This is a joint project between CREC and USDA, Fort Pierce. The objective of this project is to find poncirus hybrids that exist now that are sufficiently tolerant and of sufficient horticultural and juice quality to be used now for new planting in the presence of high levels of Huanglongbing (HLB) inoculum. We believe there is a good chance that there mature budwood exists with these properties that could be available immediately for new plantings. Although these trees are not likely to be equal in juice and horticultural qualities of the susceptible varieties of sweet oranges grown in Florida, with their tolerance to HLB they could be an acceptable crutch until better trees are developed. We surveyed the trees at the Whitney field station and found 5 lines that we thought could be acceptable for juice. Those have been propagated and are being screened for tolerance and horticultural properties. The hybrid plants are being incubated in the psyllid containment room to allow multiple psyllids to inoculate the plants with HLB. At this time, all 5 hybrids continue to appear to be tolerant to HLB. The inoculated plants are growing in the greenhouse as we wait to determine the degree of disease symptoms in each line.
We completed the first step in the analysis of Asian Citrus Psyllid proteome and compiled a website with the results:
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. Currently we have cloned 10 full-length genes in these categories in the entry vector pJET. Five of the genes were further cloned to the binary vector pBIN19plusARS and transferred to Agrobacteria. The Agro strains were sent to our collaborator Dr. Bowman’s lab to initiate citrus transformation. In the mean time, we started the process of transforming Arabidopsis to overexpress these genes and to test their defense function. T0 transformed seeds have been harvested for some constructs and will be screened for transgenic plants soon. In addition, we did extensive bioinformatics analysis to identify potential new citrus defense genes to clone and to test for their defense functions. A list of 15 genes has been generated and primers to amplify these genes have been designed. The cloning of these genes will be initiated shortly. In addition, we are continuing to generate and/or characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project, although the support of this previous project has already been terminated.
A new FT construct,FMVcDNA27, containing an FT3 cDNA insert in the pCAMBIA 2201 vector has been made with a constitutive FMV promoter that proves to be as effective at transforming citrus and tobacco as the corresponding genomic construct that has been previously used. The FMVcDNA27 construct will be used to develop a chemically inducible system for the expression of this transgene. The inducible promoter systems from the Danforth Foundation mentioned in the previous quarterly report update was not used due to unforeseen issues; therefore the work will be continued using a transcription activator-like (TAL) effector system inducible by methoxyfenozide that will activate the naturally present FT3 gene in citrus. This promoter will utilize chemical-inducible ecdysone receptor-based expression. Research has also been conducted that looks into other endogenous plant chemically inducible promoters that are not turned on by chemicals in the media used to transform plants for use in controlling the FT3 gene expression. A manuscript comparing the behavior of the three genomic clones from citrus when overexpressed in tobacco has been completed and is undergoing further review before publication. The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype has been completed and gene expression levels have been compared in a month-to-month basis using the comparative CT method from qualitative Real Time PCR. The results show a promising patter that could potentially clarify the flowering pathway and the physiological effects of these three genes as it relates to the induction of flowering. The data is being currently analyzed for statistical significance and will be cross-examined using a higher concentration of DNA to verify the results. SDS pages and western blots have been done with the synthesized FT3 protein in order to identify if the HA tag and antibody will be an effective method to test for the presence of the protein when applied to plant tissue. The synthesized protein showed high specificity the HA-antibody and therefore this method will be used to assess the presence of the FT3 synthetic protein in further studies. The same procedure was performed with the Arabidopsis FT antibody and endogenous FT in citrus, tobacco and Arabidopsis as to determine if this will be an appropriate assay for the detection of FT3 synthesized protein. Another experiment involving FT3 transgenic tobacco and the effect of plant hormones Ethylene and Gibberellin (GA) as well as a GA pathway inhibitor chemical Paclobutrazol is underway to determine an effective way of preventing precautious flowering of citrus FT3 transgenic plants in tissue culture stages.
The objectives of this project are: 1) to generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology; 2) to identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications such as RT-PCR; and 3) to create transgenic citrus cultivars with new constructs containing the resistant gene(s). The first group of 5 samples for RNA-Seq, including resistant/tolerant vs. susceptible plants are complete. The second group of 10 samples has been sequenced, and the data is subjected for detailed analysis. A couple of resistance genes are constructed for genetic engineering. We mapped the RNA-Seq data to a reference genome, C. clementina using the bioinformatics program STAR. About 85% of the raw reads could be uniquely mapped. The transfrags of each library were assembled with cufflinks and merged with cuffmerg. 24,275 genes of the originally predicted genes had been found to be expressed and a total of 10,539 novel transfrags were identified with cufflinks, which were missing from the original reference genome annotation. Some of the NBS genes were found to be expressed. For C. clementine and C. sinensis, there were 118,381 and 214,858 mRNAs or ESTs deposited in GenBank and 93 out of 607 and 221 out of 484 NBS related genes match one or more ESTs respectively. The number of ESTs varied from 1 to 25. The expression abundance of each gene was measured by FPKM. The distribution curves of density of FPKM of 5 samples are very similar, indicating that the gene expression is similar and the quality of sequencing is high. We also performed the principal component (PC) analysis study on the expressions of five samples. The plot of the first PC against the second PC showed that R2017 and R20T18 clustered together (the resistance group) and R19T23, R19T24 and R20T24 clustered together (the susceptible group). This result showed that the gene expressions were significantly different in resistant vs. susceptible citrus. Using cuffdiff, a total of 821 genes were identified as difference expressed genes (DE genes) between the two groups using both p-value and an FDR threshold of 0.01. Among them, 306 genes are up-regulated and 515 are down-regulated in resistant citrus. Using the program iAssembler, a total of 53,981 uni-transfrags were obtained. Most of the assembled uni-transfrags should be novel genes, compared with the citrus reference genome. To reveal the differences in resistance, we also identified the exon variations (SNP/INDEL). A total of 612,618 SNP/INDELs were identified using the mpileup method employed in samtools. We focused on two types of mutations that could contribute to the resistance difference. The first type of mutation is a mutation in the genes of susceptible citrus leading to pseudogenes (Type1) and the other type of mutation is a mutation in resistant citrus genes that may gain a new function (Type2). Type1 mutations should have a homozygote mutation genotype in the susceptible citrus. We identified 146 candidate genes having Type1 mutations, which produced high impact variations such as frame shifts, splice site acceptors, splice site donors, a start lost, a stop gain or stop lost, and 3,578 genes with Type2 mutations. We expect that as the number of libraries being sequencing increases, the number of candidates will be reduced to a reasonable number allowing for further validation. We identified a few LRR-PKs genes for further comparative study based on the RNA-Seq data. The results indicated sequence variations of these genes in different varieties are indeed due to SNP/indels, and some of them were annotated as putative pseudogenes because of a truncation or insertion. Further verification is underway.
Construction is completed. Parent plants will be moved into the structure over the next quarter.
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports 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 over three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, 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 are being 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. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/16th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository were planted this quarter, with half preinoculated with Liberibacter. Additional plantings are welcome from the research community.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 40 putative transgenic plants with citGRP1 are growing and will soon be tested. Forty of them were test by PCR and twenty two of them are transgenic plants with citGRP1 insertion. RNA was isolated from some of them and RT-PCR showed gene expression. More than thirty kan resistant shoots were obtained from citGRP1 transformed Hamilin. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Over 60 transgenic Carrizo with GRP2 were transferred to soil and are ready for PCR test. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Hamlin shoots with peach dormancy related gene MADS6 are in the rooting medium for rooting. Seven transgenic Hamlin with MADS6 were planted in soil. In addition, numerous putative transformants are present on the selective media transformed with different constructs. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many kanamycin resistant transformants were generated on the selective media. About twenty kanamycin resistant shoots are rooted in-vitro and one Hamlin transformant is in soil. To explore broad spectrum resistant plants, a flagellin receptor gene FLS2 from tobacco was amplified and cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus responding to HLB and other diseases. The construct pBinARSplus:nbFLS2 was used to transform Hamlin and Carrizo. Many putative transformants were generated on the selective media. About ninety transgenic shoots were rooted in rooting medium and eighty Carrizo and ten Hamlin transformants were plant in soil. Other targets identified in genomic analyses are also being pursued. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
Evaluation of existing standard cultivars (‘Temple’, ‘Fallglo’, ‘Sugar Belle’, ‘Tango’, ‘Hamlin’, and ‘Ruby’) for HLB tolerance/resistance is underway . Trees were planted in 2010, using a randomized complete block design, at Picos Farm, Ft. Pierce, FL. HLB symptom development and tree growth (diameter and height) are being monitored on a monthly basis. All of the cultivars in this trial exhibit symptoms of HLB and have tested positive for Candidatus Liberibacter asiaticus (CLas). Results to date support earlier observations that ‘Temple’ and ‘Fallglo’ are in the most tolerant group. Numerous procedures are underway to elucidate mechanisms of resistances. These methods include light, confocal, fluorescence, scanning, and transmission electron microscopy, Fourier-Transform Infrared spectroscopy and metabolite profiling using LC/MS, GC/MS and NMR to determine if there are chemical signature differences and or compounds(s) that are responsible for resistance. Another project involves the treatment of various resistant/tolerant citrus accessions and susceptible standards with various concentrations of antibiotics to generate a range of CLas titer levels. There are 9 varieties being tested: 3 resistant (‘Temple’, ‘GnarlyGlo’, and ‘Nova’); 3 tolerant (‘Jackson’, FF 5-51-2, and Ftp 6-17-48); and 3 susceptible (‘Flame’, Valencia’, ‘Murcott’). The budded plants will be evaluated for growth and HLB symptoms development over a 2-year period. Temporal progression and systemic movement of the bacteria in the inoculated plants will be determined along with HLB symptom development, and growth of the plants. Development of periclinal chimera using resistant genotypes and standard varieties is in progress. In vitro shoots have been established from nodal and internodal explants excised from mature, certified disease free plants of ‘Red Carrizo’, ‘Temple’, ‘Hamlin’, and ‘Valencia’. After root formation, chimeras will be generated using a procedure developed by Ohtsu (1994). ‘Carrizo’ and ‘Sweet Pineapple’ have been successfully approached grafted. The graft unions were cut horizontally and treated with hormones to induce callus formation. Adventitious buds are starting to develop on the cut surfaces. A technique using flavanone profiling from extracted leave are currently being developed to the layers of the resulting scions . Gus transformed ‘Carrizo’ seedlings are also being used as a marker to visualize layers. Fifty unique hybrids (USHRL advanced selections) and standard cultivars have been challenged in an Asian Citrus Psyllid (ACP) feeding trial using CLas infected ACP. HLB symptom development, growth, and titer levels will be monitored on each plant. Trees initially were exposed to no-choice feeding, then in a free-flying ACP environment, and are now in the field. ACP feeding preference will also be examined using scanning electron microscope to enumerate the amount of ACP feeding structures. One additional study has been added to the project. Screening and evaluating new scion materials is a lengthy process and require multiple testing locations. Due to the urgency to develop tolerant/resistant material, a shorter evaluation cycle and high-through put screen procedure is being developed, which it may be useful to quickly identify new sources of HLB and ACP resistance varieties that may enhance and improve citrus breeding/production in Florida.
Function of individual X. citri transcription activator like effectors (TALEs): – Xanthomonas citri strain 2090 from Florida does not contain a typical 17.5 RVD TALE essential for typical pustule formation, but does contain pthA3 15.5 RVD. We cloned this gene and expressed in Xanthomonas citri subsp. citri 306 ‘ pthA1-4 and tested transiently with the 14 EBE promoter:GUS construct in grapefruit leaves. Activity was very low for this TALE, suggesting it may play a marginal role. – Xanthomonas citri strain #93 carries two TALEs – pthC 14.5 RVD and pthC2 17.5 RVD. These were tested in the transient assay and found to weakly activate the 14 EBE promoter:GUS construct in grapefruit leaves. We used an in silico tool – Talvez (http://bioinfo.mpl.ird.fr/cgi-bin/talvez/talvez.cgi, P’rez-Quintero et al., 2013) to scan citrus genomes for TAL effector binding sites, which suggests that PthC2 has differential ability to activate host genes compared to PthC. Transformation: Whereas we observed the expected behavior of gene constructs in transient assays, we have been unable to isolate stable transgenic citrus lines with functional gene constructs. We continue to explore multiple approaches to overcome this technical issue: 1. We tested Carrizo for transformation with the 4 EBE promoter:avrGf1 and challenged with Xanthomonas citri subsp. citri strain 306 and 306 transconjugant carrying avrGf1 into young leaves. The carrizo genotype gave significant resistance for transconjugant Xcc 306: avrGf1 with pathogenicity test infiltrated at 10-3 cfu/ml bacterial suspension. 2. The 14EBE promoter construct efficiency is being tested in tomato; binary vector was engineered with NosT: ProBs314EBE :avrBs4: NosT in T-DNA. AvrBs4 when expressed in tomato results in hypersensitive reaction. The Binary construct in agrobacterium strain Agl-1 was used for transforming tomato Bonny Best and large Red Cherry varieties. Transformant screening is in progress for assessing resistance to bacterial spot disease. 3. We sent two of our constructs for testing in parallel at a contract transformation lab The facility at UC Davis compared the 14 EBE:GUS and 14 EBE:AvrG1 constructs with their standard control plasmid in both Carrizo and tobacco. They found that both of our constructs gave a far lower transformation efficiency than their standard in both tobacco and citrus, and transformants with GUS recovered so far have not showed GUS activity. These results indicate that a likely source of the difficulty we have encountered is the vector. 4. To date we have prepared four promoter constructs in another vector that we use commonly and have good success with. The first set of these transformants are in soil, and we will be able to test the integrity of the inserts by PCR in a few weeks. Preliminary histochemical screening for successful transformation in putatively transgenic shoots of grapefruit, sweet orange and Carrizo showed that transformation has been successful and that transformation was significantly higher in Carrizo than in grapefruit or sweet orange.
Current status of the research: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. In order to develop an efficient system of screening EFR mutants for their binding to elf18-CLas, and in vitro binding system was developed which could be used to screen a mutant phage display library. We demonstrated in vitro binding of both elf18-WT and elf18-CLas to fragments of EFR ectodomain, but not to the ones of related receptor FLS2. Binding of elf18-CLas was weaker than that of elf18-WT, but it was considered that improvements in binding achieved by phage display screening may not be evident considering the binding of WT EFR to elf18-CLas. In addition, recent structural information about FLS2 binding to flg22 indicates the involvement of BAK1 as a co-receptor directly binding the ligand. As the phage display system would not account for this interaction, it may yield mutants which would perturb the binding of BAK1. An alternative system based on split ubiquitin is currently in the process of being investigated, which will hopefully overcome these issues. Additional experiments have also been performed to determine which portion of elf18-CLas is non-functional, by making chimeric elf18 peptides with WT and CLas portions. Both WT-CLas and CLas-WT peptides fail to elicit ROS, indicating there are multiple issues with the function of the elf18-CLas peptide, and thus requiring further investigation. A collaboration is currently being set up with the laboratory of Prof. Chai at Tsinghua to obtain structural information surrounding elf18-WT and elf18-CLas binding to EFR, which would enable more straight-forward testable hypotheses. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Vectors are currently being constructed in the pCAMBIA backbone, under the expression of the 35S/FMV promoter. These constructs will contain: EFR; XA21; both EFR and XA21; and EFR and XA21:EFR chimeric. Cloning of these constructs should be completed in the next few weeks and will then be passed on to the Moore laboratory for transformation in citrus.
Within the last year and-a-half, researchers trying to find solution to HLB through production of transgenic plants have used every possible approach that offered some prospect for production of tolerant/resistant citrus plant. As a result, the Core Citrus Transformation Facility (CCTF) has indeed become the platform for testing the effect of different DNA sequences (‘genes’) on transgenic plants that could result in possible alteration of their ability to sustain pathogen attack. Since most of this work in its nature is theoretical, it is not well known how the introduction of certain gene into Citrus will affect production of transgenic plants. Some genes may be easily introduced into model organisms or may cause weak effect on their phenotype. However, very often situation is rather different in Citrus. From one client, CCTF has received a group of orders that all have common DNA sequence combined with other, different DNA sequences. Those orders consisted of six vectors that were supposed to be introduced into three different citrus cultivars in certain combinations. Since the time when these orders were placed and 9/20/2013, altogether 70 co-incubation experiments were performed with more than 38000 explants. Despite all this work, no transgenic plants were produced. From another research group came an order to produce plants with the gene that severely affected the phenotype of transgenic plants. Transgenic shoots were stunted and extremely bushy, making our efforts to graft them impractical. Work on this order stopped after about 30 PCR-positive shoots were produced at which time the agreement was reached with the client who will try to place new order where this gene will be controlled by an inducible promoter. The unintended consequence of the efforts of researchers to find the ‘gene’ that may render Citrus plants tolerant/resistant to HLB by using CCTF as a testing platform is low number of transgenic plants produced in the CCTF. In the last quarter, only 20 plants were produced. They belong to the following orders: pHGJ2 vector-one plant, pHGJ3 vector-seven plants, pHGJ4-three plants, pN5-five plants, pBI121-one plant, pN7-one plant, pW14 one plant, and pMED14 one plant. The second (out of ordinary) reason that contributed to low productivity within this quarter was the massive contamination in one of the common growth rooms where CCTF also keeps germinated plants. This contamination took place in June and July and wiped out 18% of our cultures from that period. Out of three orders for transgenic plants placed in the previous quarter, two were withdrawn. Within this quarter, six orders were placed. Work with two vectors is to proceed only to the early step in shoots production. For additional three vectors, the goal is to establish possible effect of introduced genes on genome activation/transformation success rate. Because of the presence of GFP as a reporter gene in these vectors, the work on these orders may also be completed at the phase of transformation when fully developed shoots are harvested from explants.
The Mature Citrus Biotechnology Facility (MCBF) continues to increase our capacity for genetic transformation of mature scion budded onto immature rootstock. To this end, we have double budded immature rootstocks with mature scion. The number of explants will be significantly increased if this double budding scheme works well. Thus far, the results are promising for grapefruit but we are still waiting for results in sweet orange. Sweet orange is not as vigorous as grapefruit. We are also increasing the number of transgenic events produced by transforming immature rootstock. Different transgenic/wild-type combinations of rootstock/scion will be tested in field studies. A number of clients have provided genetic constructs important to imparting tolerance to citrus canker. Genetic transformations of Valencia using two constructs obtained from Dr. Wang’s lab have been performed and putative transgenic shoots are regenerating which will be micro-grafted in the future. Transformations using a construct from Dr. Mou’s lab (originally provided by Dr. Dong at Duke, NC) have been used with Hamlin, two batches of Pineapple and Ray Ruby grapefruit. Mature shoot explants of Valencia will be transformed in the near future as explants become available. This construct has also been used with explants of Carrizo. Swingle and Macrophylla rootstocks will be transformed in the near future. Additional genetic constructs will be obtained from citrus researchers as the capacity of our lab increases. Molecular analyses of putative transgenics, transformed with marker genes, are underway, and these plants are expected to flower in the near future. The number of transgenes for each putative transgenic is being determined by qReal-time PCR or Southern blot. Additional laboratory equipment and consumables are being purchased and another employee hired to accomplish this objective. The temperature, photoperiod, and light source have been changed in Growth Room A to induce flowering in these transgenics. Dr. Pena indicated that thorns in mature citrus transgenics regenerated from tissue culture is normal, transitory, and a sign of vigor. During the last quarter, additional experiments were conducted with marker genes (Table 1). The number of positive shoots recovered in some experiments is relatively low, but still acceptable for mature citrus transformation. Table 1. Mature citrus transformation experiments showing transformation efficiencies. Cultivar Date Batch Plasmid Explants Positive Transformation Shoots Efficiencies (%) Ham 19 6/11/13 36 p2301 240 1 0.8 Ham 20 7/2/13 31 p2301 300 2 2.2 Pine 8 6/11/13 35B p2301 240 1 0.8 Pine 9 7/9/13 40 pE121 260 3 4.5 Val 20 5/21/13 30 pE121 810 28 2.1 Ray 2 7/30/13 37X pE121 640 7 6.7 Book chapter from previous lab manager: Orbovic, Shankar, Peeples and Hubbard (in press) Citrus Transformation using Mature Tissue. Edited by Kan Wang, IN Agrobacterium Protocols Vol 2
This quarter work has been divided among between all of the proposal objectives. For the primary objective of transformation via CPPs, a new approach in the transient expression assays has been evaluated in order to enhance the transformation protocol. The new approach has been adapted from Chugh and Eudes, 2008. Instead of 3 days of transfection of citrus seedlings, testing has been done following one hour, two hours, or three hours following incubation of the seedlings with peptide and constructs. Early data indicate that one hour is insufficient for transient expression. As another option to improve upon this method, new and different peptides are being tested, including the classic ‘Tat’ peptide penetratin, and a chimeral peptide ‘R9-Tat’. These experiments did not have as much tissue as normal, since the ‘Pineapple’ sweet orange (40 of 80, 50%) and the ‘Duncan’ grapefruit (24 of 240, 10%) both had low germination rates. The next quarter should produce more plant tissue and results from these first experiments. In addition to these new transformation events, a second reporter assay has been run on the 36 ‘Carrizo’ plants. These plants did not report the GUS gene and are presumed negative. DNA from these plants have been purified and a PCR will confirm, but the plants are probably escapes. Finally, work has been done to create plasmid constructs for a silencing experiment. The citrus analog of NPR1 has been sequenced. Using this sequence, we hypothesize that two different constructs can lead to the same desired, silenced effect. The first construct is a full length gene inserted in reverse. The second is a dual vector, where the genes will hairpin due to their complementary nature. Currently, the second vector is half finished, as only one side has been confirmed, and the full-length plasmid needs to be tried again. Finishing these constructs and more transformation on the fresh seedlings will be the focus of next quarter.
The person hired to work on the project has not arrived and therefore no significant results can be reported.
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