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. 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 the spring of 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.
Previously we standardized quantitative real time PCR assays for 18 genes (AZI1, BLI, CHI, COI1, EDR1, EDS1, EDS5, JAR1, NDR1, NPR1, NPR3, PBS1, PR1, R13032, R20540, RAR1, RdRp, and SGT1) associated with SAR and plant defense. Using this technique and based on our previous results on defense gene expression during pathogen and PAMP inoculations we selected a few genes (AZI1, CHI, EDS1, NPR1, PBS1, R13032 and RdRp) that are differentially expressed during PTI/ETI/SAR and best characterize this response in citrus. We treated 24 ‘Carrizo’ citrange AtNPR1 transgenic lines with Candidatus Liberibacter asiaticus flagellin 22 peptide (L-flg22, as a proxy for the pathogen) and analyzed the expression of these 7 genes. The idea was to determine how the transgenic lines responded L-flg22, an inducer of PTI/SAR, and which transgenic lines showed an enhanced response compared to wild type plants. Several transgenic lines showed expression levels that were much higher for most of the genes compared to the wild type plants, further indicating that AtNPR1 seems to modify the defense response in citrus. We also studied the response grapefruit plants grafted on transgenic ‘Carrizo’ AtNPR1 plants infiltrated with L-flg22. The results are being analyzed.
During the past year we developed and standardized 8 more gene expression assays for the study of defense response in citrus using real time PCR. The 18 genes we have selected are either important in the early induction and regulation of SAR (AZI1, EDR1, EDS1, EDS5, NDR1, NPR1, NPR3, PBS1, R13032, R20540, RAR1, and SGT1), are targets of the regulatory SAR pathway (BLI, CHI, PR1 and RdRp) or are components of the jasmonic acid (JA) pathway (COI1 and JAR1) that works antagonistically to SAR. Several of these genes were previously undescribed for citrus, however our microarray studies indicated that these sequences were differentially regulated by chemical and pathogen treatment. Additionally, we continued to propagate more AtNPR1 ‘Carrizo’ citrange transgenic plants. To our previous lines (854, 857, 859 and 884) we have added 757, 775, 854, 890, 896 and also more of 857, the most promising line. We also studied the response of lines 854, 857, 859, 884 and wild type (WT) to Actigard (Syngenta Corporation), a commercial version of the SAR inducer salicylic acid (SA). We studied the effect of Actigard on gene expression levels either alone or followed by treatment with a Candidatus Liberibacter asiaticus Flagellin-like peptide (L-Fgl, as a proxy for the pathogen). In general, Actigard significantly induced some SAR genes (For example: CHI, EDS1, PBS1, RAR1 and RdRp) compared to water treated plants, these same genes were induced significantly higher in transgenic lines, specially 857 and these effects were observed as early as 6 hours after treatment . We are still analyzing the effect of the L-Fgl, although it seems to repress the expression of some SAR target genes, at least in the WT plants. We have not started the HLB inoculation experiments yet as the number of plants is limited and we wanted to characterize their response before the plants were permanently confined to a containment facility. However, we will initiate this part of the research as soon as we receive the last year of funding for this project.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. Results showed that the use of the antioxidant lipoic acid significantly improves transformation efficiency in Mexican lime, and a manuscript reporting this was accepted with revision for publication in PCTOC. Recovered transgenic Mexican lime trees 1.5 years after removal from tissue culture were girdled, which induced flowering about 4 months later; with complete flowering and fruit set in less than two years. Experiments to test this with commercial sweet oranges are underway. We continued with experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency, including: kanamycin, hygromycin, mannose and phosphinothricin. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Working with Mr. Orie Lee and Faryna Harvesting, yield and fruit quality data was collected from the St. Helena project. Approximately 10 acres of trees planted 3.0 years ago include a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks. The best rootstocks identified to show positive affects on rapid tree growth with precocious bearing and good early fruit quality (higher lb. solids) were somatic hybrids Changsha mandarin+trifoliate orange 50-7, white grapefruit+trifoliate orange 50-7, and sour orange+Carrizo, and tetrazygs White#4 (Nova+HBPummelo x Succari sweet orange+Argentine trifoliate orange), Orange#13, Orange#14,Orange#18 and Orange#19 (Orange series all Nova+HBPummelo x Cleo+Argentine trifoliate orange). Trees on these rootstocks averaged more than a half-box of fruit per tree with juice brix values great than 11. These rootstocks will now be tested to determine if they can shorten juvenility in transgenic plants produced from juvenile explant. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL and Vernia sweet oranges were successfully micrografted to Carrizo citrange or experimental Tetrazyg rootstocks and are growing well in the greenhouse. These will now be clonally propagated onto the rootstocks mentioned above for further study of early flowering and transgene expression. Horticultural manipulations on these plants will include the RES (Rapid Evaluation System) growth method plus girdling. Transformation with early-flowering genes: ‘Citrus has at least 3 FT genes. Cloning and characterization of all 3 (genomic clones) has been completed. We have put them into transformation vectors with a constitutive promoter and performed transformation experiments with Carrizo and Duncan. Although only a few transgenic plants were recovered, we know the constructs work because we had previously tested them in tobacco, where we recovered early flowering phenotypes, as well as some other phenotypic alterations. Our current theory is that expression with the 35S promoter is too strong in citrus (in some cases we got flowering directly on the initial explant!). We are currently testing a poplar FT with a weaker inducible promoter (a heat shock promoter shown to be inducible in Oregon); which will hopefully solve the promoter problem.
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 antipsyllid 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 greatly improved our efficiency of screening . ‘ We are modifying the vector to express more than one anti-HLB gene. ‘ We are modifying the vector to allow addition of a second vector. ‘ We are preparing to put trees into the field for testing as soon as potential freezes are over. ‘ We continue to supply infected and healthy psyllids to the research community.
When dsRNA targeting either a psyllid cathepsin or a psyllid vacuolar ATPase gene are fed in artificial diets to the Asian citrus psyllid, an increase in psyllid mortality is realized. The oral uptake of ~300 bp dsRNA fragments matching the coding region to either psyllid Vacuolar ATPase or cathepsin can induce mortality in the Asian citrus psyllid. Comparisons were made to determine the optimal dsRNA size. Psyllids were fed either the ~300 bp dsRNAs directly or after processing to siRNAs with the Dicer enzyme. Results showed that the 300 bp dsRNAs induced greater mortality and than that observed with processed siRNAs. Furthermore, non-linear dose dependent toxicity of the ~300 bp dsRNAs suggesting complex interactions that have not yet been characterized with respect to dsRNA induced toxicity in insects.
Seed was collected from new crosses made the previous year to develop improved rootstocks and scions with tolerance to HLB and other improved traits. Selected hybrid seed was planted in the greenhouse. Promising SuperSour rootstock hybrids were identified and propagated for further testing. Cooperative work began with a commercial nursery to propagate the most promising SuperSour hybrids for large-scale commercial field trials. Fruit quality, yield, and tree size information were collected from 8 existing replicated rootstock and scion field trials. Performance data from field trials was summarized in a presentation at the Indian River Citrus Seminar. A field trial was planted in Indian River County to evaluate SuperSour selections in heavy flatwoods soil for tolerance of Phytophthora and Diaprepes weevil. A greenhouse study was completed to evaluate the tolerance of SuperSour rootstock hybrids to citrus tristeza virus. Data was collected from a replicated field trial in St. Lucie County to evaluate HLB tolerance of ten rootstocks exposed to natural infection in the field. Propagations from SuperSour rootstock hybrids were budded with ‘Hamlin’ scion to produce trees for disease testing and new rootstock field trials in the coming year. Field studies continued to assess the tolerance of different rootstocks to HLB with sweet orange scion under natural conditions, and work is beginning on a summary of those findings for publication. In coordinated research between this grant and the FCATP transgenic citrus grant to USDA, selected anti-microbial, insect resistance, and other genes were inserted into outstanding rootstock and scion cultivars to develop new cultivars with resistance to HLB and Citrus Bacterial Canker. Seed from the new USDA rootstocks US-812, US-897, US-802, and US-942 produced at the USDA Whitmore Farm was collected and provided to the Florida Citrus Nurserymen Association for commercial distribution. A study describing the tolerance of US-897 to HLB was published in the journal ‘HortScience’. In the study, both US-897 field trees naturally infected by HLB and greenhouse-grown trees that had been inoculated with Liberibacter through graft inoculation were observed to develop few or no symptoms of HLB, even though PCR clearly indicated the presence of Liberibacter infection. Typical symptoms of HLB found in other citrus, such as leaf blotchy mottle, shoot stunting and yellowing, and seed abortion were rare and not easily visible on US-897. It was noted that the HLB tolerance of US-897 probably is derived from its trifoliate orange parent. Experiments are planned to determine whether the tolerance to HLB found in US-897 results in increased tolerance for grafted trees with susceptible scions on US-897 rootstock. A greenhouse study is underway to compare the apparent HLB resistance of several different trifoliate hybrid rootstocks. Greenhouse studies continued to compare germplasm tolerance to HLB under carefully controlled conditions, and understand the impact of different factors on that tolerance. These studies will provide additional insights about how to engineer HLB resistant cultivars. Additional greenhouse and field studies are also underway to determine the most efficient methods to evaluate new citrus germplasm from crosses and transformation for resistance or tolerance to HLB. Greenhouse studies are underway to compare infection and symptom development in plants inoculated with Liberibacter by graft and Asian citrus psyllid.
1-The first objective of the second year was to build and start the operation of a plant growth room at the Citrus Research and Education Center in Florida (CREC). The growth room construction started on October 22nd 2010 and the projected finish date was February 11th 2011. There was a delay of a few weeks and the main contractor will finalize on March 25th, but the computer system contractor is still finishing the programming that will control the environmental conditions. We checked the growth room and it is working as expected however disposal of the waste stream will be a concern when the growth room is in full operation since the water that we will dispose needs to be collected in an external tank and test by the county to guarantee that we are not disposing contaminants that can affect the environment. We already started furnishing and placing equipment inside the building. Because of the delay in the construction, the growth room is not in fully operation yet. We believe this will take at least one additional month. The cost of the construction was higher than the original budget plan; the extra funding was provided by CREC and IFAS facilities as agreed initially. 2- A full time technician with nursery experience was hired after several months of searching. The process of hiring was slow. A first candidate was hired and the offer of employment was rejected due to a low salary offer. A second candidate was found but he quit two months after hiring. We hired a third technician with limited experience and he will start the first week of April. It seems like salary will be an important issue in the future to recruit and retain personal. 3- Training of the manager Dr. Zapata was completed at the IVIA under the supervision of Dr. Pena. It was emphasized during the training the improvement of transformation methods for more recalcitrant types, molecular analysis of the regenerants and plant material preparation at the greenhouse/growth room, including micrografting, phytosanitary treatments, fertilization and pruning. An annual schedule for completion of planting, transplanting, grafting and obtaining budsticks to transform was developed. 4- The Mature Transformation Laboratory was established, using an existing laboratory located at CREC. Two technicians with limited experience were hired and are currently being trained in the first tissue culture techniques, including culture media preparation, grafting, micrografting, explant preparation, culture and regeneration, etc. 5-Our selected varieties Hamlin 1-4-1, Pineapple S-F-60-3 and Valencia S-SPB-1-14-19 were subjected to cleaning through shoot tip-grafting at the Florida Department of Agriculture and Consumer Services (DOACS) with the help of Dr. Peggy Sieburth. They are kept at out lab and ready to be grafted on rootstocks when the growth room is fully operative. 6-Dr. Leandro Pena and his greenhouse and growth room manager Josep Peris made a visit of one week in March 2011 to supervise the last steps of the growth room construction before been finalized and suggested minor details to make the facility more reliable and helpful for operators. They also short-trained the tissue culture technicians in horticultural practices. They checked substrate, seed stock and nutrition issues with the manager Dr. Zapata.
The goal of this project is to transform the citrus and Arabidopsis 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. We have identified three transgenic lines overexpressing CtNPR1. These NPR1 overexpression lines were inoculated with 105 cfu/ml of Xac306 and the results showed high levels of resistance from the NPR1 overexpression lines, but not from the control plants. We also conducted growth curve analyses. Nineteen days after inoculation, the bacterial population in one of the NPR1 overexpression lines is 10,000 fold lower than that in the control plants. These results demonstrate that overexpression of CtNPR1 confers resistance to canker disease. We also graft-inoculated the NPR1 overexpression lines with greening to determine whether NPR1 is functional in greening resistance. We are in the process of monitoring Candidatus Liberibacter asiaticus populations in the inoculated plants using quantitative PCR. Five transgenic lines containing the SUC2::CtNPR1 construct, in which CtNPR1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene, have been generated. This construct may increase the expression of CtNPR1 in citrus phloem thereby maximizing the opportunity for resistance to greening. In a few weeks when the plants produce enough leaf tissue, we will perform Northern blot analyses to monitor the levels of CtNPR1 transcripts. The transgenic plants with high levels of CtNPR1 will be propagated by grafting and inoculated with greening. Finally, we have initiated microarray analyses of the CtNPR1 plants in response to Xac inoculations. The results will be reported soon. To continue our research, we request funds for the third year to achieve the following goals as originally proposed: (1) Characterization of the CtNPR1 transgenic plants inoculated with the HLB pathogen; (2) Molecular characterization of the SUC2::CtNPR1 plants; (3) inoculation of the SUC2::CtNPR1 plants with the HLB pathogen; (4) Examination of changes in hormone (abscisic acid, auxin, jasmonic acids and salicylic acids) levels in the CtNPR1 plants infected with Xac; (5) Plant maintenance.
This research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllid feeding. Both protein-based and RNAi strategies were tested by feeding psyllids artificial diets. To support the artificial diet assays, we optimized the diet composition by adding an antimicrobial agent to eliminate fungal growth that is introduced by the psyllids during the assay period. Using this approach we identified suitable buffers and optimal diet pH during the feeding period. In separate experiments, Tryspin Modulating Oostatic factor (TMOF), a mosquito decapeptide hormone, and cysteine protease inhibitor (CPI) from the Asian Citrus psayllids that was identified in our laboratory were added to artificial diets on which psyllids were allowed to feed. After 10 days of feeding, 100% mortality was observed in psyllids feeding on diets containing TMOF or CPI, whereas, 40% mortality was found in psyllids feeding on the control diets. CPI fed psyllids caused a significant higher mortality than the controls after 7 days of feeding. Based on these observations a collaborative project with Dr. Bill Dawson’s laboratory (Univ. of Florida, IFAS, CREC, Lake Alfred, FL) was initiated to use a Citrus tristeza virus (CTV) expression vector to produce TMOF and CPI in the in the citrus phloem. Clones containing the sequences of mosquito TMOF and CPI (cathepsin protease inhibitor) (both shown to be effective against psyllids) were provided to Dr. Dawson’s laboratory. Once CTV vectors are constructed and used to infect citrus plants, Dr. Dawson’s laboratory will provide us with the plants to evaluate the effect on psyllids while feeding on these plants. In parallel to these studies, we synthesized dsRNA molecules targeting 11 different psyllids essential genes encoding three different classes of proteins (alpha-tubuliln, V-ATPase, and Cathepsins). Initial feeding studies with alpha-tubulin dsRNA and V-ATPase dsRNA caused ~60% psyllids mortality as compared to only ~30% mortality for psyllids fed a control diet containing an equal amount of dsRNA not specific to the psyllid. Using this information, we have initiated experiments to produce citrus plants that express these dsRNAs in citrus phloem cells. A collaborative project with Dr. Bill Dawson’s laboratory (Univ. of Florida, IFAS, CREC, Lake Alfred, FL) was initiated to use a Citrus Tristeza Virus (CTV) expression vector to produce the psyllid dsRNAs in the citrus phloem. Clones containing the sequences of a psyllid Vacuolar ATPase and Cathepsin protease (both shown to be effective dsRNA targets) were provided to Dr. Dawson’s laboratory. Once CTV vectors are constructed and used to infect citrus plants, Dr. Dawson’s laboratory will provide us the plants to evaluate the effect on psyllids while feeding on these plants. We have also designed expression vectors for the production of transgenic plants expressing the psyllid dsRNAs through Agrobacterium-mediated plant transformation. These will be used to initiate the production of transgenic citrus plants that constitutively produce these dsRNAs in the phloem.
New lemon selections suitable for the California desert climate are needed to diversify production. Desert lemons occupy an important early-season market niche, which could be lost to international competitors. Lemons are also an important source of fruit for packinghouses located in other areas of the state. This ongoing project, which addresses the CRB ‘Production’ priority for New Variety Development, was designed to evaluate twelve lemon selections under desert conditions. The immediate objectives of the project are to provide the industry with information on the tree growth, yield, packout, and fruit quality characteristics for the lemon selections in the California desert. These include: ‘Allen Eureka’, ‘Variegated Pink-Fleshed Eureka’, ‘Corona Foothills’ (a bud sport of ‘Villafranca’), ‘Limoneira 8A Lisbon’, ‘Walker Lisbon’, ‘Femminello Santa Teresa’, ‘Interdonato’, ‘Limonero Fino 49′, Limonero Fino 95’, ‘Messina’, ‘Seedless’ lemon and ‘Yen Ben’. There are 20 trees of each variety, grouped into five replications of four trees each. All are budded to Citrus macrophylla rootstock. Leaves were collected for nutrient analysis for all 12 varieties in September, 2010; these are currently being processed and analyzed. The first harvest for 2010 took place on November 2, 2010, using professional harvesters from a local packinghouse. We collected yield from each group of four trees by counting the numbers of whole and fractional picking sacks harvested from each group. Also, we collected fruit packout and exterior fruit quality from each variety using a portable fruit line. For the first harvest, ‘Corona Foothills’, Limonero Fino 49′ and ‘Walker Lisbon’ had the greatest yields ranging from 200 to 250 lbs. per tree, while ‘Seedless’ lemon, ‘Variegated Pink-Fleshed Eureka’ and ‘Yen Ben’ had the least yield of less than 75 lbs. per tree With regards to fruit packout, ‘Messina’ had the largest size, peaking on sizes 75 and 95, while ‘Corona Foothills’, Interdonato’, ‘Limonero Fino 49’ and ‘Limonero Fino 95’ peaked on sizes 95 and 115. ‘Variegated Pink-Fleshed Eureka’ and ‘Yen Ben’ had the smallest sized fruit, peaking on size 140. There was little effect of selection upon fruit exterior quality. Samples of 20 fruit per group of four trees were collected for determination of interior fruit quality, including peel thickness, peel smoothness, percentage juice, juice pH and TSS/TA ratio. These data have not yet been analyzed. Several growers attended a Field day and saw the trees first-hand at the experimental block on September 28, 2010.
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 now planted in their research field to assess their reaction to natural inoculation with HLB. We visited these trees in October 2010 and observed no HLB symptoms, though surrounding trees showed obvious signs of disease. The tree in Guangxi that was transplanted to a protected location at the Guangxi Citrus Research Institute, was used to make several propagations, and these were deliberately inoculated by grafting infected material. These propagated trees have expressed symptoms of HLB now. They apparently are not resistant, but the question remains as to why the original source tree was not infected, and still appears symptom free. We visited with a citrus extension specialist from the Fujian Provincial Academy of Agricultural Sciences, Mr. Li, Jian, also in October 2010, to expand further our search for survivors, and to continue to learn about Chinese citrus industry adjustments in response to HLB. We visited the Pinghe County Guanximiyou (Chinese honey pummelo) production area, where we saw HLB being managed through good psyllid control, good nutrition and subsequent tree health, and the natural tolerance of this pummelo variety. We returned to specific orchards we visited previously in Guangdong and Guangxi. These were in very healthy condition in 2008, despite widespread infection of neighboring plots. The orchard in Guangdong was devastated with HLB, and we met the grower again; we were informed that fruit prices for his variety were very low and he simply stopped taking care of his trees. Orchards revisited in Guangxi were a different matter. These orchards have not only maintained their healthy condition, but they were substantially more productive and healthier in appearance than two years earlier. This confirms the utility and effectiveness of the management strategy being employed under direction of entomologist Deng of the Guangxi Citrus Research Institute. We interviewed growers, pathologists, horticulturists, and entomologists associated with these healthy orchards. The key elements outlined to us were critically timed pesticide applications, use of pathogen-free planting materials, and maintenance of tree health through good nutrition.
The objectives of this project are to develop, test and evaluate citrus rootstocks for disease and pest tolerance, and to select stocks that impart to the scion high yield, superior fruit quality, acceptable fruit size, and other essential traits. Activities involve production on new potential rootstocks by hybridization, screening these for valuable traits, and analysis of performance in replicated field trials. This report summarizes accomplishments from November 2010 to February 2011. We completed data collection for a rootstock trial with Moro blood orange scion. Yield, packline, and fruit quality analysis were completed for the Fukumoto rootstock trial at Lindcove. A trial of satsumas on 4 rootstocks was also harvested as planned. We continue to propagate trees of Washington navel on 28 rootstocks for a new trial to evaluate rootstock effects on early bearing and responses to high intensity management. This trial should be planted during summer of 2011. Seedlings for planned trials of Clementine and DaisySL mandarins are being grown at Lindcove, but these did not grow well after transplanting and we may have to start this trial again. Seeds were collected from standard rootstocks and new hybrids to be used in future rootstock trials and for future iron chlorosis and salinity tolerance screening trials. A Phytophthora citrophthora root rot resistance trial was planted in January.
TThe objectives of this project are to develop new mandarin, orange, lemon and grapefruit-type cultivars suitable for California conditions. The major approaches being used are hybridization-selection for mandarins and grapefruit, and mutation induction by gamma irradiation to obtain seedless forms of existing mandarins, oranges, and lemons. Promising selections are evaluated in field trials for tree size, yield, fruit quality, and disease susceptibility. This progress report summarizes accomplishments from November 2010 to February 2011. Hybridization during the 2010 season was quite successful and we have been harvesting and initiating culture of large numbers of new hybrid seed. No new irradiation has been conducted yet this year. Most work this year has involved evaluation of hybrids and selections from mutation breeding. Evaluation of new hybrid populations and trees grown from mutagen-treated buds is on schedule, with hundreds of trees evaluated to date. These include a population to be used to map genes influencing fruit quality and other traits. In the 6 replicated trials, detailed fruit quality studies are being conducted on DaisySL, FairchildLS, Nova IR10, KinnowLS, Encore IR6, 3 hybrid mandarins, and one grapefruit type, with Clementine and Tango as early and late season comparators. KinnowLS was released for propagation in California in late January 2010.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. A journal manuscript was submitted on research showing that use of the antioxidant lipoic acid significantly improves transformation efficiency in Mexican lime; experiments to test this with commercial sweet oranges are underway. We continued with experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency, including: kanamycin, hygromycin, mannose and phosphinothricin. ‘New publications supported by this grant:1. Dutt, M., D.H. Lee and J.W. Grosser. 2010. Bifunctional selection-reporter systems for genetic transformation of citrus: mannose and kanamycin based systems. In Vitro Cellular & Developmental Biology-Plant 46:467-476; 2.Orbovic, V., M. Dutt and J.W. Grosser. 2010. Seasonal effects of seed age on regeneration potential and transformation success rate in three citrus cultivars. Scientia Horticulturae 127: 262-266 Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Working with Mr. Orie Lee and a commercial harvesting company (w/ Frank Rogers), a plan to collect meaningful yield and fruit quality from the St. Helena project was developed – with harvest expected later this month. Approximately 10 acres of trees planted 2.8 years ago include a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks. The majority of trees have a significant yield and the trial is showing significant rootstock affects on precocious bearing and early fruit quality – the best selections from this trial will be ideal candidates for testing with juvenile transgenics. Also of interest is the cultural program being used at the St. Helena project that mimics OHS principals but with reduced input. The trees have been grown with a UF research slow-release fertilizer mix (in cooperation with Harrell’s Fertilizer) and daily irrigation. Two trees were confirmed with HLB the first year; but even with bad neighbors, there has been no detected additional spread of HLB during the past year. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL sweet oranges (a group of clones with Rhode Red quality that show high solids in young trees) were regenerated and successfully micrografted for further study of early flowering and transgene expression. Approximately 25 transgenic sweet orange trees from OLL selections were produced containing four different gene constructs. Progress was also made transforming OLL clones with the alternative embryogenic culture transformation system, as numerous transformed somatic embryos have been recovered. Progress was also made in the regeneration and characterization of plants containing the FDT transgenes for early flowering.
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