Progress 06/01/15 to 01/31/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Four scientist were trained in tomato transformation methods. An additional two scientists were trained in vector construction, gene copy number measurements, and mRNA expression measurements. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The underlying foundation of this proposal is an area of academic research indicating epigenetics changes can be used to increase yields in crops. This area of research demonstrates that suppression of a particular gene, Msh1, causes epigenetic changes, including DNA methylation changes that persist after Msh1 function is restored. Importantly, these epigenetic changes affect crop yields in a heterosis-like manner. The technical goal of this project was to evaluate four methods of inducing new epigenetic modifications into elite lines suitable for commercialization for development of higher yielding and heat tolerant tomato plants. As this epigenetic technology requires multiple generations of plants to complete, the purpose of the Phase I project was to generate the tomato lines for evaluation in a Phase II project. This Phase I project was successful in accomplishing an initial evaluation of four distinct epigenetic methods. These results are individually presented below and summarized here. The four methods are transformation of new lines, backcrossing a RNAi construct in an existing transgenic line into new lines, Virus Induced Gene Silencing (VIGS), and grafting of transgenic root stocks on shoots (scions) of new genotypes. In this Phase I study, we found the first three methods to be promising, while grafting has a surprising genotype dependence that requires the rootstock and scion to be of the same genotype, which effectively eliminates grafting for evaluation in Phase II. Tomato lines produced by these first three methods will be field tested in a proposed Phase II study to continue to advance this method of increasing yields in plants. General points to be addressed: I. A comparison of actual accomplishments with the goals established for the award: the four technical goals described in detail below were accomplished as originally intended. Some technical problems in Objective 1 (Producing transgenic RNAi plants) were encountered and solved with no major impact for the timelines for Phase II. II. Costs: the budgeted amount was spent and achieved the objectives. The company will spend some money in the interval between Phase I and Phase II to have the tomato lines ready for a Phase II proposal. III. University Subaward: Dr. Mackenzie's group at the University of Nebraska-Lincoln successfully completed their portion of the objectives (VIGS and grafting) on budget and on time. IV. No equipment was purchased for this project with funds from this SBIR grant (GRANT11743451). V. The technical objectives were to produce new epi-lines in 5 elite tomato germplasms using the following four methods: Method 1. Transgenic RNAi. A larger number of transgenic lines will be produced for a more complete evaluation of this method as individual transgenic lines vary considerably in their MSH1 suppression levels. Method 2. MSH1 VIGS. This TRV VIGS method has the advantage of avoiding plant transformation and can be rapidly implemented in most genotypes. Method 3. Grafting of MSH1-suppressed rootstocks. This alternative method is fast and has a quite different commercialization production pathway as described below. Method 4. Backcrossing of transgenic RNAi suppression lines into elite germplasm. This approach provides an alternative to the above methods for creating epi-lines in new germplasms and may benefit from selecting particularly efficacious parent lines, i.e., once a very good donor is identified, it can be used for backcrossing the RNAi transgene into many different elite germplasms. Hence, this backcrossing approach might be the most convenient for introducing epigenetic modifications into many different germplasms simultaneously. RESULTS FOR EACH METHOD IN DETAI Method 1 Transgenic RNAi. Produce 30 new MSH1-RNAi tomato transgenic lines for each of five elite germplasms. Our intermediate progress report of November 2015 indicated excellent progress on tomato transformation. We had completed 13 transformation experiments and appeared to have numerous transgenic shoots in progress. However, subsequent molecular analysis of these discovered almost all of these shoots were non-transgenic escapes: only four lines were transgenic. Two of these are suppressed for Msh1 and showing msh1-dr phenotypes. However, as we required higher numbers of lines to evaluate, we changed our vectors for transformation. The major vector change was to use a stronger promoter to express the visual marker Green Fluorescent Protein (GFP) as a GFP-NptII visual-selectable marker fusion protein. The visual presence of GFP allows us to be certain of the shoot being transgenic earlier in the process. The vectors also contained an RNAi construct for silencing the Msh1 genes, which was the purpose of these experiments. This new vector was highly successful in producing transgenic plants expressing GFP, removing any uncertainty as to whether they were transgenic or not. Transgenic tomato plant status for Five FL tomato genotypes (1/31/2016): FL7804: 18 in soil (4 oldest with fruit and confirmed for RNAi); 70 GFP+ shoots in culture FL8059: 10 in soil; 60 GFP+ shoots in culture FL8872: 0 in soil; 30 GFP+ shoots in culture FL8917: 2 in soil; 32 GFP+ shoots in culture FL8651: 6 GFP+ shoots in culture; more treated explants in selection to obtain more plants The change in transformation protocol and consequent delayed timelines for the new transformations becoming plants in soil has delayed our Msh1 mRNA knockdown analysis. This will now be done in the interval between the end of Phase I and the application for a Phase II grant. The CaMV 35S promoter/RNAi gene cassette is what Dr. Mackenzie used before in her tomato Msh1 knockdowns, so we are fairly confident in obtaining efficient knockdowns. Conclusion: Objective 1 encountered some delays due to initial non-transgenic escapes. This problem was solved with better selections and the GFP visual and the project was successful in producing a pipeline of transgenic RNAi plants for Phase II. Some of the oldest transgenic plants are showing Msh1-knockdown symptoms: altered leaf shape and delayed flowering, which are the phenotypes indicative of efficient Msh1 suppression. Method 2. MSH1 VIGS in Tomato. Wesuccessfully performed VIGS (Virus Induced Gene Silencing) on tomato line FL8059. Four of 12 plants showed a 50% reduction in Msh1 in the virus silenced plants. Progeny from the 2 plants with the most knockdown of Msh1 were then analyzed. These 'T1' plants had very mild to little difference from the control plants. The next generation 'T2' plants showed stronger Msh1-dr phenotypes. However, crossing of these plants to make 'EpiLines' did not show enhanced growth phenotypes. These experiments demonstrate VIGS of Msh1 can cause heritable epigenetic changes. However, larger number of plant lines and progeny tests are required to fully evaluate this method in Phase II. Method 3. Grafting of MSH1-suppressed rootstocks. Surprisingly, we learned that grafting between different varieties does not confer epigenetic benefits despite the fact that matching the genotypes in the rootstock and scion in a graft does accomplish this. This elucidates that transgenic rootstocks of each targeted variety are needed for grafting that variety. This is an important advancement in our understanding of the grafting process, but removes grafting as a possible method for advancement in this Phase I/Phase II project. Method 4. Backcrossing of transgenic RNAi suppression lines into elite germplasm. Crosses have been made to produce F1 seeds as the first step of backcrossing. The mature F1 seeds have been harvested and are planted in the greenhouse for a backcross to the recurring parent to move the transgene into these 5 elite germplasms. This objective was successful and appears to be a good method for moving a RNai transgene into multiple varieties for epigenetically improving tomato germplasms.
Publications
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Progress 06/01/15 to 01/31/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr Song Zhang (Epicrop Director of Plant Transformation) trained Epicrop Research Associate Mr. Daniel Bui in how to perform tomato transformations by individual mentorship. This advanced Mr. Bui's professional development as a career scientist. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We've made excellent progress in this initial stage of the project. Our plan is to continue the steps needed to complete the evaluationof the Methods outlined in the proposal.
Impacts
What was accomplished under these goals?
The technical objectives are to produce new epi-lines in 5 elite tomato germplasms using the following four methods: Method 1. Transgenic RNAi. A larger number of transgenic lines will be produced for a more complete evaluation of this method as individual transgenic lines vary considerably in their MSH1 suppression levels. Progress on Method 1: We have performed 13 independent transformation experiments with 6 tomato genotypes, FL8917, FL8059, FL8972, FL8651, FL7804 and FL8924 (a new line) using Agrobacterium EHA105 containing plasmid pMCI50 (for silencing Msh1 mRNA). The numbers of putative transgenic shoots we have obtained are 35 for FL8917, 32 for FL8059, 8 for FL8972, 27 for FL8651, 130 for FL7804 and 6 for FL8924. We have finished the transformation experiment of FL7804 (130 events). There are more transgenic shoots in progress for the other 5 lines that will be transferred to shoot elongation medium in several weeks. All the transgenic shoots are currently at shoot elongation stage and will be transferred to root induction medium soon. Method 2. MSH1 VIGS. This TRV VIGS method has the advantage of avoiding plant transformation and can be rapidly implemented in most genotypes. Progress on Method 2: Wesuccessfully performed VIGS (Virus Induced Gene Silencing) on tomato line ms FL8059. Progeny plants displayed mild msh1-dr phenotypes as expected indicating a heritable epigenetic change occurred. Method 3. Grafting of MSH1-suppressed rootstocks. Progress on Method 3: Surpisingly we learned that grafting between different varieties does not confer epigenetic benefits despite the fact that grafting does accomplish this for grafts within the same variety. This elucidates that transgenic rootstocks ofeachtargeted variety are needed for grafting that variety. Method 4. Backcrossing of transgenic RNAi suppression lines into elite germplasm. Progress on Method 4: Crosses have been made to produce F1 seeds as the first step of backcrossing.
Publications
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