Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
ENGINEERING A ‘CRISPR’ POTATO (SOLANUM TUBEROSUM L.) FOR BETTER NUTRITION AND REDUCED FOOD LOSS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1013654
Grant No.
(N/A)
Project No.
CA-D-PLS-2404-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2017
Project End Date
Sep 30, 2022
Grant Year
(N/A)
Project Director
Beckles, D.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Sciences
Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act areas of: processing, distribution, safety, marketing, and utilization of food and agricultural products; human nutrition; sustainable agriculture; molecular biology; biotechnology.The amount and properties of starch accumulated in potato tubers determines its yield, sensory properties, nutritional value, and status as a staple vegetable. Potato starch, however, has a high-glycemic index (GI) and is also highly susceptible to cold-induced sweetening (CIS). These two traits are undesirable for a number of reasons. First, high GI foods are rapidly digested and cause a spike in blood sugar levels. Their consumption is coincident with a higher occurrence of diabetes, obesity and heart disease in US and other populations, and the public is often asked to monitor their consumption of such types of food. Second, CIS is a postharvest disorder in potato characterized by an accumulation of reducing sugars during tuber storage below 12°C. When these high-sugar potatoes are fried, they become blackened, bitter, and accumulate carcinogenic acrylamide. CIS causes millions of dollars in losses annually, as cold storage is necessary to prolong tuber dormancy. The high digestibility of potato starch to sugars, either in the human gut during enzymatic breakdown, or in refrigerated potato tubers during storage, is partially determined by its molecular structure. Starch with a high percentage of branching is preferentially used by degradative enzymes as substrates. The degree of branching of potato and other starches is largely determined by two 'starch branching enzymes' (SBEs), SBE A and SBE B. Starch synthesized in the absence of SBEs has fewer branch points, and therefore provides less substrate to degradative enzymes and is degraded slowly. We propose to use a non-transgenic CRISPR-Cas9 approach to induce combinatorial knockouts of SBE A and SBE B. We plan to select lines with a 20-25% increase in starch resistant to digestion during cold storage, making it less susceptible to CIS. This starch will additionally not be digested fully in the human body, functioning as dietary fiber and promoting gut health and formation of short-chain fatty acids. Transient expression of the CRISPR system will be used to mutate the SBEs and recover plants altered in SBE sequence but with no insertion of foreign DNA. Careful phenotyping and genetic screening of the regenerated plants will enable us to find the best genetic variants with the optimal rearrangement of the starch molecule, but which maintain tuber yield. The lines will have enhanced storability, functionality, reduced toxicity after frying, and improved health outcomes, and will be publically available.
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011310100010%
2021310101010%
2031310102020%
2041310104060%
Knowledge Area
204 - Plant Product Quality and Utility (Preharvest); 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms; 202 - Plant Genetic Resources;

Subject Of Investigation
1310 - Potato;

Field Of Science
1040 - Molecular biology; 1010 - Nutrition and metabolism; 1000 - Biochemistry and biophysics; 1020 - Physiology;
Goals / Objectives
The goal of this Hatch Project is to genetically engineer two varieties of processing potatoes, 'Atlantic' and 'Russet Burbank', to increase the proportion of the resistant starch accumulated in the tuber. This will be accomplished by reducing the activity of two enzymes, starch branching enzymes A and B (SBE A, SBE B), by at least 25%, using a transient CRISPR-Cas9 expression system. Because SBEs are responsible for the synthesis of the rapidly digested (RD) component of starch, potato lines with reduced SBE function should have a higher proportion of the slowly digestible (SD) and resistant starch (RS). Potatoes so produced would have many positive aspects from a nutritional, public heath, and environmental perspective. One benefit is that high RS would behave physiologically like fiber, i.e., have a lower glycemic index when cooked and digested, and confer similar healthy attributes. High RS-potatoes should also exhibit resistance to cold-induced sweetening (CIS), which occurs during postharvest refrigerated storage. Since the starch in CIS-resistant potatoes will not be degraded to sugars, tuber yield will be preserved. Further, when fries and chips are made from these potatoes, they will not have bitter, blackened areas produced from the tuber sugar. More importantly, the carcinogen acrylamide produced in these blackened areas should also be reduced.The aims of this proposal are to develop novel potato germplasm that:1. Has at least a 25% increase in RS compared to the wild-type cultivar. This should reduce susceptibility to CIS and simultaneously lower the glycemic index in the cooked product. 2. Has no appreciable difference in yield or other quality parameters and would therefore be a net-income generator. This new potato should have reduced quantitative and qualitative losses from cold-storage. While this might otherwise reduce income by making more product available, this would be off-set by a mark-up for the healthy potatoes with high apparent fiber and reduced acrylamide.3. Is not subject to USDA-APHIS regulation under 7 C.F.R. Part 340. This line will be rapidly modified using CRISPR-Cas9, the generated plants will not contain any foreign DNA (including and especially any bacterial genes for antibiotic resistance), and will not require the laborious process of making sexual crosses for multiple generations, which can take 12-14 years.BACKGROUND AND RATIONALEMany enzymes involved in potato starch synthesis and breakdown are potential targets to increase the percentage of RS, and hence to decrease CIS and GI. A prime target is starch branching enzyme (SBE) rather than enzymes involved in starch degradation, for reasons detailed below.SBEs synthesize the highly branched, rapidly digested (RD) polymer of starch called amylopectin. Reductions in SBE function would result in a greater ratio of the less-branched starch polymer called amylose. A higher amylose or amylose-like starch content will have more RS properties. In support of this, some naturally occurring CIS-tolerant potato genotypes have been shown to have higher amylose content compared to highly susceptible ones (1). While other enzymes contribute to the determination of the amylose-to-amylopectin ratio, there is overwhelming evidence for SBEs having a predominant role in diverse species: wheat, barley, maize, rice, peas, cassava, and others. Further, there are naturally occurring low and high amylose mutants, and several are defective in their SBE complement (reviewed in 2, 3).Alpha- and β- amylases are known starch degrading enzymes, many isoforms of which are upregulated in the cold; however, they are not a good target for modification in potato, for several reasons. First, they are involved in regulating whole plant growth, and knocking them down could affect the viability of the plant and cause yield losses. Additionally, there are multiple amylases: for example, StAmy23 expression is highly correlated with CIS, but whether another amylase would be upregulated to compensate for its loss is not known. Knocking out multiple genes is time-consuming, and increases the chances of altering properties of the plant not directly related to starch storage (4, 5). Finally, in many cases, amylase activity is not highly correlated with transcription levels, but with activity of regulatory proteins. Even in those cases, the relationships are neither consistent nor linear (6). Similar problems of multiple isoforms, complex regulation, and uncertain results exist with regard to knockdown of other starch degradation enzymes, such as starch phosphorylases and glucan water dikinases, which, while they lead to significant decrease in CIS, are involved in starch and sugar use in leaves (7).Further down in the pathway are sucrose phosphate synthase, which catalyzes the formation of sucrose, and vacuolar invertase, which metabolizes sucrose into fructose and glucose. SPS transcript levels are increased by cold temperatures, increased reducing sugar content in cold-stored potato (8, 9). SPS has a significant role in plant growth. The invertase approach has already been taken by SIMPLOT, a large breeding company, and could be legally contentious.Therefore, to obtain an effective change in amylose content in potato, with minor pleiotropic effects, we propose to reduce SBE expression. In potato, loss of one or more of the SBEs often alters the ratio of amylose-to-amylopectin. The autopolyploidy that frustrates breeding efforts affords us more latitude in genetic targeting. Previous research using traditional genetic engineering approaches showed that nearly complete suppression of SBE A led to a 35% increase in amylose content (10, 11), so complete suppression of this gene is desirable. To equivalently knock out SBE B increases amylose by 115-170%, but also causes significant loss of starch with negative impacts on yield (10, 12). However, by knocking out only one or two of the four alleles of SBE B (due to tetraploidy), lines with improved amylose content over an SBE A-only knockout, yet yield nearly equal to wild type cultivars, can be recovered.EXPERIMENTAL OBJECTIVES (EO)To create potato mutants with high 'apparent fiber' and high resistance to CIS, the supporting and specific objectives of this proposal are:EO 1. DEVELOP HIGH AMYLOSE POTATO BY SUPPRESSING SBE A AND SBE B ALLELES USING CRISPR-CAS9 (YEAR I) a. Design three sgRNAs to efficiently and specifically reduce SBE A and SBE B.b. Clone the SBE-sgRNAs into the CRISPR-Cas9 constructs for transformation.c. Edit the SBE genes by transforming two potato cultivars, 'Russet Burbank' and 'Atlantic,' using a transient Agrobacterium-mediated expression.d. PCR-screen transformants for mutations in SBE A and SBE B.EO 2 ASSESS THE CONSEQUENCES OF THE SBE MODIFICATION ON QUALITY TRAITS AND YIELD UNDER CONTROLLED CONDITIONS OF TRANSFORMED POTATOES (YEAR 2)a. Select potato lines modified in SBE A and B sequence, but with no foreign DNA integrated in the genome. b. Test the modified potatoes for changes in amylose content, CIS, and for changes in starch content and starch digestibility.c. Determine changes in yield and ontogeny, compared to the transformed controls.EO 3) FIELD TESTING AND EVALUATION OF PROMISING CLONES (YEARS 3-5).1. S. H. Jansky, D. A. Fajardo Food Science & Nutrition 2, 628-633 (2014).2. I. J. Tetlow, M. J. Emes Iubmb Life 66, 546-558 (2014).3. J. Wang, et al, Front Plant Sci 8, (2017).4. H. Reinhold et al., Plant Cell 23, 1391-1403 (2011).5. A. C. Wu, Jet al, Plos One 9, (2014).6. H. L. Zhang et al., Plant Biotechnol J 12, 984-993 (2014).7. H. L. Zhang et al., Starch-Starke 69, (2017).8. U. Deiting, et al., Plant Cell Environ 21, 127-138 (1998).9. L. M. Hill, et al., Plant Cell Environ 19, 1223-1237 (1996).10. G. P. Schwall et al., Nat Biotechnol 18, 551-554 (2000).
Project Methods
In order to create potato mutants with high apparent fiber and high resistance to CIS, SBE genes will be reduced by transient expression of a CRISPR-CAS9 construct bearing SBE gRNAs. The supporting and specific objectives are as follows:EO1. DEVELOP HIGH-AMYLOSE POTATO LINES BY SUPPRESSING SBE A AND SOME SBE B ALLELES (Yr 1)Three sgRNAs for SBE A and SBE B respectively were designed. They were checked for the possibility of producing off-target effects (OTEs) using CCTop (CRISPR-Cas9 target online predictor). The PAM motifs were identified to attack the most "vulnerable" SBE domains, using the published potato genome sequence. This genomic sequence was derived from a 'synthetic' homozygous doubled-monoploid clone, and a diploid potato, so there may be polymorphisms between those lines and the tetraploids we will use, i.e., 'Russet Burbank' and 'Atlantic'. The genomic region flanking the sgRNAs was sequenced in these varieties and no heterogeneity between homeologues was identified for SBE A. Work on SBE B is ongoing.The sgRNA sequences will be synthesized by assembly PCR with plasmid pCBC-DT1T2. The optimized CRISPR-Cas9 binary plasmid pHSE401 will be digested with BsaI restriction enzyme and ligated to insert the assembly PCR product and form the plasmid we will designate pHSE401::sgSBE (3). The use of the Golden Gate assembly method means the sgRNA construct can be synthesized in one PCR reaction, and then inserted into pHSE401 in one digestion and ligation step. A. tumefaciens strain EHA105 will be transformed with pHSE401::sgSBE, and successful transformants selected by growth on hygromycin. pHSE401::sgSBE will be introduced into 2-day pre-cultured stem internodes of 10-day-old potato seedling plants using Agrobacterium. Virus-free material was obtained from the University of Idaho for this purpose. After 2 days of co-cultivation, the stem segments will be transferred to a regeneration medium lacking antibiotic. Without antibiotic selection, all calli will survive: calli where the genome was not edited, those that were edited, and those with the plasmid integrated into the genome and different combinations thereof. Lines with all copies of edited SBE A, and a variety of 'dosages' of edited SBE B would be desirable. The intensity and duration of Cas9 expression will produce many variations in dosage of both genes. Butler et al. found lines with a spectrum of dosages for the edited gene using CRISPR-Cas9 with Agrobacterium in potato.EO2. ASSESS THE CONSEQUENCE OF THE SBE MODIFICATION ON YIELD AND QUALITY TRAITS (Year 2-5)Approximately 200-500 transformants will be screened to identify a minimum of ten lines with 1) desirable modifications of SBE A and SBE B that reduce functionality and 2) no integration of pHSE401::sgSBE, as determined by amplification of three regions of the construct. The developing calli will be screened by PCR and digestion with the Surveyor nuclease to find alterations in SBE A and B sequence. This method is sensitive to single base-pair changes. While screening callus poses some risk, it is routinely done in potato tissue culture transformation and saves space, time, and money, as only calli with alterations are brought through to the plantlet stage. The plants will be subjected to a preliminary check for off-target effects (OTEs) and for partial insertion of the plasmid by PCR, which will be rejected. We will include controls: three lines that were treated similarly to the positives identified above, but which (i) have no alteration in SBEs and (ii) which lack pCBC-DT1T2::sgSBE. CRISPR-Cas9-induced edits in potato are maintained in the germline and through clonal propagation, so these non-GMO lines will be stable and otherwise identical to their commercial parents. The regenerated plants will be hardened in growth rooms before greenhouse transfer.After the regenerated mutant lines have been screened, limited tests for 1) equivalent yield, 2) higher resistance to CIS (at least 10%), and 3) a lower glycemic index (at least 20%) over the non-modified line will be done. The first tests will be done on M0 regenerants in the greenhouse. Ten biological replicates for each independent transformant will be grown (2 cultivars x 3 plasmids x 10 transformants) and the two transformed controls screened for each genotype. After 3 -5 months' growth in the greenhouse, the M1 tubers from each M0 plant will be harvested, counted, washed and weighed to collect yield data (tuber weight * tuber number). Starch digestibility will be estimated by incubating starch with 1-2 units of alpha-amylase from Aspergillus niger in a buffered solution. Assays will be done on both raw and cooked starch. As the amylase digestion proceeds, sugars are released into the solution, which is sampled over time, and the rate of production of glucose measured. Using this test, it has been proven that the kinetics of the reaction reflect amylose content.Lines with significant differences in digestibility and amylose will be our focus. We will use more expensive standardized assays for amylose and RS (i.e. starch undigestible by enzymes) determination for independent assessments of these measures. The kits from Megazyme™ assay amylose by solubilizing starch, precipitating amylopectin, and converting the remaining glucan which is amylose or 'amylose-like' to glucose for subsequent determination using a glucose oxidase reaction. The RS assay removes the easily digested starch fraction and then uses acids to break down the resistant fraction into sugars for assay. We have experience using these assays.Preliminary tests for CIS will be done on tuber disks, which will permit a high-throughput screen. We have experience examining chilling-induced changes in metabolism in tomato disks, and will apply similar protocols to this work. Disks will be placed in a simple buffer under sterile conditions in 24-well plates and 'aged' to reduce the influence of wounding-induced stress. The plates will be stored at 4 and 10°C (chilling) and at 15 and 20°C (controls). After storage for 2 weeks, the disks will be assayed for starch content and for the levels of reducing sugars, including glucose and fructose. CIS leads to 2- to 10-fold sugar increases in tuber disks, under these conditions.EO3) MULTI-STATE FIELD TESTING (Yrs 3-5). If the preliminary results from the greenhouse are promising over two generations, micropropagation of each selection will be done, using at least 5 plantlets per line. The plantlets will be cut into three sections to obtain a 3-fold increase in material. This will aid in the development of seed potatoes needed for more extensive field testing, which will be conducted in collaboration with the South Western Potato breeding consortium at Colorado State University, including Dr. David Holm (Colorado State University) and Dr. Robert Wilson (UC Davis). We will request that the clones be included in the National Chip Processing Trial (NCPT) and National Fry Processing Trial (NFPT). Clones with consistently higher resistant starch and which maintain yield in field conditions will be identified and a determination made as to whether they are commercially viable, and suitable for more extensive trialling.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Our outreach was to diverse groups, but our primary target audience was the California Potato Research Advisory Board, a small state group focused on potato improvement. We have communicated annually through oral presentation and written reports. At this stage in the project, we received funding to continue our screening process for high-resistant starch lines. This will help us maintain the tissue culture of identified lines, continue transformation and screening, and ensure that the developed pipeline remains active. We have also disseminated the project aims through laboratory instruction, training undergraduate students in the techniques needed to keep the pipeline functioning, and during formal classroom instruction. The latter including developing curriculum in BIT161A to deliver experiential-based learning on gene editing, and to highlight the importance of basic biotechnological principles to improving agriculture in the state of California. This project was also the focus of my discussions with students in the PREP program (post baccalaureate trainees), on the application of gene editing for crop improvement. I also gave guest lectures at Tuskegee University, a Historically Black College & University, describing the importance of resistant starch for groups who are socially and economically disadvantaged. The students understood how the health outcomes of their communities could be improved through higher consumption of high-fiber foods, and were especially engaged when learning about resistant starch. Many had family members who suffer from diabetes, and so there was excitement about the potential of high RS CRISPR potatoes, and the technique used to produce them. Finally, I conducted workshops in postharvest biology and technology in Thailand and Chile and had opportunities to describe this Hatch project and the important aims it seeks to address. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Six undergraduate interns worked directly on this project: Megan Higganbotham, Andrew Saluna, and Xiuhoon Giang continued working during this reporting period. Keyun Wang continued working and gave two presentations, gaining valuable experience for any future career. Sarah Kiser and Rong Qiao also joined the project and gained laboratory skills important for students studying genetics and molecular biology. Young Scholar Program participant Chloe Fuson, a high schooler, also worked on the starch profiles of the transformants. Master's student Jingwei Yu gave multiple presentations on this project and is a strong candidate for competitive Ph.D. programs based on the skills gained directing this project. Ph.D. student Emma Shipman served as Graduate Associate Instructor for BIT161A in Winter 2020, with Jingwei Yu as a Teaching Assistant. Together they updated the curriculum to include the work in this Hatch Project, teaching the students about CRISPR-Cas9 and the design of single guide RNAs. How have the results been disseminated to communities of interest?MS student Jingwei Yu presented this research at online symposia and conferences, and undergraduate interns Andrew Saluna and Keyun Wang presented posters at UC Davis via video. We reported to the California Potato Research Advisory Board our ongoing progress and communicate with the Southwest Potato Breeding Group to ensure that the academic and potato breeding community are also informed. The PI also gave presentations in various forums locally in Davis, or to a wider audience nationally, and overseas, as this work serves as a breeding/variety development project, and as a generator of basic science that feeds back into biotechnological improvement. What do you plan to do during the next reporting period to accomplish the goals?As this reporting period ends, a critical task is to secure sufficient funding to maintain, continue, and expand the project. We will need to recruit more personnel to work on this project efficiently, a task the difficulty of which is exacerbated by limitations placed by COVID-19. Our experiments will expand to include more postharvest treatment of tubers and minitubers from developed lines and collaborations with field researchers. In 2020 we moved from development of a pipeline and preliminary results to a preliminary assessment of the lines generated by our work. In 2021, we will continue to focus on evaluating our SBE-altered lines in multiple ways, to support their introduction to growers' fields and then the commercial market. Identifying lines with optimal amounts of amylose, resistant starch and resistance to cold-induced sweetening that could overcome the loss of yield will be prioritized. Additionally, with this robust pipeline, we expect to readily examine other genes related to functional starch attributes in commercial potato. This project can become a strong source of CRISPR'ed potato varieties that can be evaluated for functionality and introduction to farmers and the broader market. This type of nonconventional breeding program is valuable as a public resource provided by a land-grant university, enabling growers of all sizes to take advantage of new technologies and advancements without depending on private companies.

Impacts
What was accomplished under these goals? The aim of this project is to generate transgene-free CRISPR-edited potato lines with increased resistant starch (RS) content. Potato with increased RS will offer better health outcomes to consumers and is predicted to resist cold-induced sweetening (CIS) postharvest. Our target gene is Starch Branching Enzyme (SBE), which, when disabled, leads to high-RS phenotypes through increases in amylose. Therefore, this project has the potential to positively impact public health and reduce agricultural waste and loss by acting on a single target. Our approach was to transform potato callus derived from three genotypes i.e., 'Desiree', a lab model, and 'Atlantic', and 'Russet Burbank': two commercial cultivars. Agrobacterium transformed with CRISPR plasmids carrying sgRNAs targeting SBE was used to inoculate stem internodes. It was important to ensure that the generated germplasm would have commercial potential and be acceptable for use by both conventional farmers and those taking organic or non-GMO approaches. Our lines are non-GMO according to the USDA's standards, with no integration of foreign genetic material. It was also important that we develop a plan to identify successful transformants, i.e., those without plasmid, but with altered starch, as quickly and cheaply as possible, to accelerate progress. Therefore, we included a DsRed fluorescent reporter gene in the plasmid to first identify calli that initially expressed the CRISPR construct, and then after a second screen, to identify calli that did not incorporate it into the genome. We also developed a reliable iodine plantlet screen to select individuals with altered starch. Our accomplishments this year included: A. We determined the starch temporal-spatial profiles in potato tissue culture plantlets to establish a baseline understanding of starch mobilization for our double screening system: We looked at the diurnal and tissue-specific patterns of starch accumulation in seedlings at four developmental stages (Tissue Culture Day 10, 21, 35 and Hardening Day 10) in the three potato cultivars 'Desiree' (DES), 'Atlantic' (ATL), and 'Russet Burbank' (RB). There is little published work on starch accumulation dynamics in tissue culture plantlets and such knowledge was critical for our ability to accurately and sensitively assess changes in starch levels and compositions in our regenerated lines. We showed that in the early stage of tissue culture, plantlets accumulated high levels of starch, presumably fueled by the high concentration of sucrose in the media, but starch content decreased as the period progressed. The amount of starch in leaves vs. stem, and diurnal fluctuations, were found to vary by genotype. B. We showed that our double screening system was robust for identifying potato germplasm with lesions in SBE genes: 43 plantlets (DES=13, ATL=16, RB=15) were selected based on DsRed fluorescence and iodine staining and sequenced. Over 84% were edited, with an average 54.6% mutation rate at the targeted sites achieved (DES=34.6%, ATL=58%, RB=67.9%). Mutations in the SBE genes were inferred using Inference of CRISPR Edits (ICE, Synthego) and CRISP-ID. Mutations have been identified, from 1-bp indels to over 400 bp deletion. Most events contained mixed traces and were predicted to induce 'mild' mutations, since the plantlet has a mixture of both wildtype and edited alleles. We improved upon the sensitivity of the iodine screen developed in previous years by assigning iodine 'staining intensity scores' (Darkness, 1-5) in the edited plantlets. These values were compared to the SBE lesion 'knockout scores' (using ICE) of the same plantlets. Iodine staining intensity scores correlated well with the SBE knockout scores with r2=0.555 (p-value of <0.01), therefore, SBE gDNA lesions could explain over 55% of the dark-stained phenotype. C. We identified potato germplasm with higher amylose. Minitubers were harvested from the edited lines in the greenhouse after 3 months' growth. ATL-SBE lines increased amylose by ~15.5% on average, with the highest 25.7%. DES-SBE lines increased amylose ~7.3% on average, with the highest 13%, and RB-SBE increased amylose ~2.9% on average, with the highest 9.3%. We estimated that a 5-20% increase in amylose would give physiologically relevant increases in RS, therefore many of the genotypes were within this range. D. We identified germplasm with higher amylose and altered response to cold-induced sweetening. Potato tuber starch is digested to sugars when stored in the cold, a phenomenon known as cold-induced sweetening. Tubers with high amylose are hypothesized to have a reduced rate of starch degradation. This is important as the accumulation of sugars from starch breakdown leads to blackened, bitter regions high in acrylamide formation in baked or fried potato products, a health and sensorial concern. SBE tuber disks stored for one week in the cold had altered starch and sugar content. On average, ATL-SBE retained 9.5% more starch than WT, but 20% more sugars accumulated. DES-SBE retained 1.9% more starch than WT, and 1.7% less sugars accumulated. Certain individual lines show strong promise. A23 had 24.1% amylose increase, and retained 2.4% more starch and 3.8% less sugar after cold storage. D59 had 13% amylose increase and retained 2.43% more starch and 7.9% less sugar after cold storage. As expected, starch granule morphology was altered in the edited lines, and yield also decreased, by 25.2%, 10.1%, and 17.2% (average) in DES, ATL, and RB respectively. Yield decreases in high amylose lines do not preclude commercial use of these lines as specialty cultivars, as shown in wheat. The increases in amylose are in line with our goal; future full nutritional assessment of mature tubers for RS in addition to amylose content could show similar promise. The variety of responses, such as both increased starch retention at the same time as sugar accumulation, indicates a complex and delicate system for regulation of starch biosynthesis and degradation. This invites further study and also suggests that more nuanced control of starch content is possible with more knowledge. E. Bioinformatics analysis reveals variation in SBE genes among cultivars: To assist in future manipulation and elucidation of metabolic control by SBEs, we also characterized the native SBE isoforms in potato by exploring their evolution, structure, and for each genotype, their allelic variants, and transcriptional expression. In summary, we did extensive and rigorous testing on generated lines while maintaining the transformation pipeline and multiplying regenerated lines of interest. The comprehensive diurnal starch profile (section A) and bioinformatic information (section E) enabled the development of tools such as our iodine screen that significantly progress the project. This information is also critical for unraveling a molecular explanation for variable responses (in terms of yield, CIS resistance, sugar and starch content) across cultivars, regenerants, and treatments.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Beckles, DM (2020) Integrative approaches to studying plant responses to challenging environments: from seed to postharvest Department of Plant Sciences Seminar. August 19th 2020; 12 pm. Virtual seminar by Zoom.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Beckles, DM (2020) CRISPR Potatoes California Potato Advisory Board. Virtual Zoom, CA September 3rd 9:50 am.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Beckles, D.M. (2020) Postharvest Handling Challenges and Opportunities for reducing food losses. University of Concepci�n, Chill�n campus, Chile. January 20th-25th
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Yu, J., Beckles, D. M. (2020). Apply CRISPR to facilitate potato improvement with better nutrients and postharvest quality. University of Nebraska-Lincoln Plant Science Symposium, Innovation Campus Conference Center, Lincoln, NE. Mar 17th, 2020. (Selected for Mini-Talk. unable to attend due to the COVID-19)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Wang, K., Yu, J. & Beckles, (2020) DM. Sequence and Expression Characterization of Native Starch Branching Enzymes in Three Potato (Solanum tuberosum) Cultivars. Undergraduate Research Conference, University of California, Davis, May 7-9th. (Zoom Presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Saluna, A., Yu, J., Beckles, DM. (2020) Atlas of starch-sugar dynamics in potato (Solanum tuberosum L.) from in vitro to in vivo. Undergraduate Research Conference, University of California, Davis, May 7-9th. (Zoom Presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Beckles, DM (2019) Postharvest Chilling Injury  Plant Response to Human Intervention Department of Plant Sciences Research Symposium, [University of California, Davis, Buehler Alumni Center], UC Davis. April 6th
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Yu, J and Beckles, D.M. (2019) "Altering Potato Starch for Nutritional Benefits and Potential Enhanced Postharvest Quality". Texas A&M Genome Editing Symposium. College Station, TX. Oct 3rd. (Oral Presentation)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Yu, J and Beckles, D.M. (2019) "Altering Potato Starch for Nutritional Benefits and Potential Enhanced Postharvest Quality". Talk. Texas A&M Genome Editing Symposium. College Station, TX. Oct 3rd. (Poster presentation).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Beckles, DM (2019) Postharvest  Challenges and Opportunities 4th Annual International Training Course - Postharvest Technical Conference, Mae Fah Luang, Chiang Rai, Thailand.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Beckles, DM Progress towards understanding the molecular basis of postharvest chilling injury. National Horticultural Conference, [Nonthanburi, Thailand], November 5th.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Beckles, DM (2019) Starch - to-Sugar and back again  the critical role for starch in sugar signaling. EMSL Integrated Conference, Pacific Northwest National Laboratory, [Richland, WA], October 8th.
  • Type: Other Status: Other Year Published: 2019 Citation: Beckles, DM (2019) PREP@UCD Gene Editing for Food Security In the Future of Biology Talk series. August 2019
  • Type: Other Status: Other Year Published: 2020 Citation: Beckles, DM (2020) "Post-Harvest Strategies for Improving Agriculture" in Lecture series International Agriculture. Tuskegee University, Henderson Hall 102. March 2, 10-11 AM
  • Type: Other Status: Other Year Published: 2020 Citation: Beckles, DM (2020) Biotechnology for Improving Postharvest Physiology. Guest lecture in Class Series Physiology of Plant Growth and Development. Henderson Hall 107. March 2, 12-1 PM


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:1) We are co-founding members of the Southwest potato breeding and improvement gene editing subgroup. We've had two conference calls with the group (May 24th and December 3rd, 2019) to share the progress we have made on our work during the reporting period, The group is very interested in altering potato tuber starch and sees great potential for using CRISPR. The primary aim of these discussions was to coordinate our activities to ensure the most efficient use of resources, especially for field trials and potato starch nutrition studies using mice and human models. 2) We wrote a report summarizing our progress and plans to the California Potato Research Advisory Board (CPRAB). In my absence (due to travel), Dr. Rob Wilson UC Extension Specialist, presented our data to the CPRAB meeting on September 5th, 2019 in Fresno, CA. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several students were trained over the review period: A). Undergraduates: Sammy Ospina (McNair scholar), Andrew Saluna, Megan Higgenbottom and YSP scholar Chloe Fuson were taught basic tools in biochemistry and molecular biology, plant sterile culture, and how to conduct literature searches. This has both broadened and heightened their educational training. B). Graduate students: Jingwei Yu (MS), Emma Shipman (Ph.D.), and Hongtao Zhang (Ph.D. rotation student) were trained in biochemical and molecular biology, and sharpened their critical thinking and scientific skills. Jingwei Yu won a travel award to attend a Gene Editing Conference at Texas A&M, College Station, Texas to present the data from this work. How have the results been disseminated to communities of interest?As previously mentioned, we discussed our project to the general public on UC Davis Unfold Podcast: "The Future of food." Recorded December 11th, Mrak Hall, Broadcast October 9th https://soundcloud.com/unfoldpodcast/episode-4-the-future-of-food. It was disseminated broadly by twitter, Soundcloud, and by the Apple podcast. What do you plan to do during the next reporting period to accomplish the goals?In the next two years of the project, we aim to evaluate mature SBE-edited plants. The combination of screens i.e. Ds-Red, iodine and sequencing, should help us to narrow down the number of genotypes that we would grow to maturity in the greenhouse, for quantitative validation of RS content and yield. We will focus on 'Atlantic' and 'Russet Burbank' because of their agricultural relevance. Lines with significant RS elevation i.e. 20-40%, no integration of the transgene and minimal yield loss will be evaluated in field studies in collaboration with the Southwest potato breeding and improvement group.

Impacts
What was accomplished under these goals? The aim of this project is to produce novel potato cultivars with elevated resistant starch (RS) using CRISPR-Cas9 gene editing. RS has many of the associated benefits of fiber but with the sensory quality of starch. Increased RS intake could improve the health outcomes of consumers who dislike the texture of fiber and prefer refined starch products. To accomplish this, we plan to reduce the activity of potato SBEI and SBEII genes by transient expression of Cas9 and different combinations of SBE sgRNAs. Our aim is to recover germplasm with significant levels of RS, few pleiotropic effects on yield, and no transgenes embedded within the genome. This approach will require generating hundreds, even thousands of transformed calli, and screening them, and the regenerated plantlets for desirable genetic changes. Over the last year, the following was accomplished: A). A workflow including a) Cas9-sgRNA transformation of potato explants,b) Cas9-sgRNA transient gene expression, and c) regeneration and sequencing of transformants, has been established. DsRed fluorescence is our marker for Cas9-sgRNA expression in potato tissues. Our preliminary data show that DsRed fluoresces in stem internode calli, 7-21 days after callus induction. Unless there is stable integration, the fluorescence will not be detected after 4-weeks growth. Calli with stable fluorescence after 4 weeks are not prioritized for further study due to the likely integration of the transgene, since our aim is to produce transgene-free germplasm. We have regenerated over 200 Désirée lines, and more than 100 each of 'Russet Burbank' and 'Atlantic' lines, where DsRed fluorescence indicated that the Cas9-sgSBE complex was likely transiently expressed. 'Désirée' is a model, non-commercial cultivar used as control, while 'Russet Burbank' and 'Atlantic' are used by the potato industry. The regeneration and transformation efficiency of 'Désirée' is very high, 'Atlantic' is lower than 'Désirée', but generally higher than 'Russet Burbank.' Each line has been multiplied to permit multiple testing of events. These 'DsRed selectants' are then subjected to an iodine screen. When exposed to iodine, tissues with elevated RS levels should stain darkly (iodine-positives), and these genotypes will be prioritized for sequencing. Our visible scores showed that of the 92 'Désirée' transformants screened, 28 of them (30%) were darker than the control. 'Atlantic' tissue was darker for 16/47 (34%) and for 'Russet Burbank' it was 8/37 (22%). The percentage of potential SBE-edited positive was therefore more similar among cultivars, than their regeneration frequency. We have sequenced five 'Atlantic' and five 'Désirée' regenerants that passed the pre-screens described above. Two additional 'Désirée' transformants sequenced were 'iodine-negative' i.e. they had the same appearance as the 'non-transformed' line. These 'iodine-negative' genotypes were included as a control. We used the TIDE (https://tide.deskgen.com/) and Synthego (https://ice.synthego.com/#/) programs to assess editing efficacy across all twelve genotypes, and to identify the types of indels in the targeted regions of the SBEI and SBEII. Among the germplasm tested, indels were found in both SBEI and SBEII genes. The overall editing efficiency of these genes in 'Atlantic' and 'Désirée' was 74%. The average frequency of occurrence of indels in either gene was 15%, with the highest being 66% and the lowest 2%. The biggest deletion identified was 26 bp and the largest insertion was 4 bp. The editing frequency of SBE was very low in the 'Désirée' staining control (1%), and was much higher in the 'iodine-positive" lines which justifies our use of this staining system. Conversely, among the 'iodine-positives' there was no strong correlation between plantlet iodine-staining intensity i.e. how darkly the plantlet stained, and editing efficiency. Some of the most deeply-stained plants had a similar editing efficiency compared to those that were not as intensely stained. Our iodine-marker system may thus only report qualitative (presence or absence) and not quantitative assessments of gene editing. All regenerants that are darker than the control should therefore be sequenced. This assessment is based on a limited dataset, and would require analyzing a larger sample size, to be able to draw firmer conclusions.

Publications


    Progress 10/01/17 to 09/30/18

    Outputs
    Target Audience:The lab a presentation on CRISPR and potato genetics to students visiting UCD from Kyoto University Japan who are majoring in sustainable agriculture. I was Interviewed for an online article "Will you Eat CRISPR Produce?" Interview by Science Blogger Shelby Pope. https://medium.com/neodotlife/crispr-food-aa074305cf5c. Changes/Problems:There were changes made to our experimental approach, based on issues that arose during the course of the work. A). We are planning to transform two genotypes: 'Atlantic' and 'Russet Burbank.' Our initial aim was to amplify cultivar-specific intron sequence for our SBE gRNAs. However, PCR with primers designed to bridge exon-exon gaps did not yield any novel sequence unique to 'Russet Burbank' or 'Atlantic.' Therefore the SBE sgRNAs were designed to sequence that was highly conserved across genotypes. B). We are not using antibiotic selection, therefore to aid in the identification of Cas9-expressing callus and plantlets, we ligated the fluorescent reporter DsRed, driven by UBQ10 promoter, into the pHSE401 construct, using the InFusion cloning protocol. C). We initially proposed to test two Agrobacterium strains. In the course of this work, strain EHA105 proved to be somewhat intractable to transformation. Strain LBA4404 was successfully transformed with pHSE401, and published literature show that it works with potato. We will use this strain instead of EHA105. What opportunities for training and professional development has the project provided?Several students were trained over the review period: A). Undergraduates: Gakpe Mackenzie (McNair scholar), Justin Hom, Bichen Kou, and Ivy Chenh were trained in molecular biology techniques, plant sterile culture, literature searches, biochemistry techniques. B). Graduate students: Jingwei Yu (MS), Mary Madera (Ph.D. rotation student), and Jessie Bacha (former MS student) were trained in biochemical and molecular biology techniques. How have the results been disseminated to communities of interest?We have not completed the objectives of this Hatch project and so we have no results to disseminate per se, however, the following stakeholders were made aware of this project during the review period: A). California Potato Research Advisory Board. B). The National Potato Promotion Board. C). Start-up financiers as part of the UC Berkeley Big Ideas program early in project conception. What do you plan to do during the next reporting period to accomplish the goals?Over the next review period, our aim will be to a) complete sequencing all of CRISPR constructs, b) test the efficacy of the DsRed fluorescence on the constructs by transient expression in Agrobacterium, and c) initiate transformation of potato calli. The screen for desirable potato genotypes will likely go into the 3rd year of this project. It will be necessary to analyze hundreds of samples to find lines that have elevated resistant starch (RS), no foreign (trangenic DNA) and no significant pleiotropic effects that affect productivity and yield. Using DsRed expression should allow us to rapidly identify tissue that are transgenic i.e. those that retained the plasmid after editing, and therefore are unsuitable for use. Our rapid iodine screen should help to identify genotypes with qualitatively higher RS. Collectively, these screens should reduce the number of plantlets that we would need to grow to maturity in the greenhouse for quantitative validation of RS content and yield. We now have a Master's student working on this project which should help us to accomplish these goals more efficiently.

    Impacts
    What was accomplished under these goals? The aim of this project is to produce novel potato cultivars with elevated resistant starch (RS) using CRISPR-Cas9 gene editing. We plan to edit the potato SBE A and SBE B genes by transient expression of Cas9 and different combinations of sgRNAs. Our aim is to recover germplasm with significant levels of RS, few pleiotropic effects on yield, and no transgenes embedded within the genome. This approach will require generating hundreds of transformed calli, and screening them, and the regenerated plantlets, for desirable genetic changes. Over the last year, the following was accomplished: A). The CRISPR SBE sgRNA constructs are being developed. B). A workflow for reproducible callus regeneration of Russet Burbank and Atlantic genotypes was established. C). A protocol for the rapid identification of changes in resistant starch content in potato calli is at the early stages of development.

    Publications