Euphytica 37: 141-148 (1988) © Kluwer Academic Publishers, Dordrecht ~ Printed in the Netherlands Cryopreservation of English walnut (Juglans regia L.) Pollen J.G. Luza' and V.S. Polito Department of Pomology, University of California, Davis, CA 95616, USA; ' Fac. Agronomia, University of Chile, Casilla 1004, Santiago, Chile Received 8 September 1986; 16 March 1987 Key words: Juglans regia, walnut, pollen, cryopreservation, liquid nitrogen Summary Pollen from eight clones of English walnut (Juglans regia L.) was stored in liquid nitrogen (LN,) at —196°C for one year. Pollen was monitored for percent germination in vitro before being frozen. Pollen was frozen within two hours of anther dehiscence and subsequently at 24 hr intervals until germination assays indicated that the ability to germinate was lost. Survival after one year was evaluated by determining percent germination in vitro, Freshly collected pollen of only five of the eight clones survived LN;. By contrast, when the same pollen was held for 24 and 72 hr before freezing, pollen of all eight clones survived. Survival is correlated with moisture content (MC) of the pollen. Pollen samples with MC greater than 7.5% were killed by freezing in LN.. All pollen samples with MC between 4 and 7.5% survived as did most of the samples with MC between 3.2 and 4%. Introduction Juglans regia L. (English walnut) is a monoecious, wind-pollinated and, apparently, self-compatible (Gleeson, 1982; Forde & Griggs, 1975) species. Despite its self-compatible nature, breeding and research programs encounter difficulties having sufficient quantities of desired pollen at the time Pistillate flowers are receptive because of the di- chogamous nature of the species which has pro- tandrous and protogynous mating types (Griggs et al., 1971; Glesson, 1982; Polito & Li, 1985). Be- cause the life span of walnut pollen appears to be short under natural conditions its handling requires care with temperature, relative humidity and matu- rity, as each of these factors can affect viability. (Luza & Polito, 1985). Subfreezing temperatures have typically not been used for storage of J. regia pollen because they have been considered harmful (Crane et al., 1937; Griggs et al, 1971). There has been little careful study on methods of long-term storage pri- marily because suitable methods of viability testing have been lacking. The development of an in vitro germination method (Luza & Polito, 1985) has made it possible to evaluate methods of long-term. preservation. Storage for one or more years would be useful in breeding programs and indefinite stor- age would have value in preservation of walnut germplasm, Recently, there has been considerable interest in the possibility of storing pollen of perennial crop species at liquid nitrogen (LN;) temperature (~196°C) and Juglans nigra (Hall & Farmer, 1971; Farmer & Barnett, 1974), Persea americana (Sed- gley, 1981), Vitis vinifera (Parfitt & Almehdi, 1983), and Simmondsia chinensis (Lee et al., 1985) pollen grains showed only slight reduction in ger- mination rates after LN, exposure. However, LN, storage of Zea mays pollen142 showed considerable reduction in germination (Barnabas & Rajki, 1976). More recent studies have shown that about 50% of maize pollen can be kept viable for up toa year when water content ona fresh weight basis is reduced to about 30% of the original and the pollen is stored at -196°C (Barna- bas & Rajki, 1981). Ching & Ching (1964), and Ching & Slabaugh (1966), in attempting to deter- mine the mechanism of sub-freezing injury, showed that in Pinus monticola pollen a relation- ship existed between pollen moisture content, ice crystal formation and loss of viability. They con- cluded that the killing occurred when pollen con- tained more than 30% water. Jordan et al. (1982) found that resistance to freezing in seeds is related to water content, lettuce seeds with 5 to 13% water content were not injured by freezing to -196°C, but seed with 13 to 16% water was damaged. Water content of pollen grains at the time of dispersal varies among different families, with most recorded values in the range of 15 to 35% fresh weight (Heslop-Harrison, 1979). Moisture content (MC) in English walnut pollen varied con- siderably at the time of anther dehiscence, ranging from 4.6 to 12.1% (Luza & Polito, 1987). For many years subfreezing temperatures had been considered harmful to walnut pollen; studies with Juglans seiboldiana and J. regia pollen showed no germination after storage at sub-freezing tem- peratures (Griggs et al., 1971). However, Luza & Polito (1985) successfully preserved pollen germi- nation capacity from two of four J. regia cultivars for up to one year in LN;. ‘Membrane damage and loss of protein from the cells into extracellular media often occurs after rapid freezing by direct immersion in LN,. Glycerol has been used as a cryopreservative to provide some protection against the rigors of fast (direct immersion in LN,) or slow (1° C x min“) freezing (Westfall & Harris, 1975). However, Nath & An- derson (1975) stated that in general, freezing rates above 150°C x min“? yield more viable pollen than slow freezing rates which reduced pollen via- bility. In the same work a loss of viability was observed for pollen frozen to temperatures be- tween 0 and —50°C. Samples frozen to —100 and —196°C yielded viable pollen only when those samples were rapidly thawed, ‘The objectives of the research reported here were to examine the effect of subfreezing temper- ature on the germination of English walnut pollen and the influence of MC on pollen preservation at —196°C, Materials and methods Pollen Collection. The study was conducted on ma- terial collected from an even-aged population of four large trees for each cultivar growing in variety and election blocks at the University of California, Davis. Assays were made on eight English walnut clones; ‘Adams’, ‘Amigo’, ‘Chico’, ‘Graves Fran- quette’, ‘Meylan’, ‘Sharkey’, ‘XXX Mayette’ and 56-176. The staminate inflorescences were collect- ed at anthesis, placed in plastic bags to avoid mois- ture loss and brought into the laboratory. Pollen shed within the first 2 hr was collected directly or by shaking the dehiscing anther clusters. Pollen was cleaned by passing it through a 75 um-mesh sieve. Viability in in-vitro Culture Pollen viability was tested by germination in vitro. Pollen samples were germinated on a semi-solid medium (Luza & Polito, 1985) within 2 hr after collection and scored after 24 hr incubation. Pollen grains were considered viable if tube growth ex- ceeded one pollen grain diameter. A minimum of 500 pollen grains were counted in each of three replicate assays for each cultivar. Drying Process at Room Temperature Pollen was dried at room temperature in the lab- oratory. Germination percentages and MC were determined for pollen of each cultivar at the time of collection and at 24 hr intervals until ability to germinate was lost. MC percentage was calculated according to Barnabas & Rajki (1976) using sam- ples of approximately 400mg. Storage at -196°C (LN;) Pollen from the 8 clones was placed in 1.8ml plastic cryopreservation vials and cooled to ~196° Cat the rate of ~1°C min“ using a controlled-rate freezer. The vials were placed into a Union Carbide LN;freezer and held for 12 months. This was done at the time of collection and at 24 hr intervals using the pollen for which MC and germination were determined. At the end of the storage period the samples were transferred directly to room temper- ature. After the ice on the outside of the vials melted the pollen was removed for germination. Cryoprotectant Assay Parallel storage tests were done using a cryoprotec- tant. Aliquots of 0.5ml of glycerol at 6 and 12% were used to treat pollen before LN; storage. The vials were treated as above Results Germination and MC during drying process at room temperature Germination percentages of the eight clones at 2 hr after collection ranged between 82.4 and 95.6%. However, room-temperature storage life of the pollen varied (Fig. 1). For example ‘Meylan’ re- tained 50% pollen germination after 3 days, where- as ‘Graves Franquette’ had decreased to 50% ger- mination after 36 hr. Greater differences occurred among cultivars in the length of time required to decline to 20% viability, which occurred after 5 days with ‘Chico’ and ‘XXX Mayette’ but only 2'/ days for "Graves Franquette’. Generally, pollen germination decreased from 20 to 0% in "4, to 1'%y days. After 6 days at room temperature there was no germination by any pollen. There were large differences in MC among the cultivars tested. Two hr after collection, ‘Sharkey’ had the highest MC at 10.58% and ‘Graves Fran- quette’ was the lowest at 5.15%. The greatest loss of water occurred during the first day at room temperature. After 24 hr MC ranged between 3.66% (‘Graves Franquette’) and 7.30% (‘Shar- key’). Phe relationship between MC and pollen germi- nation is shown in Fig. 2. Both pollen germination and MC decreased with time of storage at room temperature. They did not decrease linearly with time. Ability to germinate was significantly re- duced during drying, although some pollen re- tained some viability until 3.2% MC. 143 TIME (DAYS) Adame Amigo chico Sharkey 56-176 G.Franquette XXX Mayette Fig. 1. Time required for pollen of English walnut cultivars to decline to 50%, 20% and 0% germination when held at room temperature 100 GERMINATION (%) MOISTURE CONTENT (%) TIME (DAYS) Fig, 2, Percentage germination and moisture content of English ‘walnut pollen held at room temperature. Points represent mean values for eight clones; bars = 2X SEM,144 LN; storage and germinability Pollen freshly shaken from the anthers and stored at ~196°C survived in only five of the eight cases. One of these five survivors had a germination rate 80.5% below that of the fresh pollen. The remain- ing four had survival rates from 60.3% to 77.0% (Table 1). However, when pollen was held for 24 and 72 hr before freezing, the pollen of each of the eight clones survived. Germination rates ranged from 61.1 to 101.5% of that of fresh material for pollen held 24 hr and from 58,3 t0 85.8% for pollen held 72 hr (Table 2) Influence of MC on germination after LN; storage Pollen held at room temperature dried gradually and fairly evenly. Freshly collected pollen with high MC had high germination percentages before LN; but had lowered viability on being removed from LN; (Table 1). Pollen placed into LN, on the day of collection germinated after thawing only when MC was lower than 7.5%. This was the case for pollen of ‘Amigo’, ‘Graves Franquette’, ‘Mey- Jan’. ‘XXX Mayette’ and 56-176 which had MC from 5.15-7.48%. Other cultivars, with MC values ranging from 7.54 to 10.58%, showed no germina- tion after thawing. The relationship between MC and germination Table I. MC and germination percentage of freshly collected pollen from 8 English walnut cultivars atthe time of collection and after fone year storage at —196%C. before LN; storage after LN, storage MC(%) GO% SEM) G(%ESEM) Decrease (%) Cutivars Adams 9.90 8.045.8 o 100 ‘Amigo 6.63 94243.27 6.24725 308 Chico 154 2447.70 0 100 Graves Franquette Sas 91.8439 65.827. 2332 Meylan 7.48 95.62 4.44 57.64 12.56 39.75 Sharkey 10.58 83.629.12 o 100 XXX Mayette 659 9145.85 7044733 2.98 56-176 64s 9.02484 1842798 80.50 Table 2. Germination of English walnut pollen dried for 24 and 72 hr at room temperature and stored LN; for one year. Germination (% + SEM) 2a hr Th before LN, after LN; % change before LN; after LN; % change Cultivars Adams LSE $89 722% 788-117 WAS 1624 622-165 ‘Amigo 8.6% 7.02 60.24 7.04 38.9 1824 9.99 140% 595-231 Chico B24 914 264 879 127 2#15.8 24.24 697-142 G. Franquette Ost 763 S124 712-152 60 69 204 282-333 Meylan 1662 6.69 T08E1052 ~ 7.8 5382872 40421242 249 Sharkey 824 S11 8104 S61 — 82 29.0% 886 202 807 -30.3 XXX Mayette 4441167 48.4410.13 24.8 33.6% 7.50 1964 7.23 41.7 56-176 8811366 69.8410.30 15 50% 6.85 20% 308 40.0145 1ooF . a|r b L oe L . sot °° . se E 5 S oe ge of L z & cop r . ie 2 of L Zz Z 4of r a of L 20+ r rr bet ty 10 8 6 4 2 10 8 MOISTURE CONTENT (%) Fig. 3. Percent germination vs. pollen moisture content for pollen of eight clones of J. regia . Data in (a) are from pollen placed on ‘germination medium at the time of pollen collection and at 24 hr intervals until germination dropped to zero. The line represents alinear regression for data points from 5 to 3% moisture defined by the relationship G(m) = Am + B where G(m) = germination percentage and m= percent moisture; A = 26.70, B = ~68.44 and r= 0.75. Data in (b) are from the same pollen samples after storage in LN; for one year. Lines represent linear regressions from 8 to 6% moisture (A = ~41.12, B= 337.34, r= 0.87) and S to 3% moisture (A= 22.83, B= ~58.07, r= 0.75 for all the cultivars is shown in Figure 3. There was Discussion no germination either before or after freezing by pollen with less than 3.20% MC or pollen held for The results show that English walnut pollen is rela- longer than 6 days at room temperature. tively shortlived when held at ambient temper- Viable English walnut pollen, with an MC per-_ature, but viability can be preserved for up to 1 year centage not higher than 7.50% at the time of freez-___ by LN) storage if pollen is dried to a moisture ing remained viable after 1 year in LN, storage. _content of no more than 7.5%. While it is not Those pollen grains that had MC greater than _ practical to determine moisture content of pollen 7.50% at pollen shedding and dried to values be- _to be stored, results here indicated that holding tween 3.2% and 7.5% before freezing retained the _pollen for one day before placing it into LN, stor- ability to germinate in vitro. age allowed sufficient drying to retain a level of viability at least 61.8% of fresh pollen (Table 1). Effect of cryoprotectant In a previous examination of LN, storage, we Pollen of all clones was adversely affected by glyce-_(Luza & Polito, 1985) were able to retain viability rol. No germination occurred for pollen treated _for freshly collected pollen of two of four cultivars with glycerol either before or after LN, storage. tested. The two survivors had germination rates of 95 and 45% of the pollen before storage. Interest- ingly, freshly collected ‘Adams’ pollen, which had146 the highest survival rate in that trial, was among those that failed to survive LN, storage here. The earlier trial was run on freshly collected pollen without consideration of pollen moisture content and, in light of the present results, it seems likely that the failure of two samples to survive and the great reduction in germination rate in one of the two surviving samples, was a consequence of high pollen moisture contents at the time of freezing. Although the present results indicate survival for a year, theoretically, pollen stored at extremely low temperature should retain viability indefinite- ly. Assuming that at —196°C all physiological ac- tivities of the pollen have been reduced to nil, pollen stored at that temperature would retain its initial germination capacity for an indefinite period of time (Visser, 1955). English walnut pollen via- bility was highly preserved in LN, storage as com- pared with other species (Stanley & Linskens, 1974). Other species with high survival rates in- clude Juglans nigra (Farmer & Barnett, 1974), Sim- mondsia chinensis (Lee et al., 1985), apple and pear (Visser, 1955), whose pollen germinated equally well as freshly collected pollen. However, Sedgley (1981) working with Persea americana pol- len found poor preservation noting that the loss of viability may have occurred mainly during the thawing period. Ching & Ching (1964) and Ching and Slabaugh (1966), in attempting to determine the mechanism of freeze-drying injury using x-ray diffraction anal- ysis showed that in Pinus monticola and Pseudot- suga menziesii pollen a relationship existed be- tween pollen MC, ice crystal formation, and loss of viability. They concluded that the killing effect ob- served in pollen containing more than 30% water may be attributed to membrane destruction result- ing from ice crystallization during freezing. Insome cases a preliminary drying treatment is necessary before freezing, but even this is not always satis- factory for some pollen, e.g., some Gramineae (Stanley & Linskens, 1974). Niklas (1985) has noted that numerous wind- pollinated species produce pollen grains of low density by filling walls with air cavities; in addition, ‘many such species have pollen grains that quickly dehydrate after release from the anthers. We have found that this is the case in walnut pollen (Luza & Polito, 1987). The MC of walnut pollen had a decisive influen- ce on pollen storage in LN;. English walnut pollen with initially high MC (7.50% or more) did not survive storage at —196°C, so a certain degree of drying must be carried out before cryogenic storage in order to assure retention of viability. The experi- ‘mental results show that pollen germination can be maintained if the original MC of the pollen is re- duced to a value between 7.50 and 3.20% by gentle drying before the dry pollen is then stored at 196°C. Similar observations have been made by Barnabas & Rajki (1976, 1981). They reported that mature maize pollen contains 45-60% water de- pending on water supply to the plant and relative humidity of the air. They showed that if water content of the pollen was reduced by drying to below 30% and if the pollen was stored in this state in LN,, in the majority of experiments the pollen retained viability even after long periods of stor- age. Rates of freezing and thawing have been shown to be critical in the optimum preservation of en- zymatic activity of cryogenic stored pollen. (Davies & Dickinson, 1971; Anderson & Nath, 1975; Nath & Anderson, 1975; Sedgley, 1981; Lee et al., 1985). Nath & Anderson (1975) found that slow freezing procedures led to a reduction in viable lily and maize pollen but the reduction was not as pro- nounced as that produced by slow thawing which rendered pollen immediately nonviable. This may indicate that thawing damage is primarily structur- al involving disruption of the internal structures of the cell by ice crystallization and they inferred that small ice crystals formed at rapid freezing rates cause less structural damage to the sub-cellular organelles and membrane system in the pollen grain. Sedgley (1981) reported the rapid freezing of Persea pollen was very successful because there was less possibility of ice crystal damage. Studies with Simmondsia (Lee et al., 1985) indicated that rates of freezing (0.5° min! to 100°C min!) and rates of thawing (79°C min“ to 83°C min“) in cryogenic pollen storage did not affect the viability of pollen as determined by in-vitro germination assay.Shivanna & Heslop-Harrison (1981) have point- ed out that germinability of pollen is determined principally by the state of the vegetative cell mem- brane. Ability to maintain turgor is an absolute prerequisite for germination, and a cell with leak- ing membranes can not become turgid. They did not exclude other factors contributing to low ger- mination rates such as loss of enzyme activity. Low temperature may extend germinability because it reduce perturbations that might otherwise destroy the special relationship of membrane components and so limit the chance of restoring an intact system on rehydration. This seems a more likely explana- tion than that the effect is on the rate of metabolic processes such as the consumption of reserves in respiration. ‘The moisture content of J. regia pollen was found here to be the most important single factor affecting germinability after LN, storage. The freezing rate, used in this study, combined with rapid thawing maintained high pollen germinabil- ity. Under the conditions used here, glycerol was not successful for freeze-preservation of English walnut pollen. It was not necessary to use a cryo- preservative in order to preserve pollen germin- ability. A satisfactory method for storing walnut pollen from one season to the next would enable breeders to have pollen available for crossing cultivars with different flowering times (Griggs et al., 1971). In addition, it may permit shipment of pollen to use whenever pollen is needed. In addition, the exten- sion of walnut breeding programs throughout the world has created a growing need for the exchange of germplasm. Results of these studies indicate that the pollen of English walnut cultivars may be stored at ~196°C if its MC is controlled before freezing. In practice this may be done by allowing the pollen to dry for 24 hr after collection. References Anderson, J.0. &J. Nath, 1975. The effects of freezing prese- vation of some pollen enaymes. 1. Freeze-thaw stress. Cry: biology 12: 160-168 Barnabas, B. & E. Rajki, 1976. Storage of maize (Zea mays L.) 147 pollen at ~196"C in liquide nitrogen. Euphytiea 25: 747-752 Barnabas, B. & E, Raji, 1981, Fertility of deep-frozen maize (Zea mays L.) pollen. Ann Bot 8: 861-864, Ching, TM. & K.K. Ching, 1968, Freee-deying pine pollen Plant Physiol 39: 705-709. Ching, TEM. & WH. Slabaugh, 1966, Xray difration analysis of ce crystal in coniferous pollen, Cryobiology 2: 321-227 Crane, HLL., C.A. Reed & MAN. 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Cryo- biology 19: 435-442. Lee, C.W., J.C. Thomas & S.L, Buchamann, 1985, Factors lectin in-vitro germination and storage of Jojoba pollen. J ‘Amer Soc Hort Sei 110: 671-676, Liza, J.G. & VS, Polito, 185. Invitro germination and storage cof English walnut pollen. Scent Horte 27 303-316 Liza, J.G, & VIS. Polito, 1987. Effect of desiccation and con- troled rehydration on germination in vir of pollen of walnut Cuslans spp). Plant, Cell, and Environ 10: 487-492. Nath, J .0. Anderson, 1975. Effect of freezing and freeze- deying on the viability and storage of Lilium longflorum L. and Zea mays L. pollen, Cryobiology 12: 81-88 Niklas, K.1., 1985. Wind pollination, A. study in controlled chaos, American Scientist 73: 462-470, Parfit, D.E. & A.A. Almchd, 1983, Cryogenic storage of grape pollen. Amer J Ena Vitic 4: 27-28, Polto, VS. & N.Y. Li, 1985. Pisillate ower diferetiaton in English walnut (Juglans regia L.): A developmental basis for heterodichogamy. Scient Hortic 26: 333-338 Sedgley, M., 1981. Storage of avocado pollen. Euphytica 30: 595-599, Shivanna, K.R. & J, Heslop-Harrison, 1981. Membrane state ‘and polen viability. Ann Bot 47 759-770 Stanley, RG. & H.F Linskens, 1974, Pollen biology, biochem- ry and management. Springer-Verlag, New York, Heidel- berg, Berlin, 307 pp. Visser, T, 1985. Germination and storage of pollen. Meded148 Landbouwhogesch Wageningen 55: 1-68. vatives to prevent motility loss and freeze-thaw damage to the Westfall, F.D. & G.C. Harti, 1975. The ability of ryopreser- acrosome of chicken spermatozoa. Cryobiology 12: 89-92.