MULTIPLICATION AND EVALUATION OF TISSUE CULTURE DERIVED WALNUT PLANTS. Gale McGranahan, Walt Tulecke, Chuck Leslie

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1 MULTIPLICATION AND EVALUATION OF TISSUE CULTURE DERIVED WALNUT PLANTS Gale McGranahan, Walt Tulecke, Chuck Leslie ABSTRACT The purpose of this project was to use and improve current walnut tissue culture methodology to generate plants from somatic embryos and from mature Juglans regia cultivars. Ninty-eight somatic embryo derived plants are now in soil. Fiftyfour of these are derived from the same embryogenic line. Plants from the same line are phenotypically uniform with the exception of 15% which exhibit thickened leaves, and abnormal bud development. This variant does not appear to be associated with lack of chilling. Modifications in the procedure for obtaining plants from embryogenic lines has improved the rate of survival from embryo to potted plant from 5 to 13%. Modifications included omission of cold treatment, pretreatment of selected embryos with GA3 and addition of activated charcoal to basal medium used for germlnation. Ten mature cuitivars were initiated into culture. Chandler is now in the multiplication phase and Sunland and Vina are approaching that phase. Problems encountered in micropropagation of mature J. regia cultivars included inadequate explant sterilization, latent contamination and 'slow decline'. Slow decline is characterized by gradual leaf chlorosis, leaf abscission and eventual death. Inadequate sterilization was overcome by rinsing explants for 2 hrs in running tap water prior to sterilization and by using individual rather than batch sterilization procedures. Antibiotic supplements to the medium were not effective in elminating contamination. Slow decline, which may be a result of lack of juvenility, was unaffected by several media adjustments. It was somewhat overcome by rapid transfer. OBJECTIVE This project had 2 main objectives. The first was to use the technology developed for somatic embryogenesis to obtain sufficient numbers of somatic embryo derived walnut plants to determine whether somaclonal variation will be an important factor in genetic improvement of walnuts. The second was to develop the methodology to micropropagate mature Persian walnut cultivars on their own roots. PROCEDURE A. Somatic Embryo Derived Plants A procedure for obtaining somatic embryos was reported briefly in Walnut Research Reports, 1984 and in detail in Plant Science (Tulecke, W. and G. McGranahan, Somatic embryogenesis and plant regeneration from cotyledons of walnut, Juglans regia L. Plant Science 4:57-63). The procedure is summarized in Table 1. Very few of the original selected embryos survive the entire procedure -11-

2 to become established plants. Major losses occur due to embryo death in cold storage, failure to develop roots and shoots and failure to survive transplanting to soil In order to efficiently obtain somatic embryo derived plants modifications in the established procedure were examined. These included omission of the cold treatment, media adjustments to enhance root and apex developoment and changes in the soil transfer procedures. These are described in results and discussion0 The repetitively embryogenic line SFA was used in all studies. B. Micropropagation of Mature Persian Cultivars Procedures for micropropagating Juglans have been described (McGranahan, G., J. Driver and Wo Tulecke. Tissue Culture of Juglans in: Bonga J. and D. Durzan (eds) Tissue Culture in Forestry Vol. 2 Martinus Ni jhoff, Netherlands (in press)). The methods described have been successfully applied to juvenile J. regia, J. hindsii Iparadox' and pterocarya sp, to mature 'paradox' and to suckers of mature Jo hindsii. Results with mature J. regia have been inconsistent and largely unsuccessful. Problems encountered in culturing mature J. regia include difficulties in obtalning sterile explant material, 'latent' contamination possibly from internal contaminants, and 'slow decline' due to unknown causes. Slow decline is characterized by gradual chlorosis, leaf abscission and death. To overcome these problems, modifications in the sterilization procedure (including use of antibiotics, Rifampicin (100 mg/ml) and Tetracycline (100 mg/ml), frequency of transfer, timing of excision of shoots from original explants and hormone levels (BAP at 2,5 mg/l) were investigated. Ten cultivars were used (Sunland, Vina, Eureka, Chandler, Howard, Amigo, Scharsch Franquette, Chico, Hartley and ). All material had been grafted on to juvenile rootstock (2nd leaf) and half had been sprayed with BAP (100 mg/l) and GA3 (50 mg/l) at weekly intervals and repeatedly cut back as previously descrlbed (McGranahan et. al.). RESULTS AND DISCUSSION A. Somatic Embryo Derived Plants A total of 504 embryos were selected from the SF4 repetitively embryogenic culture line were given cold treatment (8-10 wks at 4 C, dark) and 216 were given no cold treatment. Omission of cold treatment. increased the number of embryos that germinated and survived from 5% to 13% (Table 2)0 Eight of the total surviving plants were "deformed". They exhibited thickened leaves and abnormal buds. Initially, these symptoms were thought to be a result of lack of chilling but 3/8 of the deformed plants had been given the standard cold treatment. Rooting efficiency declined with removal of cold treatment. Several changes in media and embryo handling were examined to correct this problem. Of these, placement of selected embryos on basal medj,..y.:m.. supplemented with 20 mg/l GA3 for 1 week in the dark immediately prior to transfer to light, was the most effective for root initiation. This procedure increased rooting frequency to 75%, approximately a 4 fold increase 0 Root extension growth and quality was markedly

3 improved by addition of 5% charcoal to the basal medium used for root growth in the light. Reliable apex development and shoot extension remains a serious obstacle to efficient production and has not been satisfactorily corrected by either media adjustments or culturing procedures. Survival during transfer from steriie in vitro conditions to nonsterile soil was also low (10%)0 Soil sterilization, fumgicide applications, and selection for increased root size prior to transplant failed to impact this problem. By selecting only plants with stem elongation and/or leaf development in vitro, transfer survival was increased to 66%. This reinforces the importance of apex development in vitro. Currently, 98 somatic embryo derived plants are in soil. Their origins are listed in Table 3. Our next objective is to determine if varlation is present in these plants. This may be evaluated through isozyme analysis, chromosome counts and replicated trials. Heritable variation can only be assessed by selfing plants and evaluating progeny. The plants we have now appear phenotypically uniform with the exception of the deformed plants. B. Micropropagation of Mature Cultivars. Explants were collected 11 tlmes between 2/12/85 and 10/18/85. Each cultivar was collected at least twlce with average of 44 explants per collection. Vina, Chandler and Sunland were collected 7, 5 and 4 times respectively. Percent survival after 2 months in culture was increased from 3% to 58% in Vina by altering sterilization method from batch to individual sterilizatlon and rinsing for 2-3 hrs in running tap water prior to sterilizationo Antibiotics were effective in increasing survival at 2 wks from 61% (SE = 4.9) to 91% (SE = 1.8). However, the antibiotics produced increasing phytoxicity after 3 days and when explants were removed from media with antibiotics, contamination eventually reappeared. The slow decline which may be a symptom of a suboptimal medium was not positively influenced by raisln BAP levels (2x and 5x standard), addition of charcoal to mediurn, or reducing medlurn to half strength. Treatments that had a positlve effect on long term survival included maintaining the shoot on the original explant as long as possible before excision and rapld transfer (transfer to fresh medium every other day). At present Chandler is in multiplication phase and Sunland and Vina are approaching multiplication phase. Explants of the latter two clones have been dlstributed to Plant Research Laboratory. Many questions remain to be answered in micropropagation of mature J. regia cultivars. Because we, have been successful in obtaining apparently clean explants we feel the major problem interfering with micropropagation is slow decline. Slow decline, characterized by -13-

4 gradual leaf yellowing and abscission followed by microplant death, has been noted only in mature cultivars. If slow decline is related to lack of juvenility it could be attacked through several routes, the first being rejunvenation in vivo. Serial grafting or stimulation of growth through GA/BAP sprays and pruning are possibilities that have been successful in other crops. To obtain more than 2 generations of grafting/year, techniques for grafting in the greenhouse on very young seedlings would have to be developed 0 A better method for rejunvenation in vivo would be the development of apomictic seedlings. This would also-provide material to study the basic differences between juvenile and mature material of the same cultivar. (Dr. Vito Polito plans to submit a proposal on apomixis in walnuts.) Rejuvenation in vitro is another potential means of overcoming slow decline. Serial micrografting onto multiplying J. regia (juvenile) or paradox shoots in culture or onto developing embryos (derived through somatic embryogensis or sterile nut culture) appears feasible. J. Driver of Plant Research Laboratory has indicated a willingness to pursue in vitor rejuvenation. Embryogenic cultures and juvenile J. regia cultures have been released to him for this purpose. The final option for micropropagating mature cultivars is to develop media specific for mature cultivars. This would require developing matrices of numerous media components. There is potential of success using this method but the time and-labor involved are prohibitive at this time. FUTURE RESEARCH DIRECTIONS Somatic embryogenesls is a powerful tool in genetically improving walnuts. The potential of somaclonal variation will be unknown until plants derived from somatic embryos are thoroughly evaluated. Because somatic embryo derived plants appear phenotypically uniform and because somatic embryogenesis has been used in other crop species to regenerate plants from transformed sources, we propose the following: 1. To continue evaluation of somatic embryo derived plants. 2. To develop technology to regenerate plants from additional cell and organ sources. 3. To concentrate specifically on sources which may play a role in obtaining transformed plants. -14-

5 open 'l'able1. Procedures for obtaining somatic embryos and plants from walnut cotyledon tissue. Procedure Condition Time (weeks) Excise cotyledon Conditioning medium Basal medium Select somatic embryos at cotyledon stage Break apical dormancy Leaf and root growth Transfer to peat plugs Hardening of plants Aseptic technique Dark Dark Dark, basal medium Cold, 2 C, dark 16-h photoperiod 1/2 strength basal Non-sterile, reduced humidity soil 6-12, after full bloom , several transfers 2-4, repeated transfers Total time Table 2. Somatic Embryo Regeneration Treatment Embryos Germinated Surviving Initiated Embryos Plants in Soil No. No. % total No. % total - Cold treatmenty No cold treatment Total YCold treatment = 8-10 wks at 4 C, dark. Table 3. Origin of somatic embryo derived plants 1 Origin number Origin number Scharch Franquette (6) 55 J. hindsii 3 P.I (endosperm) 12 pterocarya sp x Gustine 11 Early Ehrhart 3 Payne 4 Other Franquettes 10 TOTAL 98 Iplants obtained from endosperm or cotyledons of, pollinated seed. -15-