Plant Improvement

Consolidation of citrus breeding plant material to vacate space for Stage I and Stage II field trials and to exploit tolerant germplasm for gene editing strategies

1. Please state project objectives and what work was done this quarter to address them:

Objective 1: Destroy declining plant material with no direct use to the citrus industry to make space for Stage I and Stage II trials (Years 1 and 2).
Since the previous quartly report, more acreage has been abandoned, pushed and destroyed at CREC, to make space for Stage I and Stage II trials. Hundreds of trees were identified blocks slated for destruction as being suitable for conservation, movement with the tree spade (or other means) and sourced for budwood. As mentioned previously, these trees were identified by both drone and by visual inspection. Project personnel has collected all budwood from these blocks and it was sent for propagation at the nurserylicensed to propagate uncertified budwood. UFarm charges have conserved and saved all materials needed for data mining for tolerant genes and other variants.
Objective 2: Conserve, move, consolidate, and propagate tolerant and resistant citrus trees to a centralized location. (Years 1 and 2).
The block where consolidation will take place has been reserved for this purpose. The nursery has received more accessions than expected by this time point as conservation of useful material is imperative. When the buds take, we will move forward with tree movement with tree spade and other means. The tree spade is reported to be repaired and ready, and the invoice has been paid, but the CREC grove manager has not moved it yet.

2. Please state what work is anticipated for next quarter:

We anticipate using the USDA-ARS tree spade to move the first trees awhich have budwood transferred to the nursery for propagation in late winter/early spring. A method has been proposed by the grove manager in which the spade prunes the roots for the development of a dense root ball, where possible, with a follow up removal with the spade. In some cases trees will be pruned, allowed to flush, which may aid in development in denser roots upon tree movement. There are thousands of trees that are tagged for removal, and we are waiting for final removal of those trees.

3. Please state budget status (underspend or overspend, and why):

We are expending the grant funding in a timely manner (44% time spent versus 57% funding expended) according to the UF/IFAS grants tracking system. The budget is being expended at a rate at which all funding will be expended by the end of the project period. I believe the ratio is as such because the committed funds are used in this calculation, as in the funds have not been expended yet, but they will as the proper time comes.

Evaluate new transgenic rootstocks for HLB tolerance

Generation of non-transgenic HLB-resistant sweet orange varieties using CRISPR-Cas technology

Understanding the HLB tolerance and reduced fruit drop in Parson Brown and evaluation of other early season sweet oranges

Evaluation of Potential HLB Tolerant Grapefruit Rootstock/Scion Combinations in Florida

Evaluating Novel Gene-edited Duncan Grapefruit Mutants for Resistance to Huanglongbing (HLB)

23-031: Accelerate Establishment of Stage 2 Citrus Trials to Combat Citrus Greening Disease

Evaluate new transgenic rootstocks for HLB tolerance

Preliminary Field Trial to Evaluate the Ability of HLB Tolerant Interstocks to Protect Commercial Scions Against HLB

Understanding the HLB tolerance and reduced fruit drop in Parson Brown and evaluation of other early season sweet oranges

Comparison of field performance of citrus trees on rootstocks propagated by seedlings, cuttings, and tissue culture

Final Report (Due 5/31/2024)- Project # 23-037: Fruit, Yield, and Quality Assessment for Advanced Selections and Drone Re-flight for Hurricane-Affected Sites.

Quarterly Project # 21-025: Transgenic capable field site to assess HLB-resistant and other improved citrus

1. Please state project objectives and what work was done this quarter to address them: Project # 21-025 focuses on developing HLB-resistant and other improved citrus cultivars to mitigate the devastating effects of HLB on the Florida citrus industry. The project utilizes transgenic and non-transgenic approaches, and all experiments are conducted at the secure field site of the USDA/ARS Picos Research Farm. The goal is to provide sustainable solutions to combat HLB, safeguard the citrus industry, and support economic citrus production.This quarter, The test site received routine horticultural care, and trees were exposed to HLB and psyllid vectors to assess their resistance in a real-world environment. All BRS permits were upheld, and the site underwent inspections to ensure compliance with regulatory requirements. In addition Dr. Zhonglin Mou planted a total of 23 transgenic rootstock lines were planted in the farm on July 24, 2024. These lines include 14 NPR3 RNAi rootstock lines, three NPR3 RNAi rootstock lines top-grafted with Hamlin, and six NPR1 rootstock lines. These transgenic lines were generated through mature tissue transformation. There were planted in the field for fruit/seed production. Dr. Mou’s existing transgenics were also assessed. The NPR1 scion lines: 69 trees were planted in May 2019, 98 in May 2021, and 27 in October 2021. Among those planted in 2019, 15 were produced through mature tissue transformation, and 11 survived. These trees have flowered and are producing fruits. Two lines were chosen for propagation. Budsticks from these two lines have been submitted to DPI on July 15, 2024. Leaf and fruit samples were taken from the mature trees on July 26, 2024 for CLas titer assays. One tree from juvenile transformation has flowered this spring and currently has three fruits on the tree. Other juvenile trees have not flowered. 2. Please state what work is anticipated for next quarter:Next quarter, the team will analyze the data collected from the current field trials to evaluate the efficacy of different HLB-resistant candidates. Researchers will continue to have access to the test site for data collection and analysis purposes. Discussions with Soilcea are still continuing as the USDA continues to review contract documents for the planting of CRSPR trees for trial at this site in a true Florida citrus growing region. Upcoming  meetings with Dr. Manjul Dut will be held during the end of August to discuss additional plantings at the site and plans for evlautating existing plantings. 3. Please state budget status (underspend or overspend, and why):This quarter, the project is ontrack for spending. We expect that we will be on track for spending for the remainder of the contract period.   4. Please show all potential commercialization products resulting from this research, and the status of each: The trangenic test site is consistently monitiored and researchers are contacted when their population(s) of trees show promising results for commericalization. This research has the potential to yield HLB-resistant transgenic and non-transgenic citrus cultivars suitable for commercial production. Some materials may be immediately suitable for commercial citrus production. The University of Florida has shown increased interest in the commricalization of some transgenic scions planted in the field that show promising single tree data. The data gathered from the field trials will guide recommendations for advancing materials to larger replicated trials, ensuring the development of commercially viable HLB-resistant citrus varieties.     

Generation of non-transgenic HLB-resistant sweet orange varieties using CRISPR-Cas technology

1. Please state project objectives and what work was done this quarter to address them: The purpose of this project is to generate non-transgenic HLB resistant Valencia and Hamlin sweet orange plants using CRISPR-Cas technology. Objective 1. Generate non-transgenic HLB resistant/tolerant Valencia and Hamlin sweet orange plants by mutation of HLB susceptibility genes. In total, six putative S genes were proposed to be edited and we have lately added more targets. Constructs needed for CRISPR genome editing are being made. Multiple edited lines were generated for ACD2 gene. However, further confirmation demonstrated none of them was biallelic/homozygous mutant. We are genenerating more lines for ACD2 and other target genes. To speed up the process, we have further optimized the transgene-free CRISPR genome editing using Cas12a/crRNA ribonucleoprotein. We have developed an efficient co-editing strategy for generating transgene-free, gene-edited plants via Agrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFP in planta.  Specifically, CBE/gRNA was used to base edit the ALS gene to confer resistance to herbicide chlorsulfuron as a selection marker, which has no negative effects on plant phenotypes; Cas12a/crRNA was used for editing genes(s) of interest; GFP was used for selecting transgene-free transformants. Using this approach, transgene-free genome-edited plants can be relatively easily generated for citrus in the T0 generation.  Whole genome sequencing further confirmed transgene-free and absence of off-target mutations in the edited plants. We are also using this strategy for genome editing of our target genes. This has been published by Nat. Plants (9, 1591–1597, 2023. https://doi.org/10.1038/s41477-023-01520-y). Among the 8 target genes, we have done transformation again on 5/18/23, 5/25/23, 6/25/23, 7/27/23, 8/17/23, 11/16/23, 12/07/23, 1/18/24, 2/15/2024, 2/22/2024, 3/7/2024, 4/11/2024, 4/25, 5/9, 5/23, 6/6, 6/20, 7/5, 7/19. The transformants done before August 2023 have successufully generated shoots for target genes. Deep sequencing of the edited protoplasts showed a mutation rate of 16-78%. Two targets have near zero mutation rates. We have successfully generated non-transgenic lines for two of the S genes. The plants have been confirmed to be 100% biallelic mutants and have been grafted on US-42 in the greenhouse. A third target gene has generated more than 20 shoots and been micrografted. In addition, we have generated Ray Ruby embryogenic protoplasts which are being tested for efficacy of transformation, editing, and regeneration. Objective 2. Generate cisgenic genome modified Valencia and Hamlin sweet orange plants by knock-in the gene encoding MaSAMP from Microcitrus. We are optimizing the knock-in method using the CRISPR technology. We have made some progress in knock-in methodology using non-transgenic approach. We have conducted multiple transformations useing the developed method for knockin with MaSAMP. We have demonstrated the knock-in works using GFP as an insert. We have conducted knock-in for MaSAMP on 5/18/23, 8/17/23, 11/16/23, 12/07/23, 1/18/24, 2/15/2024, 2/22/2024, 3/7/2024, 4/11/2024, 5/23, 6/20, 7/19 which are under regeneration. We are also testing to improve the insertion using priming editing technology.  2. Please state what work is anticipated for next quarter:More and more regenerated shoots have been generated. We will conduct more micrografting which will be repoted in the greenhouse. We will propogate and conduct CLas inoculation after we have acqauire 6 biological replicates per line for the non-transgenic genome edited plants.  3. Please state budget status (underspend or overspend, and why):On schedule.     

Consolidation of citrus breeding plant material to vacate space for Stage I and Stage II field trials and to exploit tolerant germplasm for gene editing strategies

1. Please state project objectives and what work was done this quarter to address them: Objective 1: Destroy declining plant material with no direct use to the citrus industry to make space for Stage I and Stage II trials (Years 1 and 2).Since project planning and the beginning of the project period, several thousand citrus trees have been pushed and destroyed at CREC, a portion of which is for making space for Stage I and Stage II trials.  Of these several thousand trees, approximately thousands are slated to be pushed and destroyed since the beginning of the last reporting period.  Hundreds of trees to up to 1,000 have been identified as being suitable for conservation, movement with the tree spade (or other means) and sourced for budwood.  Trees were identified by both drone and by visual inspection.  The nursery is aware of the need to collect and propagate uncertified budwood and they have given the go-ahead to transfer budwood immediately.  UFarm charges have conserved and saved all materials needed for data mining for tolerant genes and other variants. Objective 2: Conserve, move, consolidate, and propagate tolerant and resistant citrus trees to a centralized location. (Years 1 and 2).The block where consolidation will take place has been reserved for this purpose.  The nursery will receive the first budwood on Tuesday 8.6.24.  When the buds take, we will move forward with tree movement with tree spade and other means.  We do not have possession of the tree spade.  The tree spade has been repaired and we are waiting for the UF/IFAS payment to process before bringing it to the CREC to begin practicing moving trees of the appropriate size.  2. Please state what work is anticipated for next quarter: We anticipate using the USDA-ARS tree spade to move the first trees and transferring budwood to the nursery for propagation, which will start on this coming Tuesday.  1,000 budeyes from 84 accessions are planning to be transferred to the nursery.  We also anticipate removing thousands of additional citrus trees with no immediate value to the citrus industry to continue to make space for Stage I and II trials as well as breeding populations.    3. Please state budget status (underspend or overspend, and why): We are no longer underspent (29% time spent versus 40% funded expended) according to the UF/IFAS grants tracking system. The budget is being expended at a rate at which all funding will be expended by the end of the project period.   The payment has been made to the contractor, and we are on track for spending.