CLONAL PROPAGATION OF WALNUT ROOTSTOCK GENOTYPES FOR GENETIC IMPROVEMENT 2010

CLONAL PROPAGATION OF WALNUT ROOTSTOCK GENOTYPES FOR GENETIC IMPROVEMENT 2010 Wes Hackett, Chuck Leslie, Joe Grant, Bruce Lampinen, Gale McGranahan, Kathy Anderson, Bob Beede, Rick Buchner, Janet Caprile, Carolyn DeBuse, Janine Hasey, Nicolas Manterola, Reid Robinson, Dan Kluepfel, Greg Browne, and Mike McKenry ABSTRACT This year we produced more than 3800 liner-sized plantlets of 21 genotypes for use in greenhouse pest and disease screening and for growing in nurseries to a size large enough to graft and use in orchard trials. In addition, we produced over 1300 liner plantlets of 29 lines transformed for resistance to crown gall for greenhouse re-testing of susceptibility to gall formation. A field plot, established at UC Davis for horticultural evaluation of these lines, was partly grafted to Chandler and continues under observation for horticultural and disease performance. Six transgenic rootstock lines which show complete immunity to crown gall based on greenhouse screens will be proposed for regulatory approval and further testing in nursery and orchard trials. A new soil mix for growing liner plantlets, consisting of peat:perlite (50:50) plus lime and micronutrients, and which gives better growth than UC mix, was developed. A stageof-development and time-of-planting experiment shows that planting leafy, quiescent liner plantlets in late summer is the best practice based on survival and size of the resulting trees after growth in the nursery. A non-replicated field trial of 16 clonal rootstock/ scion combinations suggests, based on scion growth over two seasons, that there may be some unexpected rootstock/scion interactions in walnut. Field trials of clonal rootstocks continue to be developed by farm advisors in both replant situations and new orchard plantings at diverse locations to test the horticultural performance of rootstocks selected for disease and pest resistance and virus tolerance. Clonal rootstocks are now in commercial production by several laboratories and nurseries. GOAL AND OBJECTIVES The goal of this project is to provide the California walnut industry with new clonal rootstocks selected or designed to combat the most threatening pests and diseases. The overall objective is to devise clonal methods of propagation for candidate genotypes and to provide clonal plantlets so that they can be evaluated in greenhouse and field replicated disease and pest challenge tests. PROCEDURES AND RESULTS Propagation methods: We have used two approaches to clonally propagate candidate rootstock genotypes with nematode, crown gall, Phytophthora, Armillaria or blackline tolerance or resistance: A. Tissue culture micropropagation with in vitro and ex vitro rooting of microshoots. B. Dormant hardwood cuttings on bottom heated beds. California Walnut Board 65 Walnut Research Reports 2010

Tissue culture micropropagation: This year we produced over 3800 liner plantlets of 21 genotypes for replicated disease and pest screening tests by Greg Browne, Dan Kluepfel and Mike McKenry (Table 1). We also produced about 1400 liner plantlets of 28 lines transformed for putative resistance to crown gall plus non-transformed control lines for use in greenhouse screening re-tests for crown gall resistance (Table 1). Note that survival of rooted microshoots is high (60-80%) for most genotypes except for English, black and some backcross genotypes. To alleviate this poor survival of some genotypes we are working on methods using etiolated shoots of English and backcross genotypes to improve rooting. If successful this should also improve survival. We also provided 40 liner plants of each of 18 genotypes for use by Greg Browne for screening for Phytophthora citricola and P cinnamomi resistance in large plant screens. This past year s production plus hold-over plantlets from the previous year s production gives us a current inventory of about 4800 liner plantlets ready for immediate use in 2011 (Table 2). Growing liner plantlets in peat-perlite soil mix: To facilitate more rapid screening for Phytophthora resistance by Greg Browne, we developed a new system for growing liner plantlets in peat:perlite (50:50) plus lime( 84 gm/ft3) potting mix in 2 3/8 x 2 3/8 x 3 ¾ inch deep Anderson pots rather than a UC mix with equal parts of peat:sand:firbark in 1 x 7 inch Tree- Tainers. The new system facilitates easy inoculation of the soil with cultured Phytophthora species. We thought this change to a new soil mix would be a straight forward process but it turned out that the plantlets in the peat-perlite plus lime developed a crinkled leaf symptom with reduced growth. Analysis of soil samples showed that the ph of the peat-perlite plus lime mix was about 7.2 while that of the UC mix was 6.6. Consultation with plant nutrition experts suggested that the crinkled leaf symptoms could be due to a micronutrient deficiency, possibly nickel based on similar symptoms known to occur in pecan. These two pieces of information led us to hypothesize that the crinkled leaf symptoms we observed were due to micronutrient deficiency induced by high ph, possibly a nickel deficiency, even though the Hoagland fertilizer solution being used contained micronutrients. To test this hypothesis we conducted an experiment in which we supplemented the peat:perlite plus lime by adding a commercially available blend of micronutrients (Micro-Max, 17 gm/ft 3 ) or lowered the ph to 5.7 by reducing the amount of lime by 50% (42 gm/ft 3 ). As shown in Table 3, the crinkled leaf symptom was eliminated by supplementing the peat:perlite medium with micronutrients and was reduced by lowering the ph. In addition, plantlets grown in peat-perlite plus lime plus Micro-Max are observed to grow more vigorously than those in UC mix. Using the improved peat-perlite mix, we have now provided Greg Browne with about 100 liners plantlets of five rootstock genotypes with varying degrees of resistance or susceptibility to P. cinnamomi and P. citricola which he has used to validate the use of small plants for screening for resistance. We have also produced an additional 250 plantlets in the new peat-perlite mix for his use as needed. Use of small liner sized plantlets for Phytophthora screening should reduce the resources needed for production of plants and the time required for Greg to screen for resistance. Screening small liner-sized plantlets for crown gall resistance: Based on the procedure we described in our 2009 report, we are now routinely using liner-sized plantlets from cold storage in screening trials for crown gall resistance. Table 4 shows the screening results of diverse genotypes and putatively crown gall resistant transgenic lines. We evaluated material using three measures of galling: gall score (estimate of the percent of the stem circumference that the gall surrounds), gall volume (gall height x width x depth) and the ratio of gall volume to stem California Walnut Board 66 Walnut Research Reports 2010

diameter. Note that a very high percentage of the lines with the inserted transgene for resistance show immunity or near immunity to galling, while the control lines form very large galls. Based on these results and preliminary observations of vegetative vigor, we are proposing six lines (J1 1A, J1 19a, J1 20A, RR4 4A, RR4 10A, and RR4 12A) for regulatory approval and plan to propagate these for further testing in nursery and orchard trials. We are also using the small plant screen to try to establish the relative susceptibility or resistance of black, English, paradox and backcross genotypes to crown gall. Based on both gall size and gall ratio it appears that we can detect some differences in galling severity between genotypes, with wingnut exhibiting the least severe galling followed by RX1. The black walnut selection W17 showed the most severe galling followed by UZ229, WIP3 and PX1, although the results for WI7 and for the English genotypes are based on a limited number of replicates. Hardwood cuttings: Hardwood cuttings were used to clonally propagate for re-testing 15 open pollinated seedlings from mother trees representing 3 species from the USDA National Repository that had no previous gall formation or only small gall formation 3 months (2 or 3 growing seasons) after inoculation with a virulent strain of Agrobacterium tumefaciens. A total of about 180 cuttings were made in early February using the protocol described in the 2009 report. A total of 120 rooted cuttings were grown in 1.5 liter Cetap pots for re-testing by Dan Kluepfel s laboratory. Nursery trial - determining optimum stage of plant development and time of year for transplanting liners to the nursery: Optimizing survival and growth of micropropagated liner plantlets in the nursery row is very important for successful commercial deployment of disease resistant walnut rootstocks to the California walnut industry. Because liner plantlets are propagated in tissue culture laboratories and then greenhouse grown, they may not be at the optimal stage of development and well acclimatized for transplanting to an outdoor nursery environment at the traditional time for transplanting (late spring- summer). We tested the effect of the developmental state of the liner plantlets and the time of transplanting to the nursery row both on survival and growth, using what we think are near optimal cultural practices. Drip-tape lines directly below the rootball provided irrigation frequently to keep the rootball at near optimal moisture at all times during new root establishment. Fertigation was provided weekly during establishment and during the periods of growth. The liner planting times were late summer (9/14/09), late fall (12/10/09), and mid-spring (1/29/10). For each planting time, one set of plantlets was given a cold treatment (42-45F) for five to eight weeks to ensure that the dormancy requirement was fulfilled and that the plantlets had maximum potential for growth. It was intended that a second group of plantlets would go quiescent in the greenhouse (leafy but no new leaves being formed) and a third group would be actively growing in the greenhouse at the time of transplanting. Due to the condition of the plants received from the laboratory supplier, the latter two conditions were difficult to attain, so most greenhouse-treated plants were leafy and quiescent for the 9/14/09 and 12/10/09 planting times and actively growing for the 4/29/10 planting time. Table 5 shows the results for survival and trunk diameter taken two inches above the soil of dormant plants on 12/10/10. Notice that survival was generally better for the 9/14/09 and the 4/29/10 planting dates than for the 12/10/09 planting date. Survival was 90-100% for the best California Walnut Board 67 Walnut Research Reports 2010

pre-treatments and 0% for the worst. Cold pre-treatment tended to decrease survival for the 9/14/09 and 12/10/09 planting dates but had little effect on the 4/29/10 planting date. This poorer survival for the cold- treated late summer and late fall plantings may be because the cold-treated plantlets had more growth potential and were still actively growing and susceptible to injury when the first frost conditions occurred. For plantlet growth as measured by trunk diameter, the 9/14/09 planting date was substantially better than the 4/29/10 planting date for VX211 and Vlach and marginally better for RX1. Based on both plantlet survival and plantlet growth, the best practice, as indicated by these results, is to plant quiescent leafy plantlets in late summer. The superior growth of the late-summer-planted plantlets (9/14/09) maybe due to the establishment of a root system during the fall which provides a larger root system for water and nutrient uptake the next spring as compared with the liner plantlets that are planted in spring after the danger of frost is past (4/29/10). Field Trials: Rootstock/scion interactions: A non-replicated trial consisting of 16 clonal rootstock/scion combination using bare-root nursery size trees derived from root-grafts was established in the spring of 2008 at the Plant Pathology Experimental Fields at UC Davis. Each combination consisted of from three to 11 trees with a total of 94 trees. All but two of the combinations had five or more trees. The trees were planted in three rows six feet apart with 18 inch spacing between trees in the rows on 3/8/08. Trees of each combination were planted consecutively in the rows with no replication or blocking. Trees were unpruned for the duration of the experiment. The plot was furrow irrigated and fertilized using standard Plant Pathology Department cultural practices. Trunk diameter (just above the graft union) and height measurements were made on 7/29/10. The data presented in Tables 6 and 7 suggest, as expected, that rootstock clone has an effect on both trunk diameter and height of the scion cultivar grafted on it. Surprisingly, all three scion cultivars appear to be larger on RX1 than on VX211, the most vigorous clonal rootstock for which we have data. On the date that the growth data were taken, the Howards on RX1 had darker green leaf color than the Howards on VX211, which exhibited more yellowish colored leaves. Interestingly, the UX022 rootstock produced the largest trees of Chandler and Tulare but the smallest trees of Howard. This preliminary data suggests that there may be unexpected rootstock/scion interactions in walnut. Crown gall resistance rootstock trial: A field trial of lines transformed for resistance to crown gall, non- transformed back ground genotypes as controls, and other rootstock genotypes of interest, has been established on the UCD campus and was partially budded to Chandler this year. This trial continues to be evaluated for horticultural performance and natural occurrence of crown gall. This block includes budded trees of the six best genotypes recently selected to move forward in the testing and potential release process. Scion and rootstock material from this trial have been, and will continue to be, used for work now in progress to assess any possible transgraft union movement of DNA, RNA or other macromolecules. Farm advisor/grower county rootstock trials: Currently established clonal rootstock field trials in grower orchards in the various counties are summarized in Table 8. Most of the initial clonal rootstock field trials were established in replant situations but farm advisors have now established a number of clonal rootstock trials at newly California Walnut Board 68 Walnut Research Reports 2010

planted orchard sites in diverse counties, several with replicated plot designs, and with a variety of scions and soil types. Rick Buchner established a trial of RX1, VX211, and Vlach in a Tehama County orchard (H. Crain) in 2009 using nursery-grown trees developed from commercially produced liners. The clonal rootstock genotypes were planted in a replicated design and budded to Howard (see separate report in this volume). Joe Grant and Joe Conant established plantings near Wheatland of RX1, VX211, and Vlach grafted or budded to Ivanhoe, Sexton, Howard, and selection 91-077-40. A portion of these plantings are under power lines and evaluations will include observation of rootstock and scion effects on tree height. Carolyn DeBuse has planted a replicated clonal rootstock trial in Solano County (Cilker) using trees grown at Suchan Nursery from liners produced at UCD.This trial is on a good soil site and includes RX1, VX211, Burbank and Vlach (see report in this volume). A VX211 planting in Kings County (Verboon) established last year by Bob Beede includes VX211 grafted to Tulare in a fumigation treatment trial with Vlach planted in the buffer rows. Three trials of cherry leafroll-tolerant rootstocks have been established by Janet Caprile and Joe Grant and are currently under evaluation. These are located in Contra Costa County (see Caprile report in this volume) and San Joaquin County (see San Joaquin County section of this report). Several additional rootstock trials to evaluate performance of English cultivars on their own roots have been established by farm advisors in Butte, Yuba, and Stanislaus counties. Observations for these have been reported in recent volumes. See also summary of rootstock trial locations in Table 8. San Joaquin County rootstock trials - Joe Grant: Concar Ranch Trial: Performance of clonal Paradox and blackline-tolerant walnut rootstocks in San Joaquin County Project leader: Joe Grant Cooperating personnel: Brett Lagorio, Concar Ranch, Linden Bonilla Nursery, Oakdale Location: North of Flood Road, East of Escalon-Bellota Road, approximately 5 miles east of Linden, San Joaquin County Approximate GIS location: 38 1 32 N 120 59 0 W Soil at the site is Redding Gravelly Loam. The site was previously farmed as irrigated pasture. Prior to planting the soil was ripped (two directions) to six foot depth. There is a moderately compacted sandstone layer (4 thick) at approximately four foot soil depth. Scion variety is Chandler. Rootstocks include Own-rooted Chandler, RX1 clonal Paradox, VX211 clonal Paradox, WIP3, Vlach (June-budded and standard budded), AZ025, and seedling Paradox (June-budded, J. hindsii x J. regia per Bonilla Nursery). Spacing is 17 x 22. Irrigation since planting is by single-line above-ground drip. California Walnut Board 69 Walnut Research Reports 2010

Experimental design: Randomized complete block design with five four- to eight-tree replications. All trees were hand-planted 2/18/2008 as finished bare-root nursery trees. First- through thirdyear vegetative growth was very good. All trees had developed multiple 6 to 8 foot tertiary scaffolds by end of 2010 season. Own-rooted Chandler trees have noticeably more upright growth habit than those on other rootstocks. Trunk diameters after the second and third growing season were largest for own-rooted Chandlers, smallest for VX211, with other rootstocks intermediate. Mean trunk diameters (in inches), taken 24 above the soil line at the Concar Rootstock trial: Rootstock 12/8/2009 12/10/2010 Own-rooted Chandler 2.7 a 4.3 a RX1 2.5 b 3.8 b Paradox seedling (J. hindsii x J. regia) 2.4 bc 3.7 bcd Vlach (Standard budded) 2.4 cd 3.6 cd WIP3 2.4 cd 3.8 bc Vlach (June-budded) 2.4 cd 3.6 cde AZ025 2.3 de 3.5 de VX211 2.3 e 3.4 f Chiappi Farms Trial: Performance of RX1 clonal Paradox walnut rootstock in San Joaquin County Project leader: Joe Grant Cooperating personnel: Tony Chiappe, Chiappe Farms, Stockton, CA Greg Browne, USDA-ARS, Davis Burchell Nursery, Oakdale, CA Location: South Highway 4, east of Hewitt Road, approximately 1.8 miles west of Farmington, San Joaquin County Approximate GIS location: 37 55 39 N 121 1 59 W Soil at the site is Archerdale Clay Loam. The site was previously planted to walnuts which died from Phytophthora root rot (isolated and identified from root and soil samples as P. cinnamomi), and were removed one or two years prior to planting. Tree sites were pre-plant fumigated with 1 pound methyl bromide. Rootstocks include RX1 clonal Paradox and seedling Paradox (J. hindsii x J. regia per Burchell Nursery). Tree site spacing is 28 x 28. Each tree site is planted to two trees - one RX1 and one Paradox seedling - spaced about 2 apart, paired roughly by tree size at planting. Irrigation is by impact sprinklers. Experimental design: Randomized complete block design with ten ten-tree (paired tree) replications. All trees were hand-planted in May 2008 as bare-root un-grafted nursery whips and budded in August 2010 to Serr. California Walnut Board 70 Walnut Research Reports 2010

There was no first-year tree mortality, though some seedling trees appeared weak and grew noticeably more poorly than RX1 trees. Tree growth, as measured by trunk circumference increment, was similar for the two rootstocks: Trunk circumference (cm) of trees at Chiappi Farms Rootstock Trial 7/15/2010 12/10/2010 % Change Paradox seedling 5.4 7.4 37.9 RX1 5.5 7.7 39.7 Anderson Barngrover Trial Project leader: Joe Grant Cooperating personnel: Dave Taylor, Anderson Barngrover Ranch, Linden, CA Location: Comstock Rd, San Joaquin County Planted 2004, 12 X 24 hedgerow, Chandler scions Trial includes WIP 3, WIP, and WIP6. The number of trees is variable. English seedling trees produced by Dave Taylor are inter-planted between the clonal rootstock. Observations for 2010: trunk circumferences taken 1 foot above soil WIP3 (n=11 trees). trunk circ. = 53.5 cm One of the 12 original trees has died. WIP 5 (n=3). trunk circ. = 56.3 cm. No trees have died since planting WIP 6 (n=4). trunk circ. = 44.4 cm. No trees have died since planting English seedling trees (n=19). trunk circ. = 50.0 cm. No trees have died since planting Based on visual observation while walking the plot in December 2010, the scion portion of the English trees looked 20% larger/more vigorous than WIP3 and 40% more than WIP 6. The WIP5 certainly appeared not more vigorous than the English seedlings. Generally WIP clones appeared to be less vigorous, especially WIP6. Nursery Propagation and Commercialization: We are prepared to provide cultures of microshoots to any laboratory or nursery that wants them for licensed production of plants. We can also provide microshoots of Vlach, a public domain clone, to any laboratory or nursery that wants to produce it. Appendix 1 of this report includes a list of laboratories currently licensed for in vitro production of clonal rootstocks RX1 and VX211 and for sale of clonal rootstock plantlets as liners for nursery or orchard planting. California Walnut Board 71 Walnut Research Reports 2010

Table 1. (part 1). Greenhouse survival of rooted clonal microshoots 2010. # Rooted # Survived % Survival PDS Paradox AX1 102 56 55 AZ025 81 62 77 Burbank 147 70 48 DAR 51 35 69 Px1 155 91 59 RX1 752 629 84 Vlach 454 327 72 VX211 800 591 74 2542 1861 73 Transgenic Crown Gall Resistant J1 1A 99 75 76 J1 2A 44 16 36 J1 3A 79 48 61 J1 7A 277 141 51 J1 10A 24 13 54 J1 11A 150 105 70 J1 12A 79 53 67 J1 13A 131 84 64 J1 17A 118 74 63 J1 19A 75 58 77 J1 20A 38 17 45 J1a control 134 65 49 J21 2A 27 13 48 J21 3A 22 11 50 J21 4A 20 10 50 J21 5a 73 33 45 J21 6A 52 36 69 J21a control 74 27 36 RR4 1A 59 32 54 RR4 2B 23 10 43 RR4 3A 76 43 57 RR4 4A 32 20 63 RR4 6A 3 0 0 RR4 6C 35 19 54 RR4 8A 94 66 70 RR4 10A 88 64 73 RR4 11A 288 180 63 RR4 12A 39 30 77 RR4 control 49 25 51 2302 1368 59 California Walnut Board 72 Walnut Research Reports 2010

Table 1 (cont.). Greenhouse survival of rooted clonal microshoots 2010. # Rooted # Survived % Survival CLRV Tolerant Backcross 84-116-1 298 77 26 WIP2 91 32 35 WIP3 41 8 20 WIP4 71 47 66 WIP6 66 16 24 567 180 32 English Chandler 127 34 27 Serr 22 3 14 149 37 25 Black W17 267 107 40 Wingnut/hybrids WNBxGRZ 1a 99 52 53 WNxW 10.05 b 151 148 98 250 200 80 Nematode selections #21 1423 1077 76 B2-12 126 111 88 RX032 341 243 71 UZ 229 3 3 100 1893 1434 76 Total 7970 5187 65 California Walnut Board 73 Walnut Research Reports 2010

Table 2 (part 1). Inventory of chilled or partially chilled (as of 12/1/10) clonal rootstock plant available for use in disease and pest resistance trials and field trials in 2011. Genotype Plants with full chilling Plants with partial chilling Re-grown after chilling Total chilled plants available Paradox AX1 8 8 0 16 AZ025 40 30 0 70 Burbank 10 0 0 10 DAR 15 0 0 15 PX1 44 6 6 56 RX1 293 173 251 717 Vlach 287 37 24 348 VX211 488 131 93 712 Black W17 39 51 0 90 Wingnut and hybrid WNBxGRZ 1a 16 0 0 16 Wingnut 10.05 0 90 0 90 CLRV tolerant backcross 84-116-1 28 20 0 48 WIP3 8 4 0 12 WIP4 29 0 2 31 Nematode selections # 21 952 238 0 1190 B2-12 96 51 0 147 RX032 185 41 0 226 UZ229 3 2 61 66 English Chandler 9 0 0 9 Serr 0 0 0 0 Sunland 0 0 0 0 Tulare 0 0 0 0 Vina 0 0 0 0 California Walnut Board 74 Walnut Research Reports 2010

Table 2 (cont.). Inventory of chilled or partially chilled (as of 12/1/10) clonal rootstock plant available for use in disease and pest resistance trials and field trials in 2011. Genotype Plants with full chilling Plants with partial chilling Re-grown after chilling Total chilled plants available Transgenic crown gall resistant J1 1A 69 0 0 69 J1 3A 27 0 0 27 J1 4A 3 0 0 3 J1 7A 93 0 0 93 J1 10A 0 0 0 0 J1 11A 76 0 0 76 J1 13A 113 0 19 132 J1 17A 3 0 0 3 J1 19A 102 0 0 102 J1 20A 0 0 15 15 J1a control 47 0 12 59 J21 2A 11 1 0 12 J21 3A 0 0 0 0 J21 4A 0 0 12 12 J21 5A 2 4 0 6 J21 6A 20 0 0 20 J21a control 17 0 0 17 RR4 1A 19 0 0 19 RR4 4A 14 0 0 14 RR4 6C 4 0 0 4 RR4 8A 52 0 0 52 RR4 10A 39 23 42 104 RR4 11A 164 0 0 164 RR4 12A 0 2 2 4 RR4 control 16 4 0 20 Total Available 3441 916 539 4896 California Walnut Board 75 Walnut Research Reports 2010

Table 3. Effect of lime and micronutrient soil amendments on severity of foliage distortion symptoms following growth of clonal walnut plants in the greenhouse in a 50:50 peat:perlite soil mix. Treatment Severity of Symptoms Peat:Perlite + Lime (84g/ft 3 ) + + + + Peat:Perlite + Lime (42g/ft 3 ) + + Peat:Perlite + Lime (84g/ft 3 ) + MicroMax (17g/ ft 3 ) - Plantlets grown in 50:50 peat:perlite supplemented with and 84g/ft 3 lime plus MicroMax (left), 50:50 peat:perlite with 42g/ft 3 lime (center), or 50:50 peat:perlite with 84g/ft 3 lime (right). California Walnut Board 76 Walnut Research Reports 2010

Table 4. Galling response of transgenic and non-transgenic genotypes to stab inoculations using Agrobacterium strain EC1. Genotype of Galls Evaluated Mean Gall Score Mean Gall volume Gall vol./stem diameter ratio Non-transgenic Rootstock AX1 55 3.31 1834 355 Burbank 6 3.17 986 191 Chandler 9 3.33 869 194 Chandler SE1 6 3.00 1162 203 DAR 24 3.58 3071 502 Px1 44 3.70 3866 635 RX1 43 2.92 882 158 Tulare 5 3.00 2461 338 UZ229 36 3.78 4804 859 Vlach 43 3.35 1756 360 VX211 33 2.85 2058 355 W17 8 4.00 5962 1406 WIP1 7 3.57 1484 276 WIP2 59 3.73 2038 375 WIP3 41 3.71 3608 651 WIP4 31 3.39 1263 253 WIP6 15 3.53 1047 192 Wingnut 10.05 25 2.40 705 78 Transgenics and controls J1 1A 13 1.00 0 0 J1 2A 2 4.00 4594 1079 J1 3A 12 1.04 0 0 J1 7A 21 1.12 10 2 J1 10A 8 1.00 0 0 J1 12A 15 1.17 8 2 J1 13A 34 1.10 6 1 J1 19A 25 1.00 0 0 J1 20A 27 1.00 0 0 J1A control 29 3.34 2140 412 J21 3A 21 1.02 1 0 J21 4A 37 1.09 4 1 J21 6A 7 1.14 5 1 J21A control 7 3.29 959 269 RR4 1A 3 1.00 0 0 RR4 2B 2 1.33 24 4 RR4 4A 34 1.03 2 0 RR4 6C 7 1.07 5 1 RR4 8A 12 1.08 7 1 RR4 10A 49 1.02 1 0 RR4 11A 8 1.19 13 2 RR4 12A 33 1.02 1 0 RR4 control 38 2.80 1064 195 California Walnut Board 77 Walnut Research Reports 2010

Table 5. Number of nursery-grown liner trees surviving and mean diameters after nursery growth for 8-15 months. Data are for five replicates, ten liner plantlets per replicate, of three genotypes planted at three different times. Before transplanting, liners were either chilled for 5 to 8 weeks, actively grown in a greenhouse using half-strength Hoagland s solution, or stalled in growth by using only DI water in the greenhouse. Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Total Genotype Pre-treat date Pre-treatment Planting Date RX1 7/15/09 Cold Quiescent Hoagland s DI water VX211 7/15/09 Cold Quiescent Hoagland s DI water Vlach 7/15/09 Cold Quiescent Hoagland s DI water RX1 10/8/09 Cold Quiescent Hoagland s 9/14/09 9/14/09 9/14/09 12/10/09 7 17.1 8 22.7 10 21.1 6 23.7 5 19.3 36 20.8 9 20.3 8 21.5 10 20.7 10 21.0 10 23.4 47 21.4 10 21.4 9 22.0 10 20.8 10 22.0 10 21.9 49 21.6 4 36.5 5 43.2 3 37.1 4 25.9 1 38.2 17 36.2 8 33.9 9 35.3 10 31.4 10 30.5 10 29.8 47 32.2 9 35.1 10 40.9 10 34.6 9 33.8 9 28.6 47 34.6 0 0 0 0 0 0 10 33.9 8 34.9 9 32.5 10 26.0 10 26.7 47 30.8 10 35.4 10 31.5 10 31.9 10 26.6 9 30.9 49 31.3 0 0 0 0 0 0 2 12.9 3 17.3 1 14.8 5 18.0 1 27.4 12 18.1 California Walnut Board 78 Walnut Research Reports 2010

California Walnut Board 79 Walnut Research Reports 2010

Table 5. (cont.) Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Total Genotype Pre-treat date Pre-treatment Planting Date VX211 10/8/09 Cold Quiescent Hoagland s Vlach 10/8/09 Cold Quiescent Hoagland s DI water RX1 3/24/10 Cold Pushing Cold Quiescent Pushing VX211 3/24/10 Cold Pushing Cold Quiescent Pushing Vlach 3/24/10 Cold Small leaf Cold Large leaf Leafing 12/10/09 0 0 0 0 0 0 4 35.7 1 14.4 3 32.3 5 32.8 2 40.6 15 31.2 12/10/09 0 0 0 0 0 0 8 32.5 9 28.3 8 19.2 8 22.8 8 29.3 41 26.4 3 35.1 10 30.1 9 24.6 5 30.5 5 34.3 32 31.0 4/29/09 10 16.1 10 17.7 10 13.8 10 19.3 10 18.5 50 17.1 9 15.2 10 16.4 10 9.7 8 14.6 9 14.1 46 14.0 10 19.4 10 18.3 10 16.4 10 18.6 10 20.1 50 18.6 4/29/09 6 27.8 8 25.8 9 14.5 7 22.5 8 23.9 38 22.9 3 20.2 4 22.1 5 14.5 4 22.2 3 23.5 19 20.5 10 23.6 9 25.6 10 18.8 8 18.9 9 21.6 46 21.7 4/29/09 10 21.7 9 20.5 10 17.2 10 18.6 10 18.8 49 19.4 10 20.7 10 20.1 8 22.0 9 20.5 10 20.3 47 20.7 10 23.9 10 22.2 10 20.7 10 18.0 10 20.6 50 21.1 California Walnut Board 80 Walnut Research Reports 2010

Table 6. Mean scion diameters of standard varieties root-grafted to clonal rootstock genotypes. Diameters (mm) were taken immediately above the graft union. Planted 3/8/08. Data taken 7/29/10. Scion variety Rootstock Genotype Chandler Howard Tulare AX2 33.6 37.8 48.4 WIP3 26.2 --- 35.2 RX1 54.6 47.6 50.8 VX211 35.6 41.9 44.7 UX022 54.6 38.0 57.8 GZ2 35.3 --- 49.5 Table 7. Scion height (cm) of standard varieties root-grafted to clonal rootstock genotypes. Planted 3/8/08. Data taken 7/29/10. Scion variety Rootstock Genotype Chandler Howard Tulare AX2 131.1 178.1 239.2 WIP3 86.7 --- 157.5 RX1 208.3 245.0 219.1 VX211 150.8 175.0 216.4 UX022 247.1 134.2 257.5 GZ2 165.0 --- 221.0 California Walnut Board 81 Walnut Research Reports 2010

Table 8. Current Clonal Rootstock Field Trials. County Grower Genotypes Tehama H. Crain RX1, VX211, Vlach, Px sdlg., Date Established Comments 2009 Budded Sept 2009 New orchard Scion: Howard Butte Deseret RX1, AZ2 2006 New orchard Scion: Chandler Sutter/Yuba Conant RX1,VX211, Serr, Vlach 2007 New orchard Spot fumigated old Hartley site Sutter/Yuba Conant RX1, VX211, Vlach 2009 Power-line planting Scions: Ivanhoe, Howard, Sexton, 91-077-40 New orchard Sutter/Yuba Double Nut VX211, AZ2, NZ1, JX2 2005 Phytophthora Replants Solano Cilker RX1, VX211, Burbank, Vlach, Px sdlg. Solano UCD AX1, AZ2, NZ1,GZ1, JX2, Px1, VX211, RX1, WIP3, NCB, wingnut Contra Costa Tennant WIP2, WIP3, clonal Sunland and Vina, Paradox sdlg. San Joaquin Dondero RX1, VX211, AZ2, NZ1, JX2 2009 New orchard Budded fall 2009 Scion: Tulare 2005 Phytophthora test Infested and non-infested soil Not grafted 2006 Blackline tolerant New orchard Scion: Vina 2005 Phytophthora Replants San Joaquin Taylor WIP3, WIP5, WIP6 2005 Blackline tolerant New orchard Scion: Chandler San Joaquin Lagorio RX1, VX211, WIP3, AZ025, Vlach June-bud, Vlach-grafted, Chandler own-rooted, Px sdlg. 2007 New orchard Scion: Chandler San Joaquin Chiappi RX1, Paradox seedlings 2010 Phytophthora plot Replanted block Scion: Serr Calavaras Gotelli RX1, VX211 2007 Phytophthora, water table Replants DONE Stanislaus MJC Clonal Vina and Chandler, Vlach, 84-121, Sunland sdlg., Px sdlg. Kings Doug Verboon VX211, Px sdlg, with Vlach buffer 1999 Planted 1999 Grafted 2000 New orchard 2008 Fumigation trial treatments New orchard Scion: Tulare California Walnut Board 82 Walnut Research Reports 2010

Appendix 1. Laboratories Licensed to Produce Liners of UC Clonal Rootstock Selections Acemi Nursery License: RX1, VX211 California Seed and Plant Lab License: RX1, VX211 Test agreement: Px1 Duarte Nursery License: RX1, VX211 Test agreement: WIP3, AZ025 North American Plants License: RX1, VX211 Test agreement: WIP2, WIP3, WIP6 ProTree License: VX211, RX1 V-Tree License: VX211, RX1 Test Agreement: WIP3 VitroTech License: RX1, VX211 Test Agreement: WIP2, WIP3 California Walnut Board 83 Walnut Research Reports 2010