Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. The rationale is that by constitutive expression of an R protein, the plant innate immunity response will be at a high state of alert and will be able to mount a robust defense against infection by phloem pathogens. Overexpression of R proteins often results in lethality or in severe stunting of growth. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: The transgenic plants containing AtSUC2/snc1 and AtSUC2/ssi4 mutants, as well transgenic control plants are growing in the laboratory of Dr. Orbovic at the UF Citrus Research Facility (Lake Alfred) until they are ready for the next level experiments. Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. By further restricting expression of the R protein to a single cell that is pierced by the insect stylet, we anticipate that a defense can be mounted without a manifestation of a dwarf phenotype. Results: The vast majority of T1 and T2 transgenic Arabidopsis plants expressing snc1 and ssi4 mutant coding sequences under the control of the AtSUC2-940 promoter have wild type phenotypes. Although the AtSUC2 promoter has been reported to be phloem-specific, we have found that it often does not maintain this tissue-specific pattern of expression in transformed Arabidopsis. However, despite the likelihood of expression in tissues other than phloem, only a few transformants showed any negative developmental or growth abnormalities. This lack of a negative phenotype in Arabidopsis provides a basis for optimism for similar results in transformed citrus. Our working hypothesis is that expression of the constitutive R proteins (mutants) in the phloem will active components of the innate immunity response to provide enhanced protection from Liberibacter infection in phloem cells. In order to monitor the activation state on the innate immunity system, we will cross the R protein transformants with transformed Arabidopsis lines containing pathogen-inducible promoters driving GUS reporter genes. We cloned the PR2 (also known as BGL2), and PR5 pathogen-inducible promoters in front of the GUSplus gene in pCAMBIA 2301. They were sequenced, transformed via electroporation into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis (strain GV3101) through the floral dip protocol in order to generate stable transgenic lines. We currently await the T-1 seeds from these transformations. In parallel, we acquired BGL2-GUS (in pBI101 vector; from Dr. Xinnian Dong from the Duke University) stable transgenic line to use as an alternative donor. The introduction of our R protein constructs into reporter lines by crosspollination will be faster and more efficient than transformation by agrobacterium. Being able to monitor constitutive activation of the innate immunity system by GUS will provide a test of the hypothesis that our constructs will activate pathogen-inducible promoters and will allow us to select lines that have strict phloem-specific expression for further study.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or anti-psyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We now are making good progress: ‘ We continue to screen potential genes for HLB control and are finding peptides that reduce disease symptoms and allow continued growth of infected trees. We have about 30 new peptides that are now being screened. ‘ We have greatly improved our efficiency of screening . ‘ We have greatly improved the CTV vector. ‘ We have modified the vector to allow addition of a second anti-HLB gene. ‘ We have obtained permission and established a field test to determine whether the CTV vector and antimicrobial peptides can protect trees under field conditions. ‘ We continue to supply infected and healthy psyllids to the research community. ‘ We are testing numerous genes against greening or the psyllid for other labs.
Based on lesion number, phenotype, bacterial growth curve and cellular reaction, two cybrids of canker susceptible Red grapefruit (RG) with field tolerant Valencia orange (VO) as the cytoplasm donor,CY#3 and CY#10 have been characterized as tolerant of Xanthomonas citri subsp. citri (Xcc). The influence of mitochondrial exchange in the cybrids on the expression of nuclear genes was evaluated in an assay of the two cybrids and the parental lines (VO and RG) in the greenhouse. After inoculation of young leaves with Xcc at 108cfu/ml, tissue was collected at 4 and 24 hr post inoculation, mRNA was extracted, and the level of gene expression was determined by RT-qPCR. Several primers sets were tested to determine the differential expression among the parents (RG and VO) and the two cybrids. For the 15 nuclear genes tested, there was differential expression of several genes compared with parents. Among the changes detected were higher levels of expression of pathogenesis related proteins, enzymes related to the salicylic acid and jasmonate pathways, detoxifying enzymes and genes involved in oxidative stress response. Nuclear genes predicted to be involved in mitochondrial retrograde signaling were also affected. Several mitochondria and plastid related genes were assayed and the level of expression of genes in the cybrids differed from the parental lines including: ribulose bisphosphate carboxylase, aconitase-iron-regulated protein, ferritin-3, chloroplast precursor and ascorbate peroxidase. These results suggest that nuclear gene expression is modulated with respect to the interaction with the heterologous organelles in the cybrids. At present further characterization of the mt genome from kumquat, VO, RGF, Rough Lemon (RL), and previously obtained cybrids (RL+VO) and (RG+VO) is underway. Eleven citrus mitochondrial (mt) genes and introns were amplified with specific primers. The PCR products have been sent for sequencing. The sequence data obtained will be used for the design of specific TaqMan probes and used as gene markers. This method will allow us to monitor the mt genes transferred in the cybridization process. Production of new cybrids lines with susceptible Red grapefruit using a callus line of Meiwa kumquat as the cytoplasmic donor will be evaluated for inheritance of HR resistance using the same approaches as with the system described above.
Over the past quarter, we have made progress in the following areas: 1. We have continued testing TAL effector and promoter constructs in a Nicotiana benthamiana system to examine effector specificity for induction. 2. We have isolated TAL effectors from new citrus canker strains. Newly isolated variants will be sequenced, compared to known citrus TAL effectors, and tested for their ability to trigger our engineered resistance approach. 3. We have been continuing the molecular characterization of transgenic lines and testing of response to bacterial infiltration. 4. We have undertaken transformation of additional citrus varieties important to the Florida citrus industry, specifically sweet orange and red grapefruit. We continue to test additional promoter constructs in Duncan grapefruit. 5. We have begun planning for field trials of transgenic material.
The content of this quarterly report is similar to that of the annual report submitted in June, 2011. This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, 20 transgenic Duncan grapefruit plants have been generated, and these plants have been growing in greenhouse for 6 months and ready for canker resistance test. Since canker resistance test is straightforward, we will test all 20 transgenic plants. However, for greening resistance test, the transgenic plants will need to be propagated. We will first extract total RNA from each of the 20 plants and determine the expression levels of AtMKK7. We will choose 4 to 6 lines that highly express the transgene AtMKK7 for propagation. Six plants from each line will be used for greening resistance test. For objective 2, we have been using hypocotyls as explants for gamma irradiation mutagenesis. A total of about 75,000 hypocotyl cuttings have been irradiated in three batches with a irradiation dosage of 40 Gy. Shoots formed on the irradiated cuttings were transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Several shoots are currently growing on the selective medium. To increase the screening efficiency, we have also been using seeds for this objective. A large quantity of Ray Ruby grapefruit was obtained in the Spring of 2011 from the Indian River area. Seeds were obtained from the fruits and cleaned with Pectinase. Seeds were then treated with 8-hydroxyquinoline as a preservative to allow long-term storage of seeds at 4’C. Moisture content of the seeds was determined for future reference. Two quarts of seeds have been treated with gamma irradiation. One quart was irradiated at 50 Gy, the other at 100 Gy. Both untreated and irradiated seeds were plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots have been formed on the seeds and will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Based on the result of this batch of seeds, we will treat other seeds with either the same condition or a modified dosage. Shoots formed on these gamma irradiated seeds will be screened on the selective medium. Those shoots that are resistant to sodium iodoacetate will be grafted onto rootstocks to generate plants for resistance test.
The objectives of this project include: (1) Characterization of the transgenic citrus plants for resistance to canker and greening; (2) Examination of changes in host gene expression in the NPR1 overexpression lines in response to canker or greening inoculations; (3) Examination of changes of hormones in the NPR1 overexpression lines in response to canker or greening inoculations; (4) Overexpression of AtNPR1 and CtNPR1 in citrus by using a phloem-specific promoter. We have transformed the cloned CtNPR1 (also named CtNH1) into the susceptible citrus cultivar ‘Duncan’ grapefruit. After survey on transgene expression, we now focus on the three lines, CtNH1-1, CtNH1-3, and CtNH1-5, which showed normal growth phenotypes, but high levels of CtNH1 transcripts. The three lines were inoculated with Xac306. They all developed significantly less severe canker symptoms as compared with the ‘Duncan’ grapefruit plants. To confirm resistance, we carried out growth curve analysis. Consistent with the lesion development data, as early as 7 days after inoculation (DAI), there is a differential Xac population in the infiltrated leaves between CtNH1-1 and ‘Duncan’ grapefruit. At 19 DAI, the level of Xac in CtNH1-1 plants is 104 fold lower than that in ‘Duncan’ grapefruit. These results indicate that overexpression of CtNH1 results in a high level of resistance to citrus canker. We are planning to propagate the CtNH1 line by grafting. We are in the process of inoculating the CtNH1 lines with Candidatus Liberibacter asiaticus (Las). We have completed the SUC2::CtNH1 construct, in which CtNH1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene. The construct were transformed into ‘Duncan’ grapefruit. To date, five transgenic lines have been obtained.
We have completed Carrizo genomic DNA isolation and purification. The source plant material was obtained from the University of California Riverside Citrus Variety Collection. Libraries for 454 sequencing (for random and paired-end reads) are currently being assembled. The 454 GS FLX sequencer at the Albany location was recently updated by Roche personnel to allow longer reads (averaging >700 bp, approximately doubling read length), with final testing and training to be completed by July 20, 2011. These longer reads should result in significantly improved genome coverage relative to the 10x coverage indicated in the original proposal. The extended reads will also facilitate both contig assembly and identification/separation of the sweet orange and Poncirus genomes within Carrizo. Sequence acquisition is expected to be initiated within the next three weeks, with completion within three months. While the 454 modifications have delayed the sequencing, the resulting improvement in read length can be expected to expedite the assembly and physical map alignment phases of the project.
Huanglongbing (HLB) is a serious and devastating disease of citrus caused by Candidatus Liberibacter spp. and vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The disease has the potential to greatly limit the production of citrus in Florida and other citrus growing regions worldwide. Current chemical and classical biological control of ACP and HLB is inadequate, but identifying and incorporating traits from Citrus spp. and Citrus relatives that confer resistance to ACP is a potential strategy to manage the disease. In a study by USDA-ARS, 87 genotypes primarily in the Rutaceae (orange subfamily Aurantioideae), were assessed in the field in South Florida for resistance to natural populations of ACP. The majority of genotypes hosted all three life stages of ACP, however there were differences among genotypes in the mean ranks for eggs (F = 3.13, df = 86, P < 0.001), nymphs (F = 9.01, df = 86, P < 0.001), and adults (F = 4.21, df = 86, P < 0.001). Very low levels of ACP were found on two genotypes of Poncirus trifoliata, 'Simmon's trifoliate' and 'little-leaf'. Poncirus trifoliata, the trifoliate orange, readily forms hybrids with Citrus spp. and is commonly incorporated into rootstock varieties, so it may be useful in breeding programs as a potential source of genes that confer resistance to insects. The field experiment was followed by no-choice tests in which female ACP had the opportunity to lay eggs for six days on 46 genotypes of P. trifoliata, 35 genotypes of xCitroncirus sp. (hybrids of P. trifoliata and another parent species), three genotypes from the Citrus genera that were not represented in the field, and a control (Citrus macrophylla) to determine whether any genotypes were resistant to ACP. All genotypes of Poncirus trifoliata, except for one, and 14 of the genotypes of xCitroncirus sp. were resistant to oviposition by ACP. Currently we are testing whether development, weight, and lifespan of ACP nymphs and adults are negatively influenced by the resistant genotypes. Studies also have been initiated to compare plant volatiles of the resistant genotypes to those of susceptible genotypes to ascertain whether that is the mechanism conferring resistance of plants. Collaborators at the Fujian Academy of Agricultural Sciences in Fuzhou, China, initiated several experiments on resistance to ACP within the Rutaceae. Seventy-one genotypes of grafted pants were evaluated in a free-choice experiment conducted in a screen house. The majority of genotypes hosted all three life stages of ACP, however, eight genotypes had low levels of all life stages of ACP: Citrus sinensis (two cultivars), C. reticulata, C. unshiu (three cultivars), and C. mitis. Two additional free-choice experiments in a screen house and the field with seedling and grafted plants, respectively, with 102 genotypes yielded no differences in abundance of ACP. Six morphological structures of the leaf were also measured for five genotypes of Citrus that varied in abundance of ACP to determine whether morphology was correlated to infestation levels of ACP. Morphology was significantly different among the genotypes, but there was no correlation with abundance of ACP. Current work is investigating whether the eight genotypes with low abundance of ACP are resistant in no-choice tests.
This is the second year of a currently funded multi-investigator, multi-institution project, with the second year end time of 6/30/2011. A total of $224,000 are the current funds allocated to the second year of the project. We are requesting a 6 month no-cost-extension on this grant. There are two major reasons for this. First, there was a delay in dispersing the funds. It took an unusually long time for the funds to flow from agency to UF. Then, there was a further delay at UF establishing subaccounts at UF (CREC) and particularly with USDA, Ft. Pierce. It was October before all of the subaccounts were established. Finally,there is $24,000 in the second year budget for Dr. Machado in Brazil. He was never able to submit the necessary paperwork to receive these funds because of government restrictions. He has a student coming to the Moore lab in July for training and Dr. Machado has asked that the funds be used for her. The second reason for requesting the NCE is that there were a number of personnel changes this year. Because of this, we are requesting permission to adjust what funding is in specific categories and that we can adjust some funding between PIs. Randy Neidz (USDA) has had a post-doc working on this research until recently, and USDA post-docs are costly, so he has been funding much of the supplies used on the project from another source. He has hired a non-PhD person with tissue culture experience to continue the work on the project, but at a lower salary ($25,000 for the NCE). The rest of his funds would be primarily in materials. Jude Grosser (UF) has spent almost all of his current funding on this project. I am requesting that we be permitted to transfer $4,653 from Fred Gmitter’s subaccount to Jude and that I transfer $12,000 from the main account (originally alloted to Dr. Machado) to Dr. Grosser’s subaccount. This will give him adequate money to pay a post-doc and purchase supplies. I will use the other $12,000 originally allocated to Dr. Machado for fees and stipend for his student. I hope this is clear. If I can provide you with any further information or cost breakdown, please let me know.
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, where HLB and ACP are widespread. The first trees have been in place for more than fourteen months. Dr. Jude Grosser of UF has provided 300 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has just planted an additional 89 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place. Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been propagated for a replicated trial in collaboration with Fred Gmitter of UF and are growing well in the greenhouse. These will be planted in July 2011, and monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. An experimental attract/kill product, to disrupt citrus leaf miner (CLM) without disrupting ACP, was not effective last year. Our experience suggests CLM may significantly compromise tree growth where insecticides are avoided to permit ready transfer of Las by psyllids. CLM damage also compromises ability to view HLB symptoms. Several applications of Admire are being used to encourage an undamaged flush on transgenic trees. We are still learning how to grow trees for best assessment of HLB-resistance. In June the test site was visited by APHIS Biotechnology Regulatory Services, and we received notice that the site is in compliance with all relevant regulations.
Based on our previous results analyzing the 24 transgenic ‘Carrizo’ citrange AtNPR1 transgenic lines we selected for propagation a few more: 754, 761, 763, 769, 771, 779, 853, 858, 862 and 880 that showed an enhanced defense response compared to wild type plants. Simultaneously, we have inoculated grapefruit scions grafted on transgenic ‘Carrizo’ rootstock of lines 854, 857, 859, 884 and wild type with Candidatus Liberibacter asiaticus. The plants have been acclimating in the containment facility at the University of Florida. However, we had a mite infection that has prevented us from analyzing the response to HLB. We have prunned and treated the plants with pesticide to control the infection. As soon as the plants are rid of the mites and growing again we will start the analysys as stated in our objectives.
We have transferred the initial indexed mature material obtained from Dr. Peggy Sieburth’s lab to the growth room. As we mentioned in previous reports the material was not in excellent condition in vitro because of excess storage time and only 20 % of the material prepared during the last quarter of 2010 survived the in vitro conditions. Usually these in vitro grafted plants are grafted in rootstocks on greenhouse conditions, but since the rootstocks were not ready because they were growing in lab conditions, they were transferred to soil directly. The rootstocks produced in lab conditions were small, and they were not actively growing and they are still recovering from the stress suffered in the laboratory and only a small portion of them will be used for grafting. We started preparing new source of material from the same 3 orange types: Valencia SPB 1-14-10, Hamlin 1-4-1 and Pineapple F-60-3. The mature budsticks are coming from indexed plants from the Department of Agriculture. New rootstocks from Swingle citrumelo and C. macrophylla were planted once the growth room was ready and they are currently growing. We will need a few more months to use this material. The Growth Room was finished and we passed the local inspection, however we operated for many weeks without being able to control the growth room due to lack of computer access to the program, the greenhouse technician does not have still access to the program. There were also several inconsistencies among the different companies involved in the construction that were solved during this post construction period to be able to operate the growth room. We are still dealing with some changes in the humidifier in the small room and finalizing the drainage of water from the growth room to the outside field. Fortunately the facility is under warranty for one year and the contractors are addressing most of the problems. We addressed the need of an emergency stand by generator for the facility as well as some improvements that were not in the construction plan, that were requested initially. These improvements are still necessary to make production in the growth room reliable. We experienced already a few electricity failures in the facility due to lightening and thunderstorms that affect the electrical service, and it took several hours to fix the situation on site and put the growth room back up and running again. The temperature increases considerably during the time of the electrical outage and there was no “clean water available” since the UV light system will not work under these conditions. It will compromise the whole process if we have more than 2 days with no water and electricity.
In previous informs we have already mentioned that mature tissues from the three sweet orange genotypes we pretended to transform through this project, namely Hamlin, Valencia and Pineapple, are readily transformable, as demonstrated by positive results from molecular analyses of marker transgenes incorporated into plants established in the greenhouse from the three sweet orange types. The procedures have been transferred in detail to our lab at the CREC. We are not working any more with Hamlin at our IVIA’s lab, but Valencia and Pineapple are being routinely transformed with several transgenes of interest not related to this project, meaning that, at least for these two sweet orange types, we can efficiently insert other transgenes able to modify plant phenotype and likely provide new improvement traits. For Carrizo citrange, we first established a procedure for transformation of mature tissues and then used the system to incorporate a hairpin construct aimed to induce RNA interference to silence and endogenous GA20-oxidase gene and them reducing gibberellin biosynthesis into actively growing tissues. These transgenic plants would be semidwarf and could be used as rootstocks to provide semi-dwarfing characteristics to non-transgenic scions grafted into them. A field trial assay initiated 5 years ago in Moncada (Valencia, Spain) indicates that GA20-oxidase antisense lines provide semi-dwarfing architecture (a one-third reduction in height) to mandarin scions. In theory, RNAi-inducing constructs should work better than antisense ones. We already have PCR-positive shoots for the hairpin construct in the growth room. We have initiated experiments to attempt transformation of mature tissues from Swingle citrumelo and Star Ruby grapefruit. We are also preparing new source material of Ray Ruby grapefruit to initiate transformation experiments by the end of the year. These objectives were not contemplated in the original project. For improving citrus tree management, we proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. We have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange over-expressing either FT or AP1 flowering-time genes already established in the greenhouse. We continue characterizing them in detail. In Florida, construction of the growth room has been finalized, but several important issues need to be set up to make production of healthy source plant material reliable. We are helping the manager and her team to establish a calendar for production of rootstock, clean scions, mother plants, propagations, etc. to get maximum occupation of the available space in the growth room and being able to make the maximum number of mature transformation experiments per year. We are also helping to establish substrate, fertirrigation and phytosanitary treatments.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, 20 transgenic Duncan grapefruit plants have been generated, and these plants have been growing in greenhouse for 6 months and ready for canker resistance test. Since canker resistance test is straightforward, we will test all 20 transgenic plants. However, for greening resistance test, the transgenic plants will need to be propagated. We will first extract total RNA from each of the 20 plants and determine the expression levels of AtMKK7. We will choose 4 to 6 lines that highly express the transgene AtMKK7 for propagation. Six plants from each line will be used for greening resistance test. For objective 2, we have been using hypocotyls as explants for gamma irradiation mutagenesis. A total of about 75,000 hypocotyl cuttings have been irradiated in three batches with a irradiation dosage of 40 Gy. Shoots formed on the irradiated cuttings were transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Several shoots are currently growing on the selective medium. To increase the screening efficiency, we have also been using seeds for this objective. A large quantity of Ray Ruby grapefruit was obtained in the Spring of 2011 from the Indian River area. Seeds were obtained from the fruits and cleaned with Pectinase. Seeds were then treated with 8-hydroxyquinoline as a preservative to allow long-term storage of seeds at 4’C. Moisture content of the seeds was determined for future reference. Two quarts of seeds have been treated with gamma irradiation. One quart was irradiated at 50 Gy, the other at 100 Gy. Both untreated and irradiated seeds were plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots have been formed on the seeds and will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Based on the result of this batch of seeds, we will treat other seeds with either the same condition or a modified dosage. Shoots formed on these gamma irradiated seeds will be screened on the selective medium. Those shoots that are resistant to sodium iodoacetate will be grafted onto rootstocks to generate plants for resistance test.
The Core Citrus Transformation Facility (CCTF) continues to serve the community of researchers exploring ways to improve Citrus plants and make them tolerant/resistant to diseases. CCTF does its service by producing transgenic material. Within the last quarter, the CCTF facility produced the following transgenic citrus plants (transgene in parenthesis): three Mexican lime plants (pHK vector); nine Duncan plants (ELP3 gene); one Duncan plant (MKK7 gene); four Duncan plants (p7 gene); nine Duncan plants (p10 gene); one Mexican lime and two Hamlin plants (p33 gene); six Duncan plants (SUC-CitNPR1 gene); two Duncan plants (pWG19-5 vector); two Duncan plants (pWG20-7 vector); 11 Duncan plants (pWG21-1 vector); seven Duncan plants (pWG22-1 vector); two Duncan plants (pWG24-13 vector); and three Duncan plants (pWG25-13 vector). There are additional 30 plants in soil that need to be tested for the presence of the transgene of interest. Within last three months, the CCTF facility also sustained a loss of 30 soil-adapted transgenic plants due to unknown contamination coming from the rootstock plants (probably Phytophthora). Steps have been taken to prevent this from happening again in the future, including improved greenhouse sanitation and use of more resistant rootstocks in micrografting.
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