CRYOBIOLOGY 30, 179-184 (1993)

Responses to ABA and Cold Acclimation Are Genotype Dependentfor Cryopreserved Blackberry and Raspberry Meristems 1

BARBARA M. REEDUSDA/ARS National Clonal Germplasm Repository, 33447 Peoria Road, Corvallis, Oregon 97333-2521

The meristems of cold-acclimated cultivars and species of Rubus (blackberry and raspberry) can besuccessfully cryopreserved through optimization of cryoprotectants, cooling rates, and cold acclima-tion treatments. Genotypes that do not cold acclimate are difficult to cryopreserve and new methodsare needed for this group of plants. The effects of abscisic acid (ABA) (50 110M) and 7-day coldacclimation (CA). treatments on the survival of cryopreserved apical meristems was determined forfive Rubus genotypes (Blackberries: Rubus cissoides Cunn. and Rubus hybrid cvs. Hillemeyer andSilvan, and Raspberries: R. parvifolius L., and R. parvijlorus Nutt.). ABA alone did not significantlyimprove the survival of any ofrth.~ ~~.~types tested. The combination of ABA and CA significantlyimproved the survival of R. ~,';;but neither was effective alone. Cold acclimation with orwithout ABA significantly improved the survival of meristems of 'Hillemeyer,' 'Silvan,' and R. parvi-folius L. Survival of R. parvijlorus Nutt. meristems was not significantly affected by any ofthe treatments. In contrast to cytokinin effects in other culture systems, the cytokinin benzyladeninedid not significantly affect survival of 'Hillemeyer' and showed no interaction with ABA or CA.Murashige and Skoog-based recovery medium produced higher survival rates than did Anderson'smedium for the nine genotypes tested. Overall, genotype played a large role in the survival of cryo-preserved Rubus meristems. In this system ABA does not substitute for CA, but in some genotypesmay interact with CA to increase survival through a synergistic effect. c> 1993 Academic Press. Inc.

Meristems of many Rubus genotypes canbe cryopreserved using optimum cryopro-tectants, freezing rates, and cold acclima-tion treatments (11). Improved cryopreser-vation methods are needed for many tropi-cal plants and those that do not respond tocold acclimation. Cold acclimation can beinduced by abscisic acid (ABA) in cell cul-tures of many plant species (2, 8, 10). Max-imum hardiness is often obtained within 7days of ABA treatment and is indicated byincreases in starch grains, lipid bodies,sugar content, and dry weight similar tothat produced by cold acclimation (16).

Cytokinins sometimes counteract or alterthe normal action of ABA (3, 7). For exam-

Received February 26, 1992; accepted October 15,1992.

1 The technical assistance of Carolyn Paynter and

Patricia Buckley is greatly appreciated. The use oftrade names in this publication does not imply en-dorsement by the u.s. Department of Agriculture.

pIe, freezing tolerance induced by ABA canbe reduced or totally inhibited by the pres-ence of kinetin in the growth medium, butthis effect varies with genotype (10). Pre-sent methods of Rubus cryopreservationuse a growth medium containing the cyto-kinin benzyladenine (BA) and have moder-ate survival rates (11). The interactive ef-fect of cytokinins, cold acclimation, andABA in the Rubus cryopreservation systemis unk!lown.

Survival and the rate of shoot develop-ment from meristems following cryopreser-vation are dependent on the culture me-dium used in the recovery period (19). Vari-ations among genotypes indicate that asingle recovery medium is not always suit-able for all members of a genus (18, 19). Themedium on which a plant is grown also in-fluences its vigor in culture and may affectthe tolerance of meristems to cryopreserva-tion (12).

This study compared ABA and cold ac-

179

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All rights of reproduction in any form reserved.

180 BARBARA M. REED

climation pretreatments for improving thesurvival of cryopreserved Rubus meristemsfrom five genotypes; examined the interac-tion between ABA treatment and the cyto-kinin BA in the growth medium; and com-pared the effect of two commonly used re-covery media on meristem survival ratesfor nine genotypes.

ment, meristems were transferred to 0.25ml liquid medium in 1.2-ml plastic cryo-tubes on ice. The cryoprotectant PGD (4), amixture of 10% each of polyethylene glycol(MW 8000, Sigma, St. Louis MO), glucose,and DMSO in water, was added dropwiseup to 1.2 ml over 30 min. A 30-min equili-bration period on ice was followed by re-moval of excess cryoprotectant down to 1mI. Samples were frozen to -35 at 0.8°C/min in a programmable controlled-temperature cooling chamber (Cryomed,New Baltimore, MI) and then immersed inliquid nitrogen (LN) for 1 h. Vials werethawed for one min. in 45°C water, thencooled in 23°C water until the ice was com-pletely melted. The cryoprotectant wasdrained from the vials and replaced withliquid MS medium. Meristems weredrained on sterile filter paper and plated onrecovery medium for regrowth. Ten ortwenty treated meristems and five controls(not frozen) were tested for each replicateof each accession with three or four repli-cates per experiment.

Recovery. Meristems were plated on 2 mlmedium in sterile 24-well plates (Costar,Cambridge, MA) for regrowth under stan-dard growth room conditions. Total sur-vival (shoots and callus) was evaluated at 1month for the formation of shoots or callus.Shoot survival included only those mer-istems exhibiting organized shoot growth 1month after thawing. The basal MSmediumwas used in all except one recovery exper-iment in which both MS and Anderson'smedium were used.

Histology. For histological study, mer-istems were fixed in pH 6.8 phosphate-buffered (0.02 M) 3% glutaraldehyde for1-3 days at 4°C, dehydrated with an ace-tone:water series, infiltrated and embeddedin .Spurr's low-viscosity epon mixture(Polysciences, Warrington, fA). Sections10-15 ~m thick were stained with toluidineblue or methylene blue. .

Data analysis. Data were analyzed withMSTAT software (Michigan State Univer-

MATERIALS AND METHODS

Media. Micropropagated shoots of Ru-bus accessions (species and cultivars) weremultiplied in Magenta GA 7 boxes (MagentaCorp., Chicago, IL) on pH 5.7 base me-dium: Murashige and Skoog (MS) (9) saltsand vitamins with 3% sucrose, I mg N6-BA,0.05 mg indo1e-3-butyric acid, 0.01 mg GA3(Sigma, St. Louis, MO) and 3.5 g BiTekagar (Difco, Detroit, MI), and 1.45 g Gelrite(Kelco, San Diego, CA) per liter. After au-toclaving, ABA (:tcistrans isomer, Sigma)(50 ~M) in dimethyl sulfoxide (DMSO,Sigma) was added to the medium, and anequivalent amount of DMSO was added tothe non-ABA treatments (15). The finalconcentration of DMSO in the medium was0.1%. For recovery, the same MS formula-tion or Anderson's medium (1) with thesame growth regulator concentrations and 3g agar and 1.25 g Gelrite were used.

Pretreatment. In vitro plants were cold.,acclimated (CA) for 1 week in a growthchamber with 22°C 8 h days (20 ~E M-2S-l) and -1°C 16 h nights (11). Regulargrowth room conditions were maintained at25°C with a 16/8 h (light/dark) photoperiod(30 ~E M-2 S-l). Apical meristems weredissected from in vitro plants pretreated for1 week with the appropriate medium andenvironmental condition. Dissected mer-istems (0.8 mm) of each treatment weregrown for 48 h on basal medium with 3.5 gagar and 1.75 g Gelrite per liter and with 5%DMSO under the same growth regulatorand environmental conditions as the parentplants. ABA concentrations used were de-termined in preliminary experiments.

Cryopreservation. Following pretreat-

181RESPONSES TO ABA AND COLD ACCLIMATION IN Rubus MERISTEMS

sity, East Lansing, MI) using factorial anal-ysis and mean separation with Duncan'smultiple range test.

RESULTS ~ND DISCUSSION

Effects of Cold Acclimation and ABA

Cold acclimation significantly improvedsurvival of most Rubus genotypes eitherwith or without ABA; however, a 7-day 5 x10-5 M ABA pretreatment produced nosignificant increases in total or meristemsurvival for plants grown under warm con-ditions (Table 1). Factorial analysis showedthat the interaction of pretreatment and ge-notype was significant (P ~ 0.01) for totalsurvival percentages. The effect of pre-treatment was also significant (P ~ 0.01) forthe percentage shoot survival. The additionof 50 ~M ABA failed to produce significantincreases in total survival for any of thewarm-grown or for three of five cold-acclimated plants. 'Hillemeyer' and R. cis-soides showed significantly greater totaland shoot survival with CAI + ABA than

with any other treatment. This appears tobe a synergistic effect for R. cis so ides sinceit was unaffected by CA alone or by ABAunder warm conditions but total survivaland shoot survival tripled following pre-treatment with the combination of ABAand CA. CA with or without ABA in-creased survival over warm-grown plants infour genotypes and for CA alone with R.parviflorus (Table 1).

The response of Rubus meristems differsmarkedly from the effect of ABA on cellcultures. The induction of cold acclimationby ABA treatment has been observed forcell cultures of winter wheat (ABA 7.5 x10-5 M) (2), birdsfoot trefoil (ABA 10-4,10-5, 10-6 M) (8), and alfalfa (ABA 5 x10-5 M) (10). Bromegrass cells treated withABA at warm or cold temperatures devel-oped more freezing resistance than cellscultured at 3°C without ABA (14). Ishikawaet al. (6) found 25 to 30°C to be the optimumtemperature range for 7.5 x 10-5 M ABA-induced acclimation of Bromus inermis sus-pension cultures. In the present study thesurvival of Rubus plantlets grown at 25°C

TABLE IPercentage Survival after Cryopreservation of Rubus Meristems Grown with 0.44 jjM Benzyladenine (BA)

and Pretreatments of 50 f!.M Abscisic Acid (ABA) and/or I Week Cold Acclimation (CA), 4 WeeksAfter Thawing

Percentage survival following thawing*

Warm/-ABA Warm/+ABA CAI- ABA CAI + ABAGenotype

182 BARBARA M. REED

was not improved by 7-day ABA pretreat-ment before cryopreservation at the sameconcentration. This lack of acclimationmay be due to inhibition of transport to api-cal buds in stress.ed plants (5) or inherentdifferences in response to ABA by diversegenera (10). Further studies of this re-sponse are necessary to determine if this istruly a CA/ ABA synergism as well as themode of action.

TABLE 2The Influence of Cold Acclimation, Benzyladenine

(BA 0.44 110M) and Abscisic Acid (ABA 50 11M)Pretreatments on the Percentage Shoot Survival ofRubus Hybrid Cv. Hillemeyer Meristems Frozen inLiquid Nitrogen, Thawed, and Grown for 4 Weeks

BAfABAtreatment

-BAf-ABA-BAf+ABA+BAf-ABA+BAf+ABA

Percentage survivalfollowing thawing*

Grown at 25°C Cold acclimated

5.0 ~ 3.5 b 42.0 ~ 12.1 a13.8 ~ 4.3 b 52.0 ~ 11.3 a7.2 ~ 4.1 b 40.5 ~ 11.6 a7.3 ~ 3.0 b 43.8 ~ 13.5 a

Interactions Between BA and ABA

Results f°t: blackberry cv. Hillemeyershowed that BA does not inhibit the sur-vival of cryopreserved meristems. Only thepretreatment temperature was significant inthe survival of 'Hillemeyer' meristems fol-lowing cryopreservation in LN. BA did notsignificantly change the survival percentagewith or without either cold acclimation orABA, and interactions were not apparent ineither case. Total survival (shoots and cal-lus) following CA (mean 71%) was signifi-cantly greater than that for warm-grownmeristems (mean 14.8%) in all cases. Shootsurvival was also significantly greater forCA plants of all pretreatments, but the ef-fects of BA and ABA were not significantfor meristems within a temperature treat-ment (Table 2). In contrast to these results,freezing tolerance of alfalfa cell cultures re-quires the deletion of kinetin from thegrowth medium and the use of both low-temperature and ABA treatments (10).

Note. Survival reported as meristems with shootgrowth at 4 weeks after thawing (N = 4 experiments of20 meristems each). Survival of unfrozen controls was100%. Mean separation by Duncan's multiple rangetest (P ~ 0.05). Means followed by the same letter arenot significantly different.

* Mean :f: standard error.

Differences in survival were significant forfive of the nine individual genotypes (P ~0.05) and in all cases survival washigher on MS medium than on Anderson'smedium.

TABLE 3Survival following Cryopreservation of Meristems ofRubus Species and Cultivars Pretreated with I-WeekCold Acclimation and Grown for 4 Weeks on Two

Types of Culture Medium

56.2 ~ 9.8 a

62.1 ~ 3.1 a

33.3 ~ 15.6 a33.3 ~ 25.4 a

Recovery

Medium Effects

Recovery medium experiments showedsignificant effects for medium type (P ~0.001) and genotype (P ~ 0.01), but inter-actions were not significant. Overall, mer-istems of the nine Rubus genotypes had sig-nificantly higher survival rates (mean42.2%) when grown on MS medium than onAnderson's medium (mean 19.3%) (P ~0.001). Genotype effects varied consider-ably from blackberry cv. Hillemeyer withgood survival on both mediatp R. hirsutuswith low survival rates on both (Table 3).

30.0 ~ 17.3 a3.3 ~ 1.7 a

39.7:t 23.3 a10.7:t 3.2 a

1.1:tl.1b 28.3:t 6.0 a

11.8:!: 4.4 b24.7 :!: 14.4 b

19.3 :!: 3.7 b

51.1 :!: 6.7 a65.4:!: 4.8 a

42.2:!: 5.0 a

HillemeyerKotataMandarinORUS 1362R. grabowskii Weihe

ex. Gunther et at.R. hirsutus Thunb.R. leucodermis Doug.

ex Tor. & GrayR. ulmifolius f

bellidiflorus SchottSilvan

Mean of Medium Types

Note. Survival as meristems at 4 weeks after thawing (N =3 experiments of 20 meristems each). Survival ef unfrozencontrols was 100%. Means in rows followed by the same letterare not significantly different [Duncan's multiple range test (P~ 0.05»).

* Mean :to standard error.

183RESPONSES TO ABA AND COLD ACCLIMATION IN Rubus MERISTEMS

Rubus is a diverse genus and the re-sponse of species and cultivars to standardmedia formulations varies considerably(13). This is consistent for the genotypestested in this study. Most grow better onMS medium although some will also growmoderately well on Anderson's medium,which contains lower nitrogen and micro-nutrient concentrations than MS and in-cludes the cytokinin adenine sulfate as aregular component. We have not tested theeffect of adenine sulfate in pretreatmentmedium on survival of cryopreserved mer-istems. Different survival rates among ge-notypes have in some cases been shown tobe due to the recovery medium (17-19).The medium on which a plant is grown in-fluences its vigor in culture and a subopti-mum medium may contribute to decreasedsurvival following cryopreservation (12).

however, meristems grown on MS-basedmedium had higher survival in general thanthose on Anderson's medium. In contrastto cytokinin effects in other culture sys-tems, BA did not significantly affect sur-vival and produced no interaction withABA or CA. Cold acclimation significantlyimproved survival of four genotypes andsurvival of R. cissoides was tripled by com-bined ABA and CA pretreatments. It ap-pears that in this system, ABA does notsubstitute for CA pretreatments but insome genotypes it may interact with CA toincrease survival through a synergistic ef-fect.

Histology.Histological examination of frozen and

control meristems showed normal meriste-matic growth on those that formed shoots.Any callus formed by recovering meristemsremained as callus and did not regenerateshoots, thus preserving the clonal integrityof the sample (data not shown). Callus pro-duction in cryopreserved Rubus may resultfrom wounding during excision or fromfreeze-induced injury. In other studies, theamount of callus production appears to begenetically controlled but may also be dueto growth regulators present in the medium(18). Genetic control seems likely with Ru-bus since the amount of callus productionwas high in all treatments for some geno-types and low for others. Improved recov-ery of shoots from cryopreserved mer-istems through media improvements hasbeen proposed for several genera (18, 19).

REFERENCES

1. Anderson, W. C. Tissue culture propagation ofred and black raspberries. Acta Hort. 112, 13-20 (1979).

2. Chen, T. H. H., and Gusta, L. V. Abscisic acid-induced freezing resistance in cultured plantcells. Plant Physiol. 73, 71-75 (1983).

3. Cowan, A. K., and Railton, I. D. Cytokinins andancymidol inhibit abscisic acid biosynthesis inPersea gratissima. J. Plant Physiol. 130, 273-277 (1987).

4. Finkle, B. J., and Ulrich, J. M. Effects of cryo-protectants in combination on the survival offrozen sugarcane cells. Plant Physiol. 63, 598-604 (1979).

5. Hartung, W. Effect of water stress on transport of(2_14C) abscisic acid in intact plants of Phaseo-Ius coccineus L. Oecologia 26, 177-183 (1976).

6. Ishikawa, M., RobertsQn, A. J., and Gusta, L. V.Effect of temperature, light, nutrients and de-hardening on abscisic acid induced cold hardi-ness in Bromus inermis Leyss suspension cul-tured cells. Plant Cell Physiol. 31,51-59 (1990).

7. Khan, A. A. Primary, preventive and permissiveroles of hormones in plant systems. Bot. Rev.41, 391-420 (1975).

8. Keith, C. N., and McKersie, B. D. The effect ofabscisic acid on the freezing tolerance of calluscultures of Lotus corniculatus L. Plant Physiol.80, 766-770 (1986).

9. Murashige, T., and Skoog, F. A revised mediumfor rapid growth and bioassays with tobacco tis-sue cultures. Physiol. Plant. 15,474-494(1962).

10. Off, W., Singh, J., and Brown, D. C. W. Induc-tion of freezing tolerance in alfalfa cell suspen-sion cultures. Plant- Cell Rep. 4, 15-18 (1985).11.

Reed, B. M. Cold acclimation as a method to im-

CONCLUSIONS

The effect of genotype played a large rolein the surv'ival of Rubus meristems follow-ing cryopreservation. Recovery media testsshowed differential genotype' response;

184 BARBARA M. REED

16. Tanino, K. K., Chen, T. H. H., Fuchigami,L. H., and Weiser, C. J. Metabolic alterationsassociated with abscisic acid-induced frost har-diness in bromegrass suspension culture cells.Plant Cell Physiol. 31, 505-511 (1990).17.

Towill, L. E. Survival at ultra-low temperaturesof shoot tips from Solanum tuberosum groupsAndigena, Phureja, Stenotomum, Tuberosum,and other tuber-bearing Solanum species. Cryo-Lett. S, 319-326 (1984).

18. Towill, L. E. Survival of shoot tips from mint spe-cies after short-term exposure to cryogenic con-ditions. HortScience 23, 839-841 (1988).

19. Withers, L: A., Benson, E. E., and Martin, M.Cooling rate/culture medium interactions in thesurvival and structural stability of cryopre-served shoot-tips of Brassica napus. Cryo-Lett.9,114-119 (1988).

prove survival of cryopreserved Rubus mer-istems. Cryo-Lett. 9, 166-171 (1988).

12. Reed, B. M. Survival of in vitro-grown apical mer-istems of Pyrus following cryopreservation.HortScience 25, 111-113 (1990).

13. Reed, B. M. Multiplication of Rubus germplasmin vitro: A screen of 256 accessions. Fruit Var.J. 44, 141-148 (1990).

14. Robertson,A. J.,Gusta,L. V., Reaney, M. J. T.,and Ishikawa, M. Protein synthesis in brome-grass (Bromus inermis Leyss) cultured cellsduring the induction of frost tolerance by abscis-ic acid or low temperature. Plant Physiol. 84,1331-1336 (1987).

15. Schmitz, R. Y., and Skoog, F. The use of dimeth-yl sulfoxide as a solvent in the tobacco bioassayfor cytokinins. Plant Physiol. 45, 537-538(1970).