CLONAL PROPAGATION OF WALNUT ROOTSTOCK GENOTYPES FOR GENETIC IMPROVEMENT 2011

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1 CLONAL PROPAGATION OF WALNUT ROOTSTOCK GENOTYPES FOR GENETIC IMPROVEMENT 2011 Chuck Leslie, Wes Hackett, Reid Robinson, Joe Grant, Bruce Lampinen, Kathy Anderson, Bob Beede, Rick Buchner, Janet Caprile, Carolyn DeBuse, Rachel Elkins, Janine Hasey, Kourosh Vahdati, Dan Kluepfel, Greg Browne, Mike McKenry, and John Preece ABSTRACT This year we produced more than 9700 liner-sized plantlets of 83 genotypes for use in greenhouse and field pest and disease resistance screening, growth in nurseries, and development of orchard trials. These included 6200 plants of 33 new Juglans microcarpa x Serr paradox genotypes and 230 plants of eleven additional J. cathayensis x Serr or Serr x J. cathayensis hybrid clones. Microshoots rooted and acclimated in the greenhouse were grown to appropriate size and condition for pathogen testing and provided to cooperators for greenhouse and field pest and disease trials. Our production included over 900 plantlets of 16 transgenic lines exhibiting resistance to crown gall for use in greenhouse, field, and orchard trials. Six lines have been chosen for further testing in nursery and orchard trials and one was selected as the leading candidate for pursuit of regulatory approval. The first field trial of transgenic crown gall resistant lines was established in 2008 at UC Davis and grafted to Chandler. Trees in this trial were used this year to supply bark and nut material for studies of trans-graft union movement of macromolecules. In greenhouse stab tests little variation was seen among clonal plantlets of 16 J. microcarpa paradox genotypes but seedlings of wingnut as accession DPTE 1.09 showed considerable differences with six of ten tested seedlings, stab tested four times each, producing no galls. Hardwood cuttings were used to produce clonal replicates of several seedling selections exhibiting potential crown gall resistance, methods for more rapid greenhouse germination of black walnut seed for paradox seedling production were examined, and we are attempting to develop methods for budding or grafting small container-grown plants. Field trials of clonal rootstocks continue to be established state-wide by farm advisors and we continue to supply plant material, assistance, and technical support to commercial laboratories and nurseries producing and selling improved clonal rootstocks. 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 and 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 103 Walnut Research Reports 2011

2 Tissue culture micropropagation: This year we successfully rooted microshoots of 78 individual genotypes (Table1) and produced more than 9700 fully acclimated greenhouse plants (Table 2). In addition to controls, PDS clones, English cultivars, CLRV tolerant selections, and crown gall resistant transgenics, we produced over 6600 plants of 44 new paradox genotypes initiated into culture last year from immature zygotic embryos. These include 33 J. microcarpa (DJUG 29.11) x Serr, six J. cathayensis (DJUG 11.3) x Serr, and five Serr x J. cathayensis (selection #21) paradox hybrid genotypes (Table 2) Plants produced were grown to appropriate size and condition for use in replicated disease and pest screening tests by Greg Browne, Dan Kluepfel and Mike McKenry. Approximately 4700 liner plants are currently available in our inventory for use in trials this coming year (Table 3). In addition, approximately 3200 plants were delivered or allocated during past year for a variety of testing purposes including greenhouse Phytophthora trials by Greg Browne, Armillaria experiments by Kendra Baumgartner, Mike McKenry s field trials for nematode response, crown gall testing by Dan Kluepfel and ourselves, and plants sent to Burchell Nursery to grow for a root development study by Bruce Lampinen and Louise Comas (Table 4). Plants produced also included about 900 plantlets of 16 transgenic lines expressing an RNAi construct for resistance to crown gall plus non-transformed controls for use in greenhouse, nursery, and orchard trials (Table 2). Growing liner plantlets in small containers: To facilitate more rapid screening for Phytophthora resistance we implemented a new system for growing liner plantlets. We now use smaller 1 x 5 Stubby Tree-tainer pots and a commercially available potting mix (Sunshine Mix #4) supplemented with a micronutrient mix (Micromax, 17g/ft 3 of soil). The change to Sunshine Mix from the previously used UC Mix was made to avoid including fir bark, which is unsuitable for Phytophthora testing, in the potting soil but we have also found that walnut plantlets grown in peat:perlite soils, including this particular commercial mix, show a tendency to develop a dwarfed and crinkled leaf symptom with reduced growth, similar to nickel deficiency symptoms seen in pecan known as mouse-ear. This occurs in spite of continuous fertilization with halfstrength Hoagland s solution, appears to be somewhat genotype dependent, and is more prevalent in plants grown out after chilling. RX1 has shown particular sensitivity. In last year s report we presented data showing this problem could be avoided by supplementing peat:perlite soils with a micronutrient mix and reduced by lowering the ph. In addition we have now introduced a 20 ppm foliar application of nickel sulfate to young, newly acclimated plantlets about 6 weeks after rooting. Screening small liner-sized plantlets for crown gall resistance: Using the procedures we described in our 2009 and 2010 reports, we are now routinely using liner-sized plantlets in greenhouse screening trials for crown gall resistance. We evaluate 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 diameter. Based on previous results and field observations of vegetative vigor, we proposed six lines (J1 1A, J1 19a, J1 20A, RR4 4A, RR4 10A, and RR4 12A) last year for advancement towards regulatory approval and began to propagate additional plants of these for further testing (Table 2). Table 5 shows additional greenhouse screening results completed this year for these lines and for several others for which we had limited previous data. Results provide further support to previous results and confirm our choices for advancement towards deregulation and release. Additional plants of the leading genotypes are available for greenhouse and field tests planned by Dan Kluepfel (Table 4) and for planting in nursery and orchard trials (Table 3). California Walnut Board 104 Walnut Research Reports 2011

3 We also used small plant crown gall screens to examine the relative susceptibility or resistance of 16 J. microcarpa x Serr paradox clonal genotypes and a diverse set of wingnut seedlings grown from seed of 5 NCGR wingnut accessions (Tables 6 and 7). There were no apparent differences in galling response among the J. microcarpa hybrids tested (Table 6) but wingnut results, based in most cases on 4 stab inoculations per seedling, showed considerable variation among seedlings grown from seed of NCGR accession DPTE 1.09 (Table 7). Six of the offspring of this wingnut tree developed no galls at all while the other four seedlings produced consistently large galls, including some of the largest measured in the trial. These results suggest further investigation of the crown gall resistance of wingnut accessions and of this accession in particular. Hardwood cuttings: Hardwood cuttings were used to clonally propagate for re-testing 15 open pollinated seedlings from mother trees of several species from the USDA National Clonal Germplasm 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 393 cuttings were made in early February using the protocol described in the 2009 report. A total of 85 rooted cuttings were grown in 3 liter avocado pots for re-testing by Dan Kluepfel s laboratory. Germination of black walnut seeds from paradox sources for disease resistance screening: Shell cutting and soaking is routinely used as an alternative to stratification by the Walnut Breeding Program to germinate English seedling progeny for testing. To facilitate faster production of black or paradox seedling plants for use in disease resistance screening, we compared greenhouse germination following use of: 1) a standard stratification pre-treatment or; 2) pre-treatment involving cutting off the tip of the shell on the blossom (embryo) end of nuts followed by a running water soak. Nuts harvested in the fall of 2010 from three sources were stored dry at 40 F until treated. For one group of nuts from each source, a cross-sectional cut of the shell was made at the embryo end using a jig saw, so that the tip of the embryo was just exposed and water could enter the nut. Another group of nuts was left uncut. All nuts were soaked in running water for 24 hours. One group of uncut and one group of cut nuts from each source were stratified in damp peat moss at 45 F for 8 weeks. All groups of nuts were planted in coarse vermiculite in 10 x 20 x 2 inch flats in a greenhouse with a minimum temperature of 70 F. Germination data were taken 6 weeks later. Table 8 shows that the best germination in the stratification treatment was 60% for the Sierra Gold source but varied from 30 to 60 % depending on source and treatment, with shell cutting having no apparent affect. With the un-stratified treatment, the best germination was for cut nuts, with germination of 55% for the Sierra Gold source, 43% for the James source, and 23% for the Rawlins source. Un-stratified and uncut nuts from all three sources had low germination rates, varying 0 to 8%. These results indicate that shell cutting and 24 hour water soaking may be an alternative to eight weeks of stratification treatment for obtaining near maximal germination rates for paradox seed germination and thereby saving time in carrying out the seedling disease screening process. California Walnut Board 105 Walnut Research Reports 2011

4 Developing a source of scion material for grafting micropropagated rootstock liners: Some walnut rootstock micropropagation laboratories and nurseries would like to be able to routinely graft English scions onto micropropagated clonal rootstock liners. One of the limitations to being able to do this is a source and adequate supply of suitable-diameter shoots from which to take scions or buds. Orchard-derived shoot material is generally much too large for this purpose. During the past year we have begun to evaluate methods to manipulate stock plants to obtain large numbers of small diameter shoots as a source of scion material. Our approach has been to try to increase the number of shoots being produced on a plant by hedging (pinching the shoot tips) or treating with Promalin (a mixture of cytokinin and gibberellin growth regulators) or providing a higher level of nutrition and thereby decreasing the stem diameter via competition. As shown in Table 9 we have used three types and sizes of stock plants: 1) hedged field-grown trees; 2) greenhouse-grown trees in large containers, and; 3) greenhouse-grown trees in small containers. The preliminary data shown in Table 9 indicates that even by doubling the number of shoots per scaffold in large trees, the size of the resulting shoots is not reduced much, if any, while there is some indication that increasing the shoot number on small plants by Promalin treatment can result in smaller diameter shoots of the size (less than 5 mm) needed for scions for grafting rootstock liners. Field Trials: Transgenic crown gall resistance rootstock trial: A field trial of transgenic line expressing resistance to crown gall, non-transformed background genotypes as controls, and other rootstock genotypes of interest, was established on the UCD campus in 2008 and most trees have been successfully budded to Chandler. This trial continues to be evaluated for horticultural performance and natural occurrence of crown gall. The block includes budded trees of the six best genotypes 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 trans-graft union movement of DNA, RNA or other macromolecules. Both bark and nut samples were collected for use in these evaluations this year. Farm advisor/grower county rootstock trials: Currently established clonal rootstock field trials in grower orchards in the various counties are summarized in Table 10. 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 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). Rachel Elkins initiated two field plantings in lake County this year comparing VX211, Vlach, and seedling paradox planted in freshly prepared but un-fumigated sites thought likely to subject trees to nematode pressure (see report in this volume). California Walnut Board 106 Walnut Research Reports 2011

5 Joe Grant and Joe Conant established plantings near Wheatland of RX1, VX211, and Vlach grafted or budded to Ivanhoe, Sexton, Howard, and selection A portion of these plantings are under power lines and evaluations will include observation of rootstock and scion effects on tree height. Initial observations this year suggest rootstock effects on Howard performance and further observation may give insight into current Howard yellowing problems in many orchards. Janine Hasey initiated a trial this year in Sutter County in an orchard with known and long-term Phytophthora cinnamomi problems. This replicated trial in a replant situation includes RX1 containerized and bare-root trees, VX211 bare root, and Paradox seedling trees. The trial was planted late in the spring due to wet weather, resulting in little initial growth and some tree losses (see report in this volume) Carolyn DeBuse planted a replicated clonal rootstock trial in Solano County (Cilker) in 2009 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. Trees at this location of exhibited excessive vigor which may be contributing to an observed absence on trunks of secondary axillary buds suitable for low scaffold development. Four trials which include the cherry leafroll-tolerant WIP rootstocks and own-rooted Chandler 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 also 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 10. 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: N 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. Scion variety is Chandler. Rootstocks include Own-rooted Chandler, RX1 clonal Paradox, VX211 clonal Paradox, WIP3, Vlach clonal paradox (June-budded and standard 2 year-old budded), AZ025, and seedling Paradox (June-budded, J. hindsii X J. regia per Bonilla Nursery). Spacing is 17 X 22. California Walnut Board 107 Walnut Research Reports 2011

6 Irrigation since planting is by single-line above-ground drip. The trial is configured as a randomized complete block design with five 4 to 8-tree replications. All trees were hand-planted 2/18/2008 as finished bare-root nursery trees. First- through fourth - year vegetative growth has been vigorous and uniform. Trees have been pruned and trained using the Barton multiple scaffold system. Trunk diameters after the second, third, and fourth growing seasons were largest for own-rooted Chandlers, smallest for VX211, with other rootstocks intermediate. Beginning with trunk diameter and yield measurements in 2011, data for June-budded and standard 2-year old trees were pooled because early tree growth and canopy development rate were similar for these rootstocks. In late July 2011, midday canopy photosynthetically active radiation interception values (PAR, a measure of canopy size) ranged from 39% to 45% and there were small but statistically significant differences among some rootstocks (data not shown). Nut production in 2011 did not differ among rootstocks, except for own-rooted Chandler trees, which had the lowest average yield. Avg. In-shell yield 2, lbs/a Avg. trunk diameter, inches 1 Rootstock 12/ / / Own-rooted Chandler 2.7 a 4.3 a 5.4 a 753 b RX1 2.5 b 3.8 b 4.9 b 1967 a Paradox seedling (J. hindsii X J. regia) 2.4 bc 3.7 bcd 4.7 bc 1654 a Vlach (Standard budded) 2.4 cd 3.6 cd - - Vlach (June-budded) 2.4 cd 3.6 cde - - Vlach (June & Std. budded) cd 1996 a WIP3 2.4 cd 3.8 bc 4.8 bc 1829 a AZ de 3.5 de 4.5 de 1796 a VX e 3.4 f 4.4 e 1673 a Measured 24 inches above soil line; 8% Wet basis moisture content; Data for Vlach June-budded and Vlach standard 2 year-old budded trees pooled. 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: N 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. California Walnut Board 108 Walnut Research Reports 2011

7 Experimental design: Randomized complete block design with ten ten-tree (paired tree) replications. All trees were hand-planted in March 2010 as bare-root ungrafted nursery whips and budded in August 2010 to Serr. Trees not successfully top-worked by this initial budding were grafted to Serr in spring 2011, and a small number of these trees were re-budded in August There was no first-year (2010) tree mortality. Tree growth, as measured by trunk circumference increment, was similar for the two rootstocks in 2010 (all trees measured). Because of differences in tree growth due to budding and/or grafting success, 2011 tree circumference data were only recorded for trees that had been successfully budded in 2010 and both trees of the RX1-seedling pairs had survived the 2011 season (n=51). Trunk circumference, cm* 15 July Dec Nov Paradox seedling RX * *Trunk circumference measured 30 cm above soil data include all trees. November 2011 data include only trees in intact 2010 budded seedling-rx1 pairs. Second-year tree mortality was assessed in late September By this time, 17% of trees on Paradox seedlings were dead and an additional 6% were growing very poorly compared to other trees. All trees on RX1 were alive and growing well. Root samples were collected November 3, 2011 from dead and poorly growing Paradox seedling trees and subsequently cultured on Phytophthora selective PARP medium. Phytophthora cinnamomi was isolated from one or more root pieces cultured from 63% of dead trees and 40% of poorly growing trees, indicating that P. cinnamomi infection was a principal cause of tree death and decline in the trial. Nursery Propagation and Commercialization: We continue to be 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 genotype, 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 109 Walnut Research Reports 2011

8 Table 1 (part1). Ex vitro rooting percentage for rootstock genotypes. # Attempted # Rooted % Rooted PDS Paradox AX AZ Px RX RX Vlach VX Black W Wingnut WNxW b CLRV Tolerant WIP English Chandler Serr J. microcarpa JMOP JMOP J. microcarpa x Serr JMS JMS JMS JMS 5A JMS JMS JMS JMS 11A California Walnut Board 110 Walnut Research Reports 2011

9 Table 1 (cont.). Ex vitro rooting percentage for rootstock genotypes. # Attempted # Rooted % Rooted JMS JMS JMS JMS JMS JMS JMS JMS JM JM JM JM JM JM JM JM JM JM JM JM STJM STJM STJM STJM STJM J. cathayensis x Serr JCS JCS JCS JCS S S S S S SH SH California Walnut Board 111 Walnut Research Reports 2011

10 Table 1 (cont.). Ex vitro rooting percentage for rootstock genotypes. # Attempted # Rooted % Rooted Serr x J. cathayensis #21 SC SC SBB SM SM Transgenic Crown Gall J1 1A J1 7A J1 19A J1 20A RR4 1A RR4 2B RR4 4A RR4 6C RR4 8A RR4 10A RR4 11A RR4 12A J1a control RR4 control Total California Walnut Board 112 Walnut Research Reports 2011

11 Table 2. (Part 1) Greenhouse survival of rooted clonal microshoots # Rooted # Survived % Survival PDS Paradox AX AZ DAR Px RX RX Vlach VX Black W Wingnut WNxW b CLRV Tolerant Backcross WIP WIP English Chandler Chandler Serr Tulare J. microcarpa JMOP JMOP California Walnut Board 113 Walnut Research Reports 2011

12 Table 2 (cont.). Greenhouse survival of rooted clonal microshoots # Rooted # Survived % Survival J. microcarpa x Serr JMS JMS 3A JMS JMS JMS 5A JMS JMS JMS JMS 11A JMS JMS JMS JMS JMS JMS JMS JMS JM JM JM JM JM JM JM JM JM JM JM JM STJM STJM STJM STJM STJM California Walnut Board 114 Walnut Research Reports 2011

13 Table 2 (cont.). Greenhouse survival of rooted clonal microshoots # Rooted # Survived % Survival J. cathayensis x Serr JCS JCS JCS JCS S S Serr x J. cathayensis #21 SC SC SBB SM SM Transgenic Crown Gall Resistant J1 1A J1 7A J1 10A J1 19A J1 20A J1a control J21 2A J21 5A J21 6A J21a control RR4 1A RR4 2B RR4 3A RR4 4A RR4 6C RR4 8A RR4 10A RR4 11A RR4 12A RR4 control Total Production California Walnut Board 115 Walnut Research Reports 2011

14 Table 3 (part 1). Inventory of chilled or partially chilled clonal rootstock plant available for use in disease and pest resistance trials and field trials in Genotype PDS Paradox Plants with full chilling Plants not fully chilled Total plants available AX PX RX RX Vlach VX Black W Wingnut WNxW b CLRV tolerant backcross WIP WIP WIP English Chandler Howard Serr Vina Transgenics crown gall resistant J1 1A J1 7A J1 12A J1 19A J1 20A J1a control RR4 1A RR4 2B California Walnut Board 116 Walnut Research Reports 2011

15 Table 3 (cont.). Inventory of chilled or partially chilled clonal rootstock plant available for use in disease and pest resistance trials and field trials in Plants with full chilling Plants not fully chilled Total plants available RR4 3A RR4 6C RR4 8A RR4 10A RR4 11A RR4 12A RR4 control J. microcarpa JMOP JMOP J. microcarpa x Serr JMS JMS JMS JMS 5A JMS JMS JMS JMS 11A JMS JMS JMS JMS JMS JMS JMS JMS JM JM JM JM JM JM JM JM California Walnut Board 117 Walnut Research Reports 2011

16 Table 3 (cont.). Inventory of chilled or partially chilled clonal rootstock plant available for use in disease and pest resistance trials and field trials in Plants with full chilling Plants not fully chilled Total plants available 29JM JM JM JM STJM STJM STJM STJM J, cathayensis x Serr JCS JCS JCS JCS S Serr x J. cathayensis #21 SC SBB SM SM Total Available California Walnut Board 118 Walnut Research Reports 2011

17 Table 4 (part 1). Allocation and distribution in 2011 of clonal walnut plants produced for rootstock pathogen resistance testing. Genotype Burchell Nursery for Lampinen/ Comas trial Browne for Phytophthora Tests Baumgartner Armillaria Tests McKenry Nematode tests Crown Gall Small Plant Stab Tests Kluepfel Crown Gall Tests Controls AX PX RX Vlach VX W17 black Wingnut English Chandler 74 CLRV Tolerant Backcross WIP 1 1 WIP 4 3 WIP 6 2 Nematode selection RX Transgenics and controls J1 1A 2 J1 3A 4 J1 19A 5 5 J1 20A 5 J1a control RR4 1A 6 RR4 4A 4 4 RR4 6C 2 RR4 10A 6 RR4 11A RR4 12A 3 RR4 control 22 5 California Walnut Board 119 Walnut Research Reports 2011

18 Table 4 (cont.). Allocation and distribution in 2011 of clonal walnut plants produced for rootstock pathogen resistance testing. Genotype Burchell Nursery for Lampinen/ Comas trial Browne for Phytophthora Tests Baumgartner Armillaria Tests McKenry Nematode tests Crown Gall Small Plant Stab Tests Kluepfel Crown Gall Tests J. microcarpa x Serr JMS JMS JMS JMS 5A JMS JMS JMS JMS 11A JMS JMS JMS JMS JMS JMS JMS JMS JM JM JM JM JM JM JM JM JM JM JM JM STJM STJM STJM STJM California Walnut Board 120 Walnut Research Reports 2011

19 Table 4 (cont.). Allocation and distribution in 2011 of clonal walnut plants produced for rootstock pathogen resistance testing. Crown Gall Genotype Burchell Nursery for Lampinen/ Comas trial Browne for Phytophthora Tests Baumgartner Armillaria Tests McKenry Nematode tests Small Plant Stab Tests Kluepfel Crown Gall Tests J, cathayensis x Serr JCS JCS JCS JCS S S S S S SH 5 1 3SH 8 2 Serr x J. cathayensis #21 SC 1 1 SC 5 5 SBB SM SM J. microcarpa JMOP JMOP Total Plants Allocated California Walnut Board 121 Walnut Research Reports 2011

20 Table 5. Galling response of transgenic and non-transgenic genotypes to stab inoculations using Agrobacterium strain EC1. Genotype No. of Galls Evaluated Mean Gall Score Mean Gall volume Gall vol./stem diameter ratio Standard WIP genotypes AX AZ PX VLACH RX WIP WIP WIP Transgenic selections J1 1A J1 3A J1 19A RR4 1A RR4 4A RR4 6C RR4 8A RR4 10A RR4 11A RR4 12A Controls J1a control J21a control RR4 control California Walnut Board 122 Walnut Research Reports 2011

21 Table 6. Galling response of Juglans microcarpa x Serr hybrid genotypes and controls to stab inoculations using Agrobacterium strain EC1. Genotype No. of Galls Evaluated Mean Gall Score Mean Gall volume Gall vol./stem Diameter ratio J. microcarpa x Serr JMS JMS JMS 5A JMS JMS JMS JMS JMS JMS JM JM JM JM JM STJM STJM Controls JMOP J1a control RR4 control California Walnut Board 123 Walnut Research Reports 2011

22 Table 7. Galling response of wingnut seedling genotypes to stab inoculations using Agrobacterium strain EC1. Wingnut Genotype Tree Number No. of Galls Evaluated Mean Gall Score Mean Gall volume Gall vol./stem diameter ratio DPTE DPTE DPTE DPTE DPTE California Walnut Board 124 Walnut Research Reports 2011

23 Table 8. The effect of shell cutting on germination of stratified and un-stratified Paradox seed from three sources. Seeds germinated in coarse vermiculite in a greenhouse at minimum temperature of 70 F. Seed Source Seeds Not Cut % germinated Seeds Cut % germinated Total No. Seeds Total % germinated Stratified James % % % Sierra Gold % % % Rawlins % % % Un-stratified James % % % Sierra Gold % % % Rawlins % % % Table 9. Induction of branching on Chandler trees to provide scion material for micropropagated rootstock liners. Treatments once per week for 4 weeks. Chandler Tree State Treatment Shoots/Scaffold Shoot Basal Diameter (mm) Hedged Own-Rooted Field Trees Control Promalin (450mg/l) Grafted Trees in 15 gal Containers Control Miracle Grow (2 TBs/gal) Promalin (450mg/l) Own-Rooted Trees in One Liter Containers Control Promalin (450mg/l) California Walnut Board 125 Walnut Research Reports 2011

24 Table 10. Current Clonal Rootstock Field Trials. County Grower Genotypes Tehama H. Crain RX1, VX211, Vlach, Paradox sdlg., Date Established Comments 2009 Budded Sept 2009 New orchard Scion: Howard Butte Deseret RX1, AZ New orchard Scion: Chandler Lake Valadez VX211, Vlach, Paradox seedlings Lake Valadez (Suchan) VX211, Vlach, Paradox seedlings 2011 Nematode Scion: VX211 and Vlach are grafted to Chandler; seedlings will be grafted to Chandler in Unfumigated new planting Nematode site Scion: black walnut scion for nursery trees, so will be Chandler interstems on the VX211 and Vlach 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, New orchard Sutter/Yuba Double Nut Sutter Ruzich RX1 bare root and containerized, VX211 bare root, Paradox sdlg. Solano Cilker RX1, VX211, Burbank, Vlach, Paradox sdlg. Contra Costa Tennant WIP2, WIP3, clonal Sunland and Vina, Paradox sdlg. Contra Costa Maggiore WIP3, clonal Chandler, Paradox sdlg., WIP1, WIP2 San Joaquin Dondero RX1, VX211, AZ2, NZ1, JX2 VX211, AZ2, NZ1, JX Phytophthora Replants DONE 2011 Phytophthora Replants replicated trial To be grafted 2009 New orchard Budded fall 2009 Scion: Tulare 2005 Blackline tolerant New orchard Scion: Vina 2011 Blackline tolerant New orchard Scion: Chandler 2005 Phytophthora Replants DONE San Joaquin Taylor WIP3, WIP5, WIP Blackline tolerant New orchard Scion: Chandler San Joaquin Lagorio (Concar) RX1, VX211, WIP3, AZ025, Vlach June-bud, Vlach-grafted, Chandler own-rooted, Paradox sdlg New orchard Scion: Chandler California Walnut Board 126 Walnut Research Reports 2011

25 County Grower Genotypes Date Established Comments San Joaquin Chiappi RX1, Paradox seedlings 2010 Phytophthora plot Replanted block Scion: Serr Calavaras Gotelli RX1, VX Phytophthora, water table Replants DONE Stanislaus MJC Clonal Vina and Chandler, Vlach, , Sunland sdlg., Px sdlg Planted 1999 Grafted 2000 New orchard Kings Doug Verboon VX211, Px sdlg, with Vlach buffer 2008 Fumigation trial treatments New orchard Scion: Tulare California Walnut Board 127 Walnut Research Reports 2011

26 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 North American Plants License: RX1, VX211 Test agreement: WIP2, WIP3, WIP6 ProTree License: VX211, RX1 Tissue Grown Corporation License: VX211, RX1 V-Tree License: VX211, RX1 Test Agreement: WIP3 VitroTech License: RX1, VX211 Test Agreement: WIP2, WIP3 California Walnut Board 128 Walnut Research Reports 2011