Progress 08/01/20 to 07/31/23
Outputs
Target Audience:The target audiences from this research are primarily plant scientists working on fundamental and translational research, plant breeders, and agricultural industries. These include the faculty and students in the College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa; the community of ARCS (Advancing Science in America) foundation Honolulu Chapter, and a broader community through ARCS website; American Society of Plant Biologists, American Society for Horticultural Science, American Phytopathological Society, and a broader plant science community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided training opportunities and professional development for two graduate students with one in Tropical Plant Pathology and one in Molecular Biosciences and Bioengineering, four undergraduate students, and one high school student. The graduate students acquired experience and expertise in various cutting-edge plant biology and molecular biology techniques, among which are plant genetic engineering, tissue culture, genome editing, and next-generation sequencing and analyses. They both developed critical thinking and problem-solving capabilities. They also developed research communication skills through presentations and writing theses and manuscripts. They matured into independent researchers. The undergraduate students obtained hands-on trainings in basic laboratory skills, such as plant growth, media preparation, PCR and DNA gel electrophoresis, and advanced skills in plant tissue culture and transformation. The high school student obtained hands-on lab experience and training in basic laboratory skills, and built interests and confidence in pursuing undergraduate studies in life sciences. Six out of the seven students are currently either pursuing advanced studies or a job in life sciences and/or agriculture. As such, this project has contributed to developing a talented workforce that will positively impact agriculture and other disciplines of life sciences. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest through two poster presentations in American Society of Plant Biologists (ASPB) annual meetings-Plant Biology 2022 and Plant Biology 2023, a publication in the journal " In Vitro Cellular & Developmental Biology-Plant", and a Master's thesis at University of Hawaii library (online). The results have also been presented to the faculty and students in University of Hawaii at Manoa through graduate and undergraduate students' oral presentations, and the project investigators' courses. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Establish an effective and routine papaya gene editing system via Agrobacterium-mediated transformation. Using two CRISPR systems (CRISPR/Cas9 and CRISPR/Cas12a), two papaya genes CpPDS and CpMLO6, with two guide RNAs targeting each gene for either Cas9 or Cas12a, we have established a system that allows to generate gene edited papaya plants with high efficiency via Agrobacterium-mediated transformation. This system includes methods on selection of potentially functional single guide RNAs (sgRNAs) followed by testing of their in vitro cleavage activity, construction of plasmids that express gene editing reagents, and optimized papaya transformation and regeneration. The CpPDS gene was successfully mutated using both Cas9 and Cas12a, leading to albino phenotypes. For gene editing of CpMLO6 using Cas9, we investigated the mutations in 26 transgenic plants by analyzing Sanger sequencing chromatograms using the Interference of CRISPR Edits and found that 25 plants had an insertion/deletion (indel) percentage of over 90% at both target sites, suggesting near complete mutations of the gene. For gene editing of CpMLO6 using Cas12a, we have recently obtained 18 transgenic plants and are in the process of testing the mutation efficiency. Our results suggest that this established papaya gene editing system is highly efficient and reproducible. This system allows to obtain gene edited transgenic plants in 5-6 months after transformation of hypocotyl-derived embryogenic liquid suspension culture. The time is much reduced compared to previously reported long regeneration time of 9-13 months. By using G418 instead of kanamycin for selection of transgenic embryos and shoots,timentin to inhibit the growth of Agrobacterium with concentrations adjusted at different developmental stages, and combined use of media with growth hormones based on multiple previous published papaya transformation and regeneration methods, the transformation method we established shortens the regeneration time, overcomes Agrobacterium overgrowth issues and improves rooting efficiency. The outcome from this research is of high impacts. Papaya produces nutritious fruit consumed as food and also widely used in the cosmetics, pharmaceutical and food processing industries. The highly efficient papaya gene editing system and optimized transformation system we established are expected to accelerate papaya functional genomics studies to dissect biosynthetic pathways for nutrient content and the molecular basis of plant-microbe interactions. Knowledge yielded from these studies will reveal targets for crop improvement and lead to more effective production and disease management practices. The tools we developed have enabled the editing of these gene targets to breed papaya varieties with desirable traits, such as improved nutritional values and enhanced disease resistance. A manuscript describing the methods and results from this objective is being prepared and will be submitted to an open-access scientific journal in 1-2 months. Due to the technical issues we initially encountered and the impact caused by Covid-19 pandemic, the progress of the research was delayed. We acknowledge the delay and will complete the publishing of the results as soon as possible. Objective 2: Develop DNA-free genome editing of papaya with preassembled CRISPR-nuclease ribonucleoprotein complexes. We significantly improved the isolation of high-yield, viable protoplasts from papaya leaves, obtaining an average yield of 1.11 × 108protoplasts per gram of fresh weight, which is a 7-fold increase compared to the previously published optimal yield of protoplasts. The viability of the protoplast was 89.97%. We further established an efficient genetic transfection method, with which efficient DNA-free gene editing in papaya chloroplast using CRISPR-nuclease ribonucleoprotein (RNP) complex was demonstrated for three different genes. The first example involved editing a single nucleotide frame-shift mutation in the GFPtransgene transiently expressed in papaya protoplasts that restored the wild-type translational reading frame with an efficiency of 27.88%. The editing of two endogenous papaya genes in the papaya genome,CpPDSandCpMLO6, in protoplasts was demonstrated in three different papaya cultivars, with the average mutant frequency detected using amplicon deep sequencing as 42.31% and 16.20%, respectively. Taken together, a DNA-free CRISPR-Cas9 gene editing system was successfully demonstrated in papaya protoplasts using multiple target genes. Due to technical challenges, we have not successfully regenerated gene edited protoplasts into papaya plants. However, we have modified several parameters and a resulting protocol shows promise for future work. It uses translucent non-fully expanded leaves proximal to the apical meristem as a source of protoplasts, followed by gene editing, and direct embedding into a sodium alginate hydrogel. The development of this mesophyll protoplast-based system to edit genes from papaya represents a key step toward generating DNA-free gene edited papaya plants for use in breeding programs for creating disease resistant plants. Compared with the approach as described in Objective 1, the DNA-free genome editing approach will produce the DNA-free plants in the first generation of regenerated plants without the need of waiting until the second generation, therefore shortening the breeding time. Moreover, varieties generated from DNA-free genome editing approaches are more acceptable by consumers and could more readily pass regulatory approvals. In addition, the system developed in this objective is excellent for testing the functionality of sgRNAs for papaya gene editing usingAgrobacterium-mediated transformation (Objective 1). As papaya transformation is still a relatively long process, transforming the sgRNAs confirmed to be functionalin vivowill save substantial time and effort.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Hasley, J. and Tian, M. 2022. Establishment of a genome editing system in papaya through Agrobacterium-mediated transformation. American Society of Plant Biologists (ASPB) annual meeting 2022, Portland, Oregon, July 9-13, 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Tian, M., Elias, M.J., Hasley, J.A.R, Dinulong, R.J., and Christopher, D. 2023. Genome editing of papaya. American Society of Plant Biologists (ASPB) annual meeting 2023, Savannah, Georgia, Aug 5-9, 2023.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Elias, M.J., Hasley, J., Tian, M., and Christopher D.A. 2023. Development of a mesophyll protoplast-based system for gene editing of papaya. In Vitro Cellular & Developmental Biology-Plant, 59:517-535. https://doi.org/10.1007/s11627-023-10373-1.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Elias, M.J. 2022. Development of a mesophyll protoplast-based system for gene editing of papaya. Master of Science Thesis in Molecular Biosciences and Bioengineering, University of Hawaii, December 2022. https://hdl.handle.net/10125/104593 (oai:scholarspace.manoa.hawaii.edu:10125/104593).
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Progress 08/01/21 to 07/31/22
Outputs
Target Audience:The target audiences from this research are primarily plant scientists working on fundamental and translational research, plant breeders, and agricultural industries. The target audiences for this project periodinclude the faculty and students in the College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, andAmerican Society of Plant Biologists. Changes/Problems:Due to interruption by COVID pandemic, the overall progress was delayed. A one-year no-cost extension of this project was requested by the project PIs and approved by the funding agency. What opportunities for training and professional development has the project provided?During this project period, this project continued to provide training and professional developmentof two graduate students, one PhD student in Tropical Plant Pathology (TPP) and one Master's student in Molecular Biosciences and Bioengineering (MBBE). The TPP graduate student has obtained hands-on experience in molecular cloning, sgRNA design, plant tissue culture and transformation, plant transient expression, and other various plant biology and molecular biology techniques. TPP graduate student has also got the opportunity to attend ASPB meeting, through which he presented his research progress of this project, learned new knowledge and technology, andnetworked with other plant biologists in academia and industry. The MBBE graduate studentobtained hands-on experience in sterile culture techniques, plant and bacterial aseptic growth media, growing plants in soil, light and fluorescence microscopy, protoplast preparation and plasmid DNA construction and manipulation. Both graduate students gave oral presentations of their research to the research group, the department and the college, to develop research communication skills. In addition, the project provided opportunities for three undergraduate students to get hands-on research experience, includingone undergraduate student in Biology who worked as an undergraduate research assistant hired for the project,one in MBBE through an undergraduate directed research course, and one in Biochemistry through a NIFA summer internship program. These undergraduate students got hands-on training in common lab techniques, media preparation, plant tissue culture and transformation, and sgRNAs design for gene editing. The TPP PhD student has played a major role in supervising these undergraduate students, therefore obtained skills in teaching and advising. How have the results been disseminated to communities of interest?The results have been presented tothe faculty and students in the College of Tropical Agriculture and Human Resources (CTAHR), University of Hawaii at Manoa. The research activities have been disseminated to academia and industry through a poster presentation in American Society of Plant Biologists (ASPB) annual meeting, and oral communications with other researchers. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Establish an effective and routine papaya gene editing system via Agrobacterium-mediated transformation We will continue to optimize the transformation by testing various parameters to improve the efficiency and shorten the regeneration time. Once the antibiotics-resistant embryo/calli from various ongoing transformation experiments are regenerated, we will evaluate the mutation efficiency by Sanger sequencing of the PCR fragments spanning the target sites, followed by analyzing the resultant chromatograms usingInference of CRISPR Edits (ICE)v2. Phenotypic changes of the mutant plants will be analyzed at T0 generation. The gene editedCpMLO6T0mutant plants will be propagated through micropropagation to produce sufficient numbers of plants that allow infection assays using papaya powdery mildew to evaluate the altered disease resistance. Selected T0 mutant plants will be grown into next generation to obtained transgene-free gene edited plants. Objective 2 Develop DNA-free genome editing of papaya with preassembled CRISPR-nuclease ribonucleoprotein complexes. We will test the efficiency of gene editing ofCpPDSandCpMLO6upon protoplast transfection with the corresponding RNP complexes and improve the mutation efficiency by optimizing the related parameters. We will continue to establish the method to regenerate plants from protoplasts. The protoplasts with high rate of mutations after transfection with RNP complexes will be regenerated into plants.
Impacts
What was accomplished under these goals?
Objective 1: Establish an effective and routine papaya gene editing system via Agrobacterium-mediated transformation During the previous project period, we have 1) designed sgRNAs fortwo papaya genes,phytoene desaturase (CpPDS) and Mildew Locus O 6 (CpMLO6) for gene editing using twoCRISPR/Cas systems (Cas9 and Cas12a); 2) tested the efficiency of sgRNAs byin vitrocleavage assays; 3) cloned sgRNAs into plant binary vectors to expresssgRNAs and Cas nucleases; 4) validated the functionality of sgRNAs and promoters used for papaya gene editingthrough Agrobacterium-mediated transient expression. During this project period, we have focused on establishing and optimizing papaya regeneration and transformation systems, and meanwhile establishing the gene editing systemby transforming the gene editing constructs. Papaya transformation systems were previously reported, however, with issues of agrobacterium overgrowth, difficulty in rooting and long regeneration time of 9-13 months. After analyzing the advantages and limitations of the published protocols, we designed the protocols by combined use of the methods from multiple publications instead of following the protocols exactly from the publications. We were able to regenerate fully-developed papaya plants through somatic embryogenesis with high efficiency using hypocotyl sections of 10-days-old seedlings as starting materials. We achieved this through two different approaches. For both approaches, hermaphroditic papaya seedlings grown in tissue culture were selected based on PCR assays, and the hypocotyl sections were grownon2,4-Dichlorophenoxyacetic acid(2,4-D)-containingMurashige and Skoog (MS)media to induce embryogenic calli. For the first approach, the embryogenic calli were grown at reduced concentrations of 2,4-D for the development of somatic embryos, followed by maturation on the hormone-free media and then germination on cytokinin-containing media to regenerate shoots. We tested different parameters usingIndole-3-butyric acid(IBA) to achieve high rate of root regeneration and found that exposing the shoots to IBA for about 2-3 days followed by culturing on IBA-free media produced significantly higher number of well-developed roots than growing the shoots continuously on IBA-containing media. For the second approach, the embryogenic liquid suspension cell culture was established from embryogenic calli. The liquid suspension cell culture was further grown on solid media to develop embryos and then regenerate to fully-developed plants as for the first approach. The advantage of the second approach is that the liquid suspension cell culture with 2 mg/L 2,4-D is able to maintain the embryogenic stage suitable for transformation for over 15 months and this would save at least 6-8 weeks of time to get to this stage from hypocotyl issues. We have established suspension cell cultures for two papaya varieties, Kapoho and Sunrise. We also established the Agrobacterium-mediated transformation procedures that were able to produce the transgenic events without the issues of Agrobacterium overgrowth, an issue previous papaya transformation publications reported. By transforming a construct expressing the reporter gene Ruby, we were able to visually observe Ruby-expressing tissues, suggesting the transformation procedures were effective. Using the transformation and regeneration procedures we have established, we transformed various gene editing constructs for gene editing ofCpPDSandCpMLO6using two CRISPR systems. For the transformation experiments performed earlier, the shoots for the control treatment (without the antibiotics selection for transgenic embryos) were successfully regenerated, however, with the antibiotics selection, the regeneration appeared to be much delayed or stalled. After carefully analyzing the literature and our observations, we realized that kanamycin and hygromycin, the antibiotics used to select transgenic tissues, inhibited the regeneration of papaya and should be eliminated after several weeks of selection. We are currently testing the timing and duration of antibiotics selection to obtain regenerated transgenic shoots with low rate of false positives, and also testing the use of G418 to replace kanamycin as the antibiotics to select NPTII-expressing transgenic calli for potential fast and effective regeneration of transgenic shoots. After removing of kanamycin from the transformation experiment performed earlier, we started to see regenerated shoots. The analyses of transgene integration and genome editing are underway. Objective 2 Develop DNA-free genome editing of papaya with preassembled CRISPR-nuclease ribonucleoprotein complexes. The key step of completing this objective are protoplast isolation and transfection. During the first project year, we have established a method to produce high yield of protoplasts. The method was further refined and improved during the current project year.The method now consistently produces approximately seven times higher yields of protoplasts than the previously published procedures, with an average total protoplast count of 1.11 x 108±0.07 g-1fresh weight. The use of young, fully expanded leaves on plants greater than 2.5 months old (Solo Sunrise) as a source of protoplasts was a key basis for the improvement. The freshly isolated protoplasts from the papaya cultivar, Solo Sunrise, had an average viability of 89.87±2.02%, as evaluated by staining withfluorescein diacetate (FDA). To establishan efficient method for protoplast transfection, weused a construct (plasmid) consisted of the eGFP gene controlled by the 35S promoter cloned into the Bluescript plasmid vector, and utilized a polyethylene glycol (PEG)-mediated transfection method.Fluorescence was visualized 16-18 hours post-transfection using epifluorescence and confocal microscopy. Transfection efficiencies ranged from 38.29% to 91.07% with an average transfection efficiency of 58.60±21.92%. The effectiveness of the transfection method was also confirmed by expressingPDI9-GFP, which encodes disulfide isomerase 9 (PDI9) fused in frame to the GFP gene, under the control of the 35S promoter. As expected, PD19-GFP was observedinendoplasmic reticulum(ER) of papaya protoplasts and co-localized with an ER-localization marker tagged with mCherry. To test whether the protoplast transfection system can be used to deliver CRISPR-nuclease ribonucleoprotein complexes to achieve gene editing, papaya protoplasts were co-transfected with a plasmid carrying a mutantversion of GFP (GFPm), in which a single nucleotide insertion into a functional GFP encoding gene causes a frameshift mutation leading to a deficiency of green fluorescence upon excitation, and a CRISPR/Cas9 gene editing ribonucleoprotein complex targeting the GFPm mutation site. Transfected protoplasts were visualized 36-48 hours post transfection using epifluorescence and confocal microscopy. Some of the successfully edited protoplasts resulted in the correct frameshift mutation necessary to restore GFPm to wild-type GFP. A gene editing efficiency of over 9.74±1.28%was determined by comparing the number of fluorescent protoplasts to non-fluorescing protoplasts. Currently, we have performed protoplast transfection experiments with the RNP complexes targetingCpPDSandCpMLO6, and analyses of the results are underway. Meanwhile, we started to establish the system to regenerate papaya protoplasts into plants. The development of a draft manuscript is currently underway which will detail multiple advancements in developing a method for DNA-free targeted gene editing in papaya. The manuscript will deliver an improved method for isolating high yield, highly viable protoplasts from papaya leaf mesophyll cells, an osmotically optimized polyethylene-glycol mediated transfection method using papaya protoplasts, and the application of these advancements to target and edit genes of interest within the papaya nuclear genome.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Hasley J. and Tian M. 2022. Establishment of a genome editing system in papaya through Agrobacterium-mediated transformation. American Society of Plant Biologists (ASPB) annual meeting 2022, Portland, Oregon, July 9-13, 2022.
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Progress 08/01/20 to 07/31/21
Outputs
Target Audience:The target audiences during this reporting period mainly include the faculty and students in the College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa;the community of ARCS (Advancing Science in America) foundation Honolulu Chapter, and a broader community through ARCS website. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training and profession development of two graduate students, one in Tropical Plant Pathology (TPP) and one in Molecular Biosciences and Bioengineering (MBBE), and one undergraduate student in Biology. The TPP graduate student has obtained hands-on experience in molecular cloning, sgRNA design, plant tissue culture and transformation, plant transient expression, and other various plant biology and molecular biology techniques. By sharing his research, the TPP graduate student won ARCS Foundation Honolulu Chapter Ellen M. Koenig Award. The graduate student in MBBE has got hands-on training in plant growth, sterile culture techniques, plant and bacterial aseptic growth media, light and fluorescence microscopy, protoplast and plasmid preparation, etc. From the experiments, both have been developing skills in experimental design, trouble shooting and problem solving. They have also developed research communication skills by regularly presenting their research progress during the project meetings. The undergraduate student has obtained hands-on training in basic laboratory skills, such as plant growth, media preparation, PCR and DNA gel electrophoresis.The training of these students contributes to developing a talented workforce that will positively contribute to agriculture. Also, it builds confidence that they can become scientists. Finally, it allows the students to be competitive in the job market and/or on applications to professional schools. How have the results been disseminated to communities of interest?The results have been presented tothe faculty and students in the College of Tropical Agriculture and Human Resources (CTAHR), University of Hawaii at Manoa. The research activities have been made awareto papaya industries in Hawaii and beyond through CTAHR newsletter. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Establish an effective and routine papaya gene editing system via Agrobacterium-mediated transformation We will continue withAgrobacterium-mediated stable transformation. Once the kanamycin-resistant embryogenic calli are produced, we will evaluate the mutation efficiency by Sanger sequencing of the PCR fragments spanning the target sites, followed byanalyzing the resultant chromatograms usingInference of CRISPR Edits (ICE)v2.The calli from the constructs that produce higher gene editing efficiency will be regenerated into plants, which will be subjected to detailed mutation and phenotypic analyses. In addition, we will continue to optimize the method for gene editing via Agrobacterium-mediated transient expression of gene editing reagents. Objective 2 Develop DNA-free genome editing of papaya with preassembled CRISPR-nuclease ribonucleoprotein complexes. We will continue to establish an efficient method for papaya protoplast transfection. This method will be used to co-transfect a non-functional GFP and RNP complex targeting GFP gene to develop a protocol that can be used to introduce RNP complex for high percentage of protoplasts with gene editing events. We will then use the protocol to mutate CpPDS and CpMLO6 by introducing the corresponding RNP complexes, followed by protoplast regeneration to plants. To save time, while we are establishing the method for protoplast transfection, we will establish the method for protoplast regeneration to plants in parallel.
Impacts
What was accomplished under these goals?
Objective 1: Establish an effective and routine papaya gene editing system via Agrobacterium-mediated transformation We have been using two single-copy papaya genes, phytoene desaturase (CpPDS, XP_021888908.1) and Mildew Locus O 6 (CpMLO6, XP_021909409.1), and three popular papaya varieties, Sunrise, Sunset and Kapoho, to establish the papaya gene editing system viaAgrobacterium-mediated transformation. To be able to design the sgRNAs that target the genes with 100% identity, the gDNA and cDNA ofCpPDSandCpMLO6were amplified from the above mentioned three varieties by PCR and RT-PCR, followed by Sanger sequencing. No sequence polymorphisms of these two genes were observed among different varieties. 2-4 sgRNAs for each gene and each CRISPR/Cas system (Cas9 and Cas12a) were designed, synthesized, and subjected toin vitroDNA cleavage activity assays. We were able to identify 2 sgRNAs with high cleavage activity of the target DNA for each gene and each Cas system. For Cas9-mediated gene editing, we cloned two sgRNAs of each gene to pKSE401, a binary vector previously developed for gene editing of a range of dicot plant species. For Cas12 (Cpf1)-mediated gene editing, we first modified pKSE401 to pKSE401-Cpf1 by replacing Cas9 withLachnospiraceae bacteriumCpf1 (LbCpf1), and Cas9 sgRNA scaffold and U6-26 terminator with PolyT. We then cloned the single pre-crRNA array expressing two sgRNAs of each gene to pKSE401-Cpf1 under U6-26 promoter. These constructs were transferred toAgrobacteriumstrains GV3101 and EHA105. In order to develop a gene editing system through transient expression for rapid functional analyses of papaya genes and quick evaluation of the effectiveness of gene editing reagents, we have been testing and optimizingAgrobacterium-mediated transient expression in papaya. After testing several conditions, we were able to express the GUS gene, evidenced by the detection of its activities through histostaining, following vacuum infiltration ofAgrobacteria. Using the condition that produced the highest level of GUS expression, we transiently expressed the constructs expressing Cas9 and two sgRNAs ofCpPDS. We were able to detect the expression of Cas9 and two sgRNAs in theAgrobacterium-infiltrated tissues at three and four days post infiltration (dpi) using RT-PCR, suggesting that the promoters used are appropriate in driving gene editing reagents in papaya. The gene editing efficiency was determined using the samples collected at 4 dpi by amplicon deep sequencing. We detected about 0.35% mutation frequency at each sgRNA target site. This result suggests that the promoters and sgRNAs are functional in editing of papaya genes, and therefore suitable for papaya gene editing through stable transformation. Stable transformation of papaya is underway. We have generated embryogenic suspension cell cultures for Sunrise and Sunset from hypocotyl issues of young papaya seedlings, and have performed infection of embryogenic cell culture withAgrobacteriacarrying the gene editing constructs. We are also getting ready for a transformation experiment using hypocotyl issues directly as explants. Objective 2 Develop DNA-free genome editing of papaya with preassembled CRISPR-nuclease ribonucleoprotein complexes. SgRNAs designed and tested as described above (Objective 1) have been made ready for this objective. The key step of completing this objective is protoplast isolation. We have established a method to produce high yield of protoplasts. This method produces an average of 91.82 +/-8.46% higher yields of protoplasts than any previously published procedure, with an average total protoplast count of 2.14 (+/-0.181) x 108g-1fresh weight. One key for this improvement in protoplast yield was the use of young, fully expanded leaves on more mature plants older than 2.5 months as starting material. This method is anticipated to increase the frequency and efficiency of positive gene editing events supporting Objective two. Following that, we have been establishing an efficient method for protoplast transfection. We are using constructs (plasmids) containing the green fluorescent reporter gene (eGFP) fused to the 35S constitutive promoter, and a polyethylene glycol-mediated transfection method.Preliminary experiments suggest papaya protoplasts may be more sensitive to changes in osmotic properties of the buffers. Modifying the buffer concentrations and incubation times, and using protoplasts of stronger leaves are currently undertaken.Once optimized, this method will be used to introduce preassembled CRISPR-nuclease ribonucleoprotein complexes for DNA-free genome editing of papaya.
Publications
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