genotyping transgenic plants and harvesting seeds for selecting rice varieties enriched with resistant starch

CRISPR-Based Efficient Genome Editing for Resistant Starch in Rice Breeding

Traditional breeding relies on introducing traits into crops or producing plant populations with enough variation, which requires long-term field observation1,2. Due to the limitations of traditional breeding, gene editing technology has been developed, which can precisely modify the genome of crops to obtain desired traits of plant populations3. The most widely used gene editing system in plants is CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Cas endonuclease), which relies on a programmable RNA-guided endonuclease to create targeted double-strand breaks (DSBs) in the DNA4,5. These DSBs are then repaired by the cell's natural DNA repair mechanisms6,7, often resulting in the introduction of the desired genetic changes. Although this technology has been implemented in various crops, including wheat8, maize9, soybean10, and rice11, it is predominantly used to reveal biological problems. Compared to its extensive application in elucidating plant gene functions, research on applying gene editing technologies to crop breeding remains relatively scarce12.
The process of gene editing in crops typically follows a well-defined workflow that encompasses several key steps13. The first step involves identifying the specific gene or genetic region that needs to be modified to achieve the desired trait. Secondly, a gene editing strategy is designed, involving the selection of an appropriate gene editing system (e.g., CRISPR-Cas9 or CRISPR-Cas12) and the design of specific guide RNAs to direct the endonuclease to the target site. Thirdly, the gene editing system is then incorporated into a delivery vector, which is used to introduce the editing machinery into the plant cells. These vectors can be DNA, RNA, or ribonucleoprotein (RNP) complexes. Subsequently, the gene editing vectors are delivered into plant cells using various methods, including Agrobacterium-mediated transformation, particle bombardment, or electroporation. Immediately, the transformed plant cells are cultured under appropriate conditions to generate genetically edited callus or embryogenic tissues. These tissues are then regenerated into whole plants through tissue culture techniques. The regenerated plants are subjected to rigorous molecular characterization to confirm the presence of the desired genetic changes. 
A previous article by Tsakirpaloglou et al.14 provides a broad overview of the gene editing process, from vector design to the generation of edited seedlings, but it does not delve into the detailed analysis of specific traits associated with the targeted gene nor the subsequent evaluation of agronomic performance or functional validation of the edited crops. We have gone beyond simply demonstrating the feasibility of editing a gene in rice. Our work comprehensively assesses the impact of this edit on the biochemical, molecular, and agronomic traits of the edited rice lines. This includes evaluating starch composition, a critical factor influencing grain quality and nutritional value, which has not been extensively explored in previous gene editing studies. 
Rice starch typically consists of ~20% amylose and ~80% amylopectin15. SBEIIb is an enzyme essential for amylopectin synthesis and is expressed in the endosperm16. The knockdown of OsSBEIIb by hairpin RNA (RNAi) and microRNA expression increased the resistant starch content17,18. Resistant starch is a substrate starch that cannot be digested and absorbed in the small intestine but is able to be broken down into short-chain fatty acids and gases by certain digestive bacteria in the intestines. Since it cannot be broken down quickly, it has a lower glycemic index compared to other starches and does not cause a rapid rise in blood sugar within a short period of time after eating, which can alleviate diabetes to a certain extent in diet19. In addition, resistant starch has more physiological functions, such as reducing insulin response, regulating intestinal function, preventing fat accumulation, facilitating weight control, and promoting the absorption of mineral ions. Therefore, it is being widely pursued as a new type of dietary fiber20. 
To overcome these challenges and successfully utilize gene editing technologies for breeding functional rice varieties, we have refined and optimized the operational protocols within rice. Our focus has been to meticulously analyze the design of target gene loci, carefully select the most suitable gene editing tools, and conduct rigorous phenotypic analysis throughout the breeding process. As a testament to the power and efficiency of these technologies, we present a case study showcasing the rapid development of a functional rice variety enriched with high-resistant starch. This example underscores the potential of gene editing in accelerating the breeding of functional rice, addressing the current dearth of research in this field.

Author Spotlight: Streamlining Rice Breeding with CRISPR/Cas for Obtaining Optimal Phenotypic and Agronomic Traits 

Breeding by Design for Functional Rice with Genome Editing Technologies

We are working on breeding functional rice with genome editing technologies. Our research isn't just about applying a gene editing tool. Rather, it focuses on the integration of multiple aspects, from gene selection and editing tool optimization to detailed phenotypic and agronomic trait analysis.We have gone beyond simply demonstrating the feasibility of editing the SBEIIb gene in rice. Our work comprehensively assesses the impact of this editor on the biochemical, molecular, and the agronomic traits of the edited rice. Our lab will focus on several key research questions in the future, aiming to push the limits of genetic editing technologies and crop breeding.Specifically, we are committed to contributing a wide range of foundational crop varieties that not only have high yield and exceptional quality, but also exhibit strong stress tolerance.

Genotyping Transgenic Plants and Harvesting Seeds for Selecting Rice Varieties Enriched with Resistant Starch

To begin, obtain the SG RNA and transform the Agrobacterium with the plasma DNA. Transplant the transgenic plants into pots and grow them for one month in a greenhouse. To assay the mutation type, collect two to three milligrams of fresh leaves from each tiller of a single seedling as a single sample using established protocols.Extract the genomic DNA from the collected leaf samples. Design PCR primers to amplify the SBEIIb gene region surrounding the single-guide RNA target site to obtain a 493 base pairs long amplified fragment. Sequence the PCR fragments directly using the Sanger method to identify mutations.Select the E0 lines exhibiting homozygous frame shift mutations and plant them in a greenhouse to obtain seeds. Then, harvest leaves from two-week-old seedlings and extract plant genomic DNA. Perform genomic PCR using the appropriate program to detect the presence of hygromycin, UBI, and Cascoset in the plant genome.Analyze the PCR products using gel electrophoresis to identify lines that show no bands indicating they are transgene-free E1 lines. Harvest the seeds from these selected lines. Mutant plants displayed a fully opaque, waxy appearance in their grains, unlike the translucent appearance of wild type grains.No significant differences were observed between SBEIIb mutants and wild type plants in terms of seed setting rate, grains per panicle, and yield per plant.

genotyping-transgenic-plants-harvesting-seeds-for-selecting-rice

Research

JoVE Journal

Bioengineering

53 Views. Per iniziare, ottieni l'RNA SG e trasforma l'Agrobacterium con il DNA plasmatico. Trapiantare le piante transgeniche in vaso e farle crescere per un mese in serra. Per analizzare il tipo di mutazione, raccogliere da due a tre milligrammi di foglie fresche da ciascun timone di una singola piantina come un unico campione utilizzando protocolli stabiliti.Estrarre il DNA genomico dai campioni di foglie raccolti. Progettare primer PCR per amplificare la reg...