REGENERATION SYSTEMS FOR PYRAMIDING DISEASE RESISTANCE INTO WALNUT ROOTSTOCKS John E. Preece, Kourosh Vahdati, Ana Maria Ibanez, Patty Jo Compton, Quyen Tran, Dio Gunawan, Aman Duggal, Chuck Leslie, David Tricoli, and Abhaya Dandekar ABSTRACT This study was conducted to regenerate selected walnut rootstocks adventitiously. This is an essential step to be able to produce transgenic walnut rootstocks with superior traits, such as disease resistance. A series of plant tissue culture experiments were conducted on RX1 and VX211 rootstocks with the goal of regenerating plants for future transformation studies. Leaf, floral and microshoot tip explants were placed in vitro on media with different combinations of auxins and cytokinins. Because of a wet winter, floral explants were contaminated with microbes. Leaf explants produced callus, but not organized structures. It was determined that long exposure to 2,4- D was detrimental to growth. Microshoot tip explants established and responded well in vitro on the various media tested. Some microshoot tip explants began to grow preformed shoots. However, others appeared to form organized structures other than leaves that did not produce plants. In one experiment, at least half of RX1 microshoot tips produced 3-4 roots when exposed to low amounts of indolebutyric acid (IBA). A total of 200 microshoot tips of RX1 and 100 microshoot tips of VX211 were inoculated with Agrobacterium tumefaciens to confer crown gall resistance. A portion is still living on selection medium. OBJECTIVES The specific aims and activities of this proposal 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 California Walnut Board 57 Walnut Research Reports 2011
PROCEDURES Regeneration experiments Leaves were harvested from shoot cultures of RX1, VX211 and Vlach and were cut so that individual leaflets (with their midvein cut or not) and leaflets with a portion of the main leaf vein were placed on MS medium supplemented with different levels of benzyladenine (BA) and 2,4- dichlorophenoxyacetic acid (2,4-D, Table 1), thidiazuron (TDZ) and indolebutyric acid (IBA, Table 2), or TDZ and 2,4-D (Table 3). There were 1600 leaves and female flowers of RX1 and VX211 making 20 replications. Surviving leaf explants have been subcultured for 9 months on the media described in Tables 1-3. In a separate experiment, etiolated microshoots of RX1 and VX211 were excised and cut into 16 leaf and 16 internode explants. These were placed on the 20 different combinations of TDZ and 2,4-D described in Table 3. Calluses were transferred to the light after about 2 months. Microshoot tips (apical meristematic dome plus leaf primorida, 0.5 mm diameter, 20 replications) of RX1 were placed on each of the 20 media combinations described below in Table 2. Neuman et al. (1993) showed that short term exposure of J. nigra to 2,4-D can stimulate somatic embryogenesis. Additionally, Long et al. (1995) found that TDZ stimulated regeneration in J. nigra. Therefore an experiment has been initiated testing four media and using microshoot tips as explants. Microshoot tips of RX1 (16 replications) were placed on DKW medium containing 0.1 or 1.0 µm TDZ and 1 or 10 µm 2,4-D. After 4 weeks of exposure all explants were transferred to DKW medium with no plant growth regulators. This experiment is in progress. Results are preliminary, but the 4 weeks of exposure to 2,4-D may be too long. Therefore another experiment was initiated to study the effects of pulses with 2,4-D. All 16 reps of the microshoot tip explants were placed on DKW medium containing either 1µM TDZ and 1 or 10 µm 2,4-D for 1, 2 or 3 weeks, then transferred to DKW with no plant growth regulators. In a separate experiment, 16 replications of RX1 microshoot tips were placed in vitro on DKW medium containing 0.05, 0.5, 5, or 50 µm IBA for 7 weeks. They were then transferred to DKW with no plant growth regulators. Transformation studies Microshoot tips of RX1 and VX211 were exposed to Agrobacterium tumefaciens in genetic transformation studies to insert the crown gall resistance containing binary vector (pde00.0201). A total of 200 microshoot tips of RX1 and 100 microshoot tips of VX211 were excised from green or etiolated shoots and infected with Agrobacterium containing crown gall resistance genes described earlier (Escobar et al., 2001, 2002, 2003a and 2003b). The Agrobacterium was grown overnight, spun down, washed, and re-suspended to about 0.5 OD (600 nm) in an induction medium that is part of our existing protocol. A drop of this culture was placed on the surface of the meristem. Antibiotics were used to kill the Agrobacterium after co-cultivation. California Walnut Board 58 Walnut Research Reports 2011
RESULTS AND DISCUSSION In the study where leaves harvested from shoot cultures of RX1, VX211, and Vlach and placed on the broad spectrum media described in Tables 1-3 were subcultured for 9 months. Some continue to produce brown callus tissue. Long term exposure to 2,4-D appears detrimental to growth because many of these have died. All floral tissue became contaminated because of wet weather throughout early 2011. When etiolated microshoots of RX1 and VX211 were cut into internode and leaf explants, the best callus growth was when the medium contained TDZ and no 2,4-D. If 2,4-D were present, the callus was brown. Increasing concentrations of TDZ caused more callus production. These preformed meristems have been much more responsive than leaf explants (Fig. 1). Microshoot tips will grow into shoots. To date all studies have been in darkness. Light should allow for normal microshoot growth from the microshoot tips. Some structures formed that appeared more embryo-like than leaf-like; however, they did not develop further. Callus growth eventually overtook structures. This may have been because of prolonged to the potent cytokinin TDZ (Huetteman and Preece, 1993). Generally, RX1 explants grew more vigorously and were more responsive than explants of VX211. Both have J. regia as their male parent, but RX1 has J. microcarpa and VX211 has J. hindsii as the female parent. From these large scale experiments, it can be concluded that prolonged exposure to 2,4-D is detrimental to walnut explants. Therefore, experiments have been initiated with short pulses of 2,4-D. Floral tissue should be studied in future experiments when they can be collected earlier in the year. A poster was presented recently by Paula Pijut (USDA Forest Service, Purdue University) showing adventitious shoots forming from Juglans nigra leaf pieces. Light, rather than darkness was used for incubation. Therefore, leaf explants remain of interest. Additional experiments have focused on microshoot tip explants because they are the most promising to date for regeneration and the production of plants. In the experiment where microshoot tips were cultured on media with IBA and no cytokinin, all explants exposed to 50 µm IBA died, indicating toxicity. Explants on medium with 5 µm IBA produced a highly friable callus with a slimy texture that we have not seen before. In the one run of the experiment with results to date, 5 of the 8 microshoot tip explants produced a mean of 3.4 roots/explant that produced roots when cultured on DKW containing 0.05 µm IBA. When cultured on medium with 0.5 µm IBA, 4 of the 8 explants produced a mean of 4 California Walnut Board 59 Walnut Research Reports 2011
roots on the explants that produced roots. Exposure to IBA is a typical method to stimulate root initiation. It is good to see its stimulatory effects on RX1. This will be helpful in the production of transgenic plants if microshoot tips are to be transformed. In the transformation studies, most of the microshoot tips turned brown on selection media. Some explants remained contaminated by the Agrobacterium. The remainder is alive and growing on the selection media. Most of the future studies will focus on microshoot tips as explants because they respond the best of explants that we have tested to date. However, immature flower parts remain to be tested without contamination. We will also inoculate putative RX1 embryogenic/organogenic callus and microshoot tip explants with Agrobacterium tumefaciens containing the scorable marker gene dsred (Bevis and Glick 2002, Limpens et al., 2004) a non-destructive scorable marker of transformation, which will allow us to easy monitoring of transformation and regeneration of the RX1 embryogenic/organogenic callus. Transformation efforts will focus on inserting the crown gall resistance gene into these clonal rootstocks. Various infection methods will be tried. Since microshoot tips are sensitive to all forms of stress we will also try to inhibit cell death by coinfiltration with other Agrobacterium strains like one expressing Bcl-xL as described by Khanna et al., 2007. We will also try other types of Agrobacterium that stimulate virulence to improve the transformation frequency. Additional in vitro studies will focus on pulses of plant growth regulators and nutrient media. Effects of light will be another important factor for obtaining normal shoot outgrowth from microshoot tips. In vitro studies will focus in two directions. Obtaining somatic embryogenesis and/or shoot organogenesis and stimulating preformed shoot growth from microshoot tips. LITERATURE CITED Bevis, B.J. and Glick, B. S. 2002. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nature Biotechnology 20, 83 87. 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. California Walnut Board 60 Walnut Research Reports 2011
Huetteman, C.A. and J.E. Preece. 1993. Thidiazuron: A potent cytokinin for woody plant tissue culture. Plant Cell Tissue and Organ Culture. 33:105-119. Khanna, H.K. J-Y. Paul, R.M. Harding, M.B. Dickman and J.L. Dale. 2007. Inhibition of Agrobacterium-induced cell death by antiapoptotic gene expression leads to very high transformation efficiency of banana. MPMI 20(9): 1048-1054. Limpens, E. Ramos, J., Franken, C., Raz, V., Compaan, B., Franssen, H. Bisseling, T. Geurts, R. 2004. RNA interference in Agrobacterium rhizoges-transformed roots of Arabidopsis and Medicago truncatula. Journal of Experimental Botany, Vol. 55, No. 399, pp. 983-992. Long, L.M., J.E. Preece, and J.W. Van Sambeek. 1995. Adventitious regeneration of Juglans nigra L. (eastern black walnut). Plant Cell Reports 14:799-803. 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. California Walnut Board 61 Walnut Research Reports 2011
Table 1. BA and 2,4-D factorial arrangement of treatments in a broad spectrum experiments on leaf and floral explants. BA (µm) 0.44 0.88 2.22 4.44 8.88 2,4-D (µm) 0.00 B1 B2 B3 B4 B5 2.27 B6 B7 B8 B9 B10 4.53 B11 B12 B13 B14 B15 9.06 B16 B17 B18 B19 B20 Table 2. TDZ and IBA factorial arrangement of treatments in a broad spectrum experiments on leaf and floral explants and microshoot tips. TDZ (µm) 0.0454 0.227 0.454 2.27 IBA (µm) 0.000 I1 I6 I11 I16 0.049 I2 I7 I12 I17 0.490 I3 I8 I13 I18 2.450 I4 I9 I14 I19 4.900 I5 I10 I15 I20 Table 3. TDZ and 2,4-D factorial arrangement of treatments in a broad spectrum experiments on leaf and floral explants. TDZ (µm) 0.227 0.454 2.27 4.54 9.08 2,4-D (µm) 0 T1 T5 T9 T13 T17 4.53 T2 T6 T10 T14 T18 9.06 T3 T7 T11 T15 T19 13.62 T4 T8 T12 T16 T20 California Walnut Board 62 Walnut Research Reports 2011
Figure 1. Growth from RX1 microshoot tip explants on media containing TDZ and IBA. Top left, normal shoot growth from a microshoot tip explant. Top right and bottom, mixture of leaves with trichomes and other organized structures. California Walnut Board 63 Walnut Research Reports 2011