The interest for transgenic Citrus material remains strong which keeps Core Citrus Transformation Facility (CCTF) busy at all times. This interest is exemplified by the fact that eight more orders were received in the last three months. Due to the availability of appropriate starting material needed for these orders, the work on some of them started almost immediately. CCTF also continued to service the old orders. Within this period, CCTF performed tasks associated with the production of transgenic plants within the lab and also took care of plants from different orders growing in the greenhouse. In the last three months more than 80 plants were produced and are result of work on 12 different orders (NPR1, NPR1-G, ELP3-G, ELP4-G, PR-2, MG113, MG36, X7-2, HGJ31, HGJ32, HGJ33, and HGJ34). These plants belonged to only two cultivars: Carrizo and Duncan. Most recent group of orders had to do with the ‘proof-of-concept’ experiments and the T-DNA in binary vectors contained GFP gene which reflected positively on the productivity of CCTF in this quarter. Another factor affecting productivity was that involvement of EM lab staff resulted in definition of methodology for detection of fluorescence in transgenic plants that belong to two orders. CCTF temporarily lost contact with the grower who supplied Duncan grapefruit as a source of seeds and as a result planting for new orders is not going as planned. Intense effort is under way to find another source of Duncan fruit. Initial contact was established with the employee at the Southern Gardens farm to get supply of Valencia seeds. Shipment of Carrizo seeds was received from nursery in California. Five more plants were produced for the CRDF order bringing total to more than 50. In the early phases of this project, there were regular bi-weekly meetings between Jackie Burns, and managers of both transformation facilities to discuss the on-going activities. In that period, rate of production of transgenic plants was discussed as well as choice of cultivars. There was a general understanding that there would be more transgenic rootstock cultivars produced by juvenile method than scion cultivars produced from mature tissue. Within the first 7-8 months of the project that is exactly what happened. Although initial projection was that first group of most promising transgenic rootstock plants would be propagated already, they were not. The first reason is that plants did not grow well this winter. The second reason is that according to previous agreement only a highest expressors of NPR1 should be multiplied but those plants have not been selected yet. Considering early recognition of higher rate of production of transgenic plants in CCTF, it was presumed that the work on search for high NPR1 expressors (both rootstocks and scions) would be done by mature tissue lab. This type of work involves isolation of RNA and the use of RT-PCR machine, and the members of the mature lab may be better skilled for this type of work than employees in the CCTF. Due to personnel structure in CCTF, there are some limitations on what can be accomplished there at the moment. Manager of mature tissue lab was told that her employees could use RT-PCR machine that is available in CCTF at any time. Consultations are continuing on where this work will be done. Recently, information was requested from the CCTF manager about what space would be needed (in square feet) for propagation of rootstock plants with NPR1 gene. Once selection of plants that are high NPR1 expressors is complete such information will be available.
Huanglongbing (HLB) is the most serious threat to the U.S. citrus industry. Although no known HLB-resistant citrus plants have been identified, some citrus relatives are substantially more tolerant, such as Citrus jambhiri (rough lemon) and Poncirus trifoliata. Genome analysis will improve our understanding of the HLB tolerance mechanisms. Genomic DNA from Citrus jambhiri was used to generate more than 235 M reads (2 X 100) representing about 34-fold physical coverage of the rough lemon genome. A reference-guided method was used to assemble the rough lemon genome. Compared with the haploid Clementine reference genome sequence, there were more than 2.5 million single-base differences and about 0.7 million insertion/deletion polymorphisms. The effect of these SNPs were annotated based on their position. Most of the identified SNPs were located outside of the known gene. RNA-sequencing data are being used for gene annotation. The genomes of Poncirus trifoliata and Citrus clementina, along with rough lemon, are being utilized to design the Agilent’s SureSelect probes, which will be used for target enrichment. Leaf samples of a diverse group of citrus accessions were collected, including mandarin, sweet orange, pumelo, rough lemon, and Poncirus. The methods for high throughput nuclear DNA extraction method is being tested to extract high-quality DNA for target enrichment and next generation sequencing. Poncirus trifoliata genomic sequence reads that match the nucleotide-site leucine-rich repeat (NBS-LRR) class disease resistance genes of Citrus clementina were obtained. These Poncirus sequence reads will be assembled to identify NBS-LRR genes those are homologous to C.clementina and C.sinensis. Aligned NBS-LRR gene sequences of Poncirus will be used as resistant resource pool to identify sequence variations associated with HLB disease.
We have concluded the activities of objective 1 of our proposal which have focused around finding a native citrus protein replacement for cecropin B the C-terminal component of the chimeric antimicrobial (CAP) protein. We had identified CsHAT52 using one set of bioinformatics tools and confirmed antimicrobial activity with a portion of this protein that we designated CsHAT22. Bioassay of CsHAT22 revealed a minimum inhibitory concentration (MIC) of 50 uM with Xanthomonas, 100 uM with Xylella and 300 uM with Liberibacteria crescens (Lc). Using two additional bioinformatics programs, PAGAL and SCAPEL and have successfully identified and tested 2 additional proteins, CsPPC20 and CsCHITI25 that were compared to CB and the N-terminal 21 amino acids of CB designated CBNT-21. Among the test strains used Xanthomonas was most susceptible to the peptides with CB and CBNT21 showing and MIC values 25 uM and the MIC values for CsPPC20 and CsCHITI25 were 50uM and 100uM respectively. Both Xylella and the BT-1 strain of Lc gave MIC values of 200 uM for CBNT21 against both Xylella and Lc BT-1. CsPPC20 was more active than BNT21 against Xylella giving an MIC value of150 uM and as active against Lc BT-1 giving an MIC value of 200uM. CsCHITI25 was as active as CsPPC20 against Xylella but not as active against Lc. Based on these results we have included CsPPC20 as an additional construct for testing in planta and excluded CHITI25. CTV vectors for expressing CsP14a, CsP14a-CB and CsP14a-CsHAT52 have been constructed and evaluation and testing of the efficacy of these vectors is underway. The construction of a CTV vector to express CsP14a-CsPPC20 is underway. Binary vectors for Agrobacterium-mediated transformation of CsP14a, CsP14a-CB, CsP14a-CsHAT52 and CsP14a-CsPPC20 have been completed and the transformation process of tobacco and Charrizo tissues is underway for the isolation of transgenic plants. This is being done at the plant transformation facility at UCDavis. We obtained transgenic Carrizo citrus transformed with NE-CB as a positive control. These plants are in the CRF and last week they were exposed to infected psyllids. Once transgenic Carrizo are obtained with the 4 vectors they will be transferred to the CRF for testing with infected psyllids for resistance against HLB.
Huanglongbing (HLB) is the most serious threat to the U.S. citrus industry. Although no known HLB-resistant citrus plants have been identified, some citrus relatives are substantially more tolerant, such as Citrus jambhiri (rough lemon) and Poncirus trifoliata. Genome analysis will improve our understanding of the HLB tolerance mechanisms. Genomic DNA from Citrus jambhiri was used to generate more than 235 M reads (2 X 100) representing about 34-fold physical coverage of the rough lemon genome. A reference-guided method was used to assemble the rough lemon genome. Compared with the haploid Clementine reference genome sequence, there were more than 2.5 million single-base differences and about 0.7 million insertion/deletion polymorphisms. RNA-sequencing data are being used for gene annotation. The genomes of Poncirus trifoliata and Citrus clementina, along with rough lemon, are being utilized to design the Agilent’s SureSelect probes, which will be used for target enrichment.
Improving Consumer Acceptance: 1. In efforts to reduce juvenility in citrus, transgenic Carrizo citrange have been successfully produced expressing the clementine CFT3 gene. We have very small micrografted plants flowering in the greenhouse and these plants have been evaluated by PCR to confirm presence of the CFT3 gene. A few micrografted trees flowered immediately. 2. Following the successful demonstration of the inducible cre-lox gene system, the plant transformation vector has been modified to contain our NPR1 gene and Agrobacterium mediated citrus transformation is underway to incorporate this gene. 3. Transgenic plants containing our stacked transgenes are being clonally propagated for disease resistance evaluation and the first trees will be challenged for HLB resistance in spring 2015. 4. Plants in our Indoor RES structure have not flowered this year. It is possible greenhouse temperature may have played a role in the flowering process. We will attempt to keep the greenhouse unheated this fall in hopes of initiating flowering in spring 2015 Additional Resistance Gene candidates: 1. Transgenic plants containing antimicrobial gene LIMA-B were propagated for field challenge. 2. OLL-8 sweet orange and W. Murcott were transformed with the CtNH1 gene (NPR1-like), using our protoplast/GFP transformation system. Small colonies and embryos were regenerated from OLL-8 with GFP expression.
St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 6.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). Field Day was held, approximately 168 people attended. Data was collected on % of trees removed per rootstock due to poor performance. Several diploid and tetraploid rootstocks had few trees removed after 6.5 years (0-10%). CREC scouts determined that the trial is now 92% infected, so it is now a contest to see which rootstocks can grow trees through the infection and remain productive. Several attendees commented that the trees in general looked better than last year, and that there was more fruit than last year. Greenhouse Experiments – Nutritional study: highly symptomatic trees on various rootstocks were treated with the 3x overdose of TigerSul manganese and polymer-coated sodium borate (Florikan); most trees are putting out normal healthy flush and continue to show recovery. The nutrient overdose experiment was broken down. Data is now being collected on the following parameters: New Flush (Y or N),HLB symptoms (0-7),Stem dia. (mm),Tree Height (cm),# of Leaves,PCR ‘ roots,PCR ‘ leaves, nutritional analysis ‘leaves,nutritional analysis -roots,starch content ‘ leaves,Spad Avg.,Scion Leaves Fr. wt. g,Rootstock Leaves Fr. Wt. g,Scion Leaves Surface Area,Rootstock Leaves Surface Area,Total Leaf Surface Area,Root FWt. g,Stems FWt. g,Soil pH,Young Scion Leaves Dry Wt.,Mature Scion Leaves Dry Wt. Rootstock Leaves Dry Wt.,Total Leaf Dry Wt.,Stems Dry Wt.,Feeder Roots Dry Wt.,Tap and Scaffold Root Dry wt.,and Total Root Dry Wt. Nutritional samples were sent to Waters lab for analysis. PCR is being run by Tripti Vashisth. Clear differences in growth and tree health were observed with the 3xTigerSul manganese and the 3xFlorikan Sodium Borate treatments over the controls and other treatments.
The transgenic plants to be developed for this project are now growing in two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped from the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL, to Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, in early October, 2014. An additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped to Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. The plants at both locations are growing well. In summary, a total of 15 independent transgenic lines now exist for the FLT-antiNodT fusion protein expression construct. These plants are now growing well and cuttings are being taken and rooted to produce multiple vegetatively-propagated plants for each line. This is essential to run multiple tests for the gene expression patterns and HLB resistance levels of each line.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, as proof of concept to determine the root specific nature of the promoters (RB7, C1867 or SLREO), we have incorporated the promoter-gus sequences into N. benthamiana and Carrizo citrange and several plantlets have been regenerated. Testing of these plants to confirm the root specific activity of our promoters will be performed as they become available. In addition, we have initiated experiments to incorporate the plant transformation vectors containing the GNA, APA and ASAL genes driven by either the root specific RB7 promoter or the citrus derived C1867 promoter into Carrizo citrange. Stacked constructs, each containing the GNA, APA or ASAL genes with the CpTI gene driven by the SLREO promoter have been produced and are also being incorporated into Carrizo citrange.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we have repeated NAD+ treatment experiment. Again, both soil drench and foliar spraying of NAD+ have been conducted. The plant defense activator Actogard, which is highly effective against citrus canker was included in the experiment as a control. We found that while foliar spraying did not provide significant protection against citrus canker, soil drench induced strong resistance against the pathogen. Interestingly, we observed strong systemic protection against canker by NAD+ one month after the treatment in upper new flushes. We are planning to repeat the experiment to confirm the systemic effects. Meanwhile, we are still trying to find the best approach for NAD+ application. NAD+ analogs are under test for identifying potential chemicals to control citrus canker. For objective 2, 30 transgenic lines expressing ELP3 and 22 lines expressing ELP4 have been generated. The transgenic lines have been molecularly characterized to confirm the presence and expression of the transgenes. The transgenic plants are growing in greenhouse and will be tested for canker resistance. Citrus homologs of ELP3 and ELP4 have been cloned and sequenced. We are cloning the two genes into T-DNA vector and will be transformed into the Arabidopsis elp3 and elp4 mutants, respectively, to confirm their functionality.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we have repeated NAD+ treatment experiment. Again, both soil drench and foliar spraying of NAD+ have been conducted. The plant defense activator Actogard, which is highly effective against citrus canker was included in the experiment as a control. We found that while foliar spraying did not provide significant protection against citrus canker, soil drench induced strong resistance against the pathogen. Interestingly, we observed strong systemic protection against canker by NAD+ one month after the treatment in upper new flushes. We are planning to repeat the experiment to confirm the systemic effects. Meanwhile, we are still trying to find the best approach for NAD+ application. NAD+ analogs are under test for identifying potential chemicals to control citrus canker. For objective 2, 30 transgenic lines expressing ELP3 and 22 lines expressing ELP4 have been generated. The transgenic lines have been molecularly characterized to confirm the presence and expression of the transgenes. The transgenic plants are growing in greenhouse and will be tested for canker resistance. Citrus homologs of ELP3 and ELP4 have been cloned and sequenced. We are cloning the two genes into T-DNA vector and will be transformed into the Arabidopsis elp3 and elp4 mutants, respectively, to confirm their functionality.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We have found that three different lines appear to be giving strong tolerance against HLB. We are propagating the plants for more extensive analysis.
The objectives of this project are: 1) to generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology; 2) to identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications; and 3) to create transgenic citrus cultivars with new constructs containing the resistant genes. A total of 25 samples for RNA-Seq, including resistant/tolerant vs. susceptible plants were sequenced and analyzed. We mapped the RNA-Seq data to a reference genome, C. clementina using the bioinformatics program STAR. About 85% of the raw reads could be uniquely mapped. The transfrags of each library were assembled with cufflinks and merged with cuffmerg. 24,275 genes of the originally predicted genes had been found to be expressed and a total of 10,539 novel transfrags were identified with cufflinks, which were missing from the original reference genome annotation. Some of the NBS genes were found to be expressed. For C. clementine and C. sinensis, there were 118,381 and 214,858 mRNAs or ESTs deposited in GenBank and 93 out of 607 and 221 out of 484 NBS related genes match one or more ESTs respectively. The number of ESTs varied from 1 to 25. The expression abundance of each gene was measured by FPKM. The distribution curves of density of FPKM of 6 samples are very similar, indicating that the gene expression is similar and the quality of sequencing is high. We also performed the principal component (PC) analysis study on the expressions of six samples. The results showed that the gene expressions were significantly different in resistant vs. susceptible citrus. A total of 686 differentially expressed (DE) genes between two groups using FDR threshold of 0.1 were identified. Among them, 247 genes were up-regulated and 439 were down-regulated in tolerant citrus trees. We performed Gene Ontology (GO) enrichment analysis of DE genes. Genes associated with beta-amyrin synthase, cycloartenol synthase and Camelliol C synthase were significantly up-regulated in the HLB tolerant citrus trees while terpene synthase genes (CiClev10014707, Ciclev10017785) were down-regulated in the tolerant citrus trees. Some PR-protein genes were significantly up-regulated in the resistant citrus trees, including several TIR-NBS-LRR genes. Many cell wall degradation-related genes, such as cellulose synthase/transferase, cellulase and expansins were up-regulated in the susceptible citrus trees. Some glucan hydrolase genes were also up-regulated in the resistant citrus trees. These genes may play important roles in symptom development. The DE genes were also enriched in two classes of RLKs, LRR-RLKs and DUF26-RLKs. We have experimentally verified the expressions of 14 up-regulated genes and 20 down-regulated genes on three HLB-tolerant ‘Jackson’ and three HLB-susceptible ‘Marsh’ trees using real time PCR. 11 of 14 up-regulated genes and 18 of 20 down-regulated genes were validated. We further predicted a protein-protein interaction (PPI) network of citrus using the PPIs of Arabidopsis. There are 1259 proteins and 2298 interactions in our Citrus PPI network. Among 1259 proteins, 42 proteins are differentially expressed between the HLB resistance and susceptible citrus. An interested PPI sub-network includes 14 citrus NPR1-likes proteins and three TGA proteins. There were four NPR1-like genes were significantly up-regulated in HLB resistant citrus trees and one NPR1-like gene up-regulated in HLB sensitive citrus trees. There is also one TGA gene up-regulated in HLB resistant citrus trees. Another interested sub-network includes the RPS2 protein. There were two LRR kinase receptors significantly up-regulated in HLB resistant citrus trees and four LRR kinase receptors significantly up-regulated in sensitive citrus trees. Other interested interactions are between two lipoxygenase genes, LOX2 and EIF4E, a translation initiation factor. Interestingly, the two LOX2 genes were down-regulated in the HLB resistant citrus disease trees.
The Core Citrus Transformation Facility (CCTF) continued to provide service for production of transgenic citrus plants. The work load that included: co-incubation experiments, explants incubation, shoot harvesting and inspection, PCR testing, micro-grafting, and care of plants in the greenhouse was kept at maximum level allowed by the number of holidays within the last quarter of the year. The work done by the CCTF from October through December was concentrated on older orders as five new orders were placed to CCTF from two different clients in late December. Production of transgenic rootstock plants ordered by the CRDF continued at slower pace. One out of 29 plants that were previously growing in the greenhouse died. Nineteen more ‘pot-adapted’ plants were moved from the laboratory to the greenhouse bringing the total number up to 47. The growth of first plants moved to the greenhouse was not as vigorous as expected and they were not cut into explants for propagation. That may be done in the middle of February. In the period covered by this report, CCTF produced plants for the following orders: pNPR1-17 plants, pNPR1-G-five plants, pELP3-G-12 plants, pELP4-G-five plants, pMG36-eight plants, pX7-2- five plants, pHGJ27- one plant. Three orders that are being serviced at this time require detection of transgenes in transgenic plants by microscopy. The methodology for this detection is still being worked out with the EM lab personnel. If this was solved at earlier date, the output of the facility would have been higher. Plants produced in this quarter were mostly Duncan grapefruit and Carrizo citrange with the exception of one Valencia sweet orange plant. As it was anticipated in the previous report, the quality of Duncan fruits and seeds from the CREC’s grove has deteriorated further. Before the end of January, CCTF will contact local grower to get supply of better quality Duncan fruit.
The HLB-tolerant rootstocks US-1279, US-1281, US-1282, US-1283, and US-1284 were released by USDA in September and have now been released by FDACS-DPI as clean sources for commercial use. Material for propagation or establishment of seed trees is available from DPI. Fruit yield of Hamlin trees infected by HLB on these rootstocks is 2-4 times the yield of trees on Swingle, and the trees on these rootstocks also have fruit that is larger in size and higher in sugar content. These promising new rootstock selections, along with other new HLB-tolerant rootstocks from USDA and Univ. of Florida will be used in grower-cooperator field trials with funding provided by the HLB-MAC project in 2015-17. A special permit was obtained from FDACS to establish widespread commercial field trials using clean USDA sources of many new rootstocks, including Supersour selections. Based on this permit, cooperative arrangements are being made with commercial Florida nurseries for large scale vegetative propagation of these promising new rootstocks, and trials are being established with commercial growers. Three new replicated field trials including about 100 Supersour and other promising rootstock selections were field planted this quarter. Nursery trees were prepared for planting of two new field trials with Supersour rootstocks later in 2015. About five thousand new propagations of Supersour rootstocks were prepared for greenhouse testing for disease tolerance and budding for additional field trials in 2015-6. An additional greenhouse was constructed at USDA to propagate Supersour rootstocks for field trials and is already filled with Supersour material. Greenhouse studies continued to assess Supersour tolerance of CTV, calcareous soils, and salinity. Trees were planted into the field to establish seed sources for the most promising Supersour selections. Field trials and greenhouse studies will continue as resources allow. Studies continued to examine the defense gene profiles of trifoliate orange hybrid rootstocks that are highly tolerant to HLB, so as to improve our ability to create and select rootstocks that possess this trait. Studies continued on defense-related gene expression and small RNAs associated with HLB infection, in collaboration with University of Maryland and University of California research groups. A study of localized defense gene expression in shoots and roots provided evidence of striking differences between susceptible and tolerant rootstocks that are a major advance in understanding, and yield strong insights into ways to overcome the disease. New field trials were planted to compare the effects of HLB on the most promising commercial rootstocks under the best management conditions, and to measure defense gene response. Gene expression research to develop HLB-resistant rootstocks will continue as resources allow. Work continued to create and test transgenic citrus with elevated expression of citrus defense genes that appear associated with tolerance to HLB. Three replicated tests with US-942 rootstocks that overexpress the citrus defense gene CtNDR1, are showing significant reduction in Las infection for some of the transformed clones. Monitoring and data collection continued on previous groups of transgenic plants that have been inoculated with HLB. Several transgenic rootstock selections showing increased resistance to HLB have been identified from groups transformed with other resistance genes, and are also being prepared for confirmation testing. One hundred new transgenic rootstocks were produced, targeting to increase tolerance to HLB by manipulation of the citrus resistance genes CtMPK4, CtTGA7, CtDIR1, CtERF1, CtFAD7, CtFMO1, CtAZL1, and CtNHL25. Testing of the new transgenic rootstocks will continue as resources allow.
All of the research described in the previous report is being analyzed or active research is being transferred to a new grant from CRB. The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype is concluded and analyzed. There were some surprises. RNA levels of FT3, the FT homologue from citrus that other research by us and others indicates is most closely associated with flowering, were low in leaves at bloom time, whereas we expected high levels of expression. This may indicate that high levels of FT protein are produced at that time and sent to apices and mRNA is depleted. A study of CiFT3 transgenic tobacco plants treated with various growth regulators has been performed and the data is now being analyzed. The growth hormones produced striking and individually different phenotypes in each treatment. The data includes plant height and leaf number, size, and area. The endogenous ciFT3 promoter from sweet orange was successfully cloned to be used in the transcription activator-like (TAL) effector system inducible by methoxyfenozide that will hopefully activate the naturally present FT3 gene in citrus. Large numbers of citrus seeds are being germinated for transformation studies with this construct.
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