MEDIA AND EXPLANT TRIALS AIMED AT INDUCING SOMATIC EMBRYOS FROM COMMERCIALLY IMPORTANT WALNUT CULTIVARS

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1 MEDIA AND EXPLANT TRIALS AIMED AT INDUCING SOMATIC EMBRYOS FROM COMMERCIALLY IMPORTANT WALNUT CULTIVARS Mohammed A. M. Aly, Charles A. Leslie, Robert G. Fjellstrom and Gale H. McGranahan ABSTRACT: Attempts were made during the 1990 growing season to induce somatic embryos from non-zygotic tissues of five economically important walnut cultivars. The following explants were employed: fertilized and unfertilized ovules; immature and mature catkins; abnormal "spike" flowers; and in vitro-grown leaves and roots. Modifications of the standard induction media were also examined. These included increased sucrose concentration, ethylene action inhibition, and balance and type of growth regulator. Callus was obtained from all explant types but only ovules produced somatic embryos. Somatic embryo cultures were initiated from interior tissues of two 'Sunland' ovules isolated from bagged unpollinated flowers and cultured on basal DKW medium supplemented with glutamine, casein hydrolysate, 4.6 um zeatin, 17.1 um laa and 0.45 um thidiazuron. Another somatic embryo, initiated on the exterior surface of a 'Sunland' ovule, developed to the torpedo and heart stages but then callused. Immature embryos from four suspected apomictic 'cisco' fruits were also induced to grow in vitro and two of these produced somatic embryos. Isozyme analysis showed that all of these clones, except one from each cultivar, carry the same zymotype as the maternal tissue. However, restriction fragment length polymorphism analysis demonstrated that all the clones originated from zygotic rather than maternal tissues. OBJECTIVES: Commercially important walnut cultivars may be further improved by adding traits such as disease and pest resistance to their genetic makeup. Incorporation of novel genes by genetic transformation can complement conventional breeding, but plant regeneration from genetically manipulated cells is a prerequisite to this approach. In walnut, repetitively embryogenic somatic embryos have been used as the target tissue for genetic transformation and regeneration of transgenic plants (McGranahan et al., 1988; McGranahan et al., 1990). However, somatic embryo cultures have only been obtained from immature walnut embryo tissues (Tulecke and McGranahan, 1985) and from endosperm (Tulecke et al., 1988) but not from clonal (non-zygotic) tissues of commercially desirable cultivars. The advantage to using clonal (vegetative) tissues as a source for somatic embryos is to avoid meiosis and genetic recombination;

2 producing plants that are identical to the parents except for the introduced genes. Many factors have been shown to influence the induction of somatic embryos in vitro. These include : a) Developmental and physiological stage of the explant (Jain et.al, 1989). b) Osmotic pressure of the culture medium (Chu et. ai, 1990). c) Concentration and balance of growth regulators. The concentration, type, and ratio of auxins and cytokinins used, as well as other classes of growth regulators such as abscisic acid, ethylene and gibberellic acid, may play a significant role. d) Novel auxins, e.g. picloram (Kysely and Jacobsen, 1990) e) Novel cytokinins, e.g. thidiazuron (Fiola et. ai, 1990) f) Ethylene precursors and inhibitors such as ACC (Satoh and Yang, 1989) and silver nitrate (Roustan et. ai, 1990), respectively) g) Polyamines, 1989). e.g. spermidine and spermine (Evans and Malmberg, h) Others (e.g. charcoal, liquid vs. solid medium, gelling agent, light vs. dark incubation, culture vessel, etc.). The present studies were conducted to investigate the factors controlling induction of somatic embryos from clonal explants of commercial walnut cultivars. Restriction fragment length polymorphism was used for early determination of the parentage somatic embryos. PROCEDURES: Experiments were conducted during the growing season, March-August, Catkins and fruits were collected and either immediately cultured or stored in plastic bags at 4 C for 1-30 days. Explant surface sterilization Plant materials were washed with commercial detergent, rinsed with deionized water, immersed in 70% ethanol for 10 seconds and placed in a stirred 0.1% sodium hypochlorite solution (2% commercial bleach), ph 8, (Sauer and Burroughs, 1986), for the appropriate time (Table 1). Explants were then rinsed three times in sterilized double distilled water, blotted on sterile filter paper and placed on slightly moistened filter paper during dissection to prevent desiccation. Explant types Leaves: Expanded mature leaves from 'Chandler', 'Sunland' and 'Vina' shoots grown in vitro were cut in half longitudinally through the mid rib, then transversely into 2x10 mm segments and cultured, 5 segments per petri plate. Root Segments: The root cap and segments of roots from T6 plants

3 (wingnut x walnut hybrid) growing in vitro were cultured using 5 segments/clone/plate. Male Flowers: Flowers were collected at several developmental stages from both normal catkins and abnormal, mixed sex, spikes. Normal catkins were collected at both pre-dormant (May - August) and post-dormant (March - April) stages. Post-dormant collections included both immature catkins (0.5 cm, green anthers) and mature catkins ( cm, green to yellow anthers capable of shedding pollen). Male flowersfrom abnormalspikes were collectedin June. Male flowers were surface sterilized and aseptically cultured either as whole catkins or dissected for stamen and anther culture. Explants were cultured in groups of either per 35x15 mm petri plate or per 100x15 rom petri plate. Bagged Female Flowers: Immature female flowers of 'Sunland' and 'Cisco' were bagged, presumably before they reached the receptive stage, to prevent pollination. Three weeks after anthesis, the ovules were aseptically removed and cultured, one per petri plate. Ovules and embryos of unbagged flowers were cultured as controls. Four 'Cisco' flowers, in one bag, which had not abscised and continued to grow normally for 8 weeks post anthesis were suspected to be apomictic. These were aseptically opened and their embryos were cultured immediately. Media trials: In the course of this work, numerous combinations of growth regulators were examined. In addition to previously existing media, 59 new media were tested for somatic embryo induction. These are listed in the order in which they were examined. Unless otherwise specified, the medium used is the Tulecke and McGranahan (1985) induction medium without plant growth regulators (PGR) but supplemented with the addenda shown in Table 2. Material that survived 4-6 weeks on the experimental media were usually transferred to DKW-B. Each media treatment consisted of at least three plates if sufficient material was available. Plates were individually wrapped with parafilm and incubated at C in the dark or under 16 hrs photoperiod under cool white fluorescent lamps (87 ue. m-2.s-1). Isozyme analysis: Somatic embryos clones initiated from 4 'cisco' and 3 'Sunland' ovule cultures were analyzed by horizontal starch gel electrophoresis using methods described by Arulsekar et ale (1986), to determine whether they originated from maternal or zygotic tissues. The isozymes assayed in this experiment were esterase (EST) and phosphoglucomutase (PGM). Restriction fraqment length polymorphism analysis: DNA was isolated from 1-3 grams of somatic embryo or callus tissue using the minipreparation method of Dellaporta et ale (1983). Leaves of 'Sunland' and 'cisco' were used for isolation of maternal DNA standards. After a one hour RNAse digestion with 10 ug/ml RNAse (Sigma) at 37C, extraction with 24:1 chloroform:isoamyl alcohol, and ethanol precipitation, 10 ug samples of DNA were digested with

4 40 units of EcoRI, EcoRV, or HindIII (Pharmacia) for 5 hrs, electrophoresed for 20 hrs at 1 V/cm in a 1 x TAE 0.8% agarose gel containing 0.5 ug/ml ethidium bromide and then transferred to S&S Nyrtan membranes. Prehybridizations and hybdidizations were done in heat sealed bags at 42C with 50% formamide according to S&S recommendations. Walnut probes pfp9 and pfp10 (provided by R. Fjellstrom and D. Parfitt), cloned in the vector puc18, were radiolabelled with 32p by a random priming reaction. Hybridizations were performed from 24 to 48 hrs. Membranes were washed twice for 15 min with 2x SSC (0.3 M NaCI, 0.03 M Na citrate) at 25C, 0.1% SDS, followed by two 15 min 1x SSC, 0.5% SDS washes at 40C, and a 0.5x SSC, 0.5% SDS wash for 30 min at 65C. The membranes were exposed to x-ray film for 3 days at -70C with intensifying screens. RESULTS: Leaf seament culture: Generally, segments from young leaflets did not respond in culture. Those from fully expanded mature leaves were responsive but produced only callus. The callus grew first from the cut surface of the mid rib followed by the cut edges and leaf surface. Callus grew more frequently on media with 3% and 6% sucrose (n=36/60 and 35/60) than on media with 9% and 12% sucrose (n=10/60 for both treatments). Calli derived from leaf segments cultured on media 1A-3L were similar to that on TMI medium. On media 8A-10C, the calli developed within 7-10 days and were white and granular with proembryo-like masses. Abscissic acid (media 10A-10C) suppressed the formation and development of these structures wheras silver nitrate (media 9B and 9D) seemed to stimulatethem. No further developmentcould be obtained and thus the identification of these structures as proembryos is questionable. Root-derived callus culture: Root segments were cultured on media DKW-B, TMI and 1A through 5C. Callus grew from the culturedroot segments within 7-10 days on all media. Growth rate varied but seemed to be related more to the physiological state of the explant than to medium composition. However, the callus quality did vary with the medium, however, being white and compact on all media except 4A-5C where it was white and friable. After two months in culture most of the calli were dead except for those on media 3C and 3E, which were white smooth and glistening. These were subcultured on 8C for one week and then transferred to DKW-B. One potential proembryo developed on a callus segment originally from 3C but no further development occurred. Male flowers: During initial experiments with post-dormant catkins from IEarly Ehrhardt I and ISunlandI, anthers longer than 2 mm excised from catkins 1-3 cm long, shed pollen after they were cultured on DKW-B and TMI media. Pollen expanded on these media but did not grow further or divide. The maternal tissues of these mature anthers never responded in culture. Anthers from less mature catkins ( cm) expanded in culture

5 and formed translucent callus from the maternal tissues on all media of the 3A - 3L series and occasionallyproducedpollen (which did not callus). None of the anthers grew on TMI medium or its derivatives. Anthers excised from catkins less than 0.5 cm long produced callus on all media of the 3A - 3L series, but no pollen was produced. Media 3A-3L differ from TMI medium in containing i) one hundred fold increase in auxin concentration, ii) different auxin type (IAA vs. IBA) and iii) high auxin/cytokinin ratio as opposed to high cytokinin/ auxin ratio in TMI medium (Table 2). Whole flowers excised from pre- and post-dormancy catkins and cuitured intact also expanded and opened. It was evident that tissue from catkins less than 0.5 cm length is more responsive in culture. DKW-C and TMI media do not support callus initiation, growth and development. Several proembryo-like structures were obtained infrequently from 'Sunland' male flowers excised from abnormal flower spikes and cultured on media 3C, 3D, 3E and 3F (3% sucrose) after 3-4 weeks in culture. However, these proembryo-like structures did not develop further when subcultured on any medium tested. They remained arrested at this stage and later died except for those induced on 3D media which callused. Increasing the sucrose level to 6% in media # 3D, 3E, 3F and 31 resulted in callus formation, but did not induce somatic embryogenesis. Step-wise alterations in IBA, ABA, GA, cytokinins and glutamine in the media did not induce embryogenesis or organogenesis. These results suggested that zeatin (4.6 um) and TDZ (0.45 um), separately, are more effective in inducing embryo-like structures than BAP (4.4 um) and kinetin (9.3 um) combined. Development of proembryo-like structures on media 3D, 3E and 3F suggests that silver nitrate (10 um) plays a role in embryogenesis. Thus silver nitrate was included in all subsequent media combinations. Ovule and embryo culture: Calli were obtained from several ovules of 'Chandler', 'cisco' and 'Vina' cultured on a variety of media combinations. None of these calli produced somatic embryos when subcultured on any medium. Suspected apomictic ovules: Four bagged 'cisco' fruits did not abscise when left unpollinated, but rather grew to 2 cm diameter. Their embryos were cultured on media 6D, 6G, 7A and 8C. Embryos on media 7A and 8C produced somatic embryos on the original media which continued to multiply when transferred to DKW-B medium. Embryos on media 6D and 6G developed somatic embryos only when later subcultured to DKW-B medium, and these eventually callused. Phosphoglucomutase (PGM) isozyme analysis of somatic embryos from the suspected apomictic ovules demonstrated that one of the four ovules originated from open pollinated zygotic tissue rather than maternal tissue. Esterase isozyme analysis did not produce a clear banding pattern upon staining and could not be used for analysis

6 RFLP analysis of the other three clones using DNA digested with either Hind III or EcoR V revealed that they also originated from zygotic (hybrid) tissue. Probes hybridization and were sufficient #6 to and #10 produced reliable differentiate the somatic embryos from parental material. Putatively unfertilized 'Sunland' ovules, excised from bagged flowers and cultured on DKW-B, TMI and 1A-3L, did not develop past an initial nucellus expansion. After 8 weeks the nucellus tissue was cut open and examined microscopically to determine whether or not it contained differentiating tissues. Two of these ovules, both cultured on medium #3L, contained white, solid, spherical masses. These masses were subcultured on basal medium supplemented with Cefotaxime (500 mg/l) where they produced somatic embryos. In addition, a torpedo shape somatic embryo was found on the under-side of one of the two ovules. This torpedo stage somatic embryo callused on all of the media combinations examined to date. The site of origin of this torpedo stage embryo on the outside of the ovule has strongly suggested that it was initiated from a somatic tissue of maternal type. PGM isozyme analysis demonstrated that somatic embryos from these ovules as well as the torpedo stage embryo originated from maternal tissue. RFLP analysis revealed, however, that they were of zygotic origin. Embryos from bagged fruits may have resulted from prebagging pollination, pollen that remained viable inside the bag until flowers became receptive, or poorly sealed bags. DISCUSSION The present study indicates the influence of endogenous as well as exogenous factors regulating in vitro culture and somatic embryogenesis. The developmental stage of the explant was a critical factor for survival. Mature anthers did not respond in culture. Only 1-2 romlong anthers from cm long catkins callused in vitro. Pre-dormancy and abnormal male flowers and anthers responded more consistently in culture than those from post-dormancy catkins. Zeatin (4.6 um) and thidiazuron ( um), separately, produced proembryos while kinetin (9.3 um) and benzylaminopurine (4.4 um), combined, did not. Silver nitrate (10-60 um), the ethylene action inhibitor, also may playa role in embryogenesis. The surface sterilization method used for this work produced clean explants with less tissue damage than other methods commonly used. Cold storage of catkins and female flowers for up to 30 days at 4 C did not influence the viability of the explants, nor did it induce embryogenesis. Thus, cold storage may be employed to extend the time period during which tissues are available for culturing. The use of isozyme and RFLP analyses proved valuable in detecting the tissue from which the somatic embryos originated. Walnut lacks reliable isozyme markers other than PGM but RFLP analysis using

7 several available walnut DNA probes rapidly demonstrated that somatic embryos originated from zygotic rather than maternal tissue. RFLP and fingerprinting with M13 DNA probes (which were not used in this study), may continue to prove useful for early screening as well ~s confirming the origin of the somatic embryos. REFERENCES: Arulsekar, S., G. H. McGranahan and D. E. Parfitt Inheritance of phosphoglucomutase and esterase isozymes in Persian walnut. The Journal of Heredity 77: Chu, C.C., R. D. Hill and A. L. Brule-Babel High frequency of pollen embryoid formation and plant regeneration in Triticum aestiivum L. on monosaccharide containing media. Plant Science 66: Dellaporta, S. L., J. Wood, and J. B. Hicks A plant DNA mini preparation: Version II. Plant Mol. BioI. Reporter 1 (4): Evans, P. T. and R. L. Malmberg Do polyamines have roles in plant development? Ann. Rev. Plant Physiol. Plant Mol. BioI. 40: Fiola, J. A., M. A. Hassan, H. J. Swartz, R. H. Bors and R. McNicols Effect of thidiazuron, light fluence rates and kanamycin on in vitro shoot organogenesis from excised Rubus cotyledons and leaves. Plant Cell, Tisssue and Organ Culture 20: Jain, S. M., N. Dong and R. J. Newton Somatic embryogenesis in slash pine (Pinus elliottii) from immature embryos cultured in vitro. Plant Science 65: Kysely, W. and H-J. Jacobsen Somatic embryogenesis from embryos and shoot apices. Plant Cell, Tissue and Organ Culture. 20:7-14. McGranahan, G. H., J. A. Driver and W. Tulecke Tissue culture of Juglans. In:"JM Bonga, DJ Durzan, eds., Cell and Tissue Culture in Forestry. Martinus Nijhoff, The Hague, pp McGranahan, G.H., C. A. Leslie, S. L. Uratsu, L. A. Martin, and A. M. Dandekar Agrobacterium-mediated transformation of walnut somatic embryos and regeneration of transgenic plants. Biotechnology 6: McGranahan, G. H., C. A. Leslie, S. L. Uratsu and A. M. Dandekar Improved efficiency of the walnut somatic embryo gene transfer system. Plant Cell Reports 8: Roustan, J-P, A. Latche and J. Fallot Control of carrot somatic embryogenesis by AgN03, an inhibitor of ethylene action:

8 Effect of arginine decarboxylase activity. Plant Science 67: Satoh, S. and S. F. Yang Inactivation of 1- aminocyclopropane-1-carboxylase synthase by I-Vinylglycine as related to the mechanism-based inactivation of the enzyme by S- Adenosyl-I-Methionine. Plant Physiol. 91: Sauer, D. B. and R. Burroughs Disinfection of surfaces with sodium hypochlorite. Phytopathology 76 (7): TUlecke, W. and G. McGranahan Somatic embryogenesis and plant regeneration from cotyledons of walnut, Juglans regia L. Plant Science 40: Tulecke, W., G. McGranahan and H. Ahmadi Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut, Juglans regia L. cv Manregian. Plant Cell Reports 7: Table 1: Surface sterilization time for field-grown materials: Explant Time in disinfectantz (min.) Catkins 1-2 Nuts: cm cm 5-10 Shoots z 0.1% sodium hypochlorite, ph 8, (Sauer and Burroughs, 1986)

9 Table 2: Media combinations tested for induction of somatic embryos from commercial walnut cultivars No. I Addendum (um) DKW-C I Corrected DKW (McGranahan et al., 1987) : (3% sucrose, 4.4 BAP, 0.05 IBA) DKW-C basal medium (i.e. without growth regulators) TMI DKW-B I Tulecke and McGranahan (1985) Induction medium: DKW-B mm Glu BAP KIN IBA TMB DKW-B mm Glu 020A TMI ( 6% Sucrose) 020B TMI ( 9% Sucrose) 020C TMI (12% Sucrose) 1A 1B I TMI Ag TMI Ag 2A TMI (6% Sucrose) Ag 2B TMI (6% Sucrose) Ag 3A TMB Z IM 3B TMB Z IM TDZ 3C TMB Z IM TDZ Ag *3D TMB Z IM TDZ Ag *3E TMB Z IM TDZ Ag *3F TMB Z IM TDZ Ag 3G TMB Z IM TDZ 3H TMB Z IM TDZ Ag *3I TMB Z IM TDZ Ag 3J TMB Z IM TDZ GA 3K TMB Z IM TDZ GA 3L TMB Z IM TDZ 4A TMB BAP KIN IBA 4B TMB BAP KIN IBA 4C TMB BAP KIN IBA 4D TMB BAP KIN IBA ABA 4E TMB BAP KIN IBA ABA 4F TMB BAP KIN IBA ABA 5A DKW-B IBA ABA 5B DKW-B IBA ABA 5C DKW-B IBA ABA 6A TMB Z IM Ag 6B TMB Z IM Ag 6C TMB Z IM Ag 6D TMB Z IM Ag 6E TMB Z IM Ag 6F TMB Z IM Ag 6G TMB Z IM Ag

10 f 7A TMB BAP KIN IBA Glu 7B TMB Z KIN IBA Glu 7C TMB Z KIN IBA Glu 8A TMB TDZ IAA Ag 8B TMB TDZ IAA Ag 8C TMB TDZ IAA Ag 8D TMB TDZ IAA Ag 8E TMB TDZ IAA Ag 8F TMB TDZ IAA Ag 8G TMB TDZ IAA Ag 8H TMB TDZ IAA Ag 81 TMB TDZ IAA Ag 9A TMB TDZ IAA Ag 9B TMB TDZ IAA Ag 9C TMB TDZ IAA Ag 9D TMB TDZ IAA Ag loa TMB TDZ IAA Ag ABA lob TMB TDZ IAA Ag ABA 10C TMB TDZ IAA Ag ABA IAA:Indole acetic acid BAP:Benzylaminopurine TDZ:Thidiazuron IBA:Indole 3 butyric acid KIN:Kinetin Z :Zeatin ABA:Abscisic Acid GA:Gibberellic acid Ag:Silver nitrate Glu:L-glutamine * : Same medium was prepared with 6% sucrose f : Tulecke et ale (1988)