REGENERATION SYSTEMS FOR PYRAMIDING DISEASE RESISTANCE INTO WALNUT ROOTSTOCKS John E. Preece, Ana María Ibáñez, Quyen Tran, Dio Gunawan, Chuck Leslie, David Tricoli, and Abhaya Dandekar ABSTRACT The purpose of this study was to develop an in vitro regeneration system to be able to insert selected disease resistance and other genes into walnut rootstock cultivars. Apical meristem explants of the hybrid walnut rootstocks ‘RX1’ and ‘VX211’ were pulsed with 0-30 µM 2,4-D for one week and incubated in darkness. Axillary Shoot production was progressively higher with increasing concentration of 2,4-D, with 66% of explants producing shoots when pulsed with 30 µM 2,4-D. The basal DKW medium contained cytokinins and auxin whether 2,4-D was present or not because our previous studies had showed that they were also necessary for shoot production. This establishes an apical meristem tissue culture system with sufficient survival and shoot production for transformation studies. Roots of ‘RX1’ were cultured on DKW or WPM with plant growth regulators and remained whiter and elongated on WPM that also contained activated charcoal. Nodal explants of ‘RX1’ were inoculated with Agrobacterium and are being cultured on selection medium containing kanamycin. OBJECTIVES The specific aims and activities of this study are: Specific Aim 1: Develop a meristem-based regeneration and transformation system for walnut clonal rootstocks. Activity 1: Develop a meristem-based regeneration system. Activity 2: Transformation of meristematic tissues. Specific Aim 2: Develop a regeneration system suitable for transformation of walnut clonal rootstocks based on somatic embryogenesis and/or shoot organogenesis from immature tissues of adult plants. Activity 3: Explant sources Activity 4: Tissue culture media formulations and nitrogen component Activity 5: Plant Growth Regulators Specific Aim 3: Stacking crown gall resistance into clonal Paradox rootstock clones. Activity 6: Transformation of crown gall resistance into VX211 and RX1 lines Activity 7: Testing transgenic lines of VX211 and RX1 for resistance to crown gall

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SIGNIFICANT FINDINGS Pulsing apical meristems with up to 30 µM 2,4-D for one week on DKW medium also containing 5 µM 6-benzyladenine (BA), 0.5 µM indolebutyric acid (IBA), 10 µM kinetin and then transferring to the same medium, but without 2,4-D and incubating in darkness resulted in up to 66% of explants producing axillary or shoots of undetermined origin. This rate may be improved by higher concentrations of 2,4-D; however it is sufficiently for apical meristems to be inoculated with Agrobacterium in transformation studies. Because somatic embryos and verified adventitious shoots have not been generated from apical meristem explants, an adventitious system for transformation for these rootstocks remains unavailable. Shoot apical meristems produce nodes, which in turn produce axillary buds that can be rooted and acclimatized as plants. Shoot apical meristems have been used successfully for genetic transformation of sunflower (Schrammeijer et al. 1990), sugarcane (Gambley et al. 1993), and cereal crops (Sticklen and Oraby, 2005). This is the next step in this project. Nodal and meristem explants have been inoculated with Agrobacterium in separate experiments and meristem and nodal explants will be inoculated in experiments in January 2013. Having a reliable tissue culture system for producing shoots from apical meristems of Juglans rootstocks is an important step for developing a system for transformation of walnuts that are sufficiently mature that they can be evaluated for phenotype. This may allow for a system to pyramid genes into rootstocks. PROCEDURES Specific Aim 1: Develop a meristem-based regeneration and transformation system for walnut clonal rootstocks. Activity 1: Develop a meristem-based regeneration system. Experiment 1. RXI and VX211 Walnut Shoot Regeneration from Apical Meristem Explants from In vitro Culture Shoot apices from ‘RX1’ and ‘VX211’ were harvested from in vitro culture and immediately dissected to produce explants composed of a shoot apical meristem plus leaf primordia (micro shoot tips). Meristems were placed on induction media composed of DKW basal medium supplemented with 5 µM 6-benzyladenine (BA), 0.5 µM indolebutyric acid (IBA), 10 µM kinetin, and 1 µM 2,4-D (DKW1) or 10 µM 2,4-D (DKW10), a control set of explants were placed in DKW without plant growth regulators (DKW0, Table 1). Explants were maintained on induction media for one week (Newman et al., 1993) and then transferred to DKW with the same levels of BA, IBA, and kinetin, but without 2,4-D; all were labeled as to the original 2,4-D treatment. During these 3 weeks, explants were incubated in darkness at 25ºC. Explants were then transferred to fresh media and 2 Petri dishes per 2,4-D treatment for each rootstock (ten meristems per Petri dish) were incubated in continuous darkness until the end of the experiment. Also, 2 Petri dishes per treatment combination and per each rootstock were incubated under a 16-h photoperiod (60 µmol s-1 m-2 photon flux provided by cool white fluorescent lamps) at 26ºC until the end of the experiment. There were 20 meristem explants for each rootstock x 2,4-D x light treatment combination; the experiment was repeated for a total of 240 meristems per walnut rootstock.

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Table 1. Effect of auxins and cytokinins hormones and photoperiod on embryo regeneration from ‘RX1’ and ‘VX211’ walnut shoot apical meristems DARK LIGHT Medium DKW1 DKW10 DKW0 DKW1 DKW10 DKW0 2,4-D (µM) 1 10 0 1 10 0 BA (µM) 5 5 0 5 5 0 IBA (µM) 0.05 0.05 0 0.05 0.05 0 Kinetin (µM) 10 10 0 10 10 0

Experiment 2. Effect of 2,4-D Concentration on Shoot Regeneration from ‘RXI’ and ‘VX211’ walnut meristems The focus of this experiment was shoot growth from meristem explants following one week pulses with different 2,4-D concentrations. The explants were incubated in darkness at 25ºC because the previous experiment has shown that the number of shoots regenerated is higher than when the explants are lighted. Shoot apical meristem explants from ‘RX1’ and ‘VX211’ in vitro shoot cultures were placed on DKW medium supplemented with 5 µM BA, 0.5 µM IBA, 10 µM kinetin, and 1 µM 2,4-D (DKW1), 3 µM 2,4-D (DKW3), 10 µM 2,4-D (DKW10) or 30 µM 2,4-D (DKW30, Table 2). Explants were maintained on induction media for one week and then transferred to DKW with the same levels of BA, IBA, and kinetin, but without 2,4-D; all were labeled as to the original 2,4-D treatment. Explants were transferred to fresh media every 2 weeks. There were 60 explants for each rootstock x 2,4-D combination and the experiment was repeated for a total of 240 explants per walnut rootstock.

Table 2. Effect of 2,4-D concentration on shoot regeneration from ‘RX1’ and ‘VX211’ walnut shoot apical meristems DARK Medium DKW1 DKW3 DKW10 DKW30 2,4-D (µM) 1 3 10 30 BA (µM) 5 5 5 5 IBA (µM) 0.5 0.5 0.5 0.5 Kinetin (µM) 10 10 10 10

Activity 2: Transformation of meristematic tissues. Transformation of ‘RX1’ axillary buds. Three weeks after growth began from ‘RX1’ nodal explants, shoots were excised and cut into 1.5 cm long explants containing 3 + 1 nodes and no apical bud in an attempt to insert the crown gall resistance gene contained on the binary vector (pDE00.0201). The Agrobacterium containing crown gall resistance gene (Escobar et al., 2001, 2002, 2003a and 2003b) was grown overnight, spun down, washed, and re-suspended to about 0.5 OD (600 nm) in an induction medium. All apices were excised from the excised shoots and 49 1.5 cm long axillary bud explants were

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inoculated with Agrobacterium containing the crown gall resistance genes and 69 were inoculated with a strain carrying an empty vector. Following inoculation and co-cultivation, explants were placed onto medium containing kanamycin to select for transformed shoots. Specific Aim 2: Develop a regeneration system suitable for transformation of walnut clonal rootstocks based on somatic embryogenesis and/or shoot organogenesis from immature tissues of adult plants. Activity 3: Explant sources Experiment 1. Somatic embryogenesis in root explants from ‘RX1’ walnut rootstocks Root explants from ‘RX1’ walnut rootstocks were excised to determine if they contained cells that can be induced to form somatic embryos. ‘RX1’ etiolated shoot tips from in vitro cultures of apical meristem explants were placed on DKW basal medium supplemented with 50 µM IBA for 5 days in darkness, then transferred to DKW basal medium and incubated under a 16-h photoperiod (60 µmol s-1 m-2) at 26ºC. After 14 days, 1 cm long roots were harvested from the etiolated shoots and placed on DKW supplemented with 5 µM BA, 0.5 µM IBA and 10 µM kinetin or WPM supplemented with 1 µM BA and 11 µM NAA to stimulate embryogenesis. There were 40 root explants per medium for a total of 80 explants. RESULTS AND DISCUSSION RXI and VX211 Walnut Apical Meristem Explants from in vitro Culture The percentage of meristems that survived and grew was higher when they were exposed to 10 µM 2,4-D than to 0 or 1 µM 2,4-D. Shoot and callus production were greater from meristems that where initially exposed to 10 µM 2,4-D than the lower concentrations. Detrimental effects on meristems related to longer 2,4-D exposure times was avoided by using the one week pulse. The results of this experiment led to the treatments in the next experiment, where two additional concentrations (3 and 30 µM) of 2,4-D are compared to those used in this experiment. ‘RX1’ and ‘VX211’ shoot apical meristems that were incubated under light grew into healthy green shoots after two months. Under darkness shoot apical meristems of both rootstocks were white and at first produced shoots (Fig. 1) and later callus while the shoots elongated. After 6 months, many of the etiolated shoots lost vigor and brown callus formed. No somatic embryos formed. ‘RX1’ has generally grown more vigorously than ‘VX211,’ especially when the medium contains no plant growth regulators.

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Figure 1. ‘RX1’ etiolated walnut shoots that grew from apical meristem explants that had previously been cultured for one week on DKW medium with 1 µM (left) or 10 µM 2,4-D (right) and incubated in darkness. Effect of 2,4-D Concentration on Shoot Regeneration from ‘RXI’ and ‘VX211’ walnut meristems In this experiment the shoot regeneration percentage was higher from meristems initially treated with 30 µM 2,4-D for one week than from those cultured with lower concentrations. The percent of explants with axillary or shoots of undetermined origin (adventitious or axillary, Fig. 1) was 32, 45, 55, or 66 when the culture medium contained 1, 3, 10, or 30 µM 2,4-D respectively. The rate of shoot growth with the highest concentration of 2,4-D. This leads to two planned experiments. First, pulses with higher concentrations should be tested to determine the effect on shoot production since the optimum rate remains undetermined. Second, shoot production is now sufficiently high for Juglans apical meristems to be inoculated with Agrobacterium in transformation studies. It is important that the tissue culture system that is used results in a reasonable number of shoots that can be selected and tested to determine if they are fully or partly transgenic. Transformation of ‘RX1’ axillary buds. Although still early in the experiment, all buds remain green and healthy without any sign of dying except for 3 buds that were blackened from overgrowth of the Agrobacterium. This experiment is one month old and ongoing. Somatic embryogenesis in root explants from ‘RX1’ walnut rootstocks ‘RX1’ roots on DKW medium grew callus with a soft, mushy texture that was brownish in color and surrounded the roots. No roots elongated. Roots on WPM medium also grew callus, but remained whiter than those on DKW and some of the roots elongated. No sign of embryos have observed at the time of this report. This experiment is one month old and ongoing.

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LITERATURE CITED Escobar, M.A., E.L. Civerolo, K.R. Summerfelt, and A.M. Dandekar. 2001. RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis. In: Proceedings of the National Academy of Sciences of the United States of America. 98(23): 13437-13442. Escobar, M.A., C.A. Leslie, G.H. McGranahan and A.M. Dandekar. 2002. Silencing crown gall disease in walnut (Juglans regia L.). Plant Sci. 163(3): 591-597. Escobar, M.A., E.L. Civerolo, V.S. Polito, K.A. Pinney and A.M. Dandekar. 2003a. Characterization of oncogene-silenced transgenic plants: Implications for Agrobacterium biology and post-transcriptional gene silencing. Molecular Plant Pathology 4(1): 57-65.

Escobar, M.A., and A.M. Dandekar. 2003b. Agrobacterium tumefaciens as an agent of disease. Trends Plant Sci. 2003 Aug;8(8): 380-386. Gambley, R. L., R. Ford, and G.R. Smith. 1993. Microprojectile transformation of sugarcane meristems and regeneration of shoots expressing β-glucuronidase. Plant cell reports, 12:343-346. Leslie, C.A., G.H. McGranahan M.L. Mendum, S.L. Uratsu, and A.M. Dandekar. 1997. Genetic Engineering of Walnut (Juglans Regia L.). Acta Hort. (ISHS) 442:33-42 http://www.actahort.org/books/442/442_3.htm Mendum, M.L. and G. McGranahan. 1995, Somatic embryogenesis of clonal Chandler. Walnut Research Reports: 25-27. Mendum, M.L., S. Hirbod, C. Leslie, and G. McGranahan. 2004. Somatic embryos of clonal Burbank paradox walnut. Walnut Research Reports: 47-49. Neuman, M.C., J.E. Preece, J.W. Van Sambeek, and G.R. Gaffney. 1993. Somatic embryogenesis and callus production from cotyledon explants of Eastern black walnut (Juglans nigra L.). Plant Cell Tissue and Organ Culture. 32:9-18. Schrammeijer, B., P. C. Sijmons, P. J. Elzen, and A. Hoekema 1990. Meristem transformation of sunflower via Agrobacterium. Plant Cell Reports. 9:55-60. Sticklen, M. B., and H. F. Oraby. 2005. Shoot apical meristem: A sustainable explant for genetic transformation of cereal crops. In Vitro Cellular & Developmental Biology-Plant, 41:187-200.

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