This is a 3-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, the AtMKK7 gene has been subcloned into the CTV-based expression vector and transition expression of MKK7 in citrus leaves is ongoing. The AtMKK7 gene has also been subcloned into the plant binary vector pBI1.4T (a pBI121 derivative) and transformed into citrus using the Agrobacterium-mediated approach. The AtMKK7 transgenic plants are growing. Conformation of the presence of the AtMKK7 gene in the transgenic plants by PCR and analysis of the expression levels of AtMKK7 in each transgenic line are underway. Resistance of the transgenic lines to citrus canker and greening (HLB) will be characterized when the transgenic plants are ready. For objective 2, Hamlin suspension cells have been used as starting materials for the selection. The Hamlin cell suspension culture has been scaled up in Murashige and Tucker (MT) liquid medium. Several flasks of the culture are maintained for subculture. To determine the concentrations that will be used in the selection, the Hamlin cells from the suspension culture were grown on MT medium plates supplemented with different concentrations of sodium iodoacetate ranged from 0 to 0.2 mM. Hamlin suspension cells were found to be highly sensitive to the inhibitor. A concentration of 0.1 mM of sodium iodoacetate could completely arrest their growth. Therefore, 0.1 mM of sodium iodoacetate has been used in the selection. We have tested the resistance of hypocotyls of citrus seedlings to the selective compound sodium iodoacetate and found that citrus hypocotyls are very sensitive to this inhibitor. A concentration of 0.2 mM could completely inhibit the growth of the calli generated from hypocotyls. We will use 0.2 mM of sodium iodoacetate in selection of the hypocotyl-derived calli. We have done irradiation for the first batch of Duncan grapefruit cuttings on 11/2/09. The irradiation dosage was 40G. We found that the irradiated cuttings generated significantly fewer shoots than the control and calli were formed on both irradiated cuttings and the control. The shoots and calli generated on both the irradiated cuttings and the control will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. We are preparing another batch of explants for irradiation.
Objective I: Assess community needs The PI presented a poster entitled ‘Analysis of Ca. Liberibacter asiaticus Psy62 (Las) genome sequence data and creation of the CG-HLB genome resources web site’ at the Joint Research Conference on HLB and Zebra Chip, November 2009 for the purpose of publicizing the available resources, and engaging with other researchers involved in genome analysis of Liberibacter. Objective II: Website creation and development. The Citrus Greening/HLB Genome Resources Website (http://www.citrusgreening.org/) has continued to expand. To assist CG/HLB researchers in navigating the growing number of Liberibacter-related datasets generated by 3rd party sites, links to these resources have been added to the Citrus Greening/HLB Genome Resources Website. Examples include pathway maps of Liberibacter metabolism generated by the Kyoto Encyclopedia of Genes and Genomes, and lists of genome structural features at the Genome Atlas Database. Links to other CG/HLB relevant sites including the Citrus Greening and Citrus canker publication list and web site for the International Psyllid Genome Consortium are also provided. Objective III: Bioinformatic analyses of Ca. L. asiaticus sequence data. Understanding Las biology and pathogenicity depends not only on knowing its raw genetic capability but also how and when individual genes are expressed. A list of predicted regulatory proteins encoded by the Las genome has been compiled. To initiate characterization of sites in the genome where these proteins bind (with implications for expression of downstream genes), sequences of experimentally characterized binding sites in related bacteria (e.g. Sinorhizobium, Rhizobium, and Agrobacterium) were assembled and used to create binding site models for computational analysis. These models are being applied to the Las genome sequence in order to identify candidate binding sites for three regulatory proteins: (RpoH (heat shock response), RpoD (constitutive expression), and RirA (response to iron availability). The models continue to be refined, but preliminary analyses suggest that in contrast to free-living bacteria which have distinct sets of co-regulated genes, Las has a much simpler regulatory profile. First, Las has many fewer regulatory proteins than do related free-living bacteria. Secondly, locations of predicted binding sites suggest that many genes that in free-living bacteria are tightly regulated in response to specific environmental conditions may be constitutively expressed in Las. Repetitive AT-rich sequences are also found in the promoter regions of several candidate virulence genes. It is hypothesized that their presence may enhance gene expression by allowing for easier separation of the DNA strands. The models described here can be readily applied to other Liberibacter strains and species as their genome sequences become available, with the potential to reveal sources of heat tolerance and other differences in environmental adaptation observed among isolates.
This is a 3-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (MKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, the Arabidopsis MKK7 (AtMKK7) gene has been transformed into citrus using the Agrobacterium-mediated approach. The MKK7 transgenic plants are growing. Conformation of the presence of the AtMKK7 gene in the transgenic plants by PCR and analysis of the expression levels of AtMKK7 in each transgenic line are underway. Resistance of the transgenic lines to canker and HLB will be tested. For objective 2, we have tested the resistance of hypocotyls of citrus seedlings to the selective compound sodium iodoacetate and found that citrus hypocotyls are very sensitive to this inhibitor. A concentration of 0.2 mM could completely inhibit the growth of the calli generated from hypocotyls. We will use 0.2 mM of sodium iodoacetate in selection of the hypocotyl-derived calli. We have done irradiation for the first batch of Duncan grapefruit cuttings on 11/2/09. The irradiation dosage was 40G. We found that the irradiated cuttings generated significantly fewer shoots than the control and calli were formed on both irradiated cuttings and the control. The shoots and calli generated on both the irradiated cuttings and the control will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. We are preparing another batch of explants for irradiation.
In efforts to standardize the genetic transformation protocol for Murraya paniculata, new experiments have been designed and implemented using pTLAB21 harbored in Agrobacterium tumefaciens strain EHA101, as previous efforts with standard citrus transformation strains and vectors were not successful. We have also tested pCAMBIA2301 and pGreen0029 in AGL1 for comparison. Epicotyl segments obtained from in vitro grown seedlings of Murraya were used as explants and a tissue culture medium designated as M10 (Murashige and Skoog’s (MS) standard medium supplemented with predetermined levels of BA and NAA) was used as the regeneration medium for all the transformation experiments. Various factors are being tested in efforts to develop a standard protocol for transformation, such as varying OD values of the Agrobacterium cultures, the duration of explant incubation time, duration of co-cultivation, and the amount of antibiotic used for selection of transgenic shoots and for Agrobacterium removal. At this time, no shoots have successfully been regenerated. Consequently, we are pursuing in parallel the regeneration of Murraya from axillary buds obtained from in vitro grown seedlings; this technique is being standardized with the aim of using it as a possible alternative regeneration system for future transformation experiments. Different concentrations of BA alone and in combination with NAA are being tested to induce multiple shoot regeneration from axillary buds.
Funding is now in place among all the partners of the International Citrus Genome Consortium (US, Brazil, Spain, France, and Italy) to move forward with the project to sequence a haploid citrus genome. This genome sequence, when completed, will be THE reference genome for citrus, as it will be of the highest quality technically possible. DNA samples for sequencing have been prepared, and the strict quality control standards required by the sequencing centers (JGI in the US, Genoscope in France, and IGA in Italy) have been met. DNA samples have been shipped to the three centers, and sequencing has begun at Genoscope. The Brazilian group remains in negotiations with JGI over contract language, but UF and JGI came to terms in late September 2009. Because of the various contractual delays, both here and elsewhere, and the current decreased capacity for Sanger sequencing at JGI, the ICGC goal to have the genome sequence completed and available to the citrus research community in mid-2010 will not be achieved. Meetings will be held this autumn to revisit the plan and coordination among the sequencing centers, to move forward at the quickest possible pace. Meanwhile, work has proceeded at the UF-CREC to produce sample materials needed for the microarray experiments planned, using Affymetrix GeneChips, a new array platform developed by the co-PIs at UF using Agilent technology, and for the cDNA platform available through our co-PI in Spain. To this end, two sets of plants of sweet orange, rough lemon, and Volkamer lemon, representing the more susceptible and more tolerant types respectively, have been inoculated with budwood from HLB-infected Carrizo citrange (Carrizo is resistant to CTV, so viral interaction complications will be avoided) in an environmentally controlled greenhouse. Plants have been observed for symptoms, and qPCR has indicated that we have successfully infected the plants. Samples of RNA have been prepared from all of the plants at regular intervals, to be used in microarray experiments. We are nearly finished the plan time course of RNA sample collection. Though funding to the collaborators at UCR has been delayed, they have proceeded with their objectives. The HarvEST Citrus EST database is being updated, to provide an improved database for gene expressions studies. The EST sequences from our colleagues in Brazil and Japan have all been downloaded and reassembled, increasing the number of publicly available citrus ESTs to more than 465,000. Likewise, the collaborator in Spain was delayed in receipt of the funds allocated, but he has been engaged with us in establishing experimental designs for array experiments using the cDNA platform, as well as some tissue-specific gene expression analysis that will be conducted. The plans to exploit genome sequence information for a better understanding of the interactions of citrus plants with the pathogen causing HLB are ultimately most dependent on having the genome assembled and annotated; for this reason, our main focus will be on accomplishing that goal, while continuing to establish the experiments and collecting the samples that will be used for subsequent microarray analyses and deep transcriptome sequencing.
This project was an extension in time and financial resources to the previously funded project titled “The International Citrus Genome Consortium (ICGC) sequencing project; Part 1, Sequencing” which was intended first to initiate the ICGC haploid genome sequencing project using Sanger sequencing. Those funds were allocated as a first investment intended to demonstrate good faith commitment on behalf of the Florida citrus industry. That goal was achieved, as evidenced by current funding in place for 4x coverage by our French collaborators through Genoscope, 2x coverage by our Italian colleagues through the Istituto di Genomica Applicata (IGA), 2x coverage through EMBRAPA funding of colleagues in Brazil plus 2x coverage through FCPRAC from the US together with the USDOE Joint Genome Institute (JGI). Because the timing to use the allocation first made by FCPRAC through this grant did not allow for our investment in Sanger sequencing through JGI (they require the project to be conducted at one time, not in pieces), I requested and was granted a revision to the objectives, to begin a sweet orange genome “re-sequencing project”, using next-gen sequencing (454 of Roche Diagnostics). This is a collaborative effort between my lab, Dr. W Farmerie (UF-ICBR), Dr. D. Rokhsar (JGI), and Dr. T. Harkins (Roche Diagnostics/454). This project is in concert with the ICGC sequencing initiative plans to use next-generation sequencing technologies on several diploid genomes, as an added resource to the Sanger haploid sequencing project currently underway. Currently, all sequencing runs of the sweet orange genome have been completed, including 8x coverage of 454/FLX WGS, 6x coverage 454/Titanium WGS, 9x coverage of 3 kb insert libraries paired-end sequences (PE), and 6.7x coverage of 8 kb insert libraries (PE). Combined with a 1.2x Sanger sequencing effort by JGI several years ago, we now have >30x sweet orange genome coverage. In addition, 1.2 million ESTs have been produced by one Titanium run on an RNA library from leaf tissue, to aid in subsequent assembly, gene prediction, and annotation. Six preliminary assemblies of the genome sequence have been attempted by 454 scientists, using various versions of their in-house assembly program, Newbler, and two more assembly efforts will be made using modified parameters. To this point, this work has yielded fragmented assemblies upon which gene prediction models had a difficult time to work. Roche/454 is continuing their efforts at assembly. When the challenges of assembly can be overcome, it is likely that the sweet orange genome sequence will be made available to the research community sometime in the first half of 2010, following gene prediction and annotation procedures, to make it a useful resource for subsequent research. The sequence will be housed in a new database, Tree Fruit GDR, which was funded by a recent SCRI grant to include citrus. Further, the sequence will be available also through the JGI plant genome portal, and will also be deposited with NCBI. Though funding for this project was terminated in September 2009, the work will continue to completion. This will be a valuable tool for all research projects aimed at understanding HLB infection and disease, as well as empowering for the full scope of citrus genetic improvement objectives addressed by breeding and genetics research programs.
Two trees have been found growing in HLB-ravaged orchards in Guangdong and one other in Guangxi province, that appeared to be free of HLB symptoms, while all other trees planted at the same time were either dead or declining, and replants likewise were afflicted. The trees from Guangdong were propagated at the Guangdong Institute of Fruit Tree Research facilities, and are being grown to conduct new tests of their reaction to HLB following deliberate inoculations. These original source trees have been tested twice after propagation using standard RT-PCR protocols, and they remain PCR negative for HLB; recent RT-PCR tests on the propagated trees have likewise proven to be HLB-negative. Two propagations of one of the selections have been replanted in an infected orchard location. The tree in Guangxi has been transplanted to a protected location in Guilin, at the Guangxi Citrus Research Institute; recently propagations of it have been shared with our colleagues in Guangdong and planted there in a field challenge. To expand further our search for survivors, and to continue to learn about Chinese citrus industry adjustments in response to HLB, we have established contact and good communication with a citrus extension specialist in the Fujian Provincial Academy of Agricultural Sciences, Mr. Li, Jian. This contact will provide us access to Fujian, another very seriously HLB-affected region of China. Mr. Li is very familiar with the local industry, and production areas and practices. He is aware of the goals of our collaborative project with scientists in Guangdong and Guangxi, and he is enthusiastically interested to aid us in participation. Plans are being discussed for a possible visit to Fujian sometime within the next 6 months, and follow-up visits to Guangdong and/or Guangxi. This trip seems central to encouraging the continuation of the collaboration, and to participate in planning experiments for a more in-depth analysis of the nature and underlying mechanisms of these apparent resistant phenotypes, and most importantly to confirm that the resistance persists following further propagation and inoculation with HLB.
Four phloem-specific promoter constructs have been cloned into the pCAMBIA plant transformation vectors. These are -940 and -690 5′-deletions of the Arabidopsis SUC2 promoter driving expression of GUS and GFP reporters. These are will be transformed into Arabidopsis to confirm phloem-specific expression. In parallel, these two constructs of the AtSUC2 promoter will also be used to drive expression of a series of resistance proteins (R-proteins) that have been shown to provide disease resistance when either over-expressed as wild type proteins or expressed as activation mutants. The R-proteins that are being cloned for phloem expression include the following: AtSSI4, and AtSNC1. The AtSSI4 and AtSNC1 proteins will be expressed both in the wild type and mutated forms. The others will be over-expressed in their wild type form.
Experiment set Ð To get healthy and infected material with Candidatus Liberibacter asiaticus (CLas) and Ca. L. americanus (Clam)60 seedlings (pathogen free) of sweet orange were grafted into Rangpur lime, and growth in pots with subtract. When the sprouts were 30 cm big, two infected budwoods with Ca. L. asiaticus or Ca. L. americanus were also grafted into the rootstock. Ten plants were grafted with healthy budwoods as control. Total DNA of all plants were purified and used in RT-qPCR to detect the presence of both bacteria. Five months after grafting with infected budwoods Ca. L. asiaticus was first time detected, but symptoms were observed just 220 days after grafting. On the other hand, Ca. L. americanus could be detected 90 days after grafting and symptoms were observed 120 to 150 days. All plants (10 with CLas, 10 with Clam, and 10 control) were pruned at the begin of Spring season (September) and transferred to well controlled environmental conditions (22 to 24 oC temperature and 16h/8h light/dark) in growth chambers (Conviron). The samples were collected in branches with three pair of leaves (about 30 days after pruning), and were pooled for each individual plant. 3 g of tissues (leaves and bark) have been sampled for total mRNA isolation (RNAeasy Plant Mini Ki, Qiagen). The integrity, concentration, and quality of RNA have been tested in denaturant electrophoreses 1 %, spectrophotometer and BioAnalyser (Agilent), respectively. All samples were confirmed to be bacteria positive by PCR. About 20 .g of total RNA of each sample were used in the hybridization experiments. Array set with unigenes of sweet orange Ð The database of 32,000 unigenes of sweet orange was submitted to Nimblegen for construction of the arrays. Each unigenes will be represented six times (density of 192,000 spots in two replicas per array) with different 60-mer oligo nucleotides. The complete experiments include: plants infected with CLas, plants infected with Clam (two conditions), two tissues (leaves and bark), and five biological replicas. Therefore, at least 2 (conditions) x 2 (tissues) x 5 (biological replicas) x 3 (technical replicas) = 60 (30 x 2) arrays will be produced. A first set of RNA was sent to Nimblegen (Madison) for hybridization. We are waiting for such results. A time course experiment is also underway to follow the infection process. Leaves above the grafting point were sampled 48 hours, one week and one month after grafting with infected budwoods. Total mRNA of such samples will be hybridized with the arrays by Nimblegen. The experimental design is three times of collect [48 h, 1 week and 1 month), two biological replicas, three not infected control, and two technical replicas (3 x2 x 3 x 2 = 36 (18 x 2) arrays].
The goal of this project is to transform the Arabidopsis and citrus NPR1 genes (CtNPR1 and AtNPR1), and the rice XIN31 gene into citrus, and to evaluate their resistance to both citrus canker (caused by Xanthomonas axonopodis pv. citri (Xac)) and greening diseases. The first year objectives include: (1) Molecular characterization of the transgenic plants; (2) Inoculation of the transgenic plants with Xac. (3) Inoculation of the transgenic plants with the HLB pathogen and monitoring of the bacterium in planta with quantitative PCR; (4) Transformation of SUC2::NPR1 into citrus; (5) Plant maintenance. Overall the project is going very well. Among the PCR positive plants (see the progress report submitted on July 15, 2009), we have identified three transgenic lines overexpressing CtNPR1 and AtNPR1, respectively, by using Northern blot analysis. These NPR1 overexpression lines were inoculated with a strain of Xac and the results showed high levels of resistance from the NPR1 overexpression lines, but not from the control plants, suggesting that both CtNPR1 and AtNPR1 are functional in citrus resistance to canker disease. We are currently performing growth curve analysis to confirm our observations and also comparing the function of CtNPR1 and AtNPR1 by inoculating the plants with different concentrations of Xac inoculum. In addition, we have also grafted the six NPR1 (three each of CtNPR1 and AtNPR1) overexpression lines and the control onto more root stocks to propagate the transgenic population. Our goal is to produce more than ten individuals for each line. These plants are maintained in green-houses located at the Citrus Research and Education Center in Lake Alfred, and will be used for greening inoculations starting next month.
Objective II: Website creation and development. The Citrus Greening/HLB Genome Resources Website (http://www.citrusgreening.org/), brought online in the previous quarter, has been expanded to include a ÒNewsÓ column on the home page highlighting new genome analyses (discussed in more detail under Objective III), high interest genome-related publications with links to abstracts, and events relevant to genome researchers such as the upcoming HLB/Zebra Chip Meeting. In addition, a page listing a variety of citrus greening-related on-line resources has been created, linked to the home page under ÒAdditional InformationÓ. Objective III: Bioinformatic analysis of the Ca. Liberibacter genome. A. A post-doctoral associate with training in biological systems and computational genome analysis has initiated bioinformatic characterization of extragenic sequence features in the recently published, closed genome sequence for Ca. Liberibacter asiaticus. To gain insight into co-regulated sets of genes that the bacterium may be using to exploit changing environmental niches as it moves from the psyllid vector to plant phloem and establishment in the phloem, candidate promoter sequences are being mapped genome-wide using (i) models derived from characterized promoters in related bacterial species and (ii) novel sequence motifs found present in Ca. Liberibacter. Repetitive sequences and variation in sequence composition are also being characterized. B. To better assess its metabolic capabilities and limitations, unannotated regions in the published Ca. L. asiaticus genome sequence have been carefully analyzed, revealing the presence of numerous disrupted genes. Some disruptions are consistent with adaptation to a restricted environment while others are found in genes generally believed to be essential, suggesting possible errors in the published sequence. A listing of newly annotated pseudogenes can be found in in the “Access Data” section of the Citrus Greening/HLB Genome Resources Website in both table and Genbank format, the latter suitable for viewing with the Artemis genome viewer. Objective I: Assess community needs. The PI attended a special session on citrus greening at the 2009 Annual Meeting of the American Phytopathological Society (August 1-5) and engaged in fruitful discussions with interested researchers. The PI will be presenting a poster at the upcoming Joint Research Conference on HLB and Zebra Chip with the goal of promoting genome analysis tools available through the Citrus Greening/HLB Genome Resources Website, and establishing greater connections with researchers engaged in genome sequencing and genome scale analyses of additional strains and species of Ca. Liberibacter. Sequence comparison among isolates and species is predicted to yield important insights into the adaptation of this organism to different environmental niches.
Juvenile sweet oranges grafted to several rootstocks with potential to induce scion precosity were grown for entry into our RES (rapid evaluation system) to encourage precocious flowering and fruit set. Seedlings of selected sweet oranges, including high juice quality precocious selections, were grown for entry into the RES, as needed to determine differences in minimum time to flowering/fruiting. ‘ Several key components of the Agro-transformation system have been investigated in order to improve transformation and regeneration efficiency. We have optimized the following components for efficient transformation and data has been published. o media formulations o hormonal combinations o pre transformation incubation conditions o bacterial growth conditions o co-cultivation conditions and o plantlet regeneration conditions ‘ Produced the first transgenic plants (containing the LIMA lytic peptide construct) of 5 new high quality sweet orange selections, including 3 precocious somaclones and two Rhode Red Valencia somaclones. ‘ New transformation methods are being developed in order to improve the transformation efficiency for sweet oranges and to allow for transformation of important citrus cultivars that are recalcitrant to standard Agro-transformation. A system for embryogenic callus transformation has been developed and numerous transgenic plants of OLL8 sweet orange, and specialty fruits W Murcott tangor and Ponkan tangerine have been produced. Overexpression of a FLOWERING LOCUS T (FT) gene in transgenic plants leads to accelerated flowering: Experiments with the citrus FT genes (there are three of them) continue. Many putatively transformed citrus (Carrizo) plants have been produced and are being analyzed. Shoots from one construct continue to flower in culture. The three construct. Publications: 1. Dutt, M. and J.W. Grosser. Evaluation of parameters affecting Agrobacterium-mediated transformation of Citrus. 2009. Plant Cell, Tissue and Organ Culture 98: 331-340. 2. Dutt, M., V. Orbovic, and J.W. Grosser. Cultivar dependent gene transfer into citrus using Agrobacterium. Proceedings of the Florida State Horticultural Society (accepted).
As proposed, a transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce. A new 8 acre site has been bedded, supplied with irrigation, and a ground cover established. Several acres in the far NE corner have been prepared for Dr. Dawson’s proposed field test of modified CTV expression vectors designed to produce anti-microbial peptides in citrus host plants. APHIS specified that Dr. Dawson’s site be as far from existing commercial citrus groves as possible, and recommended the NE corner of the Picos Farm. Answers have been provided to numerous questions from regulators to facilitate field testing approval. Cooperators have been made aware that the site is ready for planting. Dr. Jude Grosser of UF has initiated plans to establish ~1 acre of transgenic plants expressing genes expected to provide HLB/canker resistance. USHRL has filed papers with APHIS to conduct field trials of their transgenic plants at this site.
At the USDA, mature tissue transformation technology using in vitro derived shoots is being developed. Established greenhouse-grown source plants Ð of four populations including Valencia sweet orange, Ruby Red grapefruit, and US-942 on two different rootstocks are in place. A procedure to disinfest plant material derived from greenhouse grown plants has been developed that results in little phytotoxicity and little contamination (< 10%). Conditions suitable for in vitro shoot growth have been identified; a variety of conditions were screened including those relating to substrate, growth regulators, basal medium composition, ethylene, and ventilation. Experiments have been initiated using in vitro grown shoots. At the CREC, budwood was collected from 'Midsweet' sweet orange (Citrus sinensis L. Osb.) trees growing in the field and grafted onto Volkameriana rootstock in the greenhouse. The first flush from the grafted buds was disinfested, cut transversely into thin cell layers (1-2mm) (TCLs) and placed horizontally on MS supplemented with BA and NAA with or without the addition of Plant Preservative Mixture (PPM). PPM prevented some level of contamination, but also may have inhibited regeneration. In other experiments, Hamlin mature budwood source trees were grown on a selected complex rootstock that seems to have superior nutrient uptake, and nutrition was provided by a new granular slow release product that has been showing excellent results with nursery and field trees. Six transgenic Hamlin lines stably expressing the GFP gene were regenerated from the 1st experiment using the first flush on the grafted trees as explants. The 2nd experiment, using the 2nd flush on the same trees yielded no transgenics, indicating that the regeneration potential diminishes with sequential flushes. Similar experiments are being initiated in Brazil. In Gainesville cell penetrating peptides are being evaluated as an aid to transducing compounds into mature citrus tissue.
In Florida, first year’s milestones were: 1) Build a greenhouse for growing citrus for mature transformation. The University of Florida IFAS facilities has the specifications for the greenhouse and still are in the planning stages. The original specifications did not include a headhouse for maintaining clean entry and supply for the greenhouse. Facilities are modifying in an attempt to stay within budget. 2) Hire a manager of the mature transformation facility and allow this scientist to begin training in the Spanish laboratory. Dr. Cecilia Zapata Carrero has been hired as the mature transformation facility manager. She is now making plans to travel to Spain to begin learning the mature transformation technology. In Spain, we have to develop genetic transformation systems for mature tissues of the most important sweet orange varieties of Florida, and then transfer the methodology to Florida, once the greenhouse is constructed and implemented in Lake Alfred for this purpose. Valencia sweet orange has been highly responsive to regeneration and transformation. We have been able to produce a good number of transformants from the first experiments. The first Valencia transformants are already acclimated to the greenhouse. More important than that, the procedure has been shown to be reliable and consistent, meaning that we have been able to generate independent transformants with 1-2% transformation efficiency from each of the four experiments performed so far. This frequency is lower than that obtained with the readily transformable Pineapple sweet orange type that we use as responsive control, but we think transformation rates can be still increased for Valencia sweet orange. In any case, this result means that we are able to generate 1-2 independent transformant/s per 100 explants (about 20 shoots) agroinoculated, which we consider a very good rate considering that mature material is being used. Hamlin sweet orange is easy to transform from seedlings. However, we have been unable to prepare the type of starting plant material needed for mature transformation in the two first experiments performed, and this resulted in poor callus formation and regeneration. A third experiment is underway with important adjustments of the basic protocol. Plant material from mature Carrizo citrange has been prepared and it is ready to use in coming days for the first time. In addition, we are generating shoots from Pineapple sweet orange seedling explants transformed to ectopically over-express CsAP1 (AP1 from sweet orange) and CsFT (FT from sweet orange) flowering-time genes with the aim of modifying tree architecture. Interestingly, many shoots transformed with the CsFT construct are flowering in vitro soon after regeneration.
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