 0.498. Tests for Hardy-Weinberg equilibrium (HWE) and pairwise linkage disequilibrium of the polymorphic loci revealed that all loci except MB-SSR14 significantly departed from HWE and four pairwise combinations, viz. MB-SSR14 vs. MB-SSR42, MB-SSR42 vs. MB-SSR87, MB-SSR114 vs. MB-SSR121, and MB-SSR175 vs. MB-SSR231 significantly deviated from linkage disequilibrium. The markers are being used to study genetic diversity and genome mapping of mungbean.