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.

The first dataset of de novo transcriptome assembly of Heterotrigona itama (Apidae, Meliponinae) queen larva.

Heterotrigona itama is a species of stingless bee recently domesticated (or reared) for honey production in a few Southeast Asian countries namely Malaysia and Indonesia. Being categorized in the clade Corbiculata together with the honeybees (Apis spp.) and bumble bees (Bombus spp.), the stingless bees are highly social in which the colony members are subjected to labor division where a queen functions as the reproductive caste. In this data article, we provide a resource encompassing a transcriptome profile (de novo assembled) from H. itama queen larva - the first report of transcriptome assembly for this species. The generated data is pivotal for the characterization of important genes and biological pathways in order to further improve our understanding on the developmental biology, behavior, social structure and ecological needs of this eusocial hymenopteran insect from the molecular aspect. The raw RNA sequencing data is available at NCBI Sequence Read Archive (SAR) under the accession number SRP230250 and the assembled reads are deposited at DDBJ/EMBL/Genbank as Transcriptome Shotgun Assembly (TSA) under the accession GIIH00000000.