Plant Physiol. (1982) 69, 624-6270032-0889/82/69/0624/04/$00.50/0

Effects of Cryogenic Treatment on Plantlet Production fromFrozen and Unfrozen Date Palm Callus

Received for publication March 16, 1981 and in revised form August 3, 1981

JANE M. ULRICH, BERNARD J. FINKLE, AND BRENT H. TISSERATWestern Regional Research Center, Agricultural Research Service, United States Department ofAgriculture,Berkeley, Caitfornia 94710 (J. M. U., B. J. F.); and Fruit and Vegetable Chemistry Laboratory, AgriculturalResearch Service, United States Department ofAgriculture, Pasadena, Calfornia 91106 (B. H. T.)

ABSTRACT

Embryogenic date pahl (Phoeix dctylfera L. var. Medjool) calluscultures were treated with a cryoprotective mixture of polyethylene glycol(Carbowax 6000), glucose, and dinethylsulfoxide (10%/8%/10%, w/v);treated with the mixture, frozen to -1960C, and then thawed; or leftuntreated. Growth subs t to treatment was measured as fresh weightincrease and as the number of embryos produced during 18 weeks ofculturem. he growth of cali that were frozen and tharwed, compared to theother treatments, was gready inhIbited during the first 9 weeks of culture.Tis htio disa in subcultured tissue. In adl treatments, culturesinitiated plantlets after 9 weeks. Enzyme polymorphism, for five gene-associated enzyme systems including alcohol dehydrogenase, esterase,peroxidase, phosphoglucomutase, and pbosphoglucoisomerase, was ana-lyzed in leaves of regenerated plantlets by using starch gel electrophoresisfor separation. Isozyme patterns were similar for alH treatments.

Cryogenic storage offers an enormous potential advantage forthe preservation of plant genetic resources and experimental plantlines compared to that available through other in vivo and in vitromeans. Fruit trees and other plants that are vegetatively main-tained under field conditions for cultivar preservation and breed-ing purposes are susceptible to environmental hazards, geneticerosion, pests, and diseases (8, 30). These collections also requireconstant supervision and maintenance, expenditure of natural andfinancial resources and commitment of large areas to relativelynonproductive acreage. Moreover, field collections that may re-quire decades to accumulate are often subjected to total elimina-tion. An alternative to soil maintenance of cultivars (or of mutantor other special lines for research purposes) is the in vitro preser-vation of germplasm through serial transfers of plant tissue cul-tures. This alternative is often unsatisfactory in that many plantcultures change in genetic composition during serial subculture(6, 7, 23, 28). In addition, the tissues of many fruit tree crops canbe cultured at present only through an intervening callus stage (4,24). Plantlets produced from callus need to be grown to thefruiting stage (usually 2 to 7 years of age) to evaluate their clonalnature.Development of an alternative method to preserve plant genetic

resources is needed so that the greatest diversity of genotypes canbe retained for future breeding and genetic engineering studies.Coupling plant tissue culture with cryogenics appears to be aplausible alternative method for long-term preservation of planttissue in a genetically stable form (either as genetic resources or aswell-defined experimental lines). Frozen gene banks are exemptfrom many of the disadvantages that occur in the field and in

serial tissue cultures.Freezing and thawing of tissues may also have an adverse effect.

Bacteria undergo mutation when freeze-dried. In this case, how-ever, the mutative effect has been attributed to the drying stepand not to the freezing, which appears to be benign (2). Withfrozen plant material, using tissue squashes and other tests, nochromosomal breaks have been identified, implying genetic sta-bility through the freezing process (15). Other indices are needed,however, by which to determine the clonal stability of plant cellstaken through a freezing step. Development of a genetic qualitycontrol test to aid in the selection of clones produced throughtissue culture would be welcomed, particularly by fruit tree re-searchers. Several recent reports dealing with fruit trees haveutilized biochemical markers, such as isozymes, to verify andpredict progeny hybridization and determine taxonomic relation-ships (26, 27). These studies indicate the possibility of developinga suitable biochemical test to verify the clonal nature of trees andother plants when still in the young plantlet stage.The importance of freezing temperatures (18), cooling and

rewarming rates (20), and the use of an appropriate mixture ofcryoprotectants (5, 29, 31) on plant cells subjected to a freezingprocess is well documented. Callus cultures of date palm, sugar-cane, alfalfa, rice, soybean, and other species frozen in a mixtureof cryoprotectants (see below) have survived deep freezing in ourlaboratories; the first three examples have developed into entireplants (25; 29; and J. M. Ulrich, B. J. Finkle, D. W. Rains, and S.Stavarek, unpublished observations, respectively). The detailedeffects of cryogenic freezing on growth and, particularly, ongenetic stability are, however, not well established. These aspectsof growth will need to be extensively examined when evaluatingthe degree of success to be expected from cryogenic germplasmrepositories.We report here that date palm callus was frozen to -196°C,

thawed, and then examined in terms of subsequent growth andplantlet generation. Isozyme analysis of leaves from regeneratedplantlets was performed to detect possible genetic alterationscaused by the cryogenic treatments.

MATERIALS AND METHODS

Tissue Culture Procedure. Embryogenic callus cultures ofPhoe-nix dactylifera L. var. Medjool were initiated as previously de-scribed (24), on a modified Murashige and Skoog medium (14)containing 100 mg/L 2,4-D, 0.3% activated-neutralized charcoal,and 1% agar. Callus was derived from lateral buds dissected from5-year-old offshoots. Transferring callus to nutrient medium de-void of 2,4-D allowed plantlets to develop with distinct root andshoot apices after a few weeks in culture.

Cryogenic Treatments. Embryogenic date palm callus (0.4-0.5ml each, settled volume, placed in heavy-walled glass centrifuge

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tubes on ice) were soaked for 5 to 10 min in 1 ml cold, one-fourthstrength PGD' (29). The concentration of PGD was increased byadding an equal volume of full strength PGD and mixing gently.After 5 min, the solution above the settled cells was aspirated andthe cells made up to 1 ml with full strength PGD. Callus wascooled in a programmed freezer (Cryo-Med,2 Mt. Clemens, MI).The samples were allowed to equilibrate at 4°C for 5 min andthen cooled at 3°C per min to -4°C and then at 1°C per minafter ice crystallization was initiated by a programmed surge ofnitrogen vapor from the liquid N2 cylinder. When samples reached-30°C (after 26 min) they were transferred by hand to liquid N2(- 196°C). The tubes of callus were left for 4 min in the liquid N2and then thawed by rapidly swirling the tubes in a 40°C waterbath until the ice melted. The PGD was diluted at room temper-ature with Murashige and Skoog inorganic salts medium (mac-ronutrients) containing 3% sucrose, added gradually to minimizeosmotic shock. Callus specimens were also treated with the cryo-protective mixture without freezing, or left untreated. After wash-ing, aliquots of tissue from each treatment were cultured on solidnutrient medium.Measuring Growth and Morphogenic Potential. Treated callus

samples, weighing about 25 mg, were planted on nutrient agarmedium without 2,4-D. Callus samples were removed at 3-, 6-,and 9-week intervals to evaluate gross morphology, morphogenicpotential, and fresh weights. The morphogenic potential wasdetermined by counting the number of embryos and plantletsproduced per culture. Date embryos were counted by removingthe intact culture from the culture vessel, weighing, and transfer-ring to a 125-ml Erlenmeyer flask containing 25 ml tap water. Themixture was agitated manually to disrupt callus adhesion, and anadditional 50 ml of water was added. The resultant mixture wasfiltered through a series of stainless steel sieves (Newark WireCloth Co., Newark, NJ) with decreasing diameters of pore open-ings of 4750, 2000, 850, and 600 ,um. Approximately 250 ml waterwere subsequently poured through the sieves to facilitate sievingof embryos. The screens were removed and inverted into Petridishes, and the collected embryos were washed off with the aid of15 to 25 ml of tap water sprayed from a wash bottle.At the end of the original 9 weeks in culture, 10 mg of callus

from each treatment were subcultured to fresh medium, and datawere again taken on the growth and embryo count at the sametime intervals. Triplicate cultures were used for each treatment ateach time interval.Isozyme Analysis. The isozyme patterns of extracts of trplicate

leaf samples from each treatment were examined for enzymepolymorphism and compared with each other and with samplestaken as controls, from several field-grown date palm cultivars.The general procedures used for starch gel electrophoresis and

gel staining have been described previously (26, 27). A 0.25-cm2piece of healthy leaflet tissue was placed in a plastic weighingboat. The leaflet tissue was macerated by several blows with ahammer in the presence of 3 to 4 drops of crushing buffer, whichconsisted of 10 mm pH 7.5 Tris-citrate with 100 mm 2-mercapto-ethanol, 12% soluble PVP, 1 mm EDTA, and 3% BSA. Theresultant solution was filtered through cheese cloth and wasabsorbed onto 7- x 5-mm filter paper wicks (Whatman No. 1) forseparation in 12% starch gel slabs (Sigma). Gels were formed in120- x 160-mm Plexiglass molds and presoaked sponge-clothsserved as electrode bridges. Electrode buffer was composed of 40

' Abbreviation: PGD, cryoprotective mixture consisting (at full strength)of polyethylene glycol (Carbowax 6,000, 10% w/v), glucose (8% w/v), anddimethylsulfoxide (10% v/v).

2 Reference to a company and or product named by the Department isonly for purposes of information and does not imply approval or recom-mendation of the product to the exclusion of others which may also besuitable.

mM sodium borate (pH 9.0). Electrophoresis was conducted at5°C in a refrigerator. Following electrophoretic runs, gels weresliced into three pieces, stained, and incubated at 350C. All theobserved isozymes were found in the anodal section of the gel.Gene-associated enzyme systems analyzed included alcohol de-hydrogenase, esterase, peroxidase, P-glucoisomerase and P-glu-comutase. All gel slices were fixed in 50%o aqueous methanol.Relative mobility of bands (Rp x 100) was calculated based onmigration distance to the center of each band.

RESULTS

Effects of Cryogenic Treatments on Callus Growth and Embry-ogenesis. All callus samples regardless of treatment were initiallysimilar in appearance. They were characterized as being yellow-brown in color and were composed of nodular clumps. Culturestreated with the cryoprotectant and the untreated controls grewvery similarly during incubation. At the end of 6 and 9 weeks,untreated and PGD-treated cultures were yellow-white in colorand composed of friable clumps. An examination of fresh weightsand numbers of embryos produced per culture indicated similardevelopment after these two treatments (Table I, fresh weight).No adverse effect appeared from the administration of PGDcompared to untreated control cultures. By the ninth week ofculture, untreated and PGD-treated controls had similar growthand developmental responses and were producing green plantletswith distinct foliar leaves and primary roots of about 2 to 3 cm inlength.With callus carried through the freezing treatment, however,

rate of development was notably diminished compared to theother treatments. The number of embryos was decreased (TableI, embryos). Those embryos that survived, even though frequentlyinitially smaller in size, appeared normal in plantlet developmentand growth habit (Fig. 1).

Subculturing uniform 10-mg amounts of callus specimens tofresh medium resulted in a rapid, comparable growth of allcultures in weight, number of embryos, and morphogenic re-sponses. Thus, callus growth and morphogenic response amongthe treatments were all comparable in the subcultures, includingthe treatment subjected to - 196°C, suggesting that those cellswhich survived the freezing treatment were morphogenically un-impaired. By the ninth week, all treatments were producing abun-dant embryoids and plantlets.

Analysis of Enzyme Systems of Date Palm Cultivars and Plan-tlets. The presence ofenzyme polymorphism was observed in datepalm cultivars in an earlier study (27). The results of the enzymicanalysis of leaf enzymes reported here are tabulated according toisozyme migration rates (Rp values) for each of several date palmcultivars (Table II). Each enzyme pattern was constant withrespect to Rp values although band intensity varied. Certain Rppositions were common to all date palm cultivars; but the bandprofile for these five enzyme systems served to distinguish thecultivars. The Rp positions shown in the table and the relativeintensities (not shown) ofthe bands (see Ref. 27, Figure 1) indicatethat within the Medjool cultivar no enzyme differences wereexpressed among the leaflets of plantlets from untreated, PGD-treated, and frozen cultures.

DISCUSSIONThe freeze-thawing process has almost invariably had some

adverse effect on the resultant growth rate of cultured cells due toeither cellular kill or inhibition (3, 9). Sakai et al. (19) reportedthat 60 to 80%o of thawed strawberry runners developed normalshoots. Similarly, Seibert and Wetherbee (21) reported that about60o ofcultured carnation shoot tips differentiated following thaw-ing. Hwang and Homeland (9) found that motility of thawedEuglena was delayed by the freeze-thawing process. Nag and

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Plant Physiol. Vol. 69, 1982

Table I. Effect of Cryogenic Treatments on the Growth and Sexual Embryogenesis of Date Palm Callus TissueWithout Cryoprotectant With Cryoprotectant Liquid Nitrogen

Time inCulture Original Subculture Original Subculture Original Subculture

culture culture cultureweeks gfresh wt/culture

3 0.07 ± 0.01 0.31 ± 0.02 0.10 ± 0.01 0.17 ± 0.06 0.05 ± 0.01 0.09 ± 0.016 0.20 ± 0.02 0.57 ± 0.09 0.21 ± 0.05 0.80 ± 0.11 0.05 ± 0.01 0.48 ± 0.019 0.61 ± 0.07 1.22 ± 0.20 0.58 ± 0.12 1.19 ± 0.23 0.09 ± 0.03 1.31 ± 0.22

embryos/culture3 18±3 117±39 15±4 186±22 12±3 84±416 133± 18 439±72 148±41 523± 104 42±9 339±249 451±63 546±76 528±175 543±54 87±27 691±51

FIG. 1. Examples of plantlets derived from date palm callus subjected to various cryogenic treatments. From left to right: with solution ofcryoprotectants only; without cryoprotectants (normal control); with cryoprotectants and -1960C freezing-thawing exposure. Plantlets are about 9weeks old.

Street (15) found that low-temperature freeze-thawing selectsagainst large highly vacuolated cells compared to meristematiccells and reported a 70%o cell survival rate for cultured carrot cells.They also reported that the morphogenic potential of carrotsuspensions was unimpaired by the low-temperature treatment(15, 16). We have observed similar results with callus cultures ofalfalfa (J. M. Ulrich, B. J. Finkle, D. W. Rains, and S. Stavarek,unpublished data).

In the present study, the addition of cryoprotective mixture tothe unfrozen controls at 0°C did not adversely affect growth andmorphogenesis of callus cultures of date palm, but treatment at- 196°C decreased the subsequent rate of culture growth, com-pared to control treatments. This inhibitory effect was probablydirectly related to the number of cells injured or killed by thefreeze-thawing process. When frozen callus was subcultured infresh medium, it proliferated rapidly with no indication of mor-phogenic repression or genetic changes and differentiated to formnormal looking plants.

Variation in isozyme patterns has been reported for plantletsderived from callus of highly polyploid Saccharum spp. (7). How-ever, variation in these isozymes was not directly related todifferences in morphology and cytology. McCown et al. (13) foundthat Dianthus callus cultured under various environmental condi-tions demonstrated isozyme polymorphism. Carrot callus culturedon medium with and without 2,4-D also exhibited enzyme poly-morphism (10, 11). Cultured Acer pseudoplatanus cells showedquantitative differences in their enzyme levels during growth (22).On the other hand, Quatrano (17) failed to fmd any qualitative

differences in isozyme banding patterns between frozen (-50°C)and untreated suspension cultures of flax. Similarly, we found nopolymorphism from leaves of date palm plantlets that developedafter various cryogenic treatments of the progenitor callus tissue,including freezing at - 196°C. Thus, it appears that the process offreezing itself, even at such extremely low temperatures as- 196°C, may produce no morphogenic changes in surviving cellswhich differentiate. This conclusion is in agreement with the

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Table II. Relative Positions (Rp Values) ofIsozymesfrom Leaves of Phoenix dactylifera L. Cultivars orSpecimens

l~~.nzyme ~ Deglet Khad- Dayri L7, MedjoolEnzyme DNoegt rawya BC3Noor8rawy8 BC? ~Fielda -cb +CC LNd

5053

Alcohol dehydrogenase 56 56 56 56 56 56 56Esterase 77 77 77 77 77 77 77

86 86 86Peroxidase 50 50 50 50 50 50 50Phosphoglucoisomerase 7 7 7 7 7 7

12 12 12 12 12 1220 20 20 20 20 20 2067 67 67 67 67 67 67

Phosphoglucomutase 70 70 70 70 70 70 7073 73 73 73 73 73

a Mature leaf samples from verified cultivars grown in the field.b Specimen leaf sample from untreated Medjool cultures; C, cryoprotectant.c Specimen leaf sample from cryoprotectant-treated Medjool cultures.d Specimen leaf sample from cryoprotectant-treated Medjool cultures, frozen to - 196°C.

findings of Ashwood-Smith and Grant (2) that bacteria are ge-netically stable to freezing but subject to mutagenic effects whenthey are freeze-dried. Furthermore, results of experiments withanimal cells indicate that they undergo little metabolic or geneticchange after having been stored at -196°C, even for very longperiods (1, 12). Genetic stability would be the key to reliability inthe 'frozen storage' of germplasm. Another variety of date palmcallus reported previously was stored at -196°C for 3 monthswithout any observed changes in the patterns of survival ormorphology of resulting plantlets (25).

Date palm callus was frozen to the temperature of liquid N2(-196°C) and thawed. Revived callus initiated plantlets. Analysisof the isozymes for several enzymes in leaves from these plantletsrevealed no expressions of genetic change. The feasibility ofdeveloping dependable storage for plant tissues through cryogen-ics is further indicated by these results. However, the need stillexists to improve the range and reliability of the criteria of geneticstability in plant tissues during the successive stages of freezing,low-temperature storage, and thawing.

LITERATURE CITED

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