The procedures involved in plant transformation by Agrobacterium vectors can be divided into four areas as follows:

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Molly Sanders
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1 I I WALNUT TISSUE CULTURE - SYSTEMS FOR TRANSFORMATION G. McGranahan, C. Leslie } 1 ABSTRACT The bjective f this study is t develp a tissue culture system fr regeneratin f transfrmed walnut plants, i.e., plants cntaining freign genes. This regeneratin system is t be used in cnjunctin with wrk being dne by A.M. Dandekar (86-WMB3). Repetitively embrygenic smatic embrys appear t be a prmising rute fr genetic transfrm~tin f walnuts. Embrys were shwn t be highly infectible by tumr inducing Agrbacterium tumefaciens strains. Snicatin and wunding by scalpel cuts were shwn t be satisfactry methds fr inculatin. Methds f screening fr transfrmants cntaining a kanamycin resistance gene were examined. Variable results were btained using a brad range f kanamycin cncentratins (0-500 mg/l) n nrmal walnut smatic embrys. Sensitivity was best espressed as reduced embry prliferatin at kanamycin levels ver 50 mg/l. Ptentially transfrmed embrygenic lines, btained by inculating repetitively embrygenic smatic embrys with disarmed strains f A. tumefaciens, are currently being screened using kanamycin at 75 mg/l. OBECTIVES The gal f this prject is t develp a tissue culture system fr regeneratin f transfrmed walnut plants. Agrbacterium tumefaciens has been selected as the vectr fr intrducing freign genes int the walnut genme. T be successful the accmpanying tissue culture system requires regenerative tissues f single cell rigin at an infectible site. Our effrts have been fcused n evaluating the ptential f repetitively embrygenic smatic embrys fr this purpse. PROCEDURES The prcedures invlved in plant transfrmatin by Agrbacterium vectrs can be divided int fur areas as fllws: a. Cnstructin, selectin and prpagatin f the Agrabacterium disarmed vectrs (t be carried ut by A. Dandekar). b. Inculatin f hst cells r tissues with the selected Agrbacterium vectr ~ccultivatin). This includes selectin and maintenance f hst tissue during pre- and pst-inculatin phases and develpment f inculatin prcedures. c. Selectin f transfrmants and regeneratin f plants. Marker genes are incrprated int transfrmed cells specifically s that the regenerated transfrmed plants can be identified. Because, at least in the early stages f system develpment, the frequency f regeneratin frm transfrmed cells may be extremely lw, an early, efficient selectin prcedure is essential. d. Analysis f regenerated plants fr expressin f freign genes and T-DNA (t be carried ut by A.M. Dandekar)

2 ) ) 1 Areas a and d require the expertise f a mlecular bilgist and are being carried ut in A.M. Dandekar's lab. Prcedures used in areas band care discussed here. The first step in ur study was t examine the susceptibility f English walnut embrys f a repetitively embrygenic line t ~ tumefaciens infectin. Three age classes f A Franquette embrys (1. yung/white, 2. intermediate/cream clred and 3. ld/brwn and callusing) were inculated with tw armed (tumr inducing) A. tumefaciens strains (K12xbCGN562E (=562) and K12xbCGN167 (=167») and a sterile media cntrl, using 3 inculatin methds (scalpel cuts, syringe pricks, and snicatin). Tw replicatins f 5 embrys each per treatment were inculated and grwn in the dark n basal Driver-Kuniyuki-walnut (DKW) medium cntaining 50 pm -acetsyringne fr 48 hurs t allw infectin. Embrys were then transferred t D~l basal medium with 500 mg/l carbenicillin, an antibitic used t inhibit further A. tumefaciens grwth. The number f embrys exhibiting tumr grwth was cunted at ne mnth. Tumr tissue was islated frm these cultures and repeatedly subcultured fr 3 mnths n basal DKW with 500 mg/l carbenicillin t rid the material f residual bacterial ppulatins. Tumr tissue was then assayed fr the presence f pines by paper electrphresis in A. Dandekar's lab in rder t cnfirm infectin and transfrmatin by ~ tumefaciens. The secnd step in develpment f a transfrmatin system was t design a screening prcedure that culd be used t identify transfrmants. The ~ tumefaciens strains available t us fr this study cntained a kanamycin resistance gene as a marker. Thus, several trials were undertaken t determine the lwest kanamycin cncentratin at which nrmal walnut embrys express sensitivity and the frm in which sensitivity is expressed. Embrys derived frm cells with the intrduced kanamycin resistance gene shuld nt exhibit sensitivity at this level and therefre shuld be selectable. Filter sterilized kanamycin was added t DKW basal medium at cncentratins ranging frm 0 t 500 mg/l. Three replicatins f 3-5 embrys each were used in each trial. Treatments examined included light and dark grwing cnditins, slid and liquid media, and carbenicillin and ceftxime media supplements. The third step was t inculate embrys with 3 disarmed A. tumefaciens strains (594, 200-0, 200-N) (btained frm A. Dandekar's lab) using tw inculatin..methds (snicatin and scalpel cuts). Due t cncern regarding carbenicillin effects n embry grwth and repetitive embrygenesis, bth ceftxime and carbenicillin were used as pst-infectin bacterial inhibitrs. Embrys that frmed frm inculated embrys were multiplied as subclnes. These are currently being screened fr kanamycin resistance by culturing n 75 mg/l kanamycin in slid DKW medium in the dark. Effrts were als undertaken t increase imprve the efficiency f using smatic Nutrient levels and hrmne treatments were results. embry germinatin rates t embrys fr transfrmatin. examined as discussed in the

3 - RESULTS AND DISCUSSION Smatic embrys f walnut were successfully infected with ~ tumefaciens (Table 1). Infected embrys exhibited cream t yellw clred ndular white callus which grew rapidly in the absence f exgenus hrmnes. Damaged embrys will ccasinally frm callus n basal medium in the absence f expsure t A. tumefaciens, but this callus differs in that it is white, grws very slwly, and is grainier in texture. Observatin f hrmne auttrphic callus grwth in walnut embry tissue is indicative f increased endgenus' auxin synthesis assciated with armed A. tumefaciens vectrs and strngly suggests f ~ tumefaciens infectin and transfrmatin. Early tumr grwth was readily bserved within three weeks f inculatin. All healthy embry tissues eventually prved infectible and exhibited characteristic yellwish-white ndular callus within six weeks f infectin with the exceptin f a single embry treated with strain 562. This embry has been multiplied and is currently being tested fr pssible crwn gall resistance. Old embrys exhibited a reduced infectin rate, apparently because sme f the tissue began t die befre tumr grwth cmmenced. Syringe inculatin prduced fewer tumrs, presumably due t the smaller surface area impacted by this methd. Paper electrphresis f callus! extracts demnstrated the presence f the pine ctpine in 4 f 6 tumrs assayed frm bth 167 and 562 infected material but nt frm cntrls (Table 2). The presence f pines which are nt nrmally prduced by plants, is als indicative f freign gene insertin and expressin in walnut smatic embry tissue. Kanamycin sensitivity in plants can be expressed as decreased chlrphyll prductin, reduced grwth and reduced embry prliferatin. Reduced embry prliferatin appears t be the mst reliable f these respnses fr screening walnut smatic embrys (Table 3). Kanamycin levels f 50 mg/l and higher inhibited repetitive embrygenesis in the dark. Ceftxime at 300 mg/l did nt interfere with this respnse (Table 4) whereas bth ceftxime and carbenicillin appeared t have adverse effects r embry greening and grwth in the light. This masked the embrys respnse t kanamycin. Attempts t use liquid shake and rller tube cultures t insure a mre unifrm expsure f embrys t kanamycin, which is nt well translcated in plants, have nt yet prduced a r~liable chlrphyll screen. Embrys inqo;ulatedwith the 3 disarmed strains f A. tumefaciens have exhibited excellent grwth and multiplicatin rates nbth ceftxime and carbenicillin supplemented media. In sme plants (e.g. tmat), A. tumefaciens inhibits embry frmatin but this has nt prven a prblem in walnuts. Embrys prliferated frm this material are currently being screened fr transfrmants. As part f a cntinuing attempt t imprve the verall efficiency f the smatic embry system, several changes in the culture prcedure were examined. The sucrse level in embry germinatin medium, nrmally 3%, was tested at cncentratins between 6% and 0.375% (28 embrys/treatment)

4 ) ) Reductin f sucrse t 1.5% r less increased unifrmity f ctyledn greening and extended the time t embry senescence in the light, but did nt i~crease the embry germinatin rate. Reductin f nutrient levels is emplyed in many plants t enhance germinatin and rting. Reductin f nutrients t as lw as 1/8 standard cncentratin (17 embrys/nutrient level) had n apparent effect n embry develpment r germinatin in the light ver a 4 week perid. Incrpratin f 5 mgll gibberellic acid (GA3) in the embry germinatin medium prduced minute hypctyl elngatin «0.5 em) and abnrmal elngatin and narrwing f emergent leaves but n substantial stem elngatin r release f apical r rt meristems. Subsequent transfer f these embrys t DKW (micrprpagatin media) prduced stem elngatin in 7/20 embrys that had been expsed t GA3 and in 2/15 that had nt been expsed. Applicatin f benzy1aminpurine (BAP) t germinatin media at levels as high as 20 fld thse used fr micrprpagatin failed t enhance germinatin. Applicatin f drps f varius cncentratins f BAP and GA3 directly t embry apices als prduced n effect n embry germinatin. Inhibitrs in the ctyledns may be in part respnsible fr pr germinatin rates, as has been fund in grapes, and ctyledn remval currently shws sme prmise f increasing germinatin efficiency. In cnclusin, we have established that repetitively embrygenic smatic embrys are infectible by ~ tumefaciens. Our current wrk n this prject is aimed at imprving screening techniques fr detectin f transfrmants regenerated thrugh smatic embrygenesis. The final evaluatin f this system fr transfrmatin, hwever, will be based n whether we are successful in btaining transfrmed plants frm infected embrys. TABLE 1. Percent f embrys exhibiting tumr-like grwth ne mnth after inculatin with Agrbacterium tumefaciens. Inculatin Embry x A. tumefaciens Strain Methd Age Cntrl Snicatin y I Syringe y I Cut y I xi Y - yung, I - intermediate, 0 - ld. j

5 I I TABLE 2. Opine prductin Agrbacterium Strain Cntrl x Infectin Methd syringe snicate '"cut syringe snicate cut syringe snicate cut n = 1 cu 1ture per.. r.g.n. O. x p.ne Prductin TABLE 3. Embry prliferatin Qn kanamycin medi~. Kanamycin Cncentratin (mg/1) % embrys underging embrygenesis (n = 15) Number f new embrys j I j 11 --

6 I TABLE 4. Embry prliferatin n kanamycin meclia in the presence f 300 mg/l ceftxime. Kanamycin Number f new embrys mg/l (n = 5 cultue plates) I

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