Agrobacterium-mediated in planta transformation of cut coleoptile: a new, simplified, and tissue culture-independent method to deliver the CRISPR/Cas9 system in rice.

BACKGROUND: Agrobacterium-mediated transformation and particle bombardment are the two common approaches for genome editing in plant species using CRISPR/Cas9 system. Both methods require careful manipulations of undifferentiated cells and tissue culture to regenerate the potentially edited plants. However, tissue culture techniques are laborious and time-consuming. METHODS AND RESULTS: In this study, we have developed a simplified, tissue culture-independent protocol to deliver the CRISPR/Cas9 system through in planta transformation in Malaysian rice (Oryza sativa L. subsp. indica cv. MR 219). Sprouting seeds with cut coleoptile were used as the target for the infiltration by Agrobacterium tumefaciens and we achieved 9% transformation efficiency. In brief, the dehusked seeds were surface-sterilised and imbibed, and the coleoptile was cut to expose the apical meristem. Subsequently, the cut coleoptile was inoculated with A. tumefaciens strain EHA105 harbouring CRISPR/Cas9 expression vector. The co-cultivation was conducted for five to six days in a dark room (25 ± 2 °C) followed by rooting, acclimatisation, and growing phases. Two-month-old plant leaves were then subjected to a hygromycin selection, and hygromycin-resistant plants were identified as putative transformants. Further validation through the polymerase chain reaction verified the integration of the Cas9 gene in four putative T0 lines. During the fruiting stage, it was confirmed that the Cas9 gene was still present in three randomly selected tillers from two 4-month-old transformed plants. CONCLUSION: This protocol provides a rapid method for editing the rice genome, bypassing the need for tissue culture. This article is the first to report the delivery of the CRISPR/Cas9 system for in planta transformation in rice.

Drought-Induced Changes in The Flowering Capacity, Anthesis Quality and Seed Set in Rice (Oryza sativa L.).

Drought stress significantly reduces grain yield (GY) due to poor spikelet fertility and anthesis quality. Aim of this study was to understand the changes of flowering capacity, anthesis quality traits and seed set in the re-watered drought stressed modern high yielding drought susceptible rice cultivar, IR64 at heading (DSH) and booting (DSB) stages. The well-watered plants served as control of the experiment. Results obtained suggest that spikelet moisture content at above 80% was required to maintain optimum anthesis process in rice. Anthesis process in DSH plant was suspended when leaf relative water content (LRWC) dropped to below than 70%. Effects of drought stress on the spikelet moisture were irreversible as compared to the leaf rolling and LRWC. Hence, seed set was failed to occur at the upper rachis branches of the DSH plant. Anthesis process in the re-watered drought stress plants was resumed on the third day after re-watering with about 50% and 80% of anthers managed to dehisce in the DSH and DSB plants. Consequently, percentage of spikelet fertility and seed set in the DSH and DSB plants were increased towards the lower parts of the panicle. The GY, number of seeds, spikelet fertility, and harvest index however were significantly lower in the DSH plant (0.30 g, 13, 16.40% and 14.81) as compared to DSB plant (1.34 g, 57, 59.14% and 48.30), respectively. In addition, all interrelated traits involved in the flowering process of rice could be collectively termed as the anthesis quality traits due to their significant correlation with the grain yield and other yield components.

Transcriptome profiling of Stevia rebaudiana MS007 revealed genes involved in flower development.

Stevia rebaudiana is a medicinal plant recommended to diabetic or obese patients as an alternative sweetener owing to its low-calorie property. Previous studies have found that the stevioside level is highest at the time of flower bud formation and lowest at the time of preceding and following flower bud formation. Hence, this study aims to identify the genes involved in the flowering of local S. rebaudiana accession MS007 by investigating the transcriptomic data of two stages of growth, before flowering (BF) and after flowering (AF) that were deposited under accession number SRX6362785 and SRX6362784 at the NCBI SRA database. The transcriptomic study managed to annotate 108299 unigenes of S. rebaudiana with 8871 and 9832 genes that were differentially expressed in BF and AF samples, respectively. These genes involved in various metabolic pathways related to flower development, response to stimulus as well as photosynthesis. Pheophorbide A oxygenase ( PAO ), eukaryotic translation initiation factor 3 subunit E ( TIF3E1 ), and jasmonate ZIM domain-containing protein 1 ( JAZ1 ) were found to be involved in the flower development. The outcome of this study will help further research in the manipulation of the flowering process, especially in the breeding programme to develop photo-insensitive Stevia plant.

De novo transcriptome dataset of Stevia rebaudiana accession MS007.

Stevia rebaudiana (S. rebaudiana) is a herbaceous and perennial plant belonging to Asteraceae family. The genus stevia is well known as a natural producer of sweetener comprising non-caloric and non-carcinogenic steviol glycosides. In recent years, the capability in producing natural sweetner has increased the demand for S. rebaudiana as substitute of processed sugars. Flowering phase of S. rebaudiana has shown to affect the content of steviol glycosides in the leaves. Steviol glycosides level is the highest at the time of flower bud formation and lowest at time preceding and following flower bud formation. Therefore, sequencing and analysing the genes that are involved in flowering phase will provide platform for gene manipulation in increasing steviol glycosides content. The Stevia transcriptome data that include two stages of growth (before flowering and after flowering), were obtained using Illumina RNA-seq technology and can be accessed at NCBI Sequence Read Archive under Accession No. SRX6362785 and SRX6362784.

A review on non-syndromic tooth agenesis associated with PAX9 mutations.

Tooth agenesis in the reduction of tooth number which includes hypodontia, oligodontia and anodontia is caused by disturbances and gene mutations that occur during odontogenesis. To date, several genetic mutations that unlock the causes of non-syndromic tooth agenesis are being discovered; these have been associated with certain illnesses because tooth development involves the interaction of several genes for tooth epithelium and mesenchyme odontogenesis. Mutation of candidate genes PAX9 and MSX1 have been identified as the main causes of hypodontia and oligodontia; meanwhile, AXIN2 mutation is associated with anodontia. Previous study using animal models reported that PAX9-deficient knockout mice exhibit missing molars due to an arrest of tooth development at the bud stage. PAX9 frameshift, missense and nonsense mutations are reported to be responsible; however, the most severe condition showed by the phenotype is caused by haploinsufficiency. This suggests that PAX9 is dosage-sensitive. Understanding the mechanism of genetic mutations will benefit clinicians and human geneticists in future alternative treatment investigations.