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Cryopreservation of Transformed (Hairy) Roots ofArtemisia annua
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CRYOBIOLOGY 33, ARTICLE NO. 0011
106–117 (1996)
Cryopreservation of Transformed (Hairy) Roots of Artemisia annua K. H. TEOH,* P. J. WEATHERS,* R. D. CHEETHAM,*
,1
AND D. B. WALCERZ† *Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts 01609; and †Department of Mechanical Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts 01609 The antimalarial drug artemisinin has been found in transformed (hairy) roots of Artemisia annua. A protocol was developed to preserve A. annua hairy roots in liquid nitrogen. Root tips were excised from 7-day-old cultures and held on solid White’s medium for 24 h prior to cryoprotection. They were then treated with a cryoprotecting mixture containing 8% (v/v) dimethyl sulfoxide (Me2SO) and 20% (w/v) sucrose at 25°C for 1 h followed by cooling at 0.09°C/min to 4°C then cooling to −35°C at 0.72°C/min. Vials containing the root tips were then plunged into liquid nitrogen. After thawing in a water bath to 37°C, root tips were held in the cryoprotecting mixture for 10 min before it was diluted to 25% of its original concentration. Root tips were washed once with fresh liquid White’s medium and held for 1 h prior to culturing on White’s medium with 0.2% Gelrite and 3% (w/v) sucrose. Regrowth of root tips averaged 65%. Independent variables in this study included 1) Me2SO concentration; 2) the type and concentration of cosolutes; 3) cooling rate(s); 4) the temperature at which the sample is transferred to liquid nitrogen; 5) the age of the culture from which root tips are taken; 6) the recovery period between root tip excision and immersion in cryoprotectant; and 7) the amount of Gelrite used in the postthaw plating medium. © 1996 Academic Press, Inc.
Artemisia annua is a Chinese herb that produces the antimalarial drug artemisinin, which has been used for many centuries in Chinese traditional medicine as a treatment for fever and malaria (15). Artemisinin, commonly extracted from the aerial parts of the plant, has been found in transformed (hairy) roots in our laboratory (33). Current investigations on the use of transformed root cultures as an alternative to cell cultures (10, 11) have confirmed the potential of transformed roots for production of secondary metabolites. Among the advantages of using transformed roots are short doubling times of the biomass (12) and apparent genetic and biosynthetic stabilities (1, 19, 30). The benefits of cryopreservation are significant. Cryopreservation eliminates the need for periodic subculture which requires considerable labor, materials, and space. It also reduces risk of contamination by microorganisms and genetic drift during storage as the transformed roots are quiescent. Cryopreservation of transformed roots will be important for establishing safe patent repositories. Progress in the last decade on the cryopresReceived January 12, 1995; accepted August 19, 1995. 1 To whom correspondence should be addressed. 106 0011-2240/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
ervation of plant systems has made possible the regeneration of entire plants from frozen cells (e.g., 20, 22, 23), embryos (e.g., 18, 25), and shoot tips (e.g. 6, 21, 36) of a number of species. The cryopreservation of shoot tips of some plants such as strawberry, carnation, tomato, pea, and wild potato is now routine (27). However, cryopreservation of roots has received little attention, and the development of protocols for root cryopreservation is still in its infancy as evidenced by few reports of root cryopreservation in the literature. The situation may change with increasing interest in transformed roots as potential production systems for secondary metabolites, thus leading to the development of cryopreservation protocols useful for a variety of plant species for both germplasm conservation as well as commercial purposes. There has been only one published report on cryopreservation techniques for transformed roots. The models were beet and tobacco, and the reported regrowth rate was 10% (3). Two papers on cryopreservation of hairy roots of Panax ginseng and horseradish were presented at the 1994 conference of the Society for Cryobiology (24, 36), and they reported regrowth rates of 60% and 40%, respectively. The present research was carried out to establish a method
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for the cryopreservation of transformed roots of A. annua while adding new information to the field of root cryopreservation. MATERIALS AND METHODS
Production and Maintenance of Hairy Root Cultures Transformed roots of A. annua were produced and cultured as previously described (33). Cultures were grown in liquid White’s medium (Sigma product number W 0876, St. Louis, MO) supplemented with 3% (w/v) sucrose. (All subsequent references to White’s medium assume 3% supplemental sucrose.) Cultures were kept in 125-ml Erlenmeyer flasks on an orbital shaker at 100 rpm at 25°C in dim light. Root Tip Excision Healthy, white root tips, 2–4 mm in length, were excised with a sterile scalpel from transformed roots after a 4- to 14-day subculture interval. The tips were allowed 0–48 h of recovery time on White’s medium solidified with 2.0 g/l Gelrite (Merck, Rahway, NJ). Root tips on solid medium were maintained in a 27°C incubator in the dark. All manipulations were performed in liquid medium in a sterile hood to avoid desiccation or contamination of the fragile tissue. Experimental Units and Viability Testing The basic experimental unit in all procedures is the vial, which corresponds to a 2-ml polypropylene cryovial containing 10–15 root tips. Vials were subjected to various experimental treatments after which the component root tips were tested for viability. Viability tests are pass/ fail (no intermediate grades), and the percentage of passing roots defines the viability of the vial, which is assumed to be normally and independently distributed (NID) for statistical purposes. Two viability tests were used in this study. The first, fluorescein diacetate (FDA) staining, is relatively rapid and convenient (34); however, it only indicates membrane integrity (26) and can produce false positives when cellular
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damage is not manifested in the membrane. The second, 7+ day culturing, is a rigorous test of survival but is more laborious than FDA staining. The terms membrane integrity and regrowth testing are used for the two methods, respectively. Membrane integrity testing. Stock solution of FDA (0.2%), consisting of 2 mg of FDA dissolved in 1 ml of acetone, was diluted 20 times using liquid White’s medium to obtain 0.01% FDA solution. Root tips were suspended in 1 drop of the 0.01% FDA solution on a microscope slide for 5 min prior to viewing under a microscope with uv illumination (17). Tips that did not fluoresce brightly throughout their entire volume were considered failing. Regrowth testing. Root tips were cultured in solid, semisolid, or liquid White’s medium in a dark, 27°C incubator for at least 7 days. (Solid medium has 0.2% Gelrite, semisolid medium has 0.1% Gelrite, and liquid medium has no Gelrite.) Early signs of recovery, detected by microscopic observation, were swelling of the meristematic region, formation of a new meristem at the root tip, and doubling of the length of the root tips. Conversion of the surviving root tips to roots (regrowth) occurred after 7 days. Root tips that did not show regrowth were considered failing. Cooling Apparatus A NalgeneTM Cryo 1°C Freezing Container was used to effect controlled cooling of the vials. The container was either placed in a −68°C freezer to obtain a nominal cooling rate of 1°C/ min, or it was placed in a 4°C refrigerator to obtain a nominal cooling rate of 0.1°C/min. Exact cooling rates were measured via a thermocouple in a dummy vial. Fig. 1 shows the temperature history of vials cooled at 1°C/min from room temperature to −40°C or at 0.1°C/min to 4°C then 1°C/min to −40°C. The dotted lines in Fig. 1 are the best fit lines for the measured data. The slopes of the best fit lines show that when the container is placed in the −68°C freezer the cooling rate averages 0.8°C/min; when it is in the 4°C refrigerator the rate averages 0.09°C/min; and when it is placed in the
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FIG. 1. The temperature history of a vial of cryoprotectant in the Nalgene Cryo 1°C Freezing Container is shown for two cooling protocols. In the first protocol the container is placed directly in a −68°C freezer. In the second protocol the container is placed in a 4°C refrigerator until the vial reaches 4°C, then it is transferred to the −68°C freezer. The dotted lines are the best fit lines for the measured data. The slopes of the best fit lines show that when the container is placed in the −68°C freezer the cooling rate averages 0.8°C/min; when it is in the 4°C refrigerator the rate averages 0.09°C/min; and when it is placed in the −68°C freezer after the refrigerator the rate averages 0.72°C/min. The actual temperature differs from the best fit line by 4°C or less, which is comparable to the manufacturer’s specifications. Three replicates were run for each cooling rate to determine the repeatability of the container. The standard deviation associated with a single measurement is 0.2°C, which indicates good repeatability.
−68°C freezer after the refrigerator the rate averages 0.72°C/min. The actual temperature differs from the best fit line by 4°C or less, which is comparable to the manufacturer’s specifications. Three replicates were run for each cooling rate to determine the repeatability of the container. The standard deviation associated with a single measurement is 0.2°C, which indicates good repeatability. Experimental Procedures Eight experiments were performed to find the optimal cryopreservation procedure for the YUT6 clone of A. annua, and these are detailed in the subsequent subsections. The number of independent variables prohibited a complete factorial experiment design; instead, factors were tested individually or in limited combinations. The independent variables under study were: Me2SO concentration; the type and concentration of cosolute; cooling rate(s); the temperature at which the sample is transferred to liquid nitrogen; the age of the culture from which root
tips are taken; the recovery period between root tip excision and immersion in cryoprotectant; and the amount of Gelrite in the postthaw plating medium. Each of the experiments relied on previous results to refine the cryopreservation procedure, as will be presented under “Results and Discussion.” A ninth experiment measured the growth curve of optimally cryopreserved root tips as well as control root tips. A tenth experiment assayed both cryopreserved and control root tips after 20 days of growth for production of artemisinic compounds. Dimethyl sulfoxide (Me2SO) tolerance at room temperature. Me2SO solutions were prepared by adding 0, 8, 10, 12, 14, or 16% (v/v) Me2SO to liquid White’s medium. Excised root tips from 6- to 9-day subcultured roots were allowed no wound recovery period after excision and placed in vials containing 0.5 ml of Me2SO solution at 25°C. After 1 h, the solution was removed with a sterile Pasteur pipette and replaced with 0.5 ml of White’s medium. Root tips remained in White’s medium for 10 min
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after which they were plated on solid White’s medium for regrowth testing. Three vials were processed for each Me2SO solution. Effects of cosolutes and terminal temperature. An initial survey of cosolutes and terminal temperatures was conducted to find conditions that produced high rates of regrowth. All vials contained 10% Me2SO (v/v) in White’s medium except for the control, which contained no Me2SO. The 10% Me2SO solution in each vial was either unmodified or augmented with one of five cosolutes: 20% (w/v) sucrose, 20% (w/v) sorbitol, 20% (w/v) mannitol, 2% (v/v) glycerol, or 2% (w/v) polyvinylpyrrolidone (PVP). Thus, there were seven different solutions including the control. Excised root tips from 6- to 9-day subcultured roots were allowed no wound recovery period and held in vials containing 0.5 ml of cryoprotective solution at 25°C for 1 h. Vials were cooled at 0.8°C/min (average), and when they reached a temperature equal to the freezing point of the cryoprotective solution they were manually removed from the bath for 10–15 s and placed on the steel refrigerator rack, which effected nucleation. They were then returned to the bath and cooled to a terminal temperature of −10, −20, −30, −35, or −40°C. Vials were thawed by immersion in a 37°C water bath for 2–3 min. The cryoprotectant solution was then diluted by the addition of 0.07, 0.10, 0.13, 0.20, 0.33, and 0.67 ml of fresh
White’s medium (total volume 1.50 ml) at 10min intervals, causing the solute concentration to drop to 25% of its original value in six equal steps. The diluted cryoprotectant solution was removed with a sterile pipette and replaced with 0.5 ml of White’s medium. Root tips were held for 1 h at 25°C before being transferred to plates containing solid White’s medium for regrowth testing. Four vials were processed for each of the 35 combinations of solutions and terminal temperatures. Effects of Me2SO concentration, sucrose concentration, wound recovery period, and transfer temperature. This experiment analyzed the effects of four factors: wound recovery, transfer temperature, Me2SO concentration, and sucrose concentration, using the Graeco–Latin square design (Table 1), which assumes no interaction between factors (14). Excised root tips from 6to 9-day subcultured roots were plated on solid White’s medium for 0, 12, or 24 h prior to immersion in cryoprotectant to allow the tips time to recover from wound stress. Cryoprotectant solutions were prepared by adding 8, 10, or 12% (v/v) Me2SO and 15, 20, or 25% (w/v) sucrose to White’s medium. Root tips were immersed in vials containing 0.5 ml of cryoprotectant at 25°C for 1 h prior to cooling. Vials were cooled at 0.09°C/min (average) to 4°C then at 0.72°C/ min (average) to −35, −37, or −40°C. When the vials reached the specified transfer temperature
TABLE 1 Nine Experimental Treatments Are Defined to Investigate Four Factors, Each Having Three Levels Factors Treatment no.
Transfer temperature (°C)
Wound recovery (h)
Me2SO concentration (% v/v)
Sucrose concentration (% w/v)
1 2 3 4 5 6 7 8 9
−35 −37 −40 −35 −37 −40 −35 −37 −40
12 0 24 0 24 12 24 12 0
12 8 10 10 12 8 8 10 12
15 15 15 25 25 25 20 20 20
Note. The experimental design is a Graeco–Latin square, which in this application is synonymous with a one-ninth replication of a 34 factorial. The design assumes there are no significant interactions between factors.
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they were immediately plunged into liquid nitrogen where they were held for at least 1 h. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were tested for membrane integrity but not regrowth. Three vials were processed for each of the nine combinations of factors shown in Table 1. Recovery medium. Excised root tips from 6to 9-day cultures were allowed 24 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. Tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/ min (average) to −35°C, then plunged into liquid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid, semisolid, or liquid White’s medium for postthaw recovery, and tested for regrowth. Three vials were processed for each recovery medium. Culture age. Excised root tips from 4-, 7-, 11-, and 14-day-old cultures were allowed 24 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. Tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/min (average) to −35°C, then plunged into liquid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid White’s medium and tested for regrowth. Three vials were processed for each culture age. Wound recovery period. The effects of different wound recovery periods, which were investigated in the four-factor experiment using the membrane integrity test, were checked and expanded in this experiment using regrowth testing. Excised root tips from 7-day-old cultures were allowed 0, 24, or 48 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. Tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/ min (average) to −35°C, then plunged into liq-
uid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid White’s medium and tested for regrowth. Three vials were processed for each recovery period. Transfer temperature. The effects of different transfer temperatures, which were investigated in the four-factor experiment using the membrane integrity test, were checked and expanded in this experiment using regrowth testing. Excised root tips from 7-day-old cultures were allowed 24 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. Tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/min (average) to −10, −20, −30, −35, −37, or −40°C, then plunged into liquid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid White’s medium and tested for regrowth. Two vials were processed for each transfer temperature. Cooling rates. Excised root tips from 7day-old cultures were allowed 24 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. For slow cooling, tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/min (average) to −35°C, then plunged into liquid nitrogen. For fast cooling, tips were cooled at 0.8°C/min (average) to −35°C, then plunged into liquid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid White’s medium and tested for regrowth. Three vials were processed for each cooling rate. Growth curve characterization. Excised root tips from 7-day-old cultures were allowed 24 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. Tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/min (average) to −35°C,
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then plunged into liquid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid White’s medium for postthaw recovery. The lengths of the root tips were measured after 3, 6, 9, 11, 13, 15, 17, and 19 days and compared with the growth of excised root tips that were simply plated on solid White’s medium. One vial of 10 root tips was cryopreserved and compared with 10 control root tips. Artemisinin production. Root tips from the YU-T6 clone of A. annua were either cryopreserved as in the growth curve experiment or plated on solid White’s medium immediately after excision. Cryopreserved tips were allowed 20 days of growth after thawing, and control tips were allowed 20 days of growth after excision. Production of secondary products (artemisinin & artemisinic acid) was measured by thin layer chromatography. The detailed procedure is given by Teoh (31). One cryopreserved vial and one control vial were processed to measure production. RESULTS AND DISCUSSION
Regrowth of root tips exposed to 0–16% (v/v) Me2SO at 25°C for 1 h is shown in Fig. 2. The survival of root tips at 25°C treated with 8, 10, or 12% (v/v) Me2SO for 1 h was not significantly different (a > 0.05) from control root
FIG. 2. The percentage of root tips that demonstrated regrowth after 1-h exposure at 25°C to White’s medium with Me2SO is shown as a function of Me2SO concentration. Error bars show the standard error of the mean. Horizontal lines at the top of the graph span concentrations that are not significantly different at the a 4 0.05 level as determined by a Neumann–Keuls test.
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tips. Increasing the concentration to 14 and 16% (v/v) caused significant drops in regrowth rates. This is consistent with the literature on slow freezing of plant systems where the use of 5–10% (v/v) Me2SO alone or with other cryoprotectants for 0.5–3 h at temperatures between 0 and 25°C has proven to be nontoxic for meristems (e.g., 2, 13), hairy roots (3), shoot tips (7), and shoot apices (29). As shown in Fig. 3, root tips that were exposed for 1 h at room temperature to 10% Me2SO (v/v) in White’s medium supplemented with either 20% (w/v) sorbitol, 20% (w/v) mannitol, 2% (v/v) glycerol, or 2% (w/v) PVP and frozen as described under “Materials and Methods” showed 0% regrowth over four replicates. Root tips frozen in plain White’s medium also evinced 0% regrowth. Root tips exposed to White’s medium plus 10% Me2SO had some regrowth depending on terminal temperature, and root tips exposed to 10% Me2SO in White’s medium supplemented with 20% sucrose had high levels of regrowth at all terminal temperatures. The loss in regrowth as the terminal temperature was lowered was not significant at the 5% level. The Graeco–Latin square experimental design (Table 1) was employed to determine the influence of four parameters (transfer temperature, wound recovery time, Me2SO concentration, and sucrose concentration) on viability. The design assumes no interactions and provides statistical tests only for main effects (14). An analysis of variance (Table 2) shows that transfer temperature and wound recovery period are significant effects at the 0.05 level, and sucrose concentration may be important. Me2SO concentration is not significant, which is in contrast to the results of the first experiment (Me2SO tolerance at room temperature). This may be due to the narrower range of concentrations used in this experiment or the use of FDA staining as opposed to the more rigorous regrowth testing used in the first experiment. A plot of the main effects is shown in Fig. 4. Twelve or 24 h of recovery is significantly better than no recovery (a < 0.05), suggesting that recovery from excision stress is beneficial to
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FIG. 3. The percentage of frozen and thawed root tips that demonstrated regrowth is shown as a function of the cryoprotectant mixture and terminal temperature. Root tips were given 1-h exposure at 25°C to White’s medium with various cryoprotectant additives then cooled at 1.0°C/min to the terminal temperature. Nucleation was effected at the equilibrium freezing point. Vials were thawed by immersion in a 37°C water bath for 2–3 min and the cryoprotectant was removed with a six-step dilution procedure.
cryopreservation. This effect was also seen by Towill (32), but Benson and Hamill (3) found the opposite. In the previous experiment (effects of cosolutes and terminal temperatures) there was a decrease in regrowth as the terminal temperature went from −35 to −40°C; however, the data were not strong enough to prove significance (a 4 0.09). The results of this experiment show that a transfer temperature of −37°C was significantly better than −35°C (a < 0.05) but not significantly better than −40°C (a 4 0.08). As shown in Fig. 5, frozen–thawed root tips plated on 0.1 or 0.2% Gelrite showed 49 and
43% regrowth, respectively (the difference is not statistically significant). None of the root tips in liquid medium (0.0% Gelrite) showed regrowth, which is significantly worse than the other two cases (a < 0.05). The benefit of solid over liquid culture has been commonly noted (3, 5, 8, 35). The effect of culture age on postthaw survival was not significant. A plot of survival vs culture age is shown in Fig. 6. Fig. 7 shows that a recovery period between excision and cryopreservation is highly beneficial. This confirms the results of the four-factor experiment, which tested 12- and 24-h recovery
TABLE 2 ANOVA of the Graeco–Latin Square Experiment Source
df a
SSb
MSc
Fd
Probability
Transfer temperature Wound recovery period % Me2SO (v/v) % Sucrose (w/v) Error Total
2 2 2 2 18 26
3,161 3,561 754 1,550 6,925 15,951
1,581 1,781 377 775 385
4.1 4.63 <1 2.01
<0.05 <0.05
Note. It is assumed that there are no interactions between factors. a Degrees of freedom associated with each source term. b Sum of squares for each source term. c Mean square for each source term. d Ratio of the mean square for a source divided by the mean square for the error term.
0.18
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FIG. 4. The percentage of frozen and thawed root tips that were viable by FDA staining is shown as a function of four factors: transfer temperature, recovery time, Me2SO concentration, and sucrose concentration. The experimental design assumed no interactions between factors. Error bars show the standard error of the mean.
periods. The difference between 24 and 48 h is not significant. Up to this point, all experiments that included freezing in liquid nitrogen have used two cooling rates: 0.09°C/min from 25 to 4°C and 0.72°C/min from 4°C to the temperature at which the sample is transferred to liquid nitrogen. This allowed 210 min for equilibration above 4°C and about 40 min below 4°C. This
FIG. 5. The percentage of frozen and thawed root tips that demonstrated regrowth on liquid, semisolid, or solid medium is shown. Liquid medium (0% Gelrite) is significantly worse than semisolid or solid medium (a < 0.05). Error bars show the standard error of the mean.
two-rate protocol was compared with a single freezing rate, 0.8°C/min, which allowed only 21 min above 4°C. The two-rate protocol resulted in regrowth of 44% of the cryopreserved root tips, compared with 0% of root tips cryopreserved with the one-rate protocol. Transfer temperature was critical to the survival of root tips frozen in liquid nitrogen as
FIG. 6. The percentage of frozen and thawed root tips that demonstrated regrowth is shown as a function of subculture age. None of the differences of the means is significant at the 5% level, but an analysis of variance (Table 3) shows that age is a significant factor. Error bars show the standard error of the mean.
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FIG. 7. The percentage of frozen and thawed root tips that demonstrated regrowth is shown as a function of the recovery period between excision and cryoprotection. Twentyfour and 48 h were not significantly different from each other but were significantly better than no recovery (a < 0.05). Error bars show the standard error of the mean.
shown in Fig. 8. −35°C was optimal, which is in slight contrast to the results of the four-factor experiment which found −37°C to be better than −35°C. The difference may be due to the assay (regrowth as opposed to FDA staining in the four-factor experiment) or an interaction with other factors that was assumed to be zero in the four-factor experiment. Table 3 summarizes the factors affecting cryopreservation of transformed roots of A. an-
FIG. 8. The percentage of frozen and thawed root tips that demonstrated regrowth is shown as a function of the temperature at which they were transferred to liquid nitrogen. −35°C was significantly better than any other temperature (a < 0.05). Error bars show the standard error of the mean.
nua and gives the optimal values found in this study. The regrowth rate for optimally cryopreserved roots was 65%, which is an improvement over previous work. Benson and Hamill (3) reported regrowth rates of 10% for transformed root tips of beet and tobacco using a slow cooling method similar to the one in this study but without sucrose as a cryoprotectant. Yoshimatsu et al. (36) reported 60% regrowth for P. ginseng hairy roots using a vitrification method. Phunchindawan et al. (24) reported 40% survival of horseradish hairy roots using an encapsulation dehydration cryopreservation protocol. Growth curves for optimally cryopreserved and control root tips are shown in Fig. 9. Cryopreserved root tips are significantly shorter than the control tips at every measurement interval from day 2 to day 19; however, the shapes of the growth curves are very similar, indicating normal growth progression. In most cases recovery proceeded in an organized manner without formation of callus or any adventitious activity, thus minimizing genetic instability. In some cases recovery was preceded by formation of new meristematic regions as the old regions degenerated with time, indicating considerable damage to the meristem. In general, there was a period of about 3 days after thawing before the root tips began to show measurable growth.
FIG. 9. Growth curves for optimally cryopreserved and control root tips are shown over 19 days. Error bars are shown where measurements were taken and indicate the standard error of the mean. Data could not be pooled because the variance associated with length measurement was significantly positively correlated with root length. Cryopreserved roots were significantly shorter than control roots for all measurements, but the shape of the growth curve suggests that cryopreserved roots are “normal.”
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TABLE 3 Factors Affecting Cryopreservation of Transformed Roots of Artemisia annua and Optimal Values Found in This Study Factor
Range
Optimal value
Tissue size Tissue age
Factor was not varied 4–14 days
Period between excision and exposure to cryoprotectant Me2SO concentration in cryoprotectant Cosolute
0–48 h
Root tips 2–4 mm long 7 days (early exponential phase) 24 h
0–16% (v/v)
8%
None, glycerol, mannitol, PVP, sorbitol, sucrose 15–25%
Sucrose 20%
Factor was not varied
1 h at 25°C 0.1°C min−1 to 4°C then 1.0°C min−1 to transfer temperature
Nucleation technique
1.0°C min−1 to transfer temperature or 0.1°C min−1 to 4°C then 1.0°C min−1 to transfer temperature Factor was not varied
Transfer temperature Thawing technique
−10°C to −40°C Factor was not varied
Cryoprotectant removal
Factor was not varied
Recovery medium
White’s medium with 0.0%, 0.1% or 0.2% Gelrite
Cosolute concentration in cryoprotectant Temperature and time of exposure to cryoprotectant Cooling rate
This delay, which is not seen in control roots, may be attributed to the time required to repair cellular damage (4) or for the remaining live cells to undergo a number of divisions before measurable growth resumed, or both (9). Postthaw delay has been observed in many systems (e.g., 16, 20, 28, 32). Production of artemisinin in a sample of 10 cultured, cryopreserved roots was 0.214 mg/g, fresh weight, which is similar to a sample of 10 control roots (0.246 mg/g, fresh weight.). Note: 10 roots were required to achieve sufficient tissue mass for high pressure liquid chromatography measurement, and the results are the average for the 10 roots. Only one measurement was taken for frozen and control roots, so no statistical comparisons were possible. The finding that secondary production was not affected by cryopreservation is consis-
Cryovials were touched to refrigerator rack when they were just below the equilibrium freezing point −35°C Immersion in 37°C water bath for 2–3 min Cryoprotectant was diluted in 6 steps then replaced with White’s medium White’s medium with 0.2% Gelrite
tent with work done on transformed roots of B. vulgaris and N. rustica (3) and P. ginseng (36). CONCLUSION
This study demonstrates that cryopreservation of transformed roots of A. annua is an effective and practical technique for maintenance of genetic stock because survival rates are high (65% on average as measured by regrowth testing), secondary metabolite production is similar to unfrozen controls, and expensive equipment is not required (an alcohol bath in standard 4°C and −68°C refrigerators was used instead of a programmable freezer). A conventional, slow cooling protocol was used and a combination of Me2SO and sucrose was essential for high survival. Other important factors included recovery time between excision and cryopreservation,
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and the concentration of Gelrite in the recovery medium. A summary of findings is given in Table 3. ACKNOWLEDGMENTS Standard compounds of artemisinic acid, artemisitene, and arteannuin B as well as seeds of four strains of A. annua were kindly provided by Dr. Nancy Acton of the Division of Experimental Therapeutics, Walter Reed Army Institute of Research. This work was supported in part by NIH Grant 1R15AI34131-O1 and was completed in partial fulfillment of the requirements for the degree of Master of Science. REFERENCES 1. Aird, E. L. H., Hamill, J. D., and Rhodes, M. J. C. Cytogenetic analysis of hairy root cultures from a number of plant species transformed by Agrobacterium rhizogenes. Plant Cell Tissue Organ Cult. 15, 47–57 (1988). 2. Bagniol, S., and Engelmann, F. Effects of pregrowth and freezing conditions on the resistance of meristems of date palm (Phoenix dactylifera L. var. Bou Sthammi Noir) to freezing in liquid nitrogen. CryoLett. 12, 279–286 (1991). 3. Benson, E. E., and Hamill, J. D. Cryopreservation and post freeze molecular and biosynthetic stability in transformed roots of Beta vulgaris and Nicotiana rustica. Plant Cell Tissue Organ Cult. 24, 163–172 (1991). 4. Cella, R., Colombo, R., Galli, M. G., Nielsen, E., Rollo, F., and Sala, F. Freeze-preservation of rice cells: A physiological study of freeze-thawed cells. Physiol. Plant. 55, 279–284 (1982). 5. Chen, T. H. H., Kartha, K. K., Leung, N. L., Kurz, W. G. W., Chatson, K. B., and Constabel, F. Cryopreservation of alkaloid-producing cell cultures of periwinkle (Catharanthus roseus). Plant Physiol. 75, 726–731 (1984). 6. Demeulemeester, M. A. C., Vandenbussche, B., and De Proft, M. P. Regeneration of chicory plants from cryopreserved in vitro shoot tips. Cryo-Lett. 14, 57– 64 (1993). 7. Diettrich, B., Wolf, T., Bormann, A., Popov, A. S., Butenko, R. G., and Luckner, M. Cryopreservation of Digitalis lanata shoot tips. Plant. Med. 53, 359– 363 (1987). 8. Dussert, S., Mauro, M. C., and Engelmann, F. Cryopreservation of grape embryogenic cell suspensions. 2. Influence of post-thaw culture conditions and application to different strains. Cryo-Lett. 13, 15–22 (1992). 9. Finkle, B. J., Zavala, M. E., and Ulrich, J. M. Cryoprotective compounds in the viable freezing of plant tissues. In “Cryopreservation of Plant Cells and Organs” (K. K. Kartha, Ed.), pp. 75–113, CRC Press, Boca Raton, FL, (1985).
10. Flores, H. E. Plant roots as chemical factories. Chem. Ind. (Lond.) 374–377 (1992). 11. Flores, H. E., and Curtis, W. R. Approaches to understanding and manipulating the biosynthetic potential of plant roots. Ann. NY Acad. Sci. 665, 188–209 (1992). 12. Flores, H. E., and Filner, P. Metabolic relationships of putrescine, GABA and alkaloids in cell and root cultures of Solanaceae. In “Primary and Secondary Metabolism in Plant Cell Cultures” (K. H. Neumann, W. Barz, and E. Reinhard, Eds.), pp. 174– 185, Springer-Verlag, Berlin, (1985). 13. Haskins, R. H., and Kartha, K. K. Freeze preservation of pea meristems: Cell survival. Can. J. Bot. 58, 833–840 (1980). 14. Hicks, C. R. “Fundamental Concepts in the Design of Experiments,” pp. 79–80. Holt, Rinehart and Winston, New York, 1982. 15. Jiang, J. B., Li, G. Q., Guo, X. B., Kong, Y. C., and Arnold, K. Antimalarial activity of mefloquine and qinghaosu. Lancet 2, 285–288 (1982). 16. Jörgensen, J. Conservation of valuable gene resources by cryopreservation in some forest tree species. J. Plant Physiol. 136, 373–376 (1990). 17. Kasten, F. H. Methods for fluorescence microscopy. In “Staining Procedures” (G. Clark, Ed.), pp. 39–103. Williams & Wilkins, Baltimore, MD, 1981. 18. Lecouteux, C., Florin, B., Tessereau, H., Bollon, H., and Petiard, V. Cryopreservation of carrot somatic embryos using a simplified freezing process. CryoLett. 12, 319–328 (1991). 19. Loyola-Vargas, V. M., and Flores, H. E. Root cultures as a source of secondary metabolites of economic importance. In “Production of Secondary Metabolites from Plant Tissue Cultures and Its Biotechnological Perspectives” (V. M. Loyola-Vargas, Ed.), pp. 281–303. Centro Investigacion Cientifica Yucatan Pub., Yucatan, Mexico, 1990. 20. Lu, T. G., and Sun, C. S. Cryopreservation of millet (Setaria italica L.). J. Plant Physiol. 139, 295–298 (1992). 21. Niino, T., Sakai, A., Yakuwa, H., and Nojiri, K. Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification. Plant Cell Tissue Organ Cult. 28, 261–266 (1992). 22. Nishizawa, S., Sakai, A., Amano, Y., and Matsuzawa, T. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic cells and subsequent plant regeneration by a simple freezing method. CryoLett. 13, 379–388 (1992). 23. Panis, B. J., Withers, L. A., and De Langhe, E. A. L. Cryopreservation of Musa suspension cultures and subsequent regeneration of plants. Cryo-Lett. 11, 337–350 (1990). 24. Phunchindawan, M., Hirata, K., Kobayashi, T., Miyamoto, K., and Sakai, A. Cryopreservation of shoot primordia induced from horseradish hairy root cul-
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tures by encapsulation-dehydration method. Cryobiology 31, 580 (abstr.) (1994). Radhamani, J., and Chandel, K. P. S. Cryopreservation of embryonic axes of trifoliate orange (Poncirus trifoliata [L.] RAF.). Plant Cell Rep. 11, 204–206 (1992). Rotman, B., and Papermaster, B. W. Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc. Natl. Acad. Sci. USA 55, 134–141 (1966). Sakai, A. Cryopreservation of shoot-tips of fruit trees and herbaceous plants. In “Cryopreservation of Plant Cells and Organs” (K. K. Kartha, Ed.), pp. 135–158. CRC Press, Boca Raton, FL, 1985. Sala, F., Cella, R., and Rollo, F. Freeze-preservation of rice cells grown in suspension culture. Physiol. Plant. 45, 170–176 (1979). Seibert, M., and Wetherbee, P. J. Increased survival and differentiation of frozen herbaceous plant organ cultures through cold treatment. Plant Physiol. 59, 1043–1046 (1977). Sree Ramulu, K., Dijkhuis, P., Hanisch Ten Cate, C. H., and De Groot, B. Patterns of DNA and chromosome
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variation during in vitro growth in various genotypes of potato. Plant Sci. 41, 69–78 (1985). Teoh, K. T. Cryopreservation of transformed roots of Artemisia annua. MS thesis, Worcester Polytechnic Institute, Worcester, MA 1994. Towill, L. E. Solanum etuberosum: A model for studying the cryobiology of shoot-tips in the tuberbearing Solanum species. Plant Sci. Lett. 20, 315– 324 (1981). Weathers, P. J., Cheetham, R. D., Follansbee, E., and Teoh, T. Artemisinin production by transformed roots of Artemisia annua. Biotechnol. Lett. 16, 1281–1286 (1994). Widholm, J. M. The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol. 47, 189–194 (1972). Withers, L. A., and King, P. J. A simple freezing unit and routine cryopreservation method for plant cell cultures. Cryo-Lett. 1, 213–220 (1980). Yoshimatsu, K., Yamaguchi, H., and Shimomura, K. Cryopreservation of Panax ginseng hairy roots by vitrification method. Cryobiology 31, 580 (abstr.) (1994).
CRYOBIOLOGY 33, ARTICLE NO. 0011
106–117 (1996)
Cryopreservation of Transformed (Hairy) Roots of Artemisia annua K. H. TEOH,* P. J. WEATHERS,* R. D. CHEETHAM,*
,1
AND D. B. WALCERZ† *Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts 01609; and †Department of Mechanical Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts 01609 The antimalarial drug artemisinin has been found in transformed (hairy) roots of Artemisia annua. A protocol was developed to preserve A. annua hairy roots in liquid nitrogen. Root tips were excised from 7-day-old cultures and held on solid White’s medium for 24 h prior to cryoprotection. They were then treated with a cryoprotecting mixture containing 8% (v/v) dimethyl sulfoxide (Me2SO) and 20% (w/v) sucrose at 25°C for 1 h followed by cooling at 0.09°C/min to 4°C then cooling to −35°C at 0.72°C/min. Vials containing the root tips were then plunged into liquid nitrogen. After thawing in a water bath to 37°C, root tips were held in the cryoprotecting mixture for 10 min before it was diluted to 25% of its original concentration. Root tips were washed once with fresh liquid White’s medium and held for 1 h prior to culturing on White’s medium with 0.2% Gelrite and 3% (w/v) sucrose. Regrowth of root tips averaged 65%. Independent variables in this study included 1) Me2SO concentration; 2) the type and concentration of cosolutes; 3) cooling rate(s); 4) the temperature at which the sample is transferred to liquid nitrogen; 5) the age of the culture from which root tips are taken; 6) the recovery period between root tip excision and immersion in cryoprotectant; and 7) the amount of Gelrite used in the postthaw plating medium. © 1996 Academic Press, Inc.
Artemisia annua is a Chinese herb that produces the antimalarial drug artemisinin, which has been used for many centuries in Chinese traditional medicine as a treatment for fever and malaria (15). Artemisinin, commonly extracted from the aerial parts of the plant, has been found in transformed (hairy) roots in our laboratory (33). Current investigations on the use of transformed root cultures as an alternative to cell cultures (10, 11) have confirmed the potential of transformed roots for production of secondary metabolites. Among the advantages of using transformed roots are short doubling times of the biomass (12) and apparent genetic and biosynthetic stabilities (1, 19, 30). The benefits of cryopreservation are significant. Cryopreservation eliminates the need for periodic subculture which requires considerable labor, materials, and space. It also reduces risk of contamination by microorganisms and genetic drift during storage as the transformed roots are quiescent. Cryopreservation of transformed roots will be important for establishing safe patent repositories. Progress in the last decade on the cryopresReceived January 12, 1995; accepted August 19, 1995. 1 To whom correspondence should be addressed. 106 0011-2240/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
ervation of plant systems has made possible the regeneration of entire plants from frozen cells (e.g., 20, 22, 23), embryos (e.g., 18, 25), and shoot tips (e.g. 6, 21, 36) of a number of species. The cryopreservation of shoot tips of some plants such as strawberry, carnation, tomato, pea, and wild potato is now routine (27). However, cryopreservation of roots has received little attention, and the development of protocols for root cryopreservation is still in its infancy as evidenced by few reports of root cryopreservation in the literature. The situation may change with increasing interest in transformed roots as potential production systems for secondary metabolites, thus leading to the development of cryopreservation protocols useful for a variety of plant species for both germplasm conservation as well as commercial purposes. There has been only one published report on cryopreservation techniques for transformed roots. The models were beet and tobacco, and the reported regrowth rate was 10% (3). Two papers on cryopreservation of hairy roots of Panax ginseng and horseradish were presented at the 1994 conference of the Society for Cryobiology (24, 36), and they reported regrowth rates of 60% and 40%, respectively. The present research was carried out to establish a method
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for the cryopreservation of transformed roots of A. annua while adding new information to the field of root cryopreservation. MATERIALS AND METHODS
Production and Maintenance of Hairy Root Cultures Transformed roots of A. annua were produced and cultured as previously described (33). Cultures were grown in liquid White’s medium (Sigma product number W 0876, St. Louis, MO) supplemented with 3% (w/v) sucrose. (All subsequent references to White’s medium assume 3% supplemental sucrose.) Cultures were kept in 125-ml Erlenmeyer flasks on an orbital shaker at 100 rpm at 25°C in dim light. Root Tip Excision Healthy, white root tips, 2–4 mm in length, were excised with a sterile scalpel from transformed roots after a 4- to 14-day subculture interval. The tips were allowed 0–48 h of recovery time on White’s medium solidified with 2.0 g/l Gelrite (Merck, Rahway, NJ). Root tips on solid medium were maintained in a 27°C incubator in the dark. All manipulations were performed in liquid medium in a sterile hood to avoid desiccation or contamination of the fragile tissue. Experimental Units and Viability Testing The basic experimental unit in all procedures is the vial, which corresponds to a 2-ml polypropylene cryovial containing 10–15 root tips. Vials were subjected to various experimental treatments after which the component root tips were tested for viability. Viability tests are pass/ fail (no intermediate grades), and the percentage of passing roots defines the viability of the vial, which is assumed to be normally and independently distributed (NID) for statistical purposes. Two viability tests were used in this study. The first, fluorescein diacetate (FDA) staining, is relatively rapid and convenient (34); however, it only indicates membrane integrity (26) and can produce false positives when cellular
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damage is not manifested in the membrane. The second, 7+ day culturing, is a rigorous test of survival but is more laborious than FDA staining. The terms membrane integrity and regrowth testing are used for the two methods, respectively. Membrane integrity testing. Stock solution of FDA (0.2%), consisting of 2 mg of FDA dissolved in 1 ml of acetone, was diluted 20 times using liquid White’s medium to obtain 0.01% FDA solution. Root tips were suspended in 1 drop of the 0.01% FDA solution on a microscope slide for 5 min prior to viewing under a microscope with uv illumination (17). Tips that did not fluoresce brightly throughout their entire volume were considered failing. Regrowth testing. Root tips were cultured in solid, semisolid, or liquid White’s medium in a dark, 27°C incubator for at least 7 days. (Solid medium has 0.2% Gelrite, semisolid medium has 0.1% Gelrite, and liquid medium has no Gelrite.) Early signs of recovery, detected by microscopic observation, were swelling of the meristematic region, formation of a new meristem at the root tip, and doubling of the length of the root tips. Conversion of the surviving root tips to roots (regrowth) occurred after 7 days. Root tips that did not show regrowth were considered failing. Cooling Apparatus A NalgeneTM Cryo 1°C Freezing Container was used to effect controlled cooling of the vials. The container was either placed in a −68°C freezer to obtain a nominal cooling rate of 1°C/ min, or it was placed in a 4°C refrigerator to obtain a nominal cooling rate of 0.1°C/min. Exact cooling rates were measured via a thermocouple in a dummy vial. Fig. 1 shows the temperature history of vials cooled at 1°C/min from room temperature to −40°C or at 0.1°C/min to 4°C then 1°C/min to −40°C. The dotted lines in Fig. 1 are the best fit lines for the measured data. The slopes of the best fit lines show that when the container is placed in the −68°C freezer the cooling rate averages 0.8°C/min; when it is in the 4°C refrigerator the rate averages 0.09°C/min; and when it is placed in the
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FIG. 1. The temperature history of a vial of cryoprotectant in the Nalgene Cryo 1°C Freezing Container is shown for two cooling protocols. In the first protocol the container is placed directly in a −68°C freezer. In the second protocol the container is placed in a 4°C refrigerator until the vial reaches 4°C, then it is transferred to the −68°C freezer. The dotted lines are the best fit lines for the measured data. The slopes of the best fit lines show that when the container is placed in the −68°C freezer the cooling rate averages 0.8°C/min; when it is in the 4°C refrigerator the rate averages 0.09°C/min; and when it is placed in the −68°C freezer after the refrigerator the rate averages 0.72°C/min. The actual temperature differs from the best fit line by 4°C or less, which is comparable to the manufacturer’s specifications. Three replicates were run for each cooling rate to determine the repeatability of the container. The standard deviation associated with a single measurement is 0.2°C, which indicates good repeatability.
−68°C freezer after the refrigerator the rate averages 0.72°C/min. The actual temperature differs from the best fit line by 4°C or less, which is comparable to the manufacturer’s specifications. Three replicates were run for each cooling rate to determine the repeatability of the container. The standard deviation associated with a single measurement is 0.2°C, which indicates good repeatability. Experimental Procedures Eight experiments were performed to find the optimal cryopreservation procedure for the YUT6 clone of A. annua, and these are detailed in the subsequent subsections. The number of independent variables prohibited a complete factorial experiment design; instead, factors were tested individually or in limited combinations. The independent variables under study were: Me2SO concentration; the type and concentration of cosolute; cooling rate(s); the temperature at which the sample is transferred to liquid nitrogen; the age of the culture from which root
tips are taken; the recovery period between root tip excision and immersion in cryoprotectant; and the amount of Gelrite in the postthaw plating medium. Each of the experiments relied on previous results to refine the cryopreservation procedure, as will be presented under “Results and Discussion.” A ninth experiment measured the growth curve of optimally cryopreserved root tips as well as control root tips. A tenth experiment assayed both cryopreserved and control root tips after 20 days of growth for production of artemisinic compounds. Dimethyl sulfoxide (Me2SO) tolerance at room temperature. Me2SO solutions were prepared by adding 0, 8, 10, 12, 14, or 16% (v/v) Me2SO to liquid White’s medium. Excised root tips from 6- to 9-day subcultured roots were allowed no wound recovery period after excision and placed in vials containing 0.5 ml of Me2SO solution at 25°C. After 1 h, the solution was removed with a sterile Pasteur pipette and replaced with 0.5 ml of White’s medium. Root tips remained in White’s medium for 10 min
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after which they were plated on solid White’s medium for regrowth testing. Three vials were processed for each Me2SO solution. Effects of cosolutes and terminal temperature. An initial survey of cosolutes and terminal temperatures was conducted to find conditions that produced high rates of regrowth. All vials contained 10% Me2SO (v/v) in White’s medium except for the control, which contained no Me2SO. The 10% Me2SO solution in each vial was either unmodified or augmented with one of five cosolutes: 20% (w/v) sucrose, 20% (w/v) sorbitol, 20% (w/v) mannitol, 2% (v/v) glycerol, or 2% (w/v) polyvinylpyrrolidone (PVP). Thus, there were seven different solutions including the control. Excised root tips from 6- to 9-day subcultured roots were allowed no wound recovery period and held in vials containing 0.5 ml of cryoprotective solution at 25°C for 1 h. Vials were cooled at 0.8°C/min (average), and when they reached a temperature equal to the freezing point of the cryoprotective solution they were manually removed from the bath for 10–15 s and placed on the steel refrigerator rack, which effected nucleation. They were then returned to the bath and cooled to a terminal temperature of −10, −20, −30, −35, or −40°C. Vials were thawed by immersion in a 37°C water bath for 2–3 min. The cryoprotectant solution was then diluted by the addition of 0.07, 0.10, 0.13, 0.20, 0.33, and 0.67 ml of fresh
White’s medium (total volume 1.50 ml) at 10min intervals, causing the solute concentration to drop to 25% of its original value in six equal steps. The diluted cryoprotectant solution was removed with a sterile pipette and replaced with 0.5 ml of White’s medium. Root tips were held for 1 h at 25°C before being transferred to plates containing solid White’s medium for regrowth testing. Four vials were processed for each of the 35 combinations of solutions and terminal temperatures. Effects of Me2SO concentration, sucrose concentration, wound recovery period, and transfer temperature. This experiment analyzed the effects of four factors: wound recovery, transfer temperature, Me2SO concentration, and sucrose concentration, using the Graeco–Latin square design (Table 1), which assumes no interaction between factors (14). Excised root tips from 6to 9-day subcultured roots were plated on solid White’s medium for 0, 12, or 24 h prior to immersion in cryoprotectant to allow the tips time to recover from wound stress. Cryoprotectant solutions were prepared by adding 8, 10, or 12% (v/v) Me2SO and 15, 20, or 25% (w/v) sucrose to White’s medium. Root tips were immersed in vials containing 0.5 ml of cryoprotectant at 25°C for 1 h prior to cooling. Vials were cooled at 0.09°C/min (average) to 4°C then at 0.72°C/ min (average) to −35, −37, or −40°C. When the vials reached the specified transfer temperature
TABLE 1 Nine Experimental Treatments Are Defined to Investigate Four Factors, Each Having Three Levels Factors Treatment no.
Transfer temperature (°C)
Wound recovery (h)
Me2SO concentration (% v/v)
Sucrose concentration (% w/v)
1 2 3 4 5 6 7 8 9
−35 −37 −40 −35 −37 −40 −35 −37 −40
12 0 24 0 24 12 24 12 0
12 8 10 10 12 8 8 10 12
15 15 15 25 25 25 20 20 20
Note. The experimental design is a Graeco–Latin square, which in this application is synonymous with a one-ninth replication of a 34 factorial. The design assumes there are no significant interactions between factors.
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they were immediately plunged into liquid nitrogen where they were held for at least 1 h. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were tested for membrane integrity but not regrowth. Three vials were processed for each of the nine combinations of factors shown in Table 1. Recovery medium. Excised root tips from 6to 9-day cultures were allowed 24 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. Tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/ min (average) to −35°C, then plunged into liquid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid, semisolid, or liquid White’s medium for postthaw recovery, and tested for regrowth. Three vials were processed for each recovery medium. Culture age. Excised root tips from 4-, 7-, 11-, and 14-day-old cultures were allowed 24 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. Tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/min (average) to −35°C, then plunged into liquid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid White’s medium and tested for regrowth. Three vials were processed for each culture age. Wound recovery period. The effects of different wound recovery periods, which were investigated in the four-factor experiment using the membrane integrity test, were checked and expanded in this experiment using regrowth testing. Excised root tips from 7-day-old cultures were allowed 0, 24, or 48 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. Tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/ min (average) to −35°C, then plunged into liq-
uid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid White’s medium and tested for regrowth. Three vials were processed for each recovery period. Transfer temperature. The effects of different transfer temperatures, which were investigated in the four-factor experiment using the membrane integrity test, were checked and expanded in this experiment using regrowth testing. Excised root tips from 7-day-old cultures were allowed 24 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. Tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/min (average) to −10, −20, −30, −35, −37, or −40°C, then plunged into liquid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid White’s medium and tested for regrowth. Two vials were processed for each transfer temperature. Cooling rates. Excised root tips from 7day-old cultures were allowed 24 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. For slow cooling, tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/min (average) to −35°C, then plunged into liquid nitrogen. For fast cooling, tips were cooled at 0.8°C/min (average) to −35°C, then plunged into liquid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid White’s medium and tested for regrowth. Three vials were processed for each cooling rate. Growth curve characterization. Excised root tips from 7-day-old cultures were allowed 24 h of wound recovery on solid White’s medium prior to 1-h immersion at 25°C in 0.5 ml of cryoprotectant composed of 8% (v/v) Me2SO and 20% (w/v) sucrose in White’s medium. Tips were cooled at 0.09°C/min (average) to 4°C, then at 0.72°C/min (average) to −35°C,
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then plunged into liquid nitrogen. Nucleation, thawing, and removal of cryoprotectant were as previously described. Root tips were plated on solid White’s medium for postthaw recovery. The lengths of the root tips were measured after 3, 6, 9, 11, 13, 15, 17, and 19 days and compared with the growth of excised root tips that were simply plated on solid White’s medium. One vial of 10 root tips was cryopreserved and compared with 10 control root tips. Artemisinin production. Root tips from the YU-T6 clone of A. annua were either cryopreserved as in the growth curve experiment or plated on solid White’s medium immediately after excision. Cryopreserved tips were allowed 20 days of growth after thawing, and control tips were allowed 20 days of growth after excision. Production of secondary products (artemisinin & artemisinic acid) was measured by thin layer chromatography. The detailed procedure is given by Teoh (31). One cryopreserved vial and one control vial were processed to measure production. RESULTS AND DISCUSSION
Regrowth of root tips exposed to 0–16% (v/v) Me2SO at 25°C for 1 h is shown in Fig. 2. The survival of root tips at 25°C treated with 8, 10, or 12% (v/v) Me2SO for 1 h was not significantly different (a > 0.05) from control root
FIG. 2. The percentage of root tips that demonstrated regrowth after 1-h exposure at 25°C to White’s medium with Me2SO is shown as a function of Me2SO concentration. Error bars show the standard error of the mean. Horizontal lines at the top of the graph span concentrations that are not significantly different at the a 4 0.05 level as determined by a Neumann–Keuls test.
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tips. Increasing the concentration to 14 and 16% (v/v) caused significant drops in regrowth rates. This is consistent with the literature on slow freezing of plant systems where the use of 5–10% (v/v) Me2SO alone or with other cryoprotectants for 0.5–3 h at temperatures between 0 and 25°C has proven to be nontoxic for meristems (e.g., 2, 13), hairy roots (3), shoot tips (7), and shoot apices (29). As shown in Fig. 3, root tips that were exposed for 1 h at room temperature to 10% Me2SO (v/v) in White’s medium supplemented with either 20% (w/v) sorbitol, 20% (w/v) mannitol, 2% (v/v) glycerol, or 2% (w/v) PVP and frozen as described under “Materials and Methods” showed 0% regrowth over four replicates. Root tips frozen in plain White’s medium also evinced 0% regrowth. Root tips exposed to White’s medium plus 10% Me2SO had some regrowth depending on terminal temperature, and root tips exposed to 10% Me2SO in White’s medium supplemented with 20% sucrose had high levels of regrowth at all terminal temperatures. The loss in regrowth as the terminal temperature was lowered was not significant at the 5% level. The Graeco–Latin square experimental design (Table 1) was employed to determine the influence of four parameters (transfer temperature, wound recovery time, Me2SO concentration, and sucrose concentration) on viability. The design assumes no interactions and provides statistical tests only for main effects (14). An analysis of variance (Table 2) shows that transfer temperature and wound recovery period are significant effects at the 0.05 level, and sucrose concentration may be important. Me2SO concentration is not significant, which is in contrast to the results of the first experiment (Me2SO tolerance at room temperature). This may be due to the narrower range of concentrations used in this experiment or the use of FDA staining as opposed to the more rigorous regrowth testing used in the first experiment. A plot of the main effects is shown in Fig. 4. Twelve or 24 h of recovery is significantly better than no recovery (a < 0.05), suggesting that recovery from excision stress is beneficial to
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FIG. 3. The percentage of frozen and thawed root tips that demonstrated regrowth is shown as a function of the cryoprotectant mixture and terminal temperature. Root tips were given 1-h exposure at 25°C to White’s medium with various cryoprotectant additives then cooled at 1.0°C/min to the terminal temperature. Nucleation was effected at the equilibrium freezing point. Vials were thawed by immersion in a 37°C water bath for 2–3 min and the cryoprotectant was removed with a six-step dilution procedure.
cryopreservation. This effect was also seen by Towill (32), but Benson and Hamill (3) found the opposite. In the previous experiment (effects of cosolutes and terminal temperatures) there was a decrease in regrowth as the terminal temperature went from −35 to −40°C; however, the data were not strong enough to prove significance (a 4 0.09). The results of this experiment show that a transfer temperature of −37°C was significantly better than −35°C (a < 0.05) but not significantly better than −40°C (a 4 0.08). As shown in Fig. 5, frozen–thawed root tips plated on 0.1 or 0.2% Gelrite showed 49 and
43% regrowth, respectively (the difference is not statistically significant). None of the root tips in liquid medium (0.0% Gelrite) showed regrowth, which is significantly worse than the other two cases (a < 0.05). The benefit of solid over liquid culture has been commonly noted (3, 5, 8, 35). The effect of culture age on postthaw survival was not significant. A plot of survival vs culture age is shown in Fig. 6. Fig. 7 shows that a recovery period between excision and cryopreservation is highly beneficial. This confirms the results of the four-factor experiment, which tested 12- and 24-h recovery
TABLE 2 ANOVA of the Graeco–Latin Square Experiment Source
df a
SSb
MSc
Fd
Probability
Transfer temperature Wound recovery period % Me2SO (v/v) % Sucrose (w/v) Error Total
2 2 2 2 18 26
3,161 3,561 754 1,550 6,925 15,951
1,581 1,781 377 775 385
4.1 4.63 <1 2.01
<0.05 <0.05
Note. It is assumed that there are no interactions between factors. a Degrees of freedom associated with each source term. b Sum of squares for each source term. c Mean square for each source term. d Ratio of the mean square for a source divided by the mean square for the error term.
0.18
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FIG. 4. The percentage of frozen and thawed root tips that were viable by FDA staining is shown as a function of four factors: transfer temperature, recovery time, Me2SO concentration, and sucrose concentration. The experimental design assumed no interactions between factors. Error bars show the standard error of the mean.
periods. The difference between 24 and 48 h is not significant. Up to this point, all experiments that included freezing in liquid nitrogen have used two cooling rates: 0.09°C/min from 25 to 4°C and 0.72°C/min from 4°C to the temperature at which the sample is transferred to liquid nitrogen. This allowed 210 min for equilibration above 4°C and about 40 min below 4°C. This
FIG. 5. The percentage of frozen and thawed root tips that demonstrated regrowth on liquid, semisolid, or solid medium is shown. Liquid medium (0% Gelrite) is significantly worse than semisolid or solid medium (a < 0.05). Error bars show the standard error of the mean.
two-rate protocol was compared with a single freezing rate, 0.8°C/min, which allowed only 21 min above 4°C. The two-rate protocol resulted in regrowth of 44% of the cryopreserved root tips, compared with 0% of root tips cryopreserved with the one-rate protocol. Transfer temperature was critical to the survival of root tips frozen in liquid nitrogen as
FIG. 6. The percentage of frozen and thawed root tips that demonstrated regrowth is shown as a function of subculture age. None of the differences of the means is significant at the 5% level, but an analysis of variance (Table 3) shows that age is a significant factor. Error bars show the standard error of the mean.
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FIG. 7. The percentage of frozen and thawed root tips that demonstrated regrowth is shown as a function of the recovery period between excision and cryoprotection. Twentyfour and 48 h were not significantly different from each other but were significantly better than no recovery (a < 0.05). Error bars show the standard error of the mean.
shown in Fig. 8. −35°C was optimal, which is in slight contrast to the results of the four-factor experiment which found −37°C to be better than −35°C. The difference may be due to the assay (regrowth as opposed to FDA staining in the four-factor experiment) or an interaction with other factors that was assumed to be zero in the four-factor experiment. Table 3 summarizes the factors affecting cryopreservation of transformed roots of A. an-
FIG. 8. The percentage of frozen and thawed root tips that demonstrated regrowth is shown as a function of the temperature at which they were transferred to liquid nitrogen. −35°C was significantly better than any other temperature (a < 0.05). Error bars show the standard error of the mean.
nua and gives the optimal values found in this study. The regrowth rate for optimally cryopreserved roots was 65%, which is an improvement over previous work. Benson and Hamill (3) reported regrowth rates of 10% for transformed root tips of beet and tobacco using a slow cooling method similar to the one in this study but without sucrose as a cryoprotectant. Yoshimatsu et al. (36) reported 60% regrowth for P. ginseng hairy roots using a vitrification method. Phunchindawan et al. (24) reported 40% survival of horseradish hairy roots using an encapsulation dehydration cryopreservation protocol. Growth curves for optimally cryopreserved and control root tips are shown in Fig. 9. Cryopreserved root tips are significantly shorter than the control tips at every measurement interval from day 2 to day 19; however, the shapes of the growth curves are very similar, indicating normal growth progression. In most cases recovery proceeded in an organized manner without formation of callus or any adventitious activity, thus minimizing genetic instability. In some cases recovery was preceded by formation of new meristematic regions as the old regions degenerated with time, indicating considerable damage to the meristem. In general, there was a period of about 3 days after thawing before the root tips began to show measurable growth.
FIG. 9. Growth curves for optimally cryopreserved and control root tips are shown over 19 days. Error bars are shown where measurements were taken and indicate the standard error of the mean. Data could not be pooled because the variance associated with length measurement was significantly positively correlated with root length. Cryopreserved roots were significantly shorter than control roots for all measurements, but the shape of the growth curve suggests that cryopreserved roots are “normal.”
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TABLE 3 Factors Affecting Cryopreservation of Transformed Roots of Artemisia annua and Optimal Values Found in This Study Factor
Range
Optimal value
Tissue size Tissue age
Factor was not varied 4–14 days
Period between excision and exposure to cryoprotectant Me2SO concentration in cryoprotectant Cosolute
0–48 h
Root tips 2–4 mm long 7 days (early exponential phase) 24 h
0–16% (v/v)
8%
None, glycerol, mannitol, PVP, sorbitol, sucrose 15–25%
Sucrose 20%
Factor was not varied
1 h at 25°C 0.1°C min−1 to 4°C then 1.0°C min−1 to transfer temperature
Nucleation technique
1.0°C min−1 to transfer temperature or 0.1°C min−1 to 4°C then 1.0°C min−1 to transfer temperature Factor was not varied
Transfer temperature Thawing technique
−10°C to −40°C Factor was not varied
Cryoprotectant removal
Factor was not varied
Recovery medium
White’s medium with 0.0%, 0.1% or 0.2% Gelrite
Cosolute concentration in cryoprotectant Temperature and time of exposure to cryoprotectant Cooling rate
This delay, which is not seen in control roots, may be attributed to the time required to repair cellular damage (4) or for the remaining live cells to undergo a number of divisions before measurable growth resumed, or both (9). Postthaw delay has been observed in many systems (e.g., 16, 20, 28, 32). Production of artemisinin in a sample of 10 cultured, cryopreserved roots was 0.214 mg/g, fresh weight, which is similar to a sample of 10 control roots (0.246 mg/g, fresh weight.). Note: 10 roots were required to achieve sufficient tissue mass for high pressure liquid chromatography measurement, and the results are the average for the 10 roots. Only one measurement was taken for frozen and control roots, so no statistical comparisons were possible. The finding that secondary production was not affected by cryopreservation is consis-
Cryovials were touched to refrigerator rack when they were just below the equilibrium freezing point −35°C Immersion in 37°C water bath for 2–3 min Cryoprotectant was diluted in 6 steps then replaced with White’s medium White’s medium with 0.2% Gelrite
tent with work done on transformed roots of B. vulgaris and N. rustica (3) and P. ginseng (36). CONCLUSION
This study demonstrates that cryopreservation of transformed roots of A. annua is an effective and practical technique for maintenance of genetic stock because survival rates are high (65% on average as measured by regrowth testing), secondary metabolite production is similar to unfrozen controls, and expensive equipment is not required (an alcohol bath in standard 4°C and −68°C refrigerators was used instead of a programmable freezer). A conventional, slow cooling protocol was used and a combination of Me2SO and sucrose was essential for high survival. Other important factors included recovery time between excision and cryopreservation,
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and the concentration of Gelrite in the recovery medium. A summary of findings is given in Table 3. ACKNOWLEDGMENTS Standard compounds of artemisinic acid, artemisitene, and arteannuin B as well as seeds of four strains of A. annua were kindly provided by Dr. Nancy Acton of the Division of Experimental Therapeutics, Walter Reed Army Institute of Research. This work was supported in part by NIH Grant 1R15AI34131-O1 and was completed in partial fulfillment of the requirements for the degree of Master of Science. REFERENCES 1. Aird, E. L. H., Hamill, J. D., and Rhodes, M. J. C. Cytogenetic analysis of hairy root cultures from a number of plant species transformed by Agrobacterium rhizogenes. Plant Cell Tissue Organ Cult. 15, 47–57 (1988). 2. Bagniol, S., and Engelmann, F. Effects of pregrowth and freezing conditions on the resistance of meristems of date palm (Phoenix dactylifera L. var. Bou Sthammi Noir) to freezing in liquid nitrogen. CryoLett. 12, 279–286 (1991). 3. Benson, E. E., and Hamill, J. D. Cryopreservation and post freeze molecular and biosynthetic stability in transformed roots of Beta vulgaris and Nicotiana rustica. Plant Cell Tissue Organ Cult. 24, 163–172 (1991). 4. Cella, R., Colombo, R., Galli, M. G., Nielsen, E., Rollo, F., and Sala, F. Freeze-preservation of rice cells: A physiological study of freeze-thawed cells. Physiol. Plant. 55, 279–284 (1982). 5. Chen, T. H. H., Kartha, K. K., Leung, N. L., Kurz, W. G. W., Chatson, K. B., and Constabel, F. Cryopreservation of alkaloid-producing cell cultures of periwinkle (Catharanthus roseus). Plant Physiol. 75, 726–731 (1984). 6. Demeulemeester, M. A. C., Vandenbussche, B., and De Proft, M. P. Regeneration of chicory plants from cryopreserved in vitro shoot tips. Cryo-Lett. 14, 57– 64 (1993). 7. Diettrich, B., Wolf, T., Bormann, A., Popov, A. S., Butenko, R. G., and Luckner, M. Cryopreservation of Digitalis lanata shoot tips. Plant. Med. 53, 359– 363 (1987). 8. Dussert, S., Mauro, M. C., and Engelmann, F. Cryopreservation of grape embryogenic cell suspensions. 2. Influence of post-thaw culture conditions and application to different strains. Cryo-Lett. 13, 15–22 (1992). 9. Finkle, B. J., Zavala, M. E., and Ulrich, J. M. Cryoprotective compounds in the viable freezing of plant tissues. In “Cryopreservation of Plant Cells and Organs” (K. K. Kartha, Ed.), pp. 75–113, CRC Press, Boca Raton, FL, (1985).
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