We now have citrus transformants in the greenhouse at UF for all of our constructs. PCR confirmations for all plants have been completed and indicate that from 17 to 88% of the surviving plants are positive for the R gene constructs. The least successful was the snc1 constitutive mutant expressed using the AtSUC2 promoter (2 positives out of 12 total). In addition, we have 11 (out of 15) citrus transformants containing the GusPlus reporter expressed using the AtPAD4 promoter, which we have recently found to be inducible by psyllid feeding. Our working hypothesis is that restricting expression of the R proteins to the phloem will lessen the negative impact that these proteins may have on normal growth and development. Transgenic citrus plants expressing either SNC1 or SSI4 wild type proteins using the phloem-specific AtSUC2 promoter appeared to exhibit a normal growth and leaf phenotype. This result was predicted since the respective R proteins should not have been activated in the absence of pathogens, and hence, would not trigger the hypersensitive response. In contrast, however, expression of the constitutively-active ssi4 protein in a phloem-specific manner resulted in approximately 60% of the plants with an obvious negative phenotype consisting of yellowish leaves, wavy-edged leaves, reduced internodal lengths, general stunting and leaf drop. This stunted phenotype resulted in death for 6 out of the 15 original transformants (40%). Although phloem-specific expression of the ssi4 mutant protein often (60% of the plants) resulted in either death or a negative growth phenotype, phloem expression of the snc1 mutant protein produced no abnormal phenotype. However, only 2 out of 12 plants were confirmed transgenics (AtSUC2/snc1). A similar tendency for the ssi4 mutant protein constructs to show an abnormal phenotype was also seen when expressed using the wound-inducible AtPAD4 promoter. The AtPAD4 promoter-snc1 construct showed no unusual phenotype. There are two questions that remain unresolved: 1) Why are some transformants showing a negative growth phenotype and others not, and 2) Do any of the R protein constructs confer resistance to Liberibacter? We are planning experiments that will evaluate the survival of Liberibacter in our transformed citrus lines. In addition, psyllid feeding tracks are being characterized histochemically to determine the relationship between AtPAD4/GusPlus reporter expression and the location of stylet sheaths and callose induction.
Analysis of transgenic citrus lines: We confirmed the presence of transgenes in transformed citrus plants using standard PCR techniques. In one line, SUC2-snc1 mutant (20-7), it was unusually difficult to establish that the desired construct was present. It required serial dilutions of genomic templates to reduce interference of the inhibitory substances present in plant genomic DNA extracts. This interference seemed to be correlated with the presence of this specific construct. It is possible that constitutively expressed snc1 mutant may affect the production of phenolic or other interfering compounds. In order to evaluate the survival of Liberibacter asiaticus (Las) in our transformed citrus lines, we first focused on the development of an assay to detect the presence of the bacteria in heavily infected, symptomatic citrus leaves. These were obtained from the UF Lake Alfred laboratory of Dr. William Dawson. Citrus leaves were sectioned into midveins and blades in order to determine the distribution of the infecting pathogen. Original quantities of tested materials were in the range of 40 mg. The primers used for PCR detection of the Liberibacter asiaticus were based on 16S ribosomal DNA, and as plant controls, the cytochrome oxidase COX gene was used (Pelz-Stelinski et al., 2010, J. Econ. Entomol. 103, 1531-1541). We were able to detect Las and Cox amplicons in genomic DNA isolates from these relatively small quantities of transgenic citrus leaf material: either in green (asymptomatic) or yellow symptomatic Las-infected leaves. Setting up a calibrated curve for real time quantitative PCR: Next, we generated Las and Cox PCR amplicons to be used in specific standard curves in the real-time PCR reactions to determine copy numbers of respective genes. The Wingless (Wg) gene that serves as the psyllid control was obtained from the genomic DNA isolated from 10 uninfected psyllids. Real-time PCR reactions required testing multiple variables in order to fine-tune the Las-detection assay, some being the primer and amplicon concentrations. We tested a range of amplicon concentrations from 10 ng to 1 pg (=12,190,283 copies), and in later experiments, down to 12 copies of Las, 14 copies of Cox and Wg. Also, lower concentrations of PCR primers were more optimal. As a starting point, we tested the expression of AtPAD4-GUSplus transgenic plants responsive to wounding to correlate the wounding event itself with the actual psyllid feeding. Preliminary citrus wounding experiments by slit-cutting, or needle-puncturing determined that the AtPAD4 promoter was very specifically induced by wounding. We performed numerous histochemical studies, including aniline blue, acid fuchsin, toluidine blue, Evans blue as individual and with combined staining, and fluorescence techniques to detect psyllid stylet sheaths. These were performed on cross-sections identified by GUS staining spots generated in response to psyllid-feeding (=wounding). After numerous attempts we were unable to establish this technology as a useful tool to meet our overall goal of the early Liberibacter detection in citrus plants.
A series of transgenics, produced in the last several years, continue to move forward in the testing pipeline. Currently, it appears prudent to replicate plants of each transgenic event and conduct challenges that last 10-14 months. Most of these plants in our program have been transformed with AMPs driven by several constitutive and vascular specific promoters. Several D4E1 lines appear to grow substantially better than controls even when infected, but do show the presence of CLas. Initial hopes of quickly identifying truly immune transgenics have matured to current efforts focusing on identifying significant resistance. For a disease that is devastating many Florida citrus groves, it is surprisingly difficult to get a consistent high-level of HLB disease even using known susceptible plants and inoculum sources with high levels of CLas. APHIS funded a grant, written by Gloria Moore, Ed Stover and Bob Shatters, focusing on identifying methods for highly-efficient and more standardized resistance screening, including exploration of strain x genotype effects. At the request of CRDF most FL researchers conducting research examining HLB-resistance met and shared observations and information. These data were summarized and used to finalize a set of experiments which are now underway. Jude Grosser and Ron Brlansky are playing key roles in addition to the grant authors. In our program, new constructs and resulting transgenics are in process, including hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab), and a citrus promoter driving citrus defensins (designed by Bill Belknap of USDA/ARS, Albany, CA).
A transgenic test site has been prepared at the USDA/ARS USHRL Picos Farm in Ft. Pierce, to support HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for more than twenty-one months. Dr. Jude Grosser of UF has provided 550 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional 89 trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenic rootstocks in place: Dr. Kim Bowman has planted several hundred rootstock genotypes transformed with the antimicrobial peptide D4E1. An MTA is in place to permit planting of Texas A&M defensin transgenics produced by Erik Mirkov and his trees expressing the snow-drop Lectin (to suppress ACP) are now on the Stover permit. Information has been provided to complete the permit application by Eliezer Louzada of Texas A&M to plant his transgenics which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants will be 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. Additional plantings are welcome from the research community.
For this project we studied the response of ‘Carrizo’ citrange plants transformed with the Arabidopsis thaliana NPR1 gene (AtNPR1) to applications of salicylic acid (SA), L-flg22 peptide derived from Candicatus Liberibacter asiaticus (Originally this was not one of our objectives but we decided it was important to compliment our study) and HLB infection. In order to study and characterize this response we developed and standardized real time PCR assays for 21 citrus genes associated with the pathogen defense response: SA biosynthesis and signaling (AZI1, EDS5, ICS1, PAL1), PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity) (EDR1, EDS1, NDR1, PBS1, RAR1, SGT1), transcriptional regulation (NPR1, NPR3, R13032, R20540), the jasmonic acid pathway (COI1 and JAR1) and targets of the regulatory SAR pathway (BLI1, CHI1, PR1, PR1b, RdRp). A few genes (AZI1, CHI, EDS1, NDR1, NPR1, NPR2, PR1, SGT1, R13032 and RdRp) were generally differentially expressed between treated and control plants, serving as good indicators of plant defense in citrus. In total we studied 25 independent transgenic lines for their response to SA and L-flg22 to identify those with an enhanced response compared to wild type (non transgenic) plants. We also determined the expression levels of the transgene using Real time PCR. Certain lines had induction levels for some of the defense genes mentioned above several orders of magnitude higher than non transgenic lines (wild type controls). Infiltrations with L-flg22 was also very useful in discerning transgenic lines that could potentially be more tolerant to HLB due to their enhanced response. We further propagated using cuttings 20 of the most promising transgenic lines. This proved to be the most limiting part of our project as some of the lines took a long time to root and shoot (sometimes a year). Additionally some lines had high mortality rates. It would be interesting to figure out if this was due to the expression levels of the transgene. Due to limitations in space to conduct the HLB inoculation experiment we concentrated our efforts on 3 lines that showed the highest potential (much higher defense gene expression levels). We also included 2 lines with low or no expression of the AtNPR1 transgene and wild type controls. In total 26 plants are currently being studied for their response to HLB either as rootstocks of wild type scions or as wholly transgenics. This will show if transgenic AtNPR1 plants could potentially be used as rootstocks to control HLB in non-transgenic scions. We have started analyzing the plants for HLB infection. So far only those grafted on non-transgenic rootstocks have tested positive for HLB, however it is still too early to make any conclusions. Despite the fact that this project has ended we will continue monitoring the plants and will add more to the study as space and resources permit. However, several important outcomes have already been achieved with this project: 1) standardization of real time PCR assays a battery of defense genes that will allow the study of defense in citrus not only for HLB but for any other pathogen; 2) Proof that L-flg22 peptide can be used to study defense response in a shorter and more controlled way than graft inoculations; 3) AtNPR1 plants show an enhanced defense response compared to wild type plants.
The first objective of the project was to hire a Florida-based faculty scientist that could be trained under Dr. Leandro Pena in Spain, for the purpose of learning the mature tissue transformation technique and transferring the technology to Florida. The scientist (Dr. Cecilia Zapata) was hired, at the end of the first year of the three year project, and traveled to Dr. Pena’s lab at the IVIA, Spain, where she was trained in all tissue culture techniques associated with citrus mature transformation, starting with preparation of the source of material at the greenhouse and ending with the acclimatization of transformants in the greenhouse. It was emphasized that the preparation of plant material needed for mature transformation is the key to successfully and consistently obtaining mature transformants, and this can only be achieved by producing budsticks in a highly controlled and clean environment. The second objective of the project was to build a greenhouse at the Citrus Research and Education Center in Florida for the purpose of creating and growing citrus for mature transformation and to establish a Mature Transformation Laboratory. Unfortunately, the greenhouse was unable to be constructed due to miscalculations in the planning budget in the original proposal. Instead a growth room was constructed adding a clean head house structure that could be used as support of the growing area. It took approximately 7 months to construct the growth room. Up to date, almost a year after finishing the construction, we are still correcting a few problems with humidity, computer sensors and the water filtration system. Also, a generator needs to be purchased; without it, any prolonged electricity failure could jeopardize the whole project. The laboratory is fully operational. The third objective of the project was to obtain mature transgenic plants from the most important Florida citrus cultivars. We started using the growth room and plant the rootstocks at the beginning of April 2011. At the same time three (3) sweet orange varieties were indexed in vitro and micrografted; the cultivars introduced were Hamlin 1-4-1, Valencia SPB 1-14-19 and Pineapple F-60-3. A calendar was established in October 2011 and firsts mature transformation experiments were performed in November 2011, all the protocols developed at the IVIA are adjusting to our specific environmental conditions and clone specificities. In our conditions, mature Valencia was very responsive to organogenic regeneration. We obtained positive plants. Hamlin was also transformed but was less responsive to organogenic regeneration, we have a few positive plants, but we are still adjusting the protocol to improve the efficiency. Our control cultivar, Pineapple, has some quality problems with the starting material and new introductions are needed in the future, however since we don’t have too much space in the growth room, we are introducing another important cultivar for Florida. The Valencia positive plants are already growing in the growth room to be tested by PCR and Southern Blot.
We have achieved significant results in each area of focus of this project during the previous funding period. Substantial progress in developing and releasing new sweet orange and grapefruit cultivars, has resulted in the release of new sweet orange cultivars with improved juice quality, including Valquarius, a January-maturing Valencia, and a grapefruit hybrid, UF914 with potential to overcome the grapefruit juice effect. We have contributed substantially through international genetic mapping efforts in support of the International Citrus Genome Consortium project, to develop and make available new genome resources to the international research community in support of HLB research. We have developed base information on the nature of host-pathogen interactions to both HLB and citrus canker providing guidance for future research leading to development of resistant varieties. For example, through time course studies comparing tolerant rough lemon to susceptible sweet orange we have determined the underlying genetic mechanisms of tolerance and susceptibility. Proteomic analysis of infected orange revealed greater abundance of several stress related proteins, and microarray data indicated that their underlying genes were also upregulated. These studies together provide additional information for creating resistant citrus using native citrus genes, and developing platforms for disease detection at very early stages. We have planted out over 1200 plants containing various combinations of natural or synthetic genes, and various promoters, located at two permitted field trial locations, and many more are currently in greenhouse tests of disease resistance potential. Several canker tolerant transgenic grapefruit trees were identified. New transgenic plants were propagated for hot psyllid greenhouse tests and field planting. New candidate genes were identified from citrus and other plants for HLB and canker resistance; vectors were constructed for transgenic plant production. Citrus-specific promoters, transcription factors, and other elements were identified and incorporated into new constructs to produce consumer-friendly transgenic plants, by limiting foreign genes or controlling their expression in specific tissues. The effort to genetically engineer resistance to both HLB and citrus canker has been one of the highest priorities for our team. We have begun evaluations of existing plant materials in the field, exposed now for several years to endemic citrus canker and HLB, and have identified sources of tolerance or resistance to HLB and canker. Some of these hold promise as new varieties directly, while others provide breeding parents for further progress in developing disease resistance, and can serve as experimental plants to further dissect host pathogen interactions, and to highlight novel approaches toward disease resistance targets. Several new rootstock trials with more than 15,000 trees on a few hundred advanced selections have been planted throughout Florida, to assess their adaptation to evolving advanced citrus production systems, and most importantly the impact they will have on disease incidence and severity. Some of these have shown repression of HLB in greenhouse tests. Previous work to develop rootstocks with resistance or tolerance against other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) has continued, and is particularly relevant as information accumulates on the confounding effects of biotic and abiotic stresses on HLB severity and tree decline. We have held two field days, and several impromptu events, where we have highlighted for the industry our new oranges and several new rootstock candidates that appear to support reasonably normal tree growth and good yields of high-quality fruit, even in the presence of HLB infection. We have continued all processes that are integral to further development of genetically superior new rootstock and scion varieties that can enable the Florida industry to survive the disease threats that currently threaten the future. These activities include basic genetics, gene discovery, development and testing of transgenics showing resistance to HLB in the field, selection and release of new varieties, extensive field trials and industry demonstration blocks, and identification of new rootstocks supporting tree growth and production in spite of HLB.
Preliminary examination of limb, scaffold and trunk phloem of healthy and HLB infected trees indicated that in diseased trees development of callose plugging was evident at all levels and started behind the newer phloem, was present in low amounts in healthy plants, could be seen in early infection stages and was readily seen by light microscopy with aniline blue or by TEM with normal fixation. Further evaluations by phloem age are underway. Huanglongbing (HLB) or citrus greening disease, caused by Candidatus Liberibacter asiaticus, is a phloem-limited fastidious pathogen transmitted by the Asian citrus psyllid, Diaphorina citri, and appears to be an intracellular pathogen that maintains an intimate association with the psyllid or the plant throughout its life cycle. The molecular basis of the interaction of this pathogen with its hosts is not well understood. We hypothesized that during infection, Ca. L. asiaticus differentially expresses the genes critical for its survival and pathogenicity in either host. To test this hypothesis, quantitative reverse transcription PCR was utilized to compare the gene expression of Ca. L. asiaticus in planta and in psyllid. Overall, 362 genes were analyzed for their gene expression in planta and in psyllid. Among them, 263 genes were up-regulated in planta compared with in psyllid, 18 genes were overexpressed in the psyllid, and 81 genes showed similar levels of expression in both plant and psyllid. Our study indicates that Ca. L. asiaticus adjusts its expression of genes involved in transport systems, secretion system, flagella, LPS, heme biosynthesis, stress resistance, hemolysin and serralysin in a host specific manner to adapt to the distinct environment of plant and insect. To our knowledge, this is the first large-scale study to evaluate the differential expression of Ca. L. asiaticus genes in a plant host and its insect vector. Efforts to propagate transgenic plants with the beta-glucanase gene continued, resulting in more than 150 Duncan and/or Valencia plants with this transgene, controlled by either or 35S promoter, or the phloem-specific Suc2 promoter. PCR analysis on a subset of these revealed that 90% are showing the specific band for the BG gene. Additional transgenic plants are being regenerated from in vitro cultures developed by Dr. Abdullah ‘ about 23 transgenic lines with the BG gene in either OLL#20 or Jin Cheng sweet oranges. These plantlets will be micrografted to rootstocks in 2012. Transgenic plants developed earlier are being moved to the Southern Gardens ‘hot psyllid’ greenhouse for inoculation.
Continued efforts to improve transformation efficiency: ‘ Evaluation of transgene expression of transgenic citrus plants with different phloem specific promoters. Several transgenic ‘Mexican lime’ lines containing the d35s promoter and the 4 phloem specific promoters were evaluated for transgene activity. Transgene analysis was carried out using PCR, RT-PCR, q-PCR and Southern Blot analyses. Publicataion: Dutt M., Ananthakrishnan G, Jaromin MK, Brlansky RH, & Grosser JW (2012) Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants. Tree Physiology 32(1):83-93. ‘ q-PCR approach to evaluate copy number and gene expression levels. Copy number of several transgenic lines has being evaluated using gene specific TaQMAN probes. Most transgenic lines had 1 ‘ 4 copies of the transgene stably incorporated into the genome. In addition a qPR-PCR approach is being used to evaluate gene expression levels in all transgenic lines. Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Continued to grow selected precocious rootstock seedlings for subsequent budding with transgenic precocious sweet oranges (Vernia and OLL series). Several transgenic lines of our precious sweet orange and mandarin transgenic lines (B4-79, W. Murcott, B10-68 and OLL8) have been grafted onto precocious rootstock including Amblycarpa + Benton and Changsha + Benton. Grafted trees have been transplanted into airpots in a heated greenhouse for evaluation (Fig 1.), with plans to grow the trees in a RES (Rapid Evaluation System horticultural manipulation) type system in the greenhouse. We are now planning to establish a transgenic site on CREC property, and hope to be able to include a small structure to apply the RES technology (horticultural manipulation to reduce the time of juvenility) to actual transgenic plants. Transformation with early-flowering genes: ‘ We have regenerated many transgenic plants with the poplar FT behind either the 35S or heat shock promoter. Some of them are quite large now and the ones with HS promoter were maintained in a growth chamber at high temperature for several months, but none have bloomed yet. We are growing T1 tobacco with all 3 citrus FTs in order to determine phenotypes. We developed a co-transformation strategy to transform Carrizo citrange with two cassettes, one containing 35S-cft1 and the other containing AtSUC2 ‘ gus. We generated 122 transgenic Carrizo plants using the two vectors. PCR analysis revealed that 16 lines contained both cassettes. Plants have not flowered 12 months after transformation. Plants are currently being evaluated in an unheated greenhouse for cold stress in order to initiate flowering in spring 2012. Numerous transgenic plantlets of Hamlin and Carrizo were regenerated containing P27, P28, P29, PATFT and pPTFT.
Analysis of transgenic citrus lines: We confirmed the presence of transgenes in transformed citrus plants using standard PCR techniques. In one line, SUC2-snc1 mutant (20-7), it was unusually difficult to establish that the desired construct was present. Serial dilutions of genomic templates were employed to reduce interference of inhibitory substances present in plant genomic DNA extracts. This interference seemed to be correlated with this specific construct. It is possible, therefore, that constitutively expressed snc1 mutant may affect the production of phenolic or other interfering compounds. In order to evaluate the survival of Liberibacter asiaticus (Las) in our transformed citrus lines, we first focused on the development of an assay to detect the presence of the bacteria in heavily infected, symptomatic citrus leaves. Infected leaves were obtained from the UF Lake Alfred laboratory of Dr. William Dawson. These were sectioned into midveins and blades in order to determine the distribution of the infecting pathogen. Original quantities of tested materials (leaf samples) were in the range of 40 mg. PCR primers for detection of the Liberibacter asiaticus were based on 16S ribosomal DNA, and as plant controls, the cytochrome oxidase COX gene was used (Pelz-Stelinski et al., 2010, J. Econ. Entomol. 103, 1531-1541). We were able to detect Las and Cox amplicons in genomic DNA isolates from these relatively small quantities of transgenic citrus leaf material: either in green (asymptomatic) or yellow symptomatic Las-infected leaves. Setting up a calibrated curve for real time quantitative PCR: Next, we generated Las and Cox PCR amplicons to be used in standard curves in real-time PCR reactions for copy number determinations. The Wingless (Wg) gene that serves as the psyllid control was obtained from the genomic DNA isolated from 10 uninfected psyllids. Real-time PCR reactions required testing multiple variables in order to fine-tune the Las-detection assay, some being the primer and amplicon concentrations. We tested a range of amplicon concentrations from 10 ng to 1 pg (=12,190,283 copies), and in later experiments, down to 12 copies of Las and 14 copies of Cox and Wg. As a starting point, we tested the expression of AtPAD4-GUSplus transgenic plants responsive to wounding to correlate the wounding event itself with the actual psyllid feeding. Preliminary citrus wounding experiments by slit-cutting, or needle-puncturing determined that the AtPAD4 promoter was very specifically induced by wounding. We performed numerous histochemical studies, including aniline blue, acid fuchsin, toluidine blue, Evans blue as individual and with combined staining, and fluorescence antibody labeling techniques to detect psyllid stylet sheaths. These were performed on cross-sections identified by GUS staining spots generated in response to psyllid-feeding (=wounding). After numerous attempts we were unable to establish this technology as a useful tool to meet our early Liberibacter detection requirements in citrus plants.
This is the end of the second year plus a 6 month NCE for a currently funded multi-investigator, multi-institution project. Although many parts of this research were successful, it cannot be continued in its present form. The USDA group, who was receiving almost 50% of the funding, does not want to continue for a third year. The post-doc who was working on the project has left and it is difficult to get a new post-doc, particularly in Ft. Pierce, when only a year is left on the project. The group has published a paper on their successful efforts (Marutani-Hert, Mizuri, Evens, Terence,McCollum, Gregory, and Niedz, Randall. 2011. Bud emergence and shoot growth from mature citrus nodal stem segments. Plant Cell, Tissue and Organ Culture 106:81-91). The Moore laboratory is also making good progress on the use of cell penetrating peptides to get molecules into citrus without having to use Agrobacterium. In the past 6 months, we have been doing successful experiments with DNA as well as proteins. This is far from a mature technology but shows. promise.
This project sought the development of in vitro regeneration techniques for Murraya paniculata, a presumed host plant citrus relative highly favored by psyllids; these regeneration methods were then to be used to attempt first the genetic transformation of Murraya with marker genes, to optimize the transformation protocol. If successful, then insecticidal or psyllid-suppressive gene construct could be introduced. The ultimate objective was to attempt the development of a deadly trap plant for psyllids that could be deployed in citrus groves to potentially decrease psyllid populations and consequent inoculum potential. Further, such deadly trap plants could be used in the urban landscape to decrease the reservoir of uncontrolled CLas inoculum from commercial or residential areas impacting nearby citrus production areas. We were successful in developing a reasonably efficient regeneration protocol for Murraya via organogenesis, with defined levels of hormone and growth regulator supplementation as well as appropriate plant tissue management and handling techniques; a manuscript on this work is under preparation, the first ever report of in vitro regeneration of this citrus relative. We struggled, however, with the objective of achieving successful genetic transformation. One bottleneck was the unavailability of a reliable source of abundant and viable seed sources necessary to initiate the large-scale experiments that we wanted to conduct. Despite this, we explored various parameters for genetic transformation of Murraya, including assessments of shoot sensitivity to the selection agent kanamycin using untransformed shoots, determinations of bacterial growth curves, and appropriate and effective antibiotic concentrations for bacterial selection. Using the optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments were conducted after testing various plasmids and Agrobacterium strains. Various factors, including a range of OD values (cell density or concentration in liquid culture) of Agrobacterium cultures, the duration of explant incubation in bacterial cultures, duration of co-cultivation period, and the composition of co-cultivation and regeneration media were likewise tested, and we established a standardized transformation protocol. Optimal conditions for transformation using shoot tips and lateral buds, to develop an alternative method using a different tissue source should the organogenic approach prove too difficult or inefficient for transformation, were also explored. Regeneration of buds and some shoots occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth was monitored, as well as the stability and uniformity of GFP expression over time. Nearly all of these transformation events proved to be either chimeric or transient, so further production of new transgenic events was pursued. Though the project has ended, we have shared our results with ctrus transformation experts, and the work is continuing in collaboration now with the Core Citrus Transformation Facility at the UF-CREC, to attempt to further refine and improve our abilities to transform Murraya, and perhaps ultimately to produce, test, and deploy the deadly trap plants we aimed to develop, to test their value and utility as part of integrated approaches to manage HLB disease in Florida citrus.
More than two thousand transgenic plants have been produced containing various combinations of natural or synthetic genes and promoters; many of these are currently in field trial locations, greenhouse tests, or still being grown off. Additional transgenic plants have been propagated for new hot psyllid greenhouse tests, and for field planting. New candidate genes have been identified from citrus and other plants for HLB and canker resistance; vectors have been constructed for new rounds of transgenic plant production. Citrus-specific promoters, transcription factors, and other genetic elements have been identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. The sweet orange citrus genome sequence was mined to identify genes controlling anthocyanin expression, in an effort to develop visual, citrus-derived markers for genetic transformation. Several candidates genes were characterized and modified by in vitro sequence alteration techniques, and these will be tested first in grape and then citrus. More than 875 transgenic plants have now been planted with a collaborator in Martin county, and these are being monitored regularly, along with a second site in Indian River county. Canker-tolerant transgenic grapefruit lines have been found in field and greenhouse tests. New rootstock trials of advanced selections (> 15,000 trees) were planted, to assess their adaptation to advanced citrus production systems; data have been collected on their early performance. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests; rootstock hybrids are showing diverse responses when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance; four complex tetraploid rootstocks have shown some repression of HLB in greenhouse tests (one was symptom-free up to 22 months ). We continued the program to rotate new germplasm (rootstock and transgenic) through a ‘hot psyllid’ house to ensure HLB inoculation prior to approved field planting; two groups of 50 trees have been rotated through so far, scheduled for planting at a collaborators field site, under permit from DPI. A new hot greenhouse site, with SG Citrus, is now being used with >340 transgenic plants grown there in replication. More than 150 new rootstock candidates that produce nucellar seedlings have been identified using SSR markers; these rootstocks were preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora, and have been used to produce new trees for pending rootstock trials. Rootstocks developed for resistance to other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) are evaluated, as we collected data from replicated trials and plantings. Final data have been collected from a field trial of various Valencia somaclones and seedless Midsweet selections, and following final analysis the most consistently high yielding clones from each will be moved forward for release; most candidates have already moved through the DPI-Parent Tree Program. New pummelo-grapefruit seedless hybrids have been selected, some showing field tolerance to canker; their fruit have been assayed for furanocoumarin content and several with good fruit quality have been found FC-free, potentially producing grapefruit cultivars that alleviate drug interaction concerns. Patents have been issued by the US-PTO for Valquarius (SF14W-62) and Valenfresh (N7-3), very early- and late- maturing Valencia selections respectively, and licensing is in process. Patent applications and documents for release were developed for 7 new cultivars, and these were approved by the UF-IFAS Cultivar Release Committee for release and commercialization according to UF-IFAS policy.
We continue to monitor HLB and canker resistance or tolerance among the more than two thousand transgenic lines we have produced thus far, in field trials (2 locations under permit) and in greenhouse tests at the CREC and with grower-collaborators. These transgenic lines contain various combinations of natural or synthetic genes and promoters. New candidate genes continue to be identified and cloned into vectors for testing. Additional citrus-specific promoters, transcription factors, and other genetic elements are being identified and incorporated into some of the new constructs to produce more consumer friendly transgenic plants, by limiting foreign genetic elements or controlling their expression in specific tissues. A gene controlling anthocyanin expression in citrus plants (CMybA1) has been identified by mining the sweet orange citrus genome sequence and has been ‘repaired’ by in vitro sequence modification, and shown to be effective in transgenic grapevines; tests in citrus are underway. If successful, this element can be used as a natural citrus-derived marker fir genetic transformation, thereby eliminating the use of antibiotic or GFP genes, and increasing the potential acceptance of transgenic citrus products by consumers. More than 875 transgenic plants in a collaborators field site in Martin County are being monitored regularly, along with a second site in Indian River County. Canker-tolerant transgenic grapefruit lines have been found in field and greenhouse tests, including some containing a broad spectrum, ancient disease resistance gene from rice; the latter are being propagated for HLB challenge. Data are being collected on the early performance of new advanced selections in trials planted to assess adaptation to advanced citrus production systems. We have made significant progress on new rootstock candidate HLB response screening in greenhouse tests. Diverse responses of rootstocks are being noted when grafted with HLB-infected Valencia, ranging from extreme sensitivity to high levels of tolerance. Greenhouse experiments are underway examining interactions of rootstock and nutrients in severity of symptom expression. A new hot psyllid greenhouse facility is now being used with >340 transgenic plants grown there in replication, and infection rates are being determined by symptom expression and qPCR; the materials being tested represent our most advanced genetic constructs with phloem-limited promoter sequences and previously proven genes. More than 150 new rootstock candidates preselected for potential tree size control and some for tolerance of Diaprepes/Phytophthora, and have been used to produce new trees that were planted into new rootstock trials, or held for pending trials. Rootstocks developed for resistance to other maladies (CTV, blight, Phytophthora, Diaprepes, etc.) are evaluated, as we collected data from replicated trials and plantings. More new pummelo-grapefruit seedless hybrids have been identified, some showing field tolerance to canker, good fruit quality, and FC-free, potentially producing grapefruit cultivars that alleviate drug interaction concerns. Trees were propagated onto 30 new sour orange-like hybrid rootstocks, some already shown to be tolerant of CTV quick decline, for a new field trial. A new demonstration planting of advanced sweet orange selections and newly-released cultivars, selected for high yields and superior juice quality, has been established to assess production performance in the grove, but more importantly to demonstrate their performance and utility in commercial processing. Two additional trials are being prepared now with the same collaborator to be planted at other locations.
We have reported previously on the escape trees that have been identified and assessed in collaboration with colleagues in Guangdong and Guangxi provinces in China. There has been no change in the status of the trees and no additional testing has been carried out. the trees propagated remain in their respective locations. From the visit with Mr. Li, Jian, a citrus extension specialist from the Fujian Provincial Academy of Agricultural Sciences in October 2010, our search for survivors was expanded. Gmitter revisited the Pinghe County Guanximiyou (Chinese honey pummelo) production area in Fujian in summer 2011, and it was impressive to see how this region of the Chinese citrus industry adjusted their production and continued to remain profitable in response to HLB. Looking into many successful or failed situations in HLB management in China, we learned that psyllid control was the key to maintain as low HLB incidence as possible, and good nutrition and routine tree health management were considered vital. Further, the natural tolerance of this pummelo variety, and several other citrus varieties in other locations in China, also contribute to sustainable productivity and profitability. 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, as we interviewed growers, pathologists, horticulturists, and entomologists associated with these healthy orchards. These insights, unintended side benefits of this project, have been widely and repeatedly shared with citrus growers and researchers in Florida and elsewhere around the world.
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