In VitroCell.Dev.Biol.29P:65-71,April1993 © 1993TissueCultureAssociation 1054-5476/93 $01.50+0.00
IN V I T R O S H O O T M U L T I P L I C A T I O N OF E A S T E R N W H I T E C E D A R (THUJA OCCIDENTALIS) KATY A. NOUR AND TREVOR A. THORPE
Plant Physiology Research Group, Deparment of Biological Sciences, Universityof Calgary, Calgary, Alberta T2N 1N4, Canada (Received 8 June 1992; accepted 17 December 1992; editor D. I. Dunstan)
SUMMARY A protocol for clonal propagation of eastern white cedar (Thuja occidentalis L.) was enhanced by optimizing the shoot multiplication stage using unbranched in vitro-produced shoots. This was achieved by careful selection of different medium components. An optimum range of 10 to 14 axillary shoots was obtained when shoots were cultured on halfstrength Quiorin and LePoivre medium containing 10 ~tM filter-sterilized zeatin for 3 wk. Transfer of the treated shoots to cytokinin-free medium containing 0.05% activated charcoal improved both the number and quality of the axillary shoots produced. Maximum axillary bud induction was also accomplished when shoots were pulsed in 1 mM liquid, filter-sterilized zeatin for 3 h, and then transferred to half-strength Quiorin and LePoivre, charcoal-containing medium. Inclusion of 4% sucrose improved the number of axillary shoots obtained. Half strength of the major salts produced an optimum response. Shoots obtained from different cultures (1 to 5 yr old) responded similarly to the applied cytokinin; however, newly induced shoots (4 mo. old) gave a significantly higher response.
Key words: eastern white cedar; Thuja occidentalis; axillary bud breaking; axillary shoots; clonal propagation. pared to adventitious bud formation, true-to-type and genetically stable plants are more likely to be produced. Eastern white cedar (Thuja occidentalis L.) or Arbor-vitae is a member of the Cupressaceae family and is one of two Thuja species native to North America (Hosie, 1979). It is widely distributed in the eastern part of North America on swampy sites (van Gelderen and van Hoey Smith, 1989). Wood from this economically important species is used for fences, poles, shingles, boats, and canoes (Hosie, 1979). Numerous cultivars are used extensively as ornamentals in Europe and North America (Harry et al., 1987). Thuja occidentalis has been successfully micropropagated in our lab from embryonic explants through the muhistaged organogenic route (Harry et al., 1987). The number of shoots induced on embryonic explants is fairly low, but this species showed potential for axillary branching. Thus, the objective of this study was to enhance the clonal propagation potential of T. occidentalis in vitro by optimizing the shoot-multiplication stage,
INTRODUCTION The world demand for forest products is predicted to rise sharply over the next few decades (Biondi and Thorpe, 1982). However, it is generally accepted that forests are being harvested at a faster rate than they are being regenerated (Thorpe et al., 1991). Hence, active renewal of the existing forests must take place to meet the future needs of the economy and environmental quality (Durzan and Campbell, 1974). Thus, there is an urgent requirement for production of large numbers of improved, fast-growing trees. Vegetative or clonal propagation of trees offers several advantages for forest renewal and tree improvement programs. It allows for the reproduction of elite full-sib families, or individuals in a family, that exhibit significant gain due to nonadditive gene effects. In addition, at least a 10% increase in gain can be expected from planting selected clonal propagules rather than selected seed families (Thorpe et al., 1991). Clonal multiplication of superior phenotypes and valuable breeding stocks can be used for establishing tree improvement and seed production orchards (Dunstan et al., 1992). Although considerable progress has been made with somatic embryogenesis in conifers, in vitro plantlet formation still is achieved mainly through the multistaged organogenic route (Thorpe et al., 1991). Currently, most research involves manipulating the process, where the optimum requirements for each stage are empirically determined. However, the stage of shoot multiplication has received relatively little attention. This process depends on the production of additional adventitious shoots or the development of preformed axillary meristems or both (Biondi and Thorpe, 1982). Despite the relatively low rate of shoot production by this latter method com-
MATERIALSANDMETHODS
Plant Material and Culture Conditions Seeds of eastern white cedar were obtained from the Ministry of Natural Resources, Ontario, and stored at - 2 0 ° C. This seed lot had a germination rate of 66%. Before culture, seeds were imbibed overnight under running tap water. Sterilization procedure involved a 20-min treatment in 30% Javex bleach (6% NaOC1)containing 3 to 4 drops of Tween-20 per 100 ml, followed by a 5-min wash with sterile water, and a 5-min treatment in 10% hydrogen peroxide followed by three 5-min washes with sterile water. Whole embryos were excised under aseptic conditions and cultured in 65
66
NOUR AND THORPE
100- X 15-mm petri dishes containing half-strength Quoirin and LePoivre (1/2 QP) medium (1977) supplemented with 1 gM Nr-benzyladenine (BA), 3% (wt/vol) sucrose, asparagine (100 mg-hter-l), myo-inositol (100 mg'hter-1), nicotinic acid (5.0 rag.liter-i), pyridoxine-HCl (5.0 mg. liter-i), thiamine-HC1 (5.0 mg. hter-l), and 0.8% Difco Bacto agar (Difco Laboratories, Detroit, MI), according to the procedure described in Harry et al. (1987). After 20 to 25 days the embryonic explants were transferred to petri dishes containing BA-free, 1/2 QP basal medium for 1 mo. to allow for bud development. Further subcultures were into 6-oz glass jars with 100 ml of basal medium supplemented with 0.05% (wt/vol) activated charcoal (Sigma Chemical Co., St. Louis, MO, no. C-4386). On Passage 2, shoots were separated and excised from the original explants and were subeultured thereafter at monthly intervals. These shoots represented the stock cultures from which single shoots were obtained for the different experiments described below. All cultures were kept at 24 + 1 o C and a 16-h photoperiod provided by Sylvania Gro-Lux F40T12 Gro-WS lamps at a photon fluence rate of about 80 gmol. m-2 • s-1.
Experimental Design
medium containing 1, 2, 4, 6, 8, and 10% (wt/vol) sucrose for the entire experiment. Further experiments were carried out using 5 and 10 #M of the optimum cytokinin in combination with 2, 3, 4, or 5% sucrose. Shoots were cultured on the various sucrose levels for the total experimental period of plus and minus cytokinin. Major salts. The optimum level of the major salts (nitrates, phosphates, sulfates) in QP basal medium was determined by culturing the shoots on different levels of 0, i/t, 1/3, 1/2, and full strength for the entire experimental period of 2 too. using 5 and 10/zM of the best cytokinin. The minor salts and iron were maintained at half-strength. Other components such as vitamins, asparagine, and myo-inositol were maintained at the same level as described earlier. Explant age. These experiments were designed to determine whether the age of the shoot cultures from which unbranched shoots were obtained affected their response to the different treatments. Shoots from material subcultured monthly for 4 yr and 4 mo. were used to test the effect of I and 10 #M of the optimum cytokinin. In another comparison, shoots selected from 1- and 4-yr-old cultures were compared using 5 and 10 #M of the optimum cytokinin. RESULTS
For each experiment, unbranched shoots, approximately 8 to 10 mm in height were excised and used as explants. All experiments were conducted using 1/2 QP as the basal medium. Qorpak 4-oz glass jars (Fisher Scientific, Edmonton, Alberta), or baby food jars with 25 ml of medium were used. For each treatment in the different experiments, except where indicated, 20 unbranched shoots were cultured in four jars. In all experiments, unless indicated otherwise, shoots were cultured on medium containing a specific treatment for 1 mo. then transferred to cytokinin-free 1/2 QP medium containing 0.05% activated charcoal for the following month. All experiments were repeated at least twice. Analyses of variance were performed on the results of each experiment using an IBM-PC version of SAS (Version 6, SAS Institute, 1985) and the data compared using Tukey's multiple comparison test when applicable.
Effect of Various Treatments on Shoot Multiplication Cytokinins. For the selection of the best cytokinin for axillary bud development, BA, kinetin, 6-(-'y,'y dimethylallylamino)-purine (2iP), or zeatin (Sigma) were included in the medium in a concentration range of 0.001 to 10 gM. Except for zeatin, which is thermally unstable, all cytokinins were added to the medium before autoclaving, the zeatin solution was adjusted to pH 5.7 to 5.8 using 0.2 M NaOH, filter sterilized, and added to the medium after autoclaving. Thidiazuron (TDZ; NOR-AM Chemical Co., Wilmington, DE) was also tested, and was added before media autoclaving in a similar range of concentrations. Further experiments used a narrower range of cytokinin concentrations, and the most effective cytokinin was tested at 1, 2.5, 5, 7.5, 10, 15, and 20 #M. The effect of applying a combination of different cytokinins was examined by including two cytokinins in the medium in equimolar concentrations with a final concentration of 0.1 or 1.0 gM. Further experiments used 10 ttM final concentration of both cytokinins. To determine the period needed for maximum shoot bud development on the optimum cytokinin treatment, shoots were cultured on 1/2 QP medium containing the cytokinin for 3, 4, or 5 wk. Liquid pulse treatment. Shoots were pulsed in filter-sterilized 1 mM solution of the selected cytokinin (pH 5.7 to 5.8). The cytokinin solution was applied for periods of 3, 6, 9, 12, 16, or 20 h. Sterile cytokinin solution was added to 4-oz glass jars containing presterilized glass beads. The glass beads were used so that the shoots could stand upright, and only the lower portions of the unbranched shoots were submerged in the liquid solution. The glass jars were agitated continuously (50 rpm) in the light (Vogelmann et al., 1984). Afterward, shoots were transferred onto charcoal-containing medium for 1 mo. The results were used to design experiments using smaller time intervals of 1 to 6 h. Activated charcoal. Shoots cultured on medium containing various cytokinins for 1 mo. were transferred to cytokinin-free medium that was either devoid or contained 0.05% activated charcoal (Sigma no. C-4386). Sucrose. Eastern white cedar shoots were cultured on cytokinin-free
The number of shoot buds induced on the embryonic explants as a result of BA treatment was fairly low and averaged between one and two shoots per explant. These shoots generally developed from the epicotyl between the two cotyledons (Harry et al., 1987). Shoot buds were allowed to elongate by transferring the explants to BAfree medium. Further elongation occurred when shoots were excised from the original explants and transferred onto BA-free medium containing 0 . 0 5 % activated charcoal. Cytokinins. Various cytokinins applied singly varied in their degree of effectiveness in axillary bud development. Preliminary results showed that more developed axillary shoots were obtained when cytokinins were applied at 10/.tM. Higher concentrations (20 ttM) gave lower numbers of axillary shoots and some showed necrosis. Kinetin was less effective than BA and in its presence shoots exhibited poor growth and a very low multiplication rate. Both 2iP and zeatin gave better responses than BA or kinetin in both numbers and length of axillary shoots obtained (Fig. 1 A). Axillary shoot production on these various cytokinins (10 ~tM) is shown in Table 1. The lengths of the shoots were not significantly different on the various cytokinins. As can be seen in Table l, inclusion of zeatin in the medium increased axillary bud production substantially compared to the rest of the eytokinins. In addition, the axillary shoots exhibited vigorous growth and elongation (Fig. 1 A). Further testing wJith a range of concentrations (1 to 20 #M) of zeatin confirmed that the highest number of axillary shoots was obtained at a concentration of 10 ~tM (Table 2). Axillary buds developed from almost all leaf axils present on the cultured shoots (Fig. 1 B). Higher concentrations did not give a higher number of axillary shoots, and in some cases such high concentrations caused the secretion of brown exudates into the medium. The size of the axillary shoots produced was similar among the different treatments. Extending the culture period on zeatinfree, plus-activated charcoal medium for 2 too. instead of 1 did not improve the numbers or elongation of the axillary shoots. However when axillary shoots were excised and cultured on fresh medium they elongated quite rapidly, which is considered an advantage of the treatment if the shoots were to be used for further multiplication or rooting. When shoots were cultured on TDZ-containing medium, small axillary buds grew out and became visible (less than 1.0 mm).
SHOOT MULTIPLICATION IN CEDAR
67
Z~at:i
Fic. 1. A, axiUarybud production in eastern white cedar shoots in the presence of 5 and 10 #M of different cytokinins applied singly. Shoots obtained from 1- to 2-yr-old cultures were cultured on cytokinin-containing medium for 1 mo. then transferred to cytokinin-free medium for another month. B, response of eastern white cedar shoots t6 the application of various concentrations of zeatin included in the culture medium for 1 mo. Shoots were subcultured twice onto cytokinin-free, plus-activated charcoal medium. Treated shoots were obtained from 1-yr-old cultures. C, axillary bud production and elongation in eastern white cedar shoots obtained from 1-yr-old cultures when pulsed in liquid solution of 1 mM filter-sterilized zeatin for 3 to 20 h. Shoots were cultured on 1/2 QP medium, plus AC for 1 mo. D, comparison of axillary bud production in eastern white cedar when shoots obtained from 1- to 2-yr-old cultures were grown on medium containing various levels of the major salts in combination with 5 or 10 #M zeatin. Shoots were cultured on medium containing a specific level of the salts during both periods of axillary bud development (+zeatin) and axillary shoot elongation (-zeatin).
However, these failed to develop into shoots. The number of developed axillary shoots decreased as the concentration of TDZ in the medium increased. In addition, most of the shoots were brown and necrotic. Concentrations higher than 1 ~tM killed more than 90% of the cultured shoots and very few produced buds. At such high concentrations, the stems of the cultured shoots became very thick (3 to 4 mm) and quite fleshy. Similar morphology of shoots was seen even when TDZ was applied in equimolar combination with other cytokinins. Cytokinin combinations. When two cytokinins were applied simultaneously in equimolar concentrations giving a total concentration of 0.1 and 1.0 #M, few axillary buds developed; maximum numbers were always obtained when zeatin was one of the cytokinins apphed. In the next stage, when 10 #M final concentration was used (Table 3), maximum shoot numbers were obtained when zeatin was combined with kinetin or 2iP. Maximum shoot length did not differ significantly among the various treatments. However, the number of shoots were not as high as when only zeatin was applied in a final concentration of 10 #M. Shoots cultured in the presence of 5 or 10 #M zeatin for 3, 4, or 5 wk did not differ significantly in the number or length of axillary
shoots produced. Some of the shoots cultured for 5 wk on plus-zeatin medium developed friable callus on their bases (data not shown). Liquid pulsing. In a preliminary experiment, application of a high concentration of zeatin for a period of 3 to 20 h caused the development of a relatively high number of axillary buds. Shoot buds started appearing after i wk of culture on plus-activated charcoal medium. After 1 mo. axillary shoots showed vigorous growth and the whole explant was quite bushy (Fig. 1 C). Periods longer than 3 h gave lower numbers of axillary shoots. When a range of 1to 6-h pulsing periods were tried, the number of the shoots produced almost doubled when the pulse duration increased from 1 to 2 h, maximum bud development occurred with 3-h pulse (Table 4). Maximum shoot length differed significantly, with the 2- and 3-h pulses giving the longest shoots (Table 4). The pH of the zeatin solution increased from 5.5 before pulsing to about 7.5 after pulsing the shoots for 1 to 2 h. Longer pulsing periods did not change the pH significantly from 7.5, as it only reached 7.7 after 5 to 6 h of pulsing. It should be noted that the number of shoots obtained was similar and sometimes higher than that obtained when shoots were cultured in the presence of 10 #M zeatin for 1 mo. (Table 2). Activated charcoal. AxiUary bud production improved in num-
68
NOUR AND THORPE TABLE 1
TABLE 3
COMPARISON OF THE EFFECTS OF THE DIFFERENT CYTOKININS ON AXILLARY SHOOT BUD DEVELOPMENT AND ELONGATION IN EASTERN WHITE CEDAR~
NUMBER AND MAXIMUM LENGTH OF AXILLARY SHOOTS OF EASTERN WHITE CEDAR PRODUCED IN THE PRESENCE OF TWO CYTOKININS a
Cytokinin Type, 10/,tM
No cytokinin Kinetin BA 2iP Zeatin
Mean No. of Axiliary Shoots
4.3 4.4 7.6 8.0 14.7
± ± ± + +
0.44 0.71 0.63 0.87 1.41
Maximum Shoot Length. cm
as a ab b c
0.85 1.13 1.06 0.98 0.9
+ + + + +
0.09 0.13 0.08 0.07 0.09
a" a a a a
Twenty shoots were cultured on plus cytokinin medium for 1 mo. then transferred to cytokinin-free medium for another month before measurements were taken. Numbers represents means + SE. s Mean number of axillary shoots followed by different letters differ significantly (F4.95 = 23.04, P < 0.001). No significant difference in the maximum shoot length between the different cytokinins (F4,91 = 1.5, P > 0.1).
bers and quality when shoots were transferred to cytokinin-free m e d i u m that contained 0 . 0 5 % activated charcoal as o p p o s e d to charcoal-free m e d i u m . In general this r e s p o n s e occurred with the different cytokinins used, but the i m p r o v e m e n t was only significant with zeatin or 2 i P (Table 5). In contrast, shoots transferred to c h a r coal-free m e d i u m showed s t r e s s h k e s y m p t o m s of browning of the main s t e m s a n d leaves. Generally axillary shoot length improved in the p r e s e n c e of activated charcoal; however the increase was not always significant (Table 5). Sucrose. Preliminary results showed that 1 % s u c r o s e in the m e d i u m was not sufficient for satisfactory growth of the cultured shoots; with a high percentage of shoots showing browning of the leaf tips or the main s t e m s by the e n d of the culture period. With levels higher than 4 % , shoots exhibited s y m p t o m s of osmotic stress.
Mean No. of Axillary Shoots
Cytokinin Combination
No cytokinin BA + 2iP BA + KIN BA + ZTN ZTN + KIN ZTN + 2iP KIN + 2iP
2.40 3.30 4.30 5.40 7.26 7,30 4.12
+ + + + + ± +
0.54 0.36 0.47 1.04 0.74 0.81 0.64
Maximum Shoot Length, em
as ab ab bc c c ab
0.73 0.72 0.91 0.76 0.97 0.84 0.91
+ + + ± ± + ±
0.14 0.14 0.11 0.10 0.12 0.07 0.13
a~ a a a a a a
Total cytokinin concentration was 10 #M for each combination. Numbers represent means of 20 shoots -4- SE. s Means labeled by different letters are significantly different (F6,1z3 = 7.23, P < 0.001). No significant difference was detected in the maximum shoot length between the different cytokinin combinations (F6,H4 = 0.6, P > 0.5).
W h e n a range of 2 to 5 % s u c r o s e was applied in the p r e s e n c e of 5 or 10 # M zeatin, axillary shoot production improved as the level of s u c r o s e increased from 2 to 4 % (Fig. 2). However, the i n c r e m e n t was higher when 4 % was c o m b i n e d with the o p t i m u m level of 10 # M zeatin. At 5 % s u c r o s e the n u m b e r s of the shoots p r o d u c e d were reduced, a n d leaves of the main shoots b e c a m e reddish brown. In addition, most shoots h a d b u d s that failed to develop into shoots. Major salts. Shoots cultured on m e d i u m in the a b s e n c e of the major salts p r o d u c e d few axillary shoots in the p r e s e n c e of 5 or 10 # M zeatin. Most cultured shoots developed roots by the e n d of 2 mo. of culture, a n d a few developed yellow or brown leaves. Addition of t h e s e salts at 1/4 strength increased the n u m b e r of axillary shoots in the p r e s e n c e of 10 # M zeatin significantly (F~,,149 = 1 2 . 3 , P < 0 . 0 2 5 ) . However, t h e s e showed poor growth a n d development. Higher levels of 1//2 a n d full strength did not significantly increase the n u m b e r of shoots produced, but t h e s e showed better growth,
TABLE 2 APPLICATION OF VARIOUS CONCENTRATIONS OF ZEATIN AND THEIR EFFECT ON AXILLARY SHOOT BUD FORMATION AND ELONGATION IN T. OCCIDENTALIS ~ Zeatin Concentration. ttM
0.0 1.0 2.5 5.0 7.5 10 15 20
Mean No. of Axillary Shoots
2.6 5.11 7.3 10.6 9.7 12.05 10.83 10.72
_+ 0.37 + 0.58 + 0.80 + 0.77 + 0.82 + 0.7 + 0.76 + 0.8
Maximum Shoot Length, cm
as ab bc d cd d d d
0.6 0.76 0.67 1.04 0.73 0.75 0.92 0.8
+ + + + + + + +
0.09 0.08 0.09 0.12 0.10 0.09 0.08 0.08
ab" ab ab b ab ab ab ab
Shoots were cultured on plus-zeatin medium for 1 mo. then transferred to minus-zeatin, plus-activated charcoal medium for another month before measurements were taken. Numbers represent means + SE for 20 shoot replicates. s Average numbers of axillary shoots followed by the different letters are significantly different (F7,136 = 2.28, P < 0.05). c Maximum shoot length followed by different letters are significantly different (F7.136 = 21.35, P < 0.001).
TABLE 4 AXILLARY SHOOT PRODUCTION IN EASTERN WHITE CEDAR WHEN SHOOTS WERE PULSED IN LIQUID SOLUTION OF ZEATINa Pulsing Time, h
0 1 2 3 4 5 6
Mean No. of Axillary Shoots
1,83 9.20 16.45 16.84 14.2 13.65 15.45
_+ 0.44 + 1.22 + 1.1 + 1.4 _+ 0.55 + 1.24 _+ 1.23
Maximum Shoot Length, em
ab b c c bc bc c
0.95 1.32 1.65 1.36 1.00 1.42 1.14
-+ 0.26 + 0.18 + 0.21 + 0.12 -+ 0.12 + 0.16 ± 0.1
a" ab b ab a ab ab
Zeatin concentration was 1 mM, pH 5.5, followed by culture on 1/2QP, plus-AC medium for 1 mo. Shoots were obtained from 1-yr-old cultures. Numbers represent means + SE for 20 shoots. b Mean number of shoots followed by different letters are significantly different (F6,116 : 24.86, P < 0.001). c Maximum shoot length followed by different letters are significantly different (F6,122 = 3.23, P < 0.01).
69
SHOOT MULTIPLICATION IN CEDAR TABLE 5
14
THE EFFECT OF ACTIVATED CHARCOAL ON AXILLARY SHOOT PRODUCTION IN EASTERN WHITE CEDAR~
13 12 11
Mean No. of Axillary Shoot# Cytokinin Type
Kinetin BA 2iP Zeatin
-AC
2.70 2.89 3.50 5.89
+ + + +
-AC
0.49 0.57 1.25 0.9
0.6 0.6 0.45 0.65
+ + + +
0.07 0.193 0.042 0.18
1.125 0.67 0.82 0.61
+ + + +
0.54 0.362 0.082 0.28
Shoots cultured on media containing different cytokinins (10 pM) for 1 mo. before transfer to cytokinin-free medium with or without activated charcoal (AC, 0.05%) for 1 mo. Numbers represent means of 10 shoots + SE. b Significant interaction was detected between the type of cytokinin applied and the presence or absence of activated charcoal in the medium (F3,69 = 5.45, P < 0.005). Significant difference between minus-AC and plus-AC was detected only for zeatin and 2iP (F1,69 = l 1.47, P < 0.005). ° No significant interaction was detected between the type of cytokinin applied and the presence or absence of AC in the medium (F3.ss = 1.879, P > 0.1). However, the presence AC in the medium improved the maximum length of axillary shoots regardless of the type of cytokinin used (FLss = 6.09, P < 0.05).
and the leaves were green and larger in size than those grown on the lower levels (Fig. 1 D). Explant age. Four-month-old shoots produced significantly more shoots and showed more vigorous growth than the 4-yr-old shoots after induction by 1 or 10 p M zeatin (Fig. 3). However, in a subsequent study, 1- and 5-yr-old shoots treated with the same concentration (5 or 10 #M) of zeatin produced axillary shoots of equivalent number and length. DISCUSSION
The presence of cytokinins in the medium was the most critical component for inducing axiUary bud development. Our results
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Fie. 2. Effect of application of different levels of sucrose on axiUary shoot bud development in eastern white cedar. Shoots were obtained from 2-yr-old cultures. Bars represent SE. No significant interaction was detected between sucrose and zeatin level (F3,137 = 1.73, P > 0.1). Different levels of sucrose labeled by different letters showed significant difference regardless of the concentration of zeatin applied (F3,137 = 5 . 4 8 , P < 0.005).
=
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14 13 12
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-4- 0.39 2.60 + 0.35 4.00 + 0.47 6.50 + 1.24 10.90
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FIG. 3. Number (NOS) and maximum length (ML) of axillary shoots produced from eastern white cedar shoots obtained from 4-yr-old (4Y) and 4-mo.-old cultures (4M). Bars represent SE. No significant interaction was detected between explant age and zeatin concentration on the number of axillary shoots produced (F1.60 = 0.39, P > 0.5). However, explant age (F1.6o = 33.9, P < 0.001) and zeatin concentration (Fl.,o = 17.11, P < 0.001) independently affected shoot production. For maximum shoot length, no significant interaction was detected between the two factors (FLss = 0.07, P > 0.75). Explant age significantly affected maximum shoot length regardless of the zeatin concentration (FLs5 = 18.3, P < 0.001).
agree with the established fact that axillary bud development requires overcoming apical dominance (Phillips, 1975), where endogenous indole acetic acid synthesis and its basipetal transport along the shoot inhibit growth of axillary buds (Tamas, 1987). Thus, applying exogenous cytokinins reduces the auxin:cytokinin ratio in the axillary buds and results in their development. The various cytokinins and the different concentrations varied in their degree of effectiveness in axillary bud development. Zeatin and 2iP, two natural cytokinins (Brock and Kaufman, 1991), were more successful in inducing high numbers of relatively long shoots than the synthetic cytokinins (BA and kinetin), with zeatin at 10 # M being the best. Zeatin was also found to be the best cytokinin for shoot multiplication of Pinus canariensis. However, cytokinins led to vitreous, stunted, and callused axillary shoots, especially at high concentrations (Martinez Pulido et al., 1990). No vitrification was seen in eastern white cedar shoots, except that at high concentrations of zeatin or 2iP some shoots showed thickening at their bases. However, the axillary shoots themselves retained a normal phenotype. Thidiazuron failed to stimulate axillary bud development at all concentrations used. At high concentrations, it was toxic to the shoots, causing them to become brown and necrotic. These results are contrary to the reports where this compound was used as a growth promoter (Mok et al., 1982) or stimulant of shoot bud proliferation in several angiosperms (Mok et al., 1987; Fellman et al., 1987) and some gymnosperms (Goldfarb et al., 1991). However, high concentrations of TDZ were reported to decrease the number of the buds produced on Douglas fir cotyledons (Goldfarb et at., 1991) and caused stunted growth of shoots of muscadine grape (Gray and Benton, 1991). In apple shoot cultures, this growth regulator caused thickening of the main stem (van Nieuwkerk et al., 1986), an observation seen in eastern white cedar shoots as well. The number of axillary shoots obtained at 10 # M zeatin was
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NOUR AND THORPE
relatively high compared to other species. In red spruce, a maximum of only four shoots were obtained at the optimum treatment (Lu et al., 1991). In Pinus oocarpa, low concentrations of BA stimulated axillary bud production; however concentrations higher than 1 #M inhibited bud development (Baxter et al., 1989). In western larch (Larix occidentalis) optimum treatment of 0.1 #M 2iP produced two to five axillary shoots on about 70% of the treated shoots (Harry et al., 1991), but with eastern white cedar shoots we always obtained 100% response at 10 ~tM zeatin. In Pinus radiata a fourfold multiplication rate was obtained every 3 too. by decapitation of the main shoot. This method was not very successful with eastern white cedar shoots because only the two buds in the leaf axils directly below the excision point developed (data not shown). Administering liquid zeatin for short periods was more efficient in inducing axillary bud development than the conventional method of incorporating zeatin in the agar-solidified medium. These observations agree with those of Zel et al. (1988) and Burns et al. (1991). They reported axillary bud development from seedlings after 4 and 5 days of BA pulse treatment in P. sylvestris and P. ellioti, respectively. A pulse of 2 to 3 h with continuous agitation was sufficient for maximum response in Thuja occidentalis shoots. This method is valuable because it shortens the time needed for shoot multiplication by approximately 1 mo., which is a benefit for commercial micropropagation. Activated charcoal improved both quality and development of axillary shoots. These observations agree with previous reports that bud proliferation and elongation were improved in various gymnosperm species when activated charcoal was included in the medium (Patel et al., 1986; Rumary and Thorpe, 1984; Martinez Pulido et al., 1990; Harry et al., 1991). The mechanism by which activated charcoal improves growth is based on its capacity to absorb phytohormones, phenolics, and other toxins (David, 1982). Indeed, phenolic exudates were seen in the medium at the base of the shoots when charcoal was excluded. Axillary shoot development and elongation was dependent on the sucrose level in the medium. A 4% concentration gave the best results, whereas 3% sucrose was optimum for the earlier stages of bud induction and elongation from embryonic explants of T. occidentalis (Harry et al., 1987). Whether the higher sucrose requirement is needed only as a carbon-energy source or in part plays an osmotic role remains to be determined. Also, half strength of the major salts in the medium was optimum for shoot multiplication. Not unexpectedly, the absence of these salts from the medium caused poor growth of the cultured shoots and their ability to respond to the optimum zeatin treatment was highly reduced. Shoots of T. occidentalis obtained from 4-mo.-old cultures were those induced on the embryos or their primary axillary shoots. Hence they were still in their active growth and elongation stage. These shoots exhibited better response compared to those obtained from 4-yr-old cultures in terms of both numbers and length. Interestingly, the response of shoots from 1- and 5-yr-old cultures were similar. This indicates that shoot cultures of eastern white cedar could be kept for long periods for future use for multiplication without losing their morphogenic capacity, a problem often faced in some tissue culture systems (Halperin, 1986). In conclusion, this study is one of the few where the stage of shoot muhiplication has been carefully manipulated. Optimizing this stage in eastern white cedar has improved the protocol previously de-
scribed for this species (Harry et al., 1987). The original protocol allowed for the production of over 250 plantlets per embryonic explant after 1 yr in culture. The improvements reported here would lead to the formation of over 1000 plantlets in the same period; thereby increasing the possibility of the method being used for in vitro mass clonal propagation. ACKNOWLEDGEMENTS
The authors thank Dr. Indra Harry for her critical review of the mannscript. This research was supported by NSERC of Canada Operating and Strategic grants to Trevor A. Thorpe. REFERENCES Baxter, R.; Brown, S. N.; England, N. F., el al. Production of c]onal plantlets of tropical pine in tissue culture via axillary shoot activation. Can. J. For. Res. 19:1338-1342; 1989. Biondi, S.; Thorpe, T. A. Clonal propagation of forest tree species. In: Rao, A. N., ed. Tissue culture of economically important plants. Singapore: COSTED and Asian Network for Biological Sciences; 1982:197-204. Brock, T. G.; Kaufman, P. B. Growth regulators: an account of hormones and growth regulation. In: Stewart, F. C., ed. Growth and development. Vol. X. Plant physiology, a treatise. New York: Academic Press; 1991:277-338. Burns, J. A.; Schwarz, O. J.; Schlarbaum, S. E. Multiple shoot production from seedhng explants of slash pine (Pinus eUiottii, Engelm.). Plant Cell Rep. 10:439-443; 1991. David, A. In vitro propagation of gymnosperms. In: Bonga, J. M.; Durzan, D. J., eds. Tissue culture in forestry. The Hague: Martinus Nijhoff; 1982:72-108. Dunstan, D. I.; Lashta, D. P.; Kikcio, S., et al. Factors affecting recurrent shoot multiplication on in vitro cultures of 17- to 20-year-old Douglas fir trees. In Vitro Cell. Dev. Biol. 28P:33-38; 1992. Durzan, D. J.; Campbell, R. A. Prospects for the mass production of improved stock of forest trees by cell and tissue culture. Can. J. For. Res. 4:151-160; 1974. Fellman, C. D.; Read, P. E.; Hosier, M. A. Effect of thidiazuron and CPPU on meristem formation and shoot proliferation. Chemical regulation in tissue culture: an overview. HortScience 22:1197-1200; 1987. Goldfarb, B.; Howe, G. T.; Bailey, L. M., et al. A liquid cytokinin pulse induces adventitious shoot formation from Douglas-fir cotyledons. Plant Cell Rep. 10:156-160; 1991. Gray, D. J.; Benton, C. M. In vitro micropropagation and plant establishment of muscadine grape cuhivars (Vitis rotundifolia). Plant Cell Tissue Organ Cult. 27:7-14; 1991. Halperin, W. Attainment and retention of morphogenic capacity in vitro. In: Vasil, I. K., ed. Cell culture and somatic cell genetics of plants, vol. 3. New York: Academic Press; 1986:3-47. Harry, I. S.; Thompson, M. R.; Thorpe, T. A. Regeneration of plantlets from mature embryos of western larch. In Vitro Cell. Dev. Biol. 27P:89-98; 1991. Harry, I. S.; Thompson, M. R.; Lu, C-Y., et al. In vitro plantlet formation from embryonic explants of eastern white cedar (Thuja occidentalis L.). Tree Physiol. 3:273-283; 1987. Hosie, R. C. Native trees of Canada, 7th ed. Don Mills, Ont. Canada: Fitzhenry and Whiteside Ltd.; 1979:96-99. Lu, C-Y.; Harry, I. S.; Thompson, M. R., et al. Plantlet regeneration from cultured embryos and seedling parts of red spruce (Picea rubens Sarg.). Bot. Gaz. 152:42-50; 1991. Martinez Pulido, C.; Harry, I. S.; Thorpe, T. A. In vitro regeneration of plantlets of Canary Island pine (Pinus canariensis). Can. J. For. Res. 20:1200-1211; 1990. Mok, M. C.; Mok, D. W. S.; Armstrong, D. J., et al. Cytokinin activity of N-phenyl-N'-l,2,3-thiadiazol-5-ylurea. Phytochemistry 21:15091511; 1982. Mok, M. C.; Mok, D. W. S.; Turner, J. E., et al. Biological and biochemical effect of cytokinin-active phenylurea derivatives in tissue culture systems. Proceedings of the Symposium on Chemical Regulation in Tissue Culture: An overview. HortScience 22:1192-1194; 1987.
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Thorpe, T. A.; Harry, I. S.; Kumar, P. P. Application of micropropagation to fi~restry. In: Debergh, P. C.; Zimmerman, R. H., eds. Micropropagation, technology and apphcation. Dordrecht: Kluwer Academic Publishers; 1991:311-336. van Gelderen, D. M.; van Hoey Smith, J. R. P. Conifers, 2nd ed. Oregon: Timber Press; 1989:27-28. van Nieuwkerk, J. P.; Zimmerman, R. H.; Fordham, I. Thidiazuron stimulation of apple shoot proliferation in vitro. HortScience 21:516-518; 1986. Vogelmann, T. C.; Bornman, C. H.; Nissen, P. Uptake of benzyladenine in explants ofPicea abies and Pinus sylvestris. Physiol. Plant. 61:513517; 1984. Zel, J.; Gogala, N.; Camloh, M. Micropropagation of Pinus sylvestris. Plant Cell Tissue Organ Cult. 14:169-175; 1988.