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

Mohammed A. M. Aly, Charles A. Leslie, Robert G. Fjellstrom andGale H. McGranahan

ABSTRACT:

Attempts were made during the 1990 growing season to induce somaticembryos from non-zygotic tissues of five economicallyimportant 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 actioninhibition, and balance and type of growth regulator.

Callus was obtained from all explant types but only ovules producedsomatic embryos. Somatic embryo cultures were initiated frominterior tissues of two 'Sunland' ovules isolated from baggedunpollinated flowers and cultured on basal DKW medium supplementedwith glutamine, casein hydrolysate, 4.6 uM zeatin, 17.1 uM lAA and0.45 uM thidiazuron. Another somatic embryo, initiated on theexterior surface of a 'Sunland' ovule, developed to the torpedo andheart stages but then callused. Immature embryos from foursuspected apomictic 'cisco' fruits were also induced to grow invitro and two of these produced somatic embryos.

Isozyme analysis showed that all of these clones, except one fromeach cultivar, carry the same zymotype as the maternal tissue.However, restriction fragment length polymorphism analysisdemonstrated that all the clones originated from zygotic ratherthan maternal tissues.

OBJECTIVES:

Commercially important walnut cultivars may be further improved byadding traits such as disease and pest resistance to their geneticmakeup. Incorporation of novel genes by genetic transformation cancomplement conventional breeding, but plant regeneration fromgenetically manipulated cells is a prerequisite to this approach.In walnut, repetitively embryogenic somatic embryos have been usedas the target tissue for genetic transformation and regeneration oftransgenic plants (McGranahanet al., 1988; McGranahan et al.,1990). However, somatic embryo cultures have only been obtainedfrom 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. Theadvantage to using clonal (vegetative) tissues as a source forsomatic embryos is to avoid meiosis and genetic recombination;

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producing plants that are identical to the parents except for theintroduced genes.

Many factors have been shown to influence the induction of somaticembryos in vitro. These include :

a) Developmental and physiological stage of the explant (Jainet.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 cytokininsused, as well as other classes of growth regulators such asabscisic acid, ethylene and gibberellic acid, may play asignificant 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, e.g. spermidine and spermine (Evans and Malmberg,1989).

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 factorscontrollinginductionof somatic embryos from clonal explants ofcommercial walnut cultivars. Restriction fragment lengthpolymorphism was used for early determinationof the parentagesomatic embryos.

PROCEDURES:

Experiments were conducted during the growing season, March-August,1990. Catkins and fruits were collected and either immediatelycultured or stored in plastic bags at 4°C for 1-30 days.

Explant surface sterilization Plant materials were washed withcommercial detergent, rinsed with deionized water, immersed in 70%ethanol for 10 seconds and placed in a stirred 0.1% sodiumhypochlorite solution (2% commercial bleach), pH 8, (Sauer andBurroughs, 1986), for the appropriate time (Table 1). Explantswere then rinsed three times in sterilized double distilled water,blotted on sterile filter paper and placed on slightly moistenedfilter 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 longitudinallythrough the mid rib, then transversely into 2x10 mm segments andcultured, 5 segments per petri plate.

Root Segments: The root cap and segments of roots from T6 plants

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(wingnut x walnut hybrid) growing in vitro were cultured using 5segments/clone/plate.

Male Flowers: Flowers were collected at several developmentalstages 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 collectionsincluded both immature catkins (0.5 cm, green anthers) and maturecatkins (0.5 - 3.0 cm, green to yellow antherscapable of sheddingpollen). Male flowersfrom abnormalspikeswere collectedin June.Male flowers were surface sterilized and aseptically culturedeither as whole catkins or dissected for stamen and anther culture.Explants were cultured in groups of either 10-15 per 35x15 mm petriplate or 30-60 per 100x15 rompetri plate.

Bagged Female Flowers: Immature female flowers of 'Sunland' and'Cisco' were bagged, presumably before they reached the receptivestage, to prevent pOllination. Three weeks after anthesis, theovules 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 andcontinued to grow normally for 8 weeks post anthesis were suspectedto be apomictic. These were aseptically opened and their embryoswere cultured immediately.

Media trials: In the course of this work, numerous combinations ofgrowth regulators were examined. In addition to previouslyexisting media, 59 new media were tested for somatic embryoinduction. These are listed in the order in which they wereexamined. Unless otherwise specified, the medium used is theTulecke and McGranahan (1985) induction medium without plant growthregulators (PGR) but supplemented with the addenda shown in Table2. Material that survived 4-6 weeks on the experimental media wereusually transferred to DKW-B. Each media treatment consisted of atleast three plates if sufficient material was available. Plateswere individually wrapped with parafilm and incubated at 25-30.C inthe dark or under 16 hrs photoperiod under cool white fluorescentlamps (87 uE. m-2 .S-1) .

Isozyme analysis: Somatic embryos clones initiated from 4 'cisco'and 3 'Sunland' ovule cultures were analyzed by horizontal starchgel electrophoresis using methods described by Arulsekar et ale(1986), to determine whether they originated from maternal orzygotic tissues. The isozymes assayed in this experiment wereesterase (EST) and phosphoglucomutase (PGM).

Restriction fraqment length polymorphism analysis: DNA wasisolated from 1 - 3 grams of somaticembryo or callus tissue usingthe minipreparation method of Dellaporta et ale (1983). Leaves of'Sunland' and 'cisco' were used for isolation of maternal DNAstandards. 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

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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 gelcontaining 0.5 ug/ml ethidium bromide and then transferred to S&SNyrtan membranes. Prehybridizations and hybdidizations were donein heat sealed bags at 42C with 50% formamide according to S&Srecommendations. Walnut probes pFP9 and pFP10 (provided by R.Fjellstrom and D. Parfitt), cloned in the vector pUC18 , wereradiolabelled with 32p by a random priming reaction. Hybridizationswere performed from 24 to 48 hrs. Membranes were washed twice for15 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 a0.5x SSC, 0.5% SDS wash for 30 min at 65C. The membranes wereexposed to x-ray film for 3 days at -70C with intensifying screens.

RESULTS:

Leaf seament culture: Generally, segments from young leaflets didnot respond in culture. Those from fully expanded mature leaveswere responsive but produced only callus. The callus grew firstfrom the cut surface of the mid rib followed by the cut edges andleaf 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 segmentscultured on media 1A-3L were similar to that on TMI medium. Onmedia 8A-10C, the calli developed within 7-10 days and were whiteand granular with proembryo-like masses. Abscissic acid (media10A-10C) suppressed the formation and development of thesestructures wheras silver nitrate (media 9B and 9D) seemed tostimulatethem. No furtherdevelopmentcould be obtainedand thusthe identification of these structures as proembryos isquestionable.

Root-derived callus culture: Root segments were cultured on mediaDKW-B, TMI and 1A through 5C. Callus grew fromthe culturedrootsegments within 7-10 days on all media. Growth rate varied butseemed to be related more to the physiological state of the explantthan to medium composition. However, the callus quality did varywith the medium, however, being white and compact on all mediaexcept 4A-5C where it was white and friable. After two months inculture most of the calli were dead except for those on media 3Cand 3E, which were white smooth and glistening. These weresubcultured on 8C for one week and then transferred to DKW-B. Onepotential proembryo developed on a callus segment originally from3C but no further development occurred.

Male flowers: During initial experiments with post-dormant catkinsfrom IEarly Ehrhardt I and ISunlandI, anthers longerthan 2 mmexcised from catkins 1-3 cm long, shed pollen after they werecultured on DKW-B and TMI media. Pollen expanded on these mediabut did not grow further or divide. The maternal tissues of thesemature anthers never responded in culture.

Anthers from less mature catkins (0.5-1.0 cm) expanded in culture

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and formed translucent callus from the maternal tissues on allmedia of the 3A - 3L seriesand occasionallyproducedpollen (whichdid not callus). None of the anthers grew on TMI medium or itsderivatives. Anthers excised from catkins less than 0.5 cm longproduced callus on all media of the 3A - 3L series, but no pollenwas produced. Media 3A-3L differ from TMI medium in containing i)one hundred fold increase in auxin concentration, ii) differentauxin type (IAA vs. IBA) and iii) high auxin/cytokinin ratio asopposed to high cytokinin/ auxin ratio in TMI medium (Table 2).

Whole flowers excised from pre- and post-dormancy catkins andcuItured intact also expanded and opened. It was evident thattissue from catkins less than 0.5 cm length is more responsive inculture. 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 andcultured on media 3C, 3D, 3E and 3F (3% sucrose) after 3-4 weeks inculture. However, these proembryo-like structures did not developfurther when subcultured on any medium tested. They remainedarrested at this stage and later died except for those induced on3D media which callused. Increasing the sucrose level to 6% inmedia # 3D, 3E, 3F and 31 resulted in callus formation, but did notinduce somatic embryogenesis. Step-wise alterations in IBA, ABA,GA, cytokinins and glutamine in the media did not induceembryogenesis or organogenesis.

These results suggested that zeatin (4.6 uM) and TDZ (0.45 uM),separately, are more effective in inducing embryo-like structuresthan BAP (4.4 uM) and kinetin (9.3 uM) combined. Development ofproembryo-like structures on media 3D, 3E and 3F suggests thatsilver nitrate (10 uM) plays a role in embryogenesis. Thus silvernitrate was included in all subsequent media combinations.

Ovule and embryo culture: Calli were obtained from several ovulesof 'Chandler', 'cisco' and 'Vina' cultured on a variety of mediacombinations. None of these calli produced somatic embryos whensubcultured on any medium.

Suspected apomictic ovules: Four bagged 'cisco' fruits did notabscise when left unpollinated, but rather grew to 2 cm diameter.Their embryos were cultured on media 6D, 6G, 7A and 8C. Embryos onmedia 7A and 8C produced somatic embryos on the original mediawhich continued to multiply when transferred to DKW-B medium.Embryos on media 6D and 6G developed somatic embryos only whenlater subcultured to DKW-B medium, and these eventually callused.

Phosphoglucomutase (PGM) isozyme analysis of somatic embryos fromthe suspected apomictic ovules demonstrated that one of the fourovules originated from open pollinated zygotic tissue rather thanmaternal tissue. Esterase isozyme analysis did not produce a clearbanding pattern upon staining and could not be used for analysis.

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RFLP analysis of the other three clones using DNA digested witheither Hind III or EcoR V revealed that they also originated fromzygotic (hybrid) tissue. Probes #6 and #10 produced reliablehybridization and were sufficient to differentiate the somaticembryos from parental material.

Putatively unfertilized 'Sunland' ovules, excised from baggedflowers and cultured on DKW-B, TMI and 1A-3L, did not develop pastan initial nucellus expansion. After 8 weeks the nucellus tissuewas cut open and examined microscopically to determine whether ornot it contained differentiating tissues. Two of these ovules,both cultured on medium #3L, contained white, solid, sphericalmasses. These masses were subcultured on basal medium supplementedwith Cefotaxime (500 mg/L) where they produced somatic embryos. Inaddition, a torpedo shape somatic embryo was found on theunder-side of one of the two ovules. This torpedo stage somaticembryo callused on all of the media combinations examined to date.The site of origin of this torpedo stage embryo on the outside ofthe ovule has strongly suggested that it was initiated from asomatic tissue of maternal type.

PGM isozyme analysis demonstrated that somatic embryos from theseovules as well as the torpedo stage embryo originated from maternaltissue. RFLP analysis revealed, however, that they were of zygoticorigin. Embryos from bagged fruits may have resulted fromprebagging pollination, pollen that remained viable inside the baguntil flowers became receptive, or poorly sealed bags.

DISCUSSION

The present study indicates the influence of endogenous as well asexogenous factors regulating in vitro culture and somaticembryogenesis. The developmental stage of the explant was acritical factor for survival. Mature anthers did notrespond in culture. Only 1-2 romlong anthers from 0.5-1 cm longcatkins callused in vitro. Pre-dormancy and abnormal male flowersand anthers responded more consistently in culture than those frompost-dormancy catkins.

Zeatin (4.6 uM) and thidiazuron (0.05-9.0 uM), separately, producedproembryos while kinetin (9.3 uM) and benzylaminopurine (4.4 uM),combined, did not. Silver nitrate (10-60 uM), the ethylene actioninhibitor, also may playa role in embryogenesis.

The surface sterilization method used for this work produced cleanexplants with less tissue damage than other methods commonly used.Cold storage of catkins and female flowers for up to 30 days at 4°Cdid not influence the viability of the explants, nor did it induceembryogenesis. Thus, cold storage may be employed to extend thetime period during which tissues are available for culturing.

The use of isozyme and RFLP analyses proved valuable in detectingthe tissue from which the somatic embryos originated. Walnut lacksreliable isozyme markers other than PGM but RFLP analysis using

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several available walnut DNA probes rapidly demonstrated thatsomatic embryos originated from zygotic rather than maternaltissue. RFLP and fingerprinting with M13 DNA probes (which werenot used in this study), may continue to prove useful for earlyscreening as well ~s confirming the origin of the somatic embryos.

REFERENCES:

Arulsekar, S., G. H. McGranahan and D. E. Parfitt. 1986.Inheritance of phosphoglucomutase and esterase isozymes in Persianwalnut. The Journal of Heredity 77:220-221.

Chu, C.C., R. D. Hill and A. L. Brule-Babel. 1990. High frequencyof pollen embryoid formation and plant regeneration in Triticumaestiivum L. on monosaccharide containing media. Plant Science66:255-262.

Dellaporta, S. L., J. Wood, and J. B. Hicks. 1983. A plant DNAmini preparation: Version II. Plant Mol. BioI. Reporter 1 (4):19-21.

Evans, P. T. and R. L. Malmberg. 1989. Do polyamines have roles inplant development? Ann. Rev. Plant Physiol. Plant Mol. BioI.40:235-269.

Fiola, J. A., M. A. Hassan, H. J. Swartz, R. H. Bors and R.McNicols. 1990. Effect of thidiazuron, light fluence rates andkanamycin on in vitro shoot organogenesis from excised Rubuscotyledons and leaves. Plant Cell, Tisssue and Organ Culture20:223-228.

Jain, S. M., N. Dong and R. J. Newton. 1989. Somatic embryogenesisin slash pine (Pinus elliottii) from immature embryos cultured invitro. Plant Science 65:233-241.

Kysely, W. and H-J. Jacobsen. 1990. Somatic embryogenesis fromembryos and shoot apices. Plant Cell, Tissue and Organ Culture.20:7-14.

McGranahan, G. H., J. A. Driver and W. Tulecke. 1987. Tissueculture of Juglans. In:"JM Bonga, DJ Durzan, eds., Cell and TissueCulture in Forestry. Martinus Nijhoff, The Hague, pp 261-271.

McGranahan, G.H., C. A. Leslie, S. L. Uratsu, L. A. Martin, and A.M. Dandekar. 1988. Agrobacterium-mediated transformation of walnutsomatic embryos and regeneration of transgenic plants.Biotechnology 6:800-804.

McGranahan, G. H., C. A. Leslie, S. L. Uratsu and A. M. Dandekar.1990. Improved efficiency of the walnut somatic embryo genetransfer system. Plant Cell Reports 8:52-516.

Roustan, J-P, A. Latche and J. Fallot. 1990. Control of carrotsomatic embryogenesis by AgN03, an inhibitor of ethylene action:

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Effect of arginine decarboxylase activity. Plant Science 67:89-95.

Satoh, S. and S. F. Yang. 1989. Inactivation of 1-aminocyclopropane-1-Carboxylase synthase by I-Vinylglycine asrelated to the mechanism-based inactivation of the enzyme by S-Adenosyl-I-Methionine. Plant Physiol. 91:1036-1039.

Sauer, D. B. and R. Burroughs. 1986. Disinfection of surfaces withsodium hypochlorite. Phytopathology 76 (7):745-749.

TUlecke, W. and G. McGranahan. 1985. Somatic embryogenesis andplant regeneration from cotyledons of walnut, Juglans regia L.Plant Science 40:57-63.

Tulecke, W., G. McGranahan and H. Ahmadi. 1988. Regeneration bysomatic embryogenesis of triploid plants from endosperm of walnut,Juglans regia L. cv Manregian. Plant Cell Reports 7:301-304.

Table 1: Surface sterilization time for field-grown materials:-------------------------------------

Explant Time indisinfectantz

(min.)-------------------------------------

CatkinsNuts:0.5-2.0 cm2.0-5.0 cm

Shoots

1-2

1-55-101-2

-------------------------------------

z 0.1% sodium hypochlorite, pH 8, (Sauer and Burroughs,1986)

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Table 2: Media combinations tested for induction of somatic embryosfrom 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-B I

DKW-C basal medium (i.e. without growth regulators)TMI Tulecke and McGranahan (1985) Induction medium:

DKW-B + 1.7 mM Glu + 4.4 BAP + 9.3 KIN + 0.05 IBATMB DKW-B + 1.7 mM Glu020A TMI ( 6% Sucrose)020B TMI ( 9% Sucrose)020C TMI (12% Sucrose)

1AI

TMI + 1.00 Ag1B TMI + 10.00 Ag

2A TMI (6% Sucrose) + 1.00 Ag2B TMI (6% Sucrose) + 10.00 Ag

3A TMB + 4.60 Z + 5.70 IM3B TMB + 0.00 Z + 5.70 IM + 0.45 TDZ3C TMB + 4.60 Z + 5.70 IM + 0.00 TDZ + 1.00 Ag

*3D TMB + 4.60 Z + 5.70 IM + 0.00 TDZ + 10.00 Ag*3E TMB + 0.00 Z + 5.70 IM + 0.45 TDZ + 1.00 Ag*3F TMB + 0.00 Z + 5.70 IM + 0.45 TDZ + 10.00 Ag3G TMB + 4.60 Z + 5.70 IM + 0.45 TDZ3H TMB + 4.60 Z + 5.70 IM + 0.45 TDZ + 1.00 Ag*3I TMB + 4.60 Z + 5.70 IM + 0.45 TDZ + 10.00 Ag3J TMB + 4.60 Z + 5.70 IM + 0.45 TDZ + 5.00 GA3K TMB + 4.60 Z + 5.70 IM + 0.45 TDZ + 10.00 GA3L TMB + 4.60 Z + 17.10 IM + 0.45 TDZ

4A TMB + 4.40 BAP + 9.30 KIN + 0.10 IBA4B TMB + 4.40 BAP + 9.30 KIN + 0.50 IBA4C TMB + 4.40 BAP + 9.30 KIN + 5.00 IBA4D TMB + 4.40 BAP + 9.30 KIN + 0.10 IBA + 40.0 ABA4E TMB + 4.40 BAP + 9.30 KIN + 0.50 IBA + 40.0 ABA4F TMB + 4.40 BAP + 9.30 KIN + 5.00 IBA + 40.0 ABA

5A DKW-B + 0.10 IBA + 40.0 ABA5B DKW-B + 0.50 IBA + 40.0 ABA5C DKW-B + 5.00 IBA + 40.0 ABA

6A TMB + 0.90 Z + 1.14 IM + 10.0 Ag6B TMB + 0.90 Z + 2.28 IM + 10.0 Ag6C TMB + 0.90 Z + 3.43 IM + 10.0 Ag6D TMB + 0.90 Z + 5.70 IM + 10.0 Ag6E TMB + 0.90 Z + 0.57 IM + 10.0 Ag6F TMB + 0.90 Z + 0.00 IM + 10.0 Ag6G TMB + 0.00 Z + 5.70 IM + 10.0 Ag

California Walnut Board Walnut Research Reports 1990

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f 7A TMB + 0.44 BAP + 0.93 KIN + 5.0 IBA + 856 Glu7B TMB + 1.37 Z + 0.00 KIN + 5.0 IBA + 856 Glu7C TMB + 0.46 Z + 0.00 KIN + 5.0 IBA + 856 Glu

8A TMB + 4.50 TDZ + 5.70 IAA + 10.0 Ag8B TMB + 9.00 TDZ + 5.70 IAA + 10.0 Ag8C TMB + 0.05 TDZ + 5.70 IAA + 10.0 Ag8D TMB + 0.05 TDZ + 5.70 IAA + 00.0 Ag8E TMB + 0.05 TDZ + 2.85 IAA + 10.0 Ag8F TMB + 0.05 TDZ + 1.43 IAA + 10.0 Ag8G TMB + 0.05 TDZ + 0.57 IAA + 10.0 Ag8H TMB + 0.05 TDZ + 0.057IAA + 10.0 Ag81 TMB + 0.05 TDZ + 0.00 IAA + 00.0 Ag

9A TMB + 0.05 TDZ + 5.70 IAA + 30.0 Ag9B TMB + 0.05 TDZ + 1.43 IAA + 30.0 Ag9C TMB + 0.05 TDZ + 5.70 IAA + 60.0 Ag9D TMB + 0.05 TDZ + 1.43 IAA + 60.0 Ag

lOA TMB + 0.05 TDZ + 5.70 IAA + 10.0 Ag + 80.0 ABAlOB TMB + 0.05 TDZ + 5.70 IAA + 10.0 Ag + 40.0 ABA10C TMB + 0.05 TDZ + 5.70 IAA + 10.0 Ag + 5.0 ABA

-------------------------------------------------------------------IAA:Indole acetic acid BAP:Benzylaminopurine TDZ:ThidiazuronIBA:Indole 3 butyric acid KIN:Kinetin Z :ZeatinABA:Abscisic Acid GA:Gibberellic acidAg:Silver nitrate Glu:L-glutamine* : Same medium was prepared with 6% sucrosef : Tulecke et ale (1988)

California Walnut Board Walnut Research Reports 1990