The goal of this project is to transform the citrus and Arabidopsis NPR1 genes (CtNPR1 and AtNPR1), and the rice XIN31 gene into citrus, and to evaluate their resistance to both citrus canker (caused by Xanthomonas axonopodis pv. citri (Xac)) and greening diseases. The first year objectives include: (1) Molecular characterization of the transgenic plants; (2) Inoculation of the transgenic plants with Xac; (3) Inoculation of the transgenic plants with the HLB pathogen, and monitoring of the bacterium in planta with quantitative PCR; (4) Transformation of SUC2::NPR1 into citrus; (5) Plant maintenance. We have identified three transgenic lines overexpressing CtNPR1. These NPR1 overexpression lines were inoculated with 105 cfu/ml of Xac306 and the results showed high levels of resistance from the NPR1 overexpression lines, but not from the control plants. We also conducted growth curve analyses. Nineteen days after inoculation, the bacterial population in one of the NPR1 overexpression lines is 10,000 fold lower than that in the control plants. These results demonstrate that overexpression of CtNPR1 confers resistance to canker disease. We also graft-inoculated the NPR1 overexpression lines with greening to determine whether NPR1 is functional in greening resistance. We are in the process of monitoring Candidatus Liberibacter asiaticus populations in the inoculated plants using quantitative PCR. Five transgenic lines containing the SUC2::CtNPR1 construct, in which CtNPR1 is driven by a phloem-specific promoter from the Arabidopsis SUC2 gene, have been generated. This construct may increase the expression of CtNPR1 in citrus phloem thereby maximizing the opportunity for resistance to greening. In a few weeks when the plants produce enough leaf tissue, we will perform Northern blot analyses to monitor the levels of CtNPR1 transcripts. The transgenic plants with high levels of CtNPR1 will be propagated by grafting and inoculated with greening. Finally, we have initiated microarray analyses of the CtNPR1 plants in response to Xac inoculations. The results will be reported soon. To continue our research, we request funds for the third year to achieve the following goals as originally proposed: (1) Characterization of the CtNPR1 transgenic plants inoculated with the HLB pathogen; (2) Molecular characterization of the SUC2::CtNPR1 plants; (3) inoculation of the SUC2::CtNPR1 plants with the HLB pathogen; (4) Examination of changes in hormone (abscisic acid, auxin, jasmonic acids and salicylic acids) levels in the CtNPR1 plants infected with Xac; (5) Plant maintenance.
This research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllid feeding. Both protein-based and RNAi strategies were tested by feeding psyllids artificial diets. To support the artificial diet assays, we optimized the diet composition by adding an antimicrobial agent to eliminate fungal growth that is introduced by the psyllids during the assay period. Using this approach we identified suitable buffers and optimal diet pH during the feeding period. In separate experiments, Tryspin Modulating Oostatic factor (TMOF), a mosquito decapeptide hormone, and cysteine protease inhibitor (CPI) from the Asian Citrus psayllids that was identified in our laboratory were added to artificial diets on which psyllids were allowed to feed. After 10 days of feeding, 100% mortality was observed in psyllids feeding on diets containing TMOF or CPI, whereas, 40% mortality was found in psyllids feeding on the control diets. CPI fed psyllids caused a significant higher mortality than the controls after 7 days of feeding. Based on these observations a collaborative project with Dr. Bill Dawson’s laboratory (Univ. of Florida, IFAS, CREC, Lake Alfred, FL) was initiated to use a Citrus tristeza virus (CTV) expression vector to produce TMOF and CPI in the in the citrus phloem. Clones containing the sequences of mosquito TMOF and CPI (cathepsin protease inhibitor) (both shown to be effective against psyllids) were provided to Dr. Dawson’s laboratory. Once CTV vectors are constructed and used to infect citrus plants, Dr. Dawson’s laboratory will provide us with the plants to evaluate the effect on psyllids while feeding on these plants. In parallel to these studies, we synthesized dsRNA molecules targeting 11 different psyllids essential genes encoding three different classes of proteins (alpha-tubuliln, V-ATPase, and Cathepsins). Initial feeding studies with alpha-tubulin dsRNA and V-ATPase dsRNA caused ~60% psyllids mortality as compared to only ~30% mortality for psyllids fed a control diet containing an equal amount of dsRNA not specific to the psyllid. Using this information, we have initiated experiments to produce citrus plants that express these dsRNAs in citrus phloem cells. A collaborative project with Dr. Bill Dawson’s laboratory (Univ. of Florida, IFAS, CREC, Lake Alfred, FL) was initiated to use a Citrus Tristeza Virus (CTV) expression vector to produce the psyllid dsRNAs in the citrus phloem. Clones containing the sequences of a psyllid Vacuolar ATPase and Cathepsin protease (both shown to be effective dsRNA targets) were provided to Dr. Dawson’s laboratory. Once CTV vectors are constructed and used to infect citrus plants, Dr. Dawson’s laboratory will provide us the plants to evaluate the effect on psyllids while feeding on these plants. We have also designed expression vectors for the production of transgenic plants expressing the psyllid dsRNAs through Agrobacterium-mediated plant transformation. These will be used to initiate the production of transgenic citrus plants that constitutively produce these dsRNAs in the phloem.
Cross protection has been used as a control strategy against virulent stem pitting strains of Citrus tristeza virus. Although it has been shown that CTV cross protection only works against CTV isolates of the same genotype, its underlying mechanism remains unknown. Cross protection can break down over time, typically as a consequence of challenge by a new virulent strain of CTV. This study of cross protection was undertaken with a model system where a parental CTV isolate called Dekopon was a mixture of three genetically different CTV strains. The Dekopon isolate protected sour orange seedlings from expression of seedling yellows (SY) symptoms which is significant because SY is associated with virulence of CTV. The parental Dekopon mixture was separated by aphid transmission and the resultant sub-isolates induced different symptoms, as expected based on individual genotypes; namely, the VT- and T3- genotypes induced strong SY in seedlings of sour orange and Duncan grapefruit; whereas the NS genotype was very mild. Various combinations of these sub-isolates failed to show cross protection or at least at the level expressed by the parental strain. Thus, the Dekopon isolate contained the essential component(s) of symptom amelioration. Deep sequencing using the Illumina platform was undertaken to analyze small interfering (si) RNAs profiles in sour orange severely affected by SY and in cross protected sour orange. An accumulation of virus-derived siRNAs mapping to the 3′-end of the CTV genome was found to be common in all three strains. Contigs from these datasets identified sequences homologous to the 282 Kb Ctv resistance locus of Poncirus trifoliata, thus, providing evidence that this region is involved in siRNA pathways. A total of 50 micro (mi) RNA (regulators of gene expression) families had significant frequency of which 8 miRNA families showed differential expression after CTV infection. Analysis of putative mRNA (chemical blueprint resulting in a protein product) targets in the EST database revealed 433 were common to sour orange plants showing protection or strong SY symptoms. Other mRNA targets were specific to cross protected plants (873) or sour orange showing severe SY (753). The different mRNA targets identified suggested that different CTV strains induced different miRNA-mRNA interactions. The experimental procedures undertaken in this research were designed to explore the siRNA pathway involved in the Citrus-CTV interactions and to identify the siRNAs and miRNAs associated with host response. The understanding gained should provide a new insights on mechanisms of CTV disease expression and cross protection.
During October ‘ December 2010 the citrus agents collectively provided 22 educational events and 61 grove visits. These activities were designed to enhance knowledge and practice adoption to combat HLB in all commercial citrus producing areas of Florida. In an effort to meet the recommendation of the National Academy of Science to develop citrus health management areas (CHMAs), the agents along with state faculty have been actively working with citrus growers to aid in the formation of the recommended pest management areas. Throughout the state, approximately 32 CHMAs are actively functioning or in various stages of development. CHMAs are being implemented in all citrus producing areas and agents are continuing to schedule meetings and other educational events to promote to pest management areas. In October of 2010, the citrus agents conducted their annual fall grower meeting series. The program was titled ‘Citrus Management Strategies in a New Disease Era’. The program series was conducted in 6 locations (Arcadia, Bartow, Ft. Pierce, Immokalee, Sebring, and Tavares) and attended by 356 growers. Program presentations were conducted and presented by the citrus agents. Presented topics included: citrus black spot; psyllid management; growing young citrus trees in the greening era; and foliar feeding and SAR for citrus trees. These presentations are currently available on the citrus agent’s web site (www.citrusagents.ifas.ufl.edu) and can be viewed upon demand. The citrus agents individually conducted or participated in 17 additional programs where HLB or topics to minimize the impact the disease has on citrus at various locations around Florida. Attendance at these programs exceeded 350 growers. The agents are documenting conditions of citrus trees that are PCR positive with HLB a photo series. This web based series is updated monthly with photos from the same selected trees to document tree conditions over time. All citrus production areas are being represented in the series. These photos can be viewed from the citrus agents web page.
Early signs of boron deficiency symptoms have begun to develop on the plants subjected to low boron fertilization. HLB infected budwood for graft inoculation is being tested for Las titers and graft inoculation will be performed as soon as the budwood status has been confirmed. Las titer and microscopy samples will be taken monthly after inoculation. Determining the mechanism of this possible HLB symptom escape will provide important information about how Las causes disease and provide a possible strategy for HLB management by citrus growers.
This project aims to provide comprehensive knowledge of the genetic and biological diversity of CTV isolates in CA and to identify and put in place mild isolates of the virus that can provide sustained protection against severe SP isolates. As the first step we are conducting molecular characterization of CTV isolates (Folimonova lab) collected from various regions in California. This approach will provide important information regarding genetic diversity of CTV populations in this region required for further selection of mild protective isolates. To this date, 53 samples received from Vidalakis lab in 2010 (as nucleic acid extracts from isolates of CCCP collection) were assayed using conventional reverse transcription PCR with CTV genotype-specific primers designed to specifically amplify sequences of the T30 or VT strains. Majority of those isolates demonstrated presence of the VT genotype, while some contained the T30 strain. Thirteen isolates appeared to contain mixed populations of both genotypes. Further analysis of those samples with primers specific to other CTV genotypes (T68, T3, T36) is in progress. The results of molecular characterization will be then evaluated along with information obtained from biological indexing of those isolates on indicator hosts in order to select mild non-stem pitting isolates as potential protective candidates against severe stem pitting isolates which share the same genotype composition. In addition to conventional reverse transcription PCR technique, we are developing a CTV genotype-specific quantitative reverse transcription PCR method that will allow us not only to determine what are the components of each isolate but also to quantify the amount of each genotype component present in isolates that contain several different isolates. Also this technique will speed up our work by allowing us to process more samples in a less amount of time. Recently we received 45 additional samples from CCTEA collection. These samples are being prepared for further characterization using the approaches described above. In order to obtain an understanding what CTV genotypes are currently present in the southern part of California where no previous eradication occurred, we are planning to conduct additional sampling of the new material from this region (Vidalakis lab). Those samples will be then used for inoculation of greenhouse trees for bioindexing characterization (Vidalakis lab) as well as sent (as nucleic acid extracts) to Folimonova lab for further molecular characterization.
The long-term goal of this project is to identify early diagnosis markers and to understand the mechanisms of plant defense responses against bacterium Candidatus Liberibacter (Ca. L.), which causes one of the most devastating citrus diseases – Citrus greening or ‘Huanglongbing’ (HLB). By examining the responses of 30 different genotypes of citrus to Florida isolates of Ca. L. asiaticus under controlled conditions in the green house, tolerant genotypes as well as sensitive genotypes of citrus have been identified (Folimonova et al., 2009). This project aims to identify important small RNAs and mRNAs that encoding signaling genes that contribute to HLB defense responses, by profiling small RNAs and mRNAs from HLB-infected tolerant and sensitive genotypes. We expect that different set of small RNAs and mRNAs will be identified from the tolerant and sensitive genotypes. HLB-induced small RNAs may target negative regulators of plant defense and HLB-suppressed small RNAs should target positive regulators of defense pathways in tolerant genotypes. mRNA profiling will help identify and validate these targets. Similarly, HLB-regulated small RNAs and mRNAs may help identify important components in pathogenicity of the greening. We chose Nules clementine as a sensitive variety and Sun Chu Cha mandarin as a tolerant one for deep sequencing analysis because both of them are mandarins and the genome sequence of a mandarin was just released which will ensure the success of our project. Currently, Dr. Folimonova’s lab is helping preparing and collecting the plant material. We will construct and sequence small RNA libraries from grafting- and Psyllid-infected Nules clementine and Sun Chu Cha mandarin use Illumina high throughput deep sequencer. Currently, we are in the processing of preparing and collecting the material to validate our small RNA results from previous studies using HLB plants that were infected by psyllid transmission. Our previous studies used only the grafting infected HLB plants from Dr. Kim Bowman’s lab at USDA lab Florida. Madam Vinous sweet orange plants (represent sensitive group) and Citrus macrophylla plants (tolerant group) that were already infected with HLB via psyllid transmission, they are at early stages of infection (about 3 months ago they we put in the psyllid room and still remain there). In addition, bacteria-regulated small RNAs often target important regulatory components in plant defense pathways. In comparison with mRNA profiling data, we will identify a series of plant regulatory genes that may contribute to HLB-tolerance and resistance responses. We propose to use high-throughput deep-sequencing approach to profile the mRNA populations of these two distinct tolerant and sensitive varieties. The newly released genome sequence of a mandarin will be essential for the data analysis. This study not only leads us to the discovery of HLB-induced small RNAs for early diagnosis marker development, but also helps us understand the pathogenicity of citrus greening, and helps us identify important components in natural defense of HLB disease.
The purpose of this California Citrus Research Board-supported project is to develop and implement methods to extend the shipping and shelf-life of fresh citrus fruits. The postharvest life of citrus fruit is often ended by fungal decay pathogens; in arid production regions of the California and Arizona, the preponderance of losses are caused by green mold and blue mold, caused by Penicillium digitatum and P. italicum, respectively. Phosphites (also termed phosphonates) are salts of phosphorous acid (H3PO3), nutritionally ineffective forms of phosphorus with an oxidation state of +5, as opposed to phosphorus compounds of oxidation state +3 that are used in fertilizers and utilized by plants. Known to control diseases caused by Oomycetes, such as brown rot of citrus, they inhibit other fungi but their activity can be modest (Guest and Grant, 1991). They merit thorough evaluation because of their relatively benign environmental aspects. Most formulations are neutral in pH and, in soil, they oxidize to phosphate. This facilitates disposal of used solutions from packinghouse tanks and drenchers, compared to solutions that contain fungicides, sodium salts, or are alkaline. Phosphites are exempt from residue tolerances for many applications. In postharvest applications to fruit inoculated the prior day, brief immersion (15-60 sec) in 1 to 3% phosphite solutions controlled both green and blue molds significantly, and were particularly effective on mandarin cultivars where another fungicide alternative treatment, sodium bicarbonate, often performs poorly. For example, a 1 min immersion at 25oC in 2% calcium or sodium phosphite reduced green mold from 63.0% among untreated ‘Murrcott’ mandarins to 12.2 and 14.5%, respectively, while 30.5% decayed after 2% sodium bicarbonate treatment. Similar treatments at 50oC with calcium or sodium phosphite further reduced green mold to 6.5 and 6.9%, respectively, while 14.3% decayed after sodium bicarbonate treatment. In most tests, heating the phosphite solution consistently improved its performance. Mixing phosphites with imazalil, thiabendazole, fludioxonil/azoxystrobin, or pyrimethanil improved the effectiveness of these fungicides. Synergy with imazalil occurred with solutions at neutral pH and not at low pH. Applications of 1 to 3% (wt/vol) solutions of calcium or potassium phosphite to run-off one week before harvest to ‘Atwood’ navel oranges reduced postharvest decay from natural or artificial infections by approximately 50%, although these very high phosphite rates, greatly above those recommended when phosphites are applied to trees for other reasons, caused some defoliation. At lower rates (less than 1%), preharvest applications of phosphite did not control postharvest Penicillium decay. Tests to control preharvest brown rot, caused by Phytopthora spp., and in progress. Brown rot is usually minor in California in most seasons, but severe outbreaks are occurring this year because of the prolonged wet conditions in groves caused by exceptionally abundant rainfall. Phosphites were effective as a postharvest treatment, but their preharvest use for postharvest decay control was not promising. Amiri and Bompeix (2011) reported similar promising results with postharvest phosphite use on pomes. Postharvest registration of potassium phosphite was granted in 2010 in the USA. Amiri, A.; Bompeix, G., 2011. Control of Penicillium expansum with potassium phosphite and heat treatment. Crop Protect. 30:222-227. Guest, D.I.; Grant, B. 1991. The complex action of phosphonates as antifungal agents. Biol. Rev. 66: 159-187.
Continued efforts to improve transformation efficiency: ‘ Experiments to test or validate the enhancing effects of various chemicals for improvement of transformation efficiency in juvenile tissues continued. A journal manuscript was submitted on research showing that use of the antioxidant lipoic acid significantly improves transformation efficiency in Mexican lime; experiments to test this with commercial sweet oranges are underway. We continued with experiments to test the effects of various antibiotics / metabolites / herbicide on the transformation efficiency, including: kanamycin, hygromycin, mannose and phosphinothricin. ‘New publications supported by this grant:1. Dutt, M., D.H. Lee and J.W. Grosser. 2010. Bifunctional selection-reporter systems for genetic transformation of citrus: mannose and kanamycin based systems. In Vitro Cellular & Developmental Biology-Plant 46:467-476; 2.Orbovic, V., M. Dutt and J.W. Grosser. 2010. Seasonal effects of seed age on regeneration potential and transformation success rate in three citrus cultivars. Scientia Horticulturae 127: 262-266 Horticultural manipulations to reduce juvenility in commercial citrus: ‘ Working with Mr. Orie Lee and a commercial harvesting company (w/ Frank Rogers), a plan to collect meaningful yield and fruit quality from the St. Helena project was developed – with harvest expected later this month. Approximately 10 acres of trees planted 2.8 years ago include a juvenile Valencia budline (Valquarius) and precocious Vernia on more than 70 rootstocks. The majority of trees have a significant yield and the trial is showing significant rootstock affects on precocious bearing and early fruit quality – the best selections from this trial will be ideal candidates for testing with juvenile transgenics. Also of interest is the cultural program being used at the St. Helena project that mimics OHS principals but with reduced input. The trees have been grown with a UF research slow-release fertilizer mix (in cooperation with Harrell’s Fertilizer) and daily irrigation. Two trees were confirmed with HLB the first year; but even with bad neighbors, there has been no detected additional spread of HLB during the past year. Transformation of precocious but commercially important sweet orange clones: ‘ Transgenic plants of precocious OLL sweet oranges (a group of clones with Rhode Red quality that show high solids in young trees) were regenerated and successfully micrografted for further study of early flowering and transgene expression. Approximately 25 transgenic sweet orange trees from OLL selections were produced containing four different gene constructs. Progress was also made transforming OLL clones with the alternative embryogenic culture transformation system, as numerous transformed somatic embryos have been recovered. Progress was also made in the regeneration and characterization of plants containing the FDT transgenes for early flowering.
Objective 1 (confirm biofilm formation by X. citri subsp. citri (Xcc) in comparison with other bacteria that are well known to form biofilms). When grapefruit, Citrus macrophylla, Swingle citrumelo, and Carrizo were spray inoculated and kept under greenhouse conditions, aggregate formation was most prevalent on Swingle and Carrizo, reduced on C. macrophylla, and greatly reduced on grapefruit. Conversely, lesions were most prevalent on grapefruit, moderate on C. macrophylla, and infrequent on Swingle and Carrizo, although all four hosts developed lesions after injection-infiltration. This suggests a host interaction between biofilm formation and Xcc infection. Expression of genes, gumD, cheZ and rpf, involved directly or indirectly in biofilm formation is under evaluation. Detection of a gumD RNA fragment previously demonstrated to be a viability marker was higher in aggregated than in planktonic bacteria which is correlated with higher survival after 3 or 7 days in aggregates compared to suspension bacteria. Expression of cheZ involved in signal transduction for cell motility was initally higher at the onset of aggregate formation (3 dpi) but decreased once the biofilm was formed. This implies a role for bacterial motility at initial stage of biofilm formation. Expression of rfp involved in quorum sensing is lower in planktonic than in aggregating cells and in biofilm after 7days. This preliminary result confirms the importance of quorum sensing in the aggregation of Xcc bacteria which may serve as a target for disruption of the Xcc infection process. Objective 2. New disinfectants were evaluated for their ability to inhibit biofilm formation or disrupt bacterial aggregates. Zixvirox and Aquazix are used as surface disinfectants for food and have reported activity against biofilms. Aquazix is a mixture of hydrogen peroxide and silver as active ingredients. Zixvirox is a mixture of hydrogen peroxide and peracetic acid. Different concentrations of these products were tested against planktonic Xcc and as inhibitors of biofilm formation. To assess the effect on planktonic bacteria, culturability on LB medium plates and viability by membrane integrity (live-dead kit) assay were measured. Zixvirox at 0.03% and Aquazix at 2% were 100% effective for killing bacteria in suspension and complete disruption of cell integrity was confirmed by live dead assay. To evaluate the effect of these products on biofilm, aggregation was observed on borosilicate plates using a non stable Gfp strain and confocal laser scanning microscopy. At sublethal concentrations, Zixvirox was not completely effective for inhibition of biofilm formation because some bacterial aggregates formed. Aquafix at sublethal concentration prevented aggregation whereas bacteria were still viable in culture suspension. Although Aquazix and Zixvirox show promise as disinfectants, further assays are needed to confirm their activity as biofilm inhibitors. The effect of peroxide, CuSO4, SOPP, NaCl and NaClO at different concentrations was evaluated after 30 minute exposure of preformed biofilms on slides. Efficacy for disruption of bacterial aggregates varied with the compound. Treatments with SOPP, NaCl and NaClO disrupted the biofilm. CuSO4 or peroxide did not disrupt and may have stimulated biofilm formation based on the observed higher aggregation. Currently, assays are underway to evaluate the viability of bacteria in aggregates after bactericide treatment of the preformed biofilms. These assays are based on respiratory activity of the aggregate and fluorescence detection of non stable Gfp expressing strains.
In FL nurseries, rootstock seed trees are located outdoors and only protected from psyllid transmission of Candidatus Liberibacter asiaticus (Las) by insecticide applications. In 2008, a survey detected two Carrizo citrange trees as HLB+. Given the potential risk for seed transmission and introduction of Las into nurseries by seed from source trees, assays of seedlings derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008 seed were collected from mature Pineapple sweet orange trees in Collier Co. and in 2009 from Murcott tangor trees in Hendry Co., FL. For Pineapple orange, 415, 723 and 439 seedlings and for Murcott, 332 seedlings were tested at least twice by qPCR using 16S primers. In 2007, a single Pineapple seedling was suspect HLB+ but upon repeated testing was negative. From nurseries in 2008, 290 seedlings were recovered from fruit located on symptomatic branches of 2 Carrizo trees, and in 2009, 125 seedlings were recovered from 2 trees of Swingle citrumelo, 649 from 4 trees of ‘Kuharske’ Carrizo, 100 from 1 tree of Cleopatra mandarin and 100 from 1 tree of Sekwasha mandarin. In 2008, one suspect HLB+ Carrizo seedling was detected but HLB+ status was not confirmed after repeated testing. In 2009, a single questionable PCR detection for Cleopatra mandarin was obtained. Subsequent detection occurred in only 75 and 33% of repeated 16S runs from two DNA extractions and the assay was negative using beta-operon primers. Despite the occasional HLB+ test results, no plants have ever developed HLB symptoms and repeated testing has never confirmed anything other than the transient presence of Las in seedlings grown from seed obtained from Las-infected trees.
Preliminary work to explore optimal parameters for genetic transformation of Murraya has been carried out, 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 recently optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments have been initiated using the plasmid pTLAB 21 harbored in Agrobacterium tumefaciens strain EHA 101. This vector contains the genes for kanamycin resistance as a selection agent and GFP (green fluorescent protein) as an easily observed marker. 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 tested, to attempt establishment of an optimal and 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 has occurred from organogenic cultures of longitudinally cut seedling epicotyl segments, following these transformation experiments. Observations of the regenerating cultures have revealed several buds and shoots displaying green fluorescence, indicating successful genetic transformation. Their growth is being monitored, as well as the stability and uniformity of GFP expression over time, and further production of new transgenic events is underway.
Progress in the project was disrupted by the unexpected departure, with no advance notice, of the previous employee responsible for the research underway. Another employee was assigned responsibility and she has made excellent efforts to retain forward momentum and to make credible progress. Seeds of Murraya paniculata were procured from the USA-ARS National Clonal Germplasm Repository for Citrus and Dates, from a commercial company (Seeds & More), and from a local source in Florida. The limited seed availability and their relatively poor quality, along with consequent low germination rates and very poor growth, have been a hurdle in the process. Such seedling materials present a major challenge for Agrobacterium based transformation efforts. Therefore, a comprehensive experiment on screening optimal conditions for in vitro seed germination was conducted. Various culture media, using Murashige and Skoog’s (MS) basal salt, with or without GA3 (3 mg/l), with varying sugar (25, and 50 g/l) and agar concentrations (4, 6 and 8 g/l), have been examined, with a goal to achieve an acceptable seed germination rate. In order to achieve the high quality (with stronger stems) of seedlings necessary for the regeneration and transformation experiments, germinated weaker seedlings were subcultured twice on MS basal medium without any plant growth regulators for another month. Stronger seedlings obtained were used as explants for regeneration and transformation experiments.
Because of successful efforts to achieve acceptable seed germination rates and to produce more vigorous seedlings in vitro, higher quality seedling explant materials have been obtained. This accomplishment has enabled work to proceed to establish a suitable and efficient protocol for in vitro regeneration of Murraya, which can then be used for the various planned transformation experiments. Research was conducted to develop a suitable and robust organogenic shoot regeneration protocol, using various parts of germinated seedlings including hypocotyls (longitudinally cut vs. no cut), epicotyls (longitudinally cut vs. no cut), roots, leaves, and cotyledons. These explants were cultured on MS basal medium supplemented with various concentrations of BA, TDZ alone, or in combination with NAA. Two suitable and reasonably effective shoot regeneration protocols have been developed and optimized for transformation experiments (a manuscript is in preparation). Simultaneously, the capacity of shoot tips and lateral buds to undergo multiple shoot formation and multiplication in vitro has been examined. Shoot tips and lateral buds were excised directly from in vitro germinated seedlings and cultured on MS medium supplemented with BA at 0, 1, 3, and 6 mg/l. Newly formed shoots were subcultured on the same medium at 3 week intervals to determine optimal conditions for maximal shoot multiplication.
Quarterly report April 2011: Reserach to explore optimal various parameters for genetic transformation of Murraya was carried out, 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 recently optimized protocol for organogenic shoot regeneration from appropriate seedling tissues, transformation experiments have been initiated using the plasmid pTLAB 21 harbored in Agrobacterium tumefaciens strain EHA 101. This vector contains the genes for kanamycin resistance as a selection agent and GFP (green fluorescent protein) as an easily observed marker. 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 tested, and we established an optimal, 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 is being pursued.
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