Based on our previous results analyzing the 24 transgenic ‘Carrizo’ citrange AtNPR1 transgenic lines we selected for propagation a few more: 754, 761, 763, 769, 771, 779, 853, 858, 862 and 880 that showed an enhanced defense response compared to wild type plants. Simultaneously, we have inoculated grapefruit scions grafted on transgenic ‘Carrizo’ rootstock of lines 854, 857, 859, 884 and wild type with Candidatus Liberibacter asiaticus. The plants have been acclimating in the containment facility at the University of Florida. However, we had a mite infection that has prevented us from analyzing the response to HLB. We have prunned and treated the plants with pesticide to control the infection. As soon as the plants are rid of the mites and growing again we will start the analysys as stated in our objectives.
We have transferred the initial indexed mature material obtained from Dr. Peggy Sieburth’s lab to the growth room. As we mentioned in previous reports the material was not in excellent condition in vitro because of excess storage time and only 20 % of the material prepared during the last quarter of 2010 survived the in vitro conditions. Usually these in vitro grafted plants are grafted in rootstocks on greenhouse conditions, but since the rootstocks were not ready because they were growing in lab conditions, they were transferred to soil directly. The rootstocks produced in lab conditions were small, and they were not actively growing and they are still recovering from the stress suffered in the laboratory and only a small portion of them will be used for grafting. We started preparing new source of material from the same 3 orange types: Valencia SPB 1-14-10, Hamlin 1-4-1 and Pineapple F-60-3. The mature budsticks are coming from indexed plants from the Department of Agriculture. New rootstocks from Swingle citrumelo and C. macrophylla were planted once the growth room was ready and they are currently growing. We will need a few more months to use this material. The Growth Room was finished and we passed the local inspection, however we operated for many weeks without being able to control the growth room due to lack of computer access to the program, the greenhouse technician does not have still access to the program. There were also several inconsistencies among the different companies involved in the construction that were solved during this post construction period to be able to operate the growth room. We are still dealing with some changes in the humidifier in the small room and finalizing the drainage of water from the growth room to the outside field. Fortunately the facility is under warranty for one year and the contractors are addressing most of the problems. We addressed the need of an emergency stand by generator for the facility as well as some improvements that were not in the construction plan, that were requested initially. These improvements are still necessary to make production in the growth room reliable. We experienced already a few electricity failures in the facility due to lightening and thunderstorms that affect the electrical service, and it took several hours to fix the situation on site and put the growth room back up and running again. The temperature increases considerably during the time of the electrical outage and there was no “clean water available” since the UV light system will not work under these conditions. It will compromise the whole process if we have more than 2 days with no water and electricity.
In previous informs we have already mentioned that mature tissues from the three sweet orange genotypes we pretended to transform through this project, namely Hamlin, Valencia and Pineapple, are readily transformable, as demonstrated by positive results from molecular analyses of marker transgenes incorporated into plants established in the greenhouse from the three sweet orange types. The procedures have been transferred in detail to our lab at the CREC. We are not working any more with Hamlin at our IVIA’s lab, but Valencia and Pineapple are being routinely transformed with several transgenes of interest not related to this project, meaning that, at least for these two sweet orange types, we can efficiently insert other transgenes able to modify plant phenotype and likely provide new improvement traits. For Carrizo citrange, we first established a procedure for transformation of mature tissues and then used the system to incorporate a hairpin construct aimed to induce RNA interference to silence and endogenous GA20-oxidase gene and them reducing gibberellin biosynthesis into actively growing tissues. These transgenic plants would be semidwarf and could be used as rootstocks to provide semi-dwarfing characteristics to non-transgenic scions grafted into them. A field trial assay initiated 5 years ago in Moncada (Valencia, Spain) indicates that GA20-oxidase antisense lines provide semi-dwarfing architecture (a one-third reduction in height) to mandarin scions. In theory, RNAi-inducing constructs should work better than antisense ones. We already have PCR-positive shoots for the hairpin construct in the growth room. We have initiated experiments to attempt transformation of mature tissues from Swingle citrumelo and Star Ruby grapefruit. We are also preparing new source material of Ray Ruby grapefruit to initiate transformation experiments by the end of the year. These objectives were not contemplated in the original project. For improving citrus tree management, we proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. We have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange over-expressing either FT or AP1 flowering-time genes already established in the greenhouse. We continue characterizing them in detail. In Florida, construction of the growth room has been finalized, but several important issues need to be set up to make production of healthy source plant material reliable. We are helping the manager and her team to establish a calendar for production of rootstock, clean scions, mother plants, propagations, etc. to get maximum occupation of the available space in the growth room and being able to make the maximum number of mature transformation experiments per year. We are also helping to establish substrate, fertirrigation and phytosanitary treatments.
This is a 4-year project with 2 main objectives: (1) Over-express the Arabidopsis MAP kinase kinase 7 (AtMKK7) gene in citrus to increase disease resistance (Transgenic approach). (2) Select for citrus mutants with increased disease resistance (Non-transgenic approach). For objective 1, 20 transgenic Duncan grapefruit plants have been generated, and these plants have been growing in greenhouse for 6 months and ready for canker resistance test. Since canker resistance test is straightforward, we will test all 20 transgenic plants. However, for greening resistance test, the transgenic plants will need to be propagated. We will first extract total RNA from each of the 20 plants and determine the expression levels of AtMKK7. We will choose 4 to 6 lines that highly express the transgene AtMKK7 for propagation. Six plants from each line will be used for greening resistance test. For objective 2, we have been using hypocotyls as explants for gamma irradiation mutagenesis. A total of about 75,000 hypocotyl cuttings have been irradiated in three batches with a irradiation dosage of 40 Gy. Shoots formed on the irradiated cuttings were transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Several shoots are currently growing on the selective medium. To increase the screening efficiency, we have also been using seeds for this objective. A large quantity of Ray Ruby grapefruit was obtained in the Spring of 2011 from the Indian River area. Seeds were obtained from the fruits and cleaned with Pectinase. Seeds were then treated with 8-hydroxyquinoline as a preservative to allow long-term storage of seeds at 4’C. Moisture content of the seeds was determined for future reference. Two quarts of seeds have been treated with gamma irradiation. One quart was irradiated at 50 Gy, the other at 100 Gy. Both untreated and irradiated seeds were plated into large glass Petri dishes as well as Magenta boxes containing water agar. Shoots have been formed on the seeds and will be transferred onto selective medium containing 0.2 mM of sodium iodoacetate. Based on the result of this batch of seeds, we will treat other seeds with either the same condition or a modified dosage. Shoots formed on these gamma irradiated seeds will be screened on the selective medium. Those shoots that are resistant to sodium iodoacetate will be grafted onto rootstocks to generate plants for resistance test.
The Core Citrus Transformation Facility (CCTF) continues to serve the community of researchers exploring ways to improve Citrus plants and make them tolerant/resistant to diseases. CCTF does its service by producing transgenic material. Within the last quarter, the CCTF facility produced the following transgenic citrus plants (transgene in parenthesis): three Mexican lime plants (pHK vector); nine Duncan plants (ELP3 gene); one Duncan plant (MKK7 gene); four Duncan plants (p7 gene); nine Duncan plants (p10 gene); one Mexican lime and two Hamlin plants (p33 gene); six Duncan plants (SUC-CitNPR1 gene); two Duncan plants (pWG19-5 vector); two Duncan plants (pWG20-7 vector); 11 Duncan plants (pWG21-1 vector); seven Duncan plants (pWG22-1 vector); two Duncan plants (pWG24-13 vector); and three Duncan plants (pWG25-13 vector). There are additional 30 plants in soil that need to be tested for the presence of the transgene of interest. Within last three months, the CCTF facility also sustained a loss of 30 soil-adapted transgenic plants due to unknown contamination coming from the rootstock plants (probably Phytophthora). Steps have been taken to prevent this from happening again in the future, including improved greenhouse sanitation and use of more resistant rootstocks in micrografting.
Huanglongbing (HLB) and Citrus Bacterial Canker present serious threats to citrus production in the US. Insertion of transgenes conferring resistance to these diseases or the HLB insect vector is a promising solution. Genes for antimicrobial peptides (AMPs) with diverse promoters are used to generate numerous transformants of rootstock and scion genotypes. New promoters and/or transgenes are being regularly introduced with more than a thousand new transformation attempts on citrus epicotyl sections each week. Plants from the initial round of scion transformations are now replicated and are being exposed to HLB, using graft inoculations and CLas infected psyllids in greenhouse and field environments. Challenge with HLB through exposure to infected ACP (D. Hall collaboration) is being conducted on a replicated set of 33 independent Hamlin transformants, 5 Valencia transformants, 4 midseason transformants, and 3 non-transformed controls. A series of promoters were tested with the GUS gene. The three vascular-specific promoters show expression only in phloem and xylem, while other promoters show broad expression in tested tissues. Sucrose synthase promoter from Arabidopsis drives high GUS expression more consistently than citrus SS promoter or a phloem promoter from wheat dwarf virus. A ubiquitin promoter from potato drives unusually consistent and high GUS activity. Use of this promoter may reduce the number of independent transformants needed. A new ubiquitin promoter from citrus (Belknap) is being tested. CLas sequence data target a transmembrane transporter (Duan collaboration),as a possible transgenic solution for HLB-resistance. In E. coli expressing the CLas translocase, two exterior epitope-specific peptides suppressed ATP uptake by 60+% and are being tested further for suppression of CLas in culture, before creating transgenes. ARS-Albany (Belknap) collaboration is providing genes from Carrizo citrange sequence generated using USDA and now CA-CRB funds, and other citrus genomic data, to permit transformation and resistance using citrus-only sequences; citrus-derived T-DNA border analogues have been shown to be effective in producing transgenic Carrizo and tobacco and are being tested in citrus scions. Sequence data are being mined for citrus AMPs and defensins to test in-vitro and ultimately in-planta. Anthocyanin production genes,give bright red shoots (UGray collaboration) and are being tested as a visual marker for transformation, as a component of a citrus-only transgenic system. Transgenes have been developed to suppress (using an RNAi strategy) a lectin-like protein produced in the phloem of HLB-infected citrus. It is possible that suppression of this protein may significantly reduce disease symptoms. High throughput evaluation of HLB resistance will require the ability to efficiently assess resistance in numerous plants. Graft-inoculation, controlled psyllid-inoculation, and ‘natural’ psyllid inoculation in the field are being compared. The first trial has been in the field for 30 months and a repeated trial has been in the field for 18 months. Leaf samples have been collected monthly and PCR analysis of CLas conducted. Several new collaborations are being explored to feed new HLB-suppressing transgenes and novel strategies into the citrus transformation pipeline.
Seed from new crosses to develop rootstocks and scions with tolerance to HLB and other improved traits were planted in the greenhouse. Fruit quality, yield, and tree size data were collected from 15 rootstock field trials. Source trees of 627 new supersour hybrids were established in the greenhouse to begin propagation. Propagations from 205 different supersour rootstock hybrids were budded to produce trees for disease testing and field trials. More than 3000 budded greenhouse trees for supersour field trials were grown to planting size. An agreement was developed to begin cooperative propagation of supersour rootstocks with a commercial nursery for widespread field trials with cooperators. One new rootstock trial with Rio Red, Hamlin, and Valencia scions was planted in St. Lucie County to evaluate tree performance with standard and new rootstocks under intensive HLB and horticultural management practices. Data was collected from three field trials at the Whitmore Farm in Lake County to study inheritance of fruit quality factors in sweet orange-type material from populations of hybrids between high quality pummelo and mandarin parents. It was apparent from one trial examining the effect of tree manipulations that most tree growth manipulations that might shorten time to fruiting will also greatly increase sensitivity to cold damage. Studies continue to assess citrus germplasm tolerance to Liberibacter – Huanglongbing (HLB) and Phytophthora/Diaprepes in the greenhouse and under field conditions. All citrus germplasm and cultivars become infected with Liberibacter, but different germplasm responds to HLB infection at different rates and with different symptom severity. Some trifoliate hybrid rootstocks, including US-802, US-812, US-897, and US-942 exhibit tolerance to HLB as seedling trees. Some hybrid selections resembling mandarin, grapefruit, and sweet orange also appear to exhibit some tolerance to HLB. A publication was prepared on differences in HLB tolerance among different rootstocks in field trials. One greenhouse experiment to evaluate supersour rootstocks for tolerance to CTV was completed, and another greenhouse experiment was initiated to compare different rapid methods for evaluating CTV tolerance of supersour selections. Promising new scion cultivars were released, including seedless Pineapple and the seedless mandarin cultivar ‘Early Pride’. Cooperative trials continued and new trials established to provide more information on new scion performance and pollination effects. The new hybrid rootstock US-942 was released for commercial use because of outstanding performance in many trials. Seed of US-942, US-897, US-812, and US-802 was provided to the Florida Citrus Nursery Association for managed distribution to commercial nurseries. Greenhouse and field studies are continuing to determine the most efficient methods to evaluate new citrus germplasm from crosses and transformation for resistance or tolerance to HLB. 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. Research is continuing to use HLB responsive genes and promoters identified in the gene expression study published last year for inducing or engineering resistance in citrus. New studies were initiated to examine gene expression and metabolic changes associated with HLB disease development and apparent resistance to Liberibacter in particular selections. A publication was prepared to document gene expression changes associated with the HLB tolerance of US-897 rootstock. A greenhouse study is underway to compare the apparent HLB resistance of several different trifoliate hybrid rootstocks. These studies will provide additional insights about how to engineer HLB resistant cultivars.
In the second year of funding, the CCTF continued to maintain and improve the quality of service it offers, proving itself as a reliable partner and integral part of the wider research community engaged in fighting HLB and canker. CCTF has become truly known and recognized beyond the community of Citrus Research and Education Center (CREC) and that is reflected in the increasing percentage of orders coming in from main campus of University of Florida in Gainesville. Within the last year, CCTF received orders to produce transgenic plants by using following vectors: p33; p7; p10; pMOG800; pAS7; pAS13*; pNAC1; pMKK7; pMOD1; pSucNPR1; pWG19-5; pWG20-7; pWG21-1; pWG22-1; pWG24-13; and pWG25-13, and pWG27-3. This is the largest number of orders received during one year since the facility opened and it clearly describes high demand for transgenic Citrus plants. At the time this report is being written, the facility has already been informed of additional five orders (from UF researchers) and another two orders for which binary vectors for insertion of customer’s genes of interest were sent to Yale University. The initial goals of this project that were reached in the first year of funding were being met throughout the second year. Despite high flux of people, the number of employees was kept constant. That allowed the number of explants processed per week to stay at about 2500. This amount of processed material per week is sufficient for production of high numbers of shoots that are being screened for presence of transgene by using different methods. And this in turn creates situation where CCTF is capable of servicing multiple orders at the same time. Application of the new PCR-based screening method that we started using last year is proving to be extremely useful. By using PCR on small shoots and detecting those that are putatively transgenic before they get micro-grafted on the rootstock plants improved CCTF productivity. Considering the fact that many orders include the use of binary vectors with no reporter gene, introduction of PCR as screening tool has brought the efficiency of the facility to a new level that permits its present output. As a result, the production stayed on high level of above 400 plants per year. More importantly, the time needed for completion of order and delivery of transgenic plants has fallen to about 10 months for many vectors. Within the last quarter, additional plants were produced for the old orders: pHK (12) and pSuperNPR1 (2). However, most of the plants were produced for newer orders: pNAC1 (26), p33 (12), pMKK7 (16), pMOD1 (5), pAS7 (7), pAS13* (4), pSucNPR1 (3), pMOG800 (1), p7+p10 (15). Similar to last year, the plants that CCTF produced in the second year of funding belong to five cultivars: sweet oranges-Hamlin and Valencia, grapefruits-Duncan and Flame, and Mexican lime. All of the new orders received in this funding period had a goal of improving tolerance and/or resistance to Citrus pathogens. The production of plants for old orders listed here also continued: pCL1; p6; pN1*; pC5*; pNPR1; pSuperNPR1; pPiTA; pCIT108p; pCIT108p3; and pHK. This decisively confirms the relevance of this project for the overall effort to produce and challenge transgenic plants as soon as possible and present them to Citrus industry as prospective candidates for tolerance and/or resistance against huanglongbing (HLB), canker, and Citrus Tristeza Virus (CTV). All current orders are for faculty presently involved in research projects funded by CRDF to battle HLB, canker or CTV. Funding for the CCTF furthers the efforts of these research groups and brings some of their results into life by producing transgenic plants that carry genes with predicted protective roles against pathogen attack. Continued funding to CCTF will allow for this situation to continue by keeping production of transgenic material at high levels and uninterrupted.
Our group continued to make excellent progress in the second year of our project. Over the last twelve months we have accomplished the following: Testing TAL effector specificity: We have synthetically assembled six TAL effector genes from X. citri strains and have establishing a system to test their activity on our broad recognition or “super” promoter in transgenic Nicotiana benthamiana plants. We have also prepared constructs with promoters containing individual TAL effector binding sites to test their activity and specificity. Testing broadness of resistance: Using the transient transformation method that we have developed, we have tested the reaction of thirty X. citri isolates on grapefruit leaves with Bs3 promoter constructs. We see a very high correlation between isolates which are capable of inducing disease in standard susceptible germplasm and recognition by our promoter constructs, indicating that the resistance constructs we have created will be able to confer broad resistance to diverse strains of citrus canker. These studies concur with the preliminary results showing that the constructs limit X. citri growth and produce HR against a number of strains. Additionally, we have generated different versions of the constructs that are designed to detect TAL effectors in all known X. citri strains. We are currently testing these, as well as X. citri strains with single TAL effectors to isolate the role of specific TAL effector proteins in the disease and resistance process. We have isolated multiple new TAL effector genes from important X. citri strains, which we will sequence and analyze in the next project year. Production and analysis of transgenic grapefruit lines: Sixteen independent transgenic lines generated in the first project year have progressed through selection, shoot formation and rooting, and are now well established in soil. These lines were verified by PCR and have been used in several experiments to examine response to pin-prick assays with X. citri and controls. We have thus far tested nine of the lines, and all but one demonstrates some degree of canker resistance, with two showing strong resistance. We have also set up additional transformations on both cotyledons and epicotyls. In total, we have initiated transformations of 6,857 explants using seven promoter constructs, and the explants are moving through tissue culture and selection, with more than 200 additional plantlets in soil. Finally, we have consulted with industry personnel to identify the most industry-relevant commercial germplasm to transform, and as a consequence of this we have obtained seed of red grapefruit and mid-season sweet orange to test in transformation assays. Other outcomes: We filed a patent application in January 2011 on canker-resistant transgenic citrus, and we have drafted an initial manuscript of our results, to be completed following the outcome of pending experiments.
USDA Ft. Pierce (Neidz) Agrobacterium-mediated transformation of mature tissue explants: Transformation of mature internode explants from greenhouse trees has been demonstrated in four citrus types including Valencia sweet orange (1 plant), Ruby Red grapefruit (1 plant), US-942 (8 plants), and Etrog citron (8 plants). Current efforts are directed toward characterizing this system for routine transgenic plant production. Source of mature tissue: Four populations of adult phase trees were maintained in the greenhouse including Valencia sweet orange/Sun Chu Sha (73 trees), Ruby Red grapefruit/US812 (62 trees), US-942 citrange rootstock/Cleo (32 trees), Calamondin (31 trees), and Etrog Arizona 861-S1 citron (67 trees). In vitro bud emergence and growth manuscript accepted for publication: A manuscript entitled, ‘Bud emergence and shoot growth from mature citrus nodal stem segments’ was accepted for publication by the journal Plant Cell, Tissue and Organ Culture. The paper documents the system developed for producing in vitro adult phase shoots from cultured nodes of greenhouse trees. Shoot regeneration from mature tissue explants: A system was developed for the production of shoots from cultured internodes from greenhouse trees. The system results in shoot and bud formation in 70-90% of the explants. A manuscript is in preparation that documents this research. New tissue culture method of Agrobacterium-mediated transformation of tissue explants: Preliminary results using alternative culture methods suggest improved transformation efficiencies. These approaches will be further explored. Mineral effect on shoots regeneration: Preliminary results suggest that mineral nutrition significantly affects in vitro culture response. The effects on transformation are currently being studied. University of Florida (Moore, Grosser, Gmitter) Efforts continue with greenhouse grown tissue (CREC) Rootstock effect on mature tissue transformation: the experiment conducted to determine if vigorous allotetraploid rootstocks could increase transformation efficiency was compromised by endogenous fungal contamination. We are now testing coconut fiber, sterile liquid nutrition, and low humidity in a clean environment for growing mature tissue explants in efforts to minimize problems with fungal contamination. Characterization of mature-tissue transgenic ‘Hamlin’ plants: recovered mature-tissue derived transgenic ‘Hamlin’ plants from previous experiments were propagated via micro-grafting for further characterization. Research continues on using cell penetrating peptides (CPPs) to deliver cargo (proteins, chemicals, plasmid) to existing citrus cells (Gainesville). Using the easily visualized GUS enzyme, we have found that we can efficiently get protein imported into a number of citrus tissues, using several different CPPs. Currently we are testing import of plasmid DNA, which should let us test clones and constructs before we do stable transformation. Based on a recent report on woody plants, we are also investigating whether we can produce cultures of rapidly proliferating cambial cells from citrus (Lee et al. 2010. Nature Biotechnology 28:1213).
We reported last quarter the cloning of the ctEDS1 gene in the binary vector pBINplusARS. We have already transformed the Arabidopsis eds1-2 mutant with this construct. The T0 seeds were harvested and will be selected for the transgenic plants in the next few weeks. Additional newly cloned genes include ctSID2, encoding the major biosynthetic enzyme for salicylic acid biosynthesis, and ctNHL1, which is a homolog of NDR1. These two genes were obtained from RACE followed by RT-PCR. The cDNA fragments of these genes are now in the pGEM T-easy vector and were confirmed with sequencing. The next step will be to clone these cDNA fragments to the binary vector pBINplusARS for plant transformation. Since the recent release of the Citrus sinensis (sweet orange) and clementine genome sequence, we have conducted extensive bioinformatics analysis on defense related genes in citrus based on published literature. Such analysis confirmed citrus defense genes that have already been cloned in my laboratory with this support. In addition, we found that most published defense genes are present in citrus with full-length sequences available. Therefore, we anticipate that our further cloning and functional characterization of citrus defense genes should be greatly expedited. We have so far selected additional 10 candidate citrus defense genes for the next round of cloning and complementation analysis.
Huanglongbing (HLB) is a serious and devastating disease of citrus caused by Candidatus Liberibacter spp. and vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The disease has the potential to greatly limit the production of citrus in Florida and other citrus growing regions worldwide. Current control of ACP and HLB is inadequate, but identifying and incorporating traits from uncultivated Citrus spp. and Citrus relatives that confer resistance to ACP is a potential strategy to manage the disease. In a study by USDA-ARS, 87 genotypes primarily in the Rutaceae orange subfamily Aurantioideae, were assessed in the field in South Florida for resistance to natural populations of ACP. The majority of genotypes hosted all three life stages of ACP, however there were differences among genotypes in the mean ranks for eggs (F = 3.13, df = 86, P < 0.001), nymphs (F = 9.01, df = 86, P < 0.001), and adults (F = 4.21, df = 86, P < 0.001). Very low levels of ACP were found on two genotypes of Poncirus trifoliata, 'Simmon's trifoliate' and 'little-leaf'. Poncirus trifoliata, the trifoliate orange, readily forms hybrids with Citrus spp. and is commonly incorporated into rootstock varieties. The field experiment was followed by no-choice tests in which female ACP had the opportunity to lay eggs for six days on five genotypes of Poncirus trifoliata, three genotypes from the Citrus genera that were not represented in the field, and a control (Citrus macrophylla) to determine whether any genotypes were resistant to ACP. Numbers of eggs on the five genotypes of P. trifoliata (means between 7-60) were lower than on the control (mean = 281.3; .2= 59.5, P < 0.001), which indicates that genotypes of P. trifoliata show some resistance to ACP. Numbers of eggs laid on the three genotypes of Citrus (means 129-200) were not significantly lower than on the control (.2= 4.37, P = 0.23). An additional 107 genotypes, including 81 genotypes of P. trifoliata and trifoliate hybrids, were planted mid-January and will be screened for resistance to oviposition by ACP in the coming months. Studies have been initiated to compare plant volatiles associated with plant genotypes that are readily colonized by the psyllid to those less colonized by the psyllid. Collaborators with the Fujian Academy of Agricultural Sciences in Fuzhou, China, initiated two experiments on resistance to ACP within the Rutaceae. Forty genotypes were evaluated in a free-choice experiment conducted in a screen house. Citrus tankan Hort. (cultivar Fuyouxuan Jiagan) was completely avoided by adults, and no eggs or nymphs were ever observed on this cultivar. No eggs or nymphs were observed on the following: C. reticulata Blanco (cultivars Bayueju, Xiang Ponkan, and Mashuiju); C. mitis (cultivars Chengshi Calamondin and Variegated Calamondin); C. sinensis (cultivars Navelia Navel orange and Skaggs Bonanza Navel Orange); C. grandis (cultivar Chandler Pummelo), and Fortunella hindsii var Chintou. The most heavily colonized genotypes included: C. reticulata Blanco (cultivar Fina Sodea Clementine); C. sinsensis (cultivar Fengcai anliucheng); and C. grandis Osbeck (cultivar HB Pummelo). A free-choice field experiment was established comparing 31 genotypes. Due to prolonged cool weather, no results have yet been obtained from this experiment. A delegation from FAAS will be visiting USDA-ARS during April or May to coordinate research efforts.
Objective 1: Transform citrus with constitutively active resistant proteins (R proteins) that will only be expressed in phloem cells. By restricting expression to phloem cells we hope to limit the negative impact on growth and development. Results: In addition to the SSI4 obtained from Arabidopsis thaliana var. Columbia genomic DNA, we created two new constructs (5-4) AtSUC2/SSI4 and AtSUC2/ssi4 mutant derived from Nossen genomic DNA (lines obtained from Dr. Klessig). Out of 49 ssi4 transgenics, 18 showed a reduced stature phenotype. However, none of the ssi4 expressing transgenics displayed a severe dwarf phenotype seen when expressed using their native promoter. We visited Dr. Orbovic’s laboratory at the UF CREC at Lake Alfred and resolved a technical problem regarding the PCR-based screening of citrus transformants. Dr. Orbovic has subsequently identified transgenic citrus for our two clones: AtSUC2/snc1 and AtSUC2/ssi4 mutants. Objective 2: Develop a method to elicit a robust plant defense response triggered by psyllid feeding. By further restricting expression of the R protein to the single cell that is pierced by the insect stylet, we anticipate that a defense can be mounted without a manifestation of a dwarf phenotype. The PAD4/ssi4 transgenics were difficult to obtain. We screened three times as many To seeds to obtain 18 plants. From these, 50% developed the “smaller stature” phenotype. Conversely, almost all transgenic plants containing SSI4 (wt) developed normally. Of the 33 transgenics with the PAD4/SNC1 construct, the majority showed no detrimental effect on growth. However, from the 37 PAD4/snc1 mutant transgenics, 10 were smaller than normal. We obtained 27 transgenics with the PAD4-reporter and analyzed GUS expression. Originally, the PAD4 promoter was selected based on literature reports of its phloem-specific expression and wound-inducibility upon the insect feeding. Our results indicated four basic groups of PAD4/GUS reporter expression in transgenic Arabidopsis leaves as follows: 1) phloem-specific expression with no wound induction; 2) universal expression with strong wound induction; 3) restricted expression with no wound induction; and 4) strong wound expression only. Conclusions: Our GUS analyses of two phloem-specific promoters (AtSUC2 and AtPAD4) revealed that transgenic plants do not maintain strict phloem-specific expression. Moreover, we found no PAD4 plants with the expected phenotype of wound-specific expression limited to phloem cells. However, substitution of the native promoters for ssi4 and snc1 genes with AtSUC2 and AtPAD4 did result in a significant reduction in the severity of the growth retardation phenotypes. Evaluation of more transformants will give us a better picture of the feasibility of utilizing these two promoters to obtain the desired expression patterns in citrus.
Once we have been able to establish at IVIA the procedures and conditions to transform mature Hamlin, Pineapple and Valencia sweet oranges, we are in conditions to transfer the basic protocols to Florida. We believe they can be reproduced with little or no modification at the new laboratory that is being set up at the CREC. As we have also developed a genetic transformation system for mature Carrizo citrange, we are now incorporating a construct of interest into this genotype. The second objective of our project was to develop genetic engineering strategies to improve citrus tree management. In this sense, we proposed to reduce endogenous gibberellin levels in transgenic rootstocks to make them dwarf or semidwarf. Such rootstocks could provide reduced size to non-transgenic scion varieties grafted into them. With this aim, we have incorporated a hairpin construct into Carrizo citrange to silence an endogenous GA20-oxidase gene and them reducing bioactive gibberellin levels in growing shoots. After Agrobacterium-mediated transformation, the explants regenerated abundant callus and showed prolific shoots formation. Around 70% of the explants regenerated shoots in the light step. At this moment there are several transgenic (PCR-positive) shoots micrografted in vitro. They are still pending of grafting on vigorous rootstocks in the greenhouse and Southern blot verification in coming weeks/months. New experiments will be run with this construct and fresh starting plant material within the next couple of months. For improving citrus tree management, we also proposed to over-express flowering-time genes in both the Carrizo citrange rootstock and the Pineapple sweet orange scion. This objective was initiated one year ago or so, and we have now at least ten independent transgenic lines of Pineapple sweet orange and Carrizo citrange expressing either FT or AP1 flowering-time genes already established in the greenhouse. We continue characterizing them in detail. The PI and his greenhouse/growth room manager, Josep Peris, travelled to Florida last March 20-27th to visit the CREC and supervise the last steps of the growth room construction before been finalized (by the end of April, we guess). We suggested to revise minor details to make the facility more reliable and helpful for operators. We also short-trained the tissue culture technicians in horticultural practices. We checked substrate, seed stock and plant nutrition issues with the manager Dr. Zapata.
A comparative study of two susceptible hosts, Duncan grapefruit (DG, Citrus paradisi), and Rough lemon (RL, C. jambhiri) and two resistant species of kumquat (Fortunella spp.), ‘Meiwa’ and ‘Nagami has been conducted to evaluate the basis for resistance to Xanthomonas citri subsp. citri (Xcc). The type of resistance occurring in kumquats is a hypersensitive response (HR) that develops within 48-72 h. This is based on the phenotype of the lesion, histological changes at the cellular level of infected tissue and early expression of genes related to programmed cell death (PCD). In kumquats but not in DG, several genes linked to PCD (lipoxygenase, glutathione transferase, metacapcase, acid chitinase and peroxidases) are expressed at 4 h post-inoculation (pi) with Xcc at 108 cfu/ml. Later at 24 h, additional genes related to plant defense (e.g. betaglucanase) are highly expressed in kumquats but less so in DG and RL and their activity continues to increase up to 48 h pi. Additional sets of genes related to PCD and the host pathogen interaction will be investigated this coming year. A cybrid is an asymmetric hybrid that contains the nucleus of one parent in combination with the mitochondrion and/or chloroplast of the cytoplasm donor parent. Twenty cybrids of highly susceptible Red grapefruit (RG) and the more tolerant Valencia orange (VO, C. sinensis) as the cytoplasm donor, were screened for their susceptibility to Xcc. Tolerance inherited from VO appeared to be quantitative based on an intermediate lesion phenotype in selected cybrids. In contrast to the callus-like lesions typical for susceptible RG, lesions were more necrotic for VO and the cybrids. This lesion phenotype indicated cell death arrested the proliferation of Xcc. Populations of Xcc at 14 days post inoculation in cybrids (7.2 Log cfu), were similar to VO (7.6 Log cfu) and one log unit lower than RG (8.4 Log cfu). Expression of genes related to host pathogen interaction in VO and cybrids differed from RG. The contrasting pattern suggested a differential interaction of genes from the nucleus with the mitochondria and chloroplast genes from the cytoplasm donor. Mitochondria and chloroplasts have a central role in stress and PCD signaling. The response of cybrids to Xcc confirms inheritance of resistance from VO may be expressed at different levels depending on whether mitochondrial and/or chloroplast genomes are transferred in the cybridization process. Field trials with several Ruby red grapefruit cybrids planted in canker-affected locations on the east coast were showing less foliar disease incidence than the adjacent Red grapefruit trees, but were severely damaged by freezes. Additional material has been planted out in canker prone locations. Production of cybrids lines using a new callus line of Meiwa kumquat as the cytoplasmic donor is underway in the Grosser lab. The resultant cybrids will be evaluated for inheritance of HR resistance using the same approaches
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