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Cold storage of shoot cultures and alginate encapsulation of shoot tips of Camellia japonica L. and Camellia reticulata Lindley
SCIENTIA HORllClJLTulM Scientia Horticulturae
71 (1997) 67-78
Cold storage of shoot cultures and alginate encapsulation of shoot tips of CumeZZiajuponica L. and Camellia reticulata Lindley A. Ballester
*,
L.V. Janeiro, A.M. Vieitez
Instituto de Inuestigaciones Agrobiokigicas de Galicia, CSIC, Apartado 122, 15080 Santiago de Compostela, Spain Accepted 25 April 1997
Abstract In vitro shoot cultures of 8 clones or cultivars of Camellia japonica L. and Camellia reticulata Lindley were stored at 2-4°C for up to 12 months. Survival frequencies near 100% were obtained during the first or second subcultures after cold storage in seven of the eight clones assayed, but the other clone could not be profitably stored for more than 6 months. When shoot tips and nodal explants of C. japonica L. (clone 2 and cv. Alba Plena) were encapsulated in alginate beads, the regrowth of shoot tips depended on the components of the gel matrix but that of nodal explants was very poor regardless of the encapsulation conditions. The best morphogenetic response of the shoot tips was obtained when the encapsulating gel was supplemented with the minerals and vitamins of Murashige and Skoog’s medium, 3% sucrose and clone-specific growth regulators. The size of the shoot tips used and the concentration of sucrose added to the gel matrix also influenced the capacity for regrowth. Encapsulated shoot tips of C. japonica L. stored at 2-4°C or 18-20°C survived for shorter periods of time than unencapsulated shoot tips stored at low temperature: after 75 days of storage, only 10% of encapsulated shoot tips survived at 2-4°C and 7% at 18-20°C. 0 1997 Elsevier Science B.V. Keywords: Camellia japonica L.; Camellia reticulata Lindley; Cold: Encapsulation;
In vitro culture; Storage
acid; IBA, indole-3Abbreviations: BA, 6benzyladenine; GA s , g ibberellic acid; IAA, indole-3-acetic butyric acid; Zip, 6-dimethylallylamino-purine; MS, Murashige and Skoog medium * Corresponding author. Tel.: +34 81 590 958; fax: f34 81 592 504; e-mail: [email protected]. 0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(97)00074-5
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1. Introduction CumeZlia (Theales, Theaceae) is a genus of tropical and subtropical trees and shrubs native to Eastern Asia. Chang and Bartolomew (1984) have carried out a major taxonomic revision, grouping some 200 recognized species in four subgenera and 20 sections. Increasing interest in ornamental camellias (mainly Camellia juponicu L., Camellia reticuluta Lindley, Camellia susanqua and Camellia suluenensis) has led to continuous efforts by researchers, growers and breeders to produce new varieties and hybrids with improved floral and growth characteristics, and to develop more efficient methods for their propagation. Protocols for micropropagation from shoot tips and nodal explants of both juvenile and mature ornamental camellias have been reported (Vieitez et al., 1992) as has the generation of adventitious shoots from leaves of C. reticulutu Lindley (San-Jose and Vieitez, 1993). The induction and development of somatic embryos of several Camellia species has recently been reviewed (Vieitez, 1995). Furthermore, such protocols are in principle applicable for ornamental camellias and tea plants not only to clonal propagation but also to artificial seed production. This has dramatically increased in recent years (Redenbaugh et al., 1993; Gray et al., 1995) leading to the establishment of gene banks for medium- or long-term storage. The low-temperature storage of germplasm produced in vitro is now a well documented alternative for the preservation of a variety of plant species (Monette, 1987; Orlikowska, 1992; Janeiro et al., 1995b). The major advantages of this approach are reduced labour and space requirements, the elimination of pathogen-related problems and the reduction of genetic erosion if optimal storage conditions are achieved (Engelmann, 1991). I n Cumellia, the effect of cold storage on embryogenic cultures of C. japonicu L. and C. reticulutu Lindley has been reported (Janeiro et al., 1995a), as have the results of attempts at long-term cryopreservation of both somatic embryos and embryonic axes of C. japonicu L. (Janeiro et al., 1996). There has been little research on the effect of encapsulation (the technology of coating somatic embryos or shoot tips in alginate beads) on the response of plant material to cold storage (Bapat et al., 1987; Bapat and Rao, 1988). Encapsulation is an aid for storage (smaller space is needed in relation to the storage of conventional in vitro cultures), but also it is important for handling and exchange of in vitro material. There is no information on the possibility of medium-term storage of either encapsulated or unencapsulated shoot tips of C. juponica L. and C. reticduta Lindley at low temperature. The objectives of this study were: (1) to study the survival and proliferation of several clones of C. japonica L. and C. reticulutu Lindley after storage of unencapsulated shoot cultures at 2-4°C for several months; (2) to study the factors affecting the encapsulation and subsequent development of shoot tips and nodal segments isolated from in vitro shoot cultures of C. juponicu L.; (3) the effect of storage on encapsulated material.
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2. Materials and methods
2.1. Cold storage of shoot cultures Shoot cultures from the species, clones or cultivars listed below were multiplied by subculturing shoot tips and nodal segments (1 cm in length) at 8-week intervals (for specific multiplication media see Janeiro, 1996). Thirty days after the last subculture or 10 days for clone 2 of C. japonica L., the 500 ml glass jars (with glass lids fixed with plastic film) containing the shoot cultures were transferred from standard conditions to cold storage. They were kept at 2-4°C in cool cabinets (340-l Sanyo Medico01 MPR 311D) with 16 h day-’ of dim light (8 pmol me2 s- I> for 3, 6, 9 or 12 months (C. japonica L. clone 2 and cv. Alba Plena), 10 months (C. japonica L. clone 1) or 12 months (C. japonica L. cv. Fimbriata Alba and C. reticulata Lindley cv. Mouchang clones 1, 3, 10 and 222). After this period, the cultures were transferred to fresh multiplication medium and kept in a growth chamber under standard growth conditions together with controls (0 month in the cold). After 8 weeks, these shoots (subculture 1) were evaluated as described below and were subcultured to produce subculture 2 which was evaluated after a further 8 weeks. For both subcultures the following parameters were evaluated: survival rate (the percentage of cultures capable of proliferating), the number of new shoots per responsive explant, the length of the longest shoot, and the multiplication coefficient, defined as the product of the proportion of explants surviving with shoot development and the mean number of 8-10 mm segments per explant (Sgnchez and Vieitez, 1991). Twelve replicate jars (containing 80 ml of multiplication medium), with 10 explants per jar, were used for each treatment. Growth conditions were provided by a growth chamber with 30 pmol rnp2 s-’ of light from Osram L4OW cool white fluorescent lamps for 16 h day-‘, with day and night temperatures of 24°C and 2O”C, respectively. 2.2. Encapsulation Shoot tips and nodal segments 5-6 mm in length were isolated at the end of a multiplication cycle from in vitro shoot cultures of C. japonica L. clone 2 and cv. Alba Plena. Tips and nodes were individually encapsulated in alginate beads by transferring them one by one with a Pasteur pipette from a 3% solution of sodium alginate in distilled water to one of the following solutions: (a) 0.1 M CaCl, .2H,O (calcium solution); (b) calcium solution with Ca-free MS medium (Murashige and Skoog, 19621, including MS vitamins; (c) calcium solution with Ca-free MS medium and 3% sucrose; (d) calcium solution with Ca-free MS medium and 3% sucrose and the growth regulators of the multiplication medium (4.40 PM BA + 0.57 FM IAA for clone 2, or 8.90 PM BA + 9.12 PM Zeatin + 9.80 /IM 2 ip + 0.049 PM IBA for cv. Alba Plena). The beads were left in the calcium solution for half an hour and then inoculated in multiplication medium, where, in accordance with conventional protocol for the multiplication of these Camellia clones, they spent 4 weeks followed by a further 4 weeks in fresh multiplication medium. At the end of this time, the survival percentage, the number of shoots per
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responsive explant and the length of the longest shoot per explant were recorded. Unencapsulated explants were used as controls. In other experiments, the effects of the size of explant were investigated using shoot tips of C. japonica L. clone 2 sized l-2 mm, 5-6 mm or lo- 11 mm, encapsulated in solution (d); and the effect of the concentration of sucrose in the encapsulating matrix were studied using 5-6 mm shoot tips of the same clone encapsulating by means of Ca solutions identical to solution (d) except that sucrose concentrations of 1.5%, 3% and 5% were employed. Twelve replicate jars (300 ml, containing 50 ml of multiplication medium), with 10 beads per jar, were used for each treatment. 2.3. Storage of encapsulated
shoot tips
Shoot tips of C. japonica L. clone 2 and cv. Alba Plena were encapsulated in beads containing 3% calcium alginate + MS + 5% sucrose + growth regulators (specific for each clone, see Section 2.2) and were then kept in sterile Petri dishes, 20 to a dish, and stored at 2-4°C or 18-20°C for 30, 60 and 75 days (clone 2) or 30 and 60 days (cv. Alba Plena). After storage, the beads were inoculated in 300 ml glass jars containing 50 ml of multiplication medium and transferred to fresh medium after 4 weeks. Unstored encapsulated explants were used as controls. At the end of the multiplication stage, the same parameters as in the experiments on the storage of unencapsulated material were recorded. Twelve replicate jars with 10 beads per jar were used for each treatment. 2.4. Statistical analysis In all experiments, treatments were arranged in a completely randomized design with subsampling and the data were statistically analyzed by one- or two-way analysis of variance (percentage data were first subjected to arcsine square root transformation), using the least significant difference (LSD) or the Tukey HSD test (Sokal and Rohlf, 1981) at the P = 0.05 level to compare means.
3. Results and discussion 3.1. Cold storage of shoot cultures In vitro shoot cultures of C. juponicu L. clone 2 were less resistant to the cold than those of cv. Alba Plena (Table 1) after more than 6 months at 2-4°C. Whereas Alba Plena had a 100% survival and unaltered morphogenetic parameters (the number of shoots produced and the length of the longest shoot were affected by subculture number, but without any clear trend), the survival percentage, number of shoots per explant and longest shoot length of clone 2 decreased significantly to very low levels. In particular, the damage caused in clone 2 by 9 months or more in the cold failed to form multiple shoots in subculture 2. In this clone necrosis was observed to spread down from the shoot tip as time elapsed.
Control 1 2 1 2 1 2 1 2
0
“b’cSignificant
F-test 104.9’ 14.lb 5.5b
LSD
13.8 8.7 6.1
F-test
156.8’ 12.2s 5.5b
P < 0.0001, respectively;
N
1.3 11.1 0.6
LSD
2.6f0.7 2.8+ 1.4 3.7+ 1.2 2.7kO.3 2.4 + 0.4 2.8kO.l 2.650.5 3.6k 1.1 3.1*0.9
50.3b 0.8b 2.lns
F-test
L
cv. Alba Plena
ns = not significant.
7.9k2.1 5.5 * 0.9 8.4 * 2.5 1.5f0.2 3.3* 1.1 1.0+0.0 1.0*0.0 1.0+0.0 1.0+0.0
Survival
Clone 2
100 100 100 100 100 100 100 100 100
cv. Alba Plena
Clone 2
100 90.6 100 68.8 39.6 29.1 1.2 14.1 1.5
(o/o)
10.5 11.1 _
LSD
37.9 + 5.9 48.1 + 3.4 58.6 + 3.2 28.9k6.1 30.3 + 1.9 20.1 k4.5 10.2* 1.3 10.2 + 2.3 10.1 +2.4
Clone 2
2.9” 0.9ns 1.3ns
F-test
N
cv. Alba Plena
68.9 i 5.4 58.9)7.3 51.3i5.4 63.2+9.1 63.5 +4.3 58.6 f 3.5 49.2 f 2.9 51.4k6.2 57.8 rf- 8.3
cv. Alba Plena
Length of longest shoot k standard error (mm)
-
_
0.7
LSD
7.9 4.9 8.4 0.8 1.3 0.3 0.07 0.2 0.07
Clone 2
9.1 -
_
LSD 4. lb l.lns 2.4ns
2.6 2.8 3.7 2.7 2.4 2.8 2.6 3.6 3.1
cv. Alba Plena
subcultures
F-test
L
Multiplication coefficient
of C. japonicu L. clone 2 and cv. Alba Plena in two successive
Clone 2
parameters Number of shoots per explant f standard error
on survival and morphogenetic
Survival
at P < 0.01, P < 0.001,
Subculture (A) Cold storage (B) AXB
Source of variation
Analvsis of Variance
12
9
6
3
Subculture
Months in cold
Table 1 Effect of cold storage (2-4°C)
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In a similar experiment in which other clones and cultivars of C. japonicu L. and C. reticulutu Lindley spent 12 months in cold storage (10 months in the case of C. juponicu L. clone l), survival percentage in subculture 1 ranged from 100% in cv. Fimbriata Alba and C. reticulutu Lindley clones 1 and 3 to 16% in C. juponicu L. clone 1. In subculture 2, however, all clones had 100% survival except C. juponicu L. clone 1, and even this clone had a survival percentage of 80% (Fig. 1). Moreover, the number of shoots per explant after 2 subcultures was close to that of control cultures (data not shown). These results show that the detrimental effects of cold storage were transitory in these clones. In all clones, surviving cultures of subculture 2 exhibited no carry-over effect of cold storage, multiplying and rooting at levels similar to those of controls in subsequent subcultures (data not shown). The results obtained in these experiments indicate that in vitro cultures of ornamental camellias are tolerant to cold storage but the degree of tolerance varies between clone or cultivar. In a germplasm bank, no general protocol can be applied, C. juponicu L. clone 2 would have to be subcultured at least every 6 months, while other clones could be left in the cold without subculture for a year or more. The causes of this differential behaviour are not known, but one may be the consistency of the shoots: clone 2 leaves were thinner and apparently less cutinized than those of other clones. One of the most important factors that can influence the success of cold storage (4°C) is the physiological state of the explants at the time of being placed in the cold
% Survival
100 90 80
70 60 50 40 30
SUbcUll
10
ture 2
culture
0 CjF
Cjl
03
CrlO Crl
Cr222
Fig. 1. Survival percentage of different Camellia clones in two successive subcultures after a prior 12.month period in cold storage. CjF, C. japonica L. cv. Fimbriata Alba; Cjl, C. japonica L. clone 1; Cr3, CrlO, Crl and Cr222, C. reticulata Lindley clones 3, 10, 1 and 222, respectively. Bars with the same letter are not significantly different at the 5% level according to the Tukey HSD test.
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(Orlikowska, 1992). The need for explants to recover from cold storage prior to the stress brought about by subculture has been shown for hybrid Populus (Son et al., 1991), Custunea and Quercus (Janeiro et al., 1995b) and Ulmus (Dorion et al., 1993), all these genera, like Camellia, also exhibit clonal variability in their response to cold storage. A possible exception to this rule is that of Populus tremulu (L.) X Populus tremuloides (L.) cv. Muhs 1, shoot tips stored at 10°C immediately after isolation had a 100% survival after 3 months storage (Hausman et al., 1994). On the basis of our previous experience, in this work Camellia cultures spent some time under standard culture conditions between the last subculture and the start of cold storage (10 or 30 days, depending on clone). As regards the biochemical basis of resistance to cold storage, Jouve et al. (1995) have suggested that an important role in cell protection and acclimatization may be played by a three-fold increase in endogenous spermidine content that they observed in wild cherry shoots stored at 10°C for 15 days. 3.2. Encapsulation In preliminary work, the optimum concentration of sodium alginate to be used for preparation of the explant beads was found to be 3%. Complexing 3% sodium alginate with 0.1 M of CaCl, . H,O afforded firm, clear, isodiametric beads suitable for handling (Fig. 2). The experiments showed that the survival percentage and development of encapsulated 5-6 mm shoot tips of C. japonica L. clone 2 improved with increasing the complexity of the matrix giving a similar survival percentage or significantly better propagating rate than those of unencapsulated controls (Table 2). Similar trends were observed with cv. Alba Plena (Fig. 3). In the experiment to evaluate the effect of gel matrix sucrose concentration on the explants, the highest concentration (5%) gave best growth in terms of the number of
Fig. 2. Camellia shoot tips encapsulated CaCl,.H,O. Scale bar = 5 mm.
in 3% sodium
alginate
matrix
after complexation
with 0.1 M
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Table 2 Effect of matrix composition Treatment
Control Alg Alg + MS Alg + MS + Sue Alg+MS+Suc+GR
on subsequent Survival
Horticulturae 71 (1997) 67-78
growth of encapsulated
shoot tips of C. japonica
L. clone 2
(%o)
Number of shoots per explant + standard error
Length of longest shoot f standard error (mm)
1OOb 55.9” 61.1” 72.1a 91.6b ***
2.6+0.2b 1.2+0.2a 1.1 +O.la 2.5 +0.5a.b 4.1 +o.3c ***
15.9 f 10.3 f 14.6& 17.0* 23.8 f ***
1.V.b 0.3” 1.2”,b 1.7b 1.5’
Alg, 3% alginate; Alg + MS, alginate + MS medium; Alg + MS + Sue, alginate + MS medium + 3% sucrose; Alg + MS + Sue + GR: alginate + MS medium + 3% sucrose + growth regulators (4.40 /*M BA + 0.57 PM IAA). Control: unencapsulated shoot tips. Within each column, values followed by the same superscript are not significantly different at the 5% level according to the Tukey HSD test. * * *F-test: significant at P < 0.0001.
shoots developed and the length of the longest shoot, and did not significantly reduce survival frequency (data not shown). The beneficial effects of including adjuvants in encapsulation matrices have also been reported for other species. Pattnaik et al. (1995) achieved efficient plant retrieval when MS mineral salts, 3% sucrose, myo-inositol, BA and GA, were present in the encapsulating axillary buds of three indigenous varieties of mulberry. A complex matrix has also been used in the production of disease-free encapsulated buds of Zingiber oficinale (Sharma et al., 1994). The presence of 5-39% sucrose in the capsule is of great importance for the emergence of shoots from encapsulated axillary buds of Bet&a
Fig. 3. Development of encapsulated 5-6 mm shoot tips of C. japonica L. cv. Alba Plena after 10 weeks in culture medium. The alginate matrix was supplemented with MS medium + 3% sucrose + 8.90 PM BA + 9.12 PM Zeatin + 9.80 PM 2 i.p. + 0.049 PM IBA. Arrows indicate encapsulation beads. Scale bar = 5 mm.
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platyphylla (Kinoshita and Saito, 1990). Absence of sucrose in the matrix inhibited the development of axillary buds of mulberry (Bapat and Rao, 1990). Encapsulated axillary buds of some species such as mulberry (Bapat and Rao, 1990; Pattnaik et al., 1995) can sprout and root when sown directly in soil. Although this would be the most desirable procedure, most published reports on encapsulated material of woody species refer only to growth on an appropriate culture medium, as it occurs in Camellia. Again, attempts to grow encapsulated explants of Japanese white birch (Kinoshita and Saito, 1990), ginger (Sharma et al., 1994) and other woody species (Piccioni and Standardi, 1995) on agar only failed in comparison with the regrowth on basal medium with growth regulators. In accordance with these results and those of the present study, we recommend encapsulating plant material in beads containing a proper basal medium, sucrose and growth regulators. When nodal explants of C. japonica L. were used instead of shoot tips, the results were very poor regardless of clone, cultivar or gel composition. Only 3.4% of encapsulated clone 2 nodes survived, and only 1.4% of encapsulated cv. Alba Plena nodes. These results indicate that only shoot tips should be encapsulated when working with C.
Fig. 4. Influence of the size of encapsulated shoot tips (C. japonica L. clone 2) on subsequent development after . 10 weeks in culture. A, shoot tips l-2 mm; B, shoot tips 5-6 mm. In both cases the alginate matrix was s”Pr llemented with MS medium + 3% sucrose + 4.40 FM BA + 0.57 PM IAA. Scale bar = 5 mm.
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juponica L. The reason for such a difference in response is not clear, but it may be related to mitotic activity being greater in the meristem of the shoot tips than in lateral buds, which are subject to apical dominance. As pointed out by Piccioni and Standardi (19951, axillary buds are quiescent. Another parameter to take into consideration, especially when working with slow growing woody species such as C. juponica L., is the size of explant to be encapsulated. In this work, clone 2 sized l-2 mm had survival rates of about 8 1% as against rates of nearly 100% for 5-6 or lo-11 mm tips regardless of whether they were encapsulated. These shoot tips also responded better than the l-2 mm tips as regards the number of new shoots produced and the length of the longest shoot (P < 0.02 and 0.0002, respectively) (Fig. 4A,B). In most work, no special study of the effect of explant size on regrowth after encapsulation has been carried out. Shoot tips as small as 2-4 mm have been used in cardamon (Ganapathi et al., 1994) and 3 mm for Japanese white birch (Kinoshita and Saito, 1990). The size of the explant is of great importance if the encapsulated material is to be used in cryopreservation programmes (Janeiro et al., 1996). In this case, the smallest explants possible should be used, yet, as we observed in Camellia, a certain minimum size is required for attainment of the highest percentages of explant regrowth. 3.3. Storage of encapsulated
shoot tips
In a preliminary experiment, in which shoot tips of C. juponicu L. clone 2 encapsulated in the most complex matrix were stored for up to 6 months at 2-4”C, only 1% of tips survived for 4 months. In view of these previous results, another experiment was carried out in which encapsulated clone 2 shoot tips were stored at either 2-4°C and 18-20°C for up to 75 days. Survival rate decreased significantly with increasing storage time regardless of storage temperature (Fig. 5). By day 75, only 10% of shoots survived at 2-4°C and 7% at 18-20°C. The number of shoots per explant and the length of the % Survival 100 80 60
30 Days of storage
0
m
Control
&Q
2-4 OC
m
60
15
Room temperature
Fig. 5. Survival rate of encapsulated shoot tips of C. japonica L. clone 2, after 8 weeks of culture in multiplication medium following different periods of storage at 2-4°C and room temperature. Unstored encapsulated shoot tips were used as controls. Bars with the same letter are not significantly different at the 5% level according to the Tukey HSD test.
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longest shoot also decreased, although not to values significantly different from those of the controls (unstored encapsulated tips). Shoot tips of cv. Alba Plena stored in the cold for up to 60 days showed similar trends: after 30 days of cold storage survival had decreased by 50%, and after 60 days only 39.5% of encapsulated shoots survived. The results on cold storage of encapsulated Camellia shoot tips contrast with those for unencapsulated shoots, which survived at 2-4°C for about 6 months (clone 2) or for at least a year (cv. Alba Plena). A similar reduction in allowable storage time for encapsulated explants has been reported for several varieties of mulberry (maximum storage time at 4°C 90 days, Pattnaik et al., 1995) and white birch (40 days, Kinoshita and Saito, 1990). The cause of the reduction in survival time may be that encapsulation gels reduce the availability of oxygen. The simple procedure outlined for in vitro conservation of Camellia at 2-4°C described in this paper appears to be a suitable method for maintaining a repository of selected genotypes, giving rise to an alternative system for germplasm conservation. To reduce labour costs and risks of handling errors, storage for long periods is preferable to storage under normal growth conditions, as storing under dim light will be less energy and space consuming than storing under light.
4. Conclusion Although encapsulated shoot tips of Camellia can be stored at 2-4°C for shorter periods of time than unencapsulated ones, the encapsulation techniques described in Section 2.2 allow new possibilities for handling, transportation and delivery of in vitro tissue cultures of these species.
References Bapat, V.A., Rao, P.S., 1988. Sandalwood plantlets from ‘synthetic seeds’. Plant Cell Rep. 7, 434-436. Bapat, V.A., Rao, P.S., 1990. In vitro growth of encapsulated axillary buds of mulberry (MOWS indica L.). Plant Cell Tissue Organ Cult. 20, 69-70. Bapat, V.A., Mhatre, M., Rao, P.S., 1987. Propagation of Morns indica (mulberry) by encapsulated shoot buds. Plant Cell Rep. 6, 393-395. Chang, H.T., Bartolomew, B., 1984. Camellias. B.T. Badsfort, London. Dorion, N., Godin, B., Bigot, C., 1993. Physiological state and clonal variability effects on low temperature storage of in vitro shoot cultures of elms ((llmus sp.). Sci. Hortic. 56, 51-59. Engelmann, F., 1991. In vitro conservation of tropical plant germplasm-a review. Euphytica 57, 227-243. Ganapathi, T.R., Bapat, V.A., Rao, P.S., 1994. In vitro development of encapsulated shoot tips of cardamon. Biotech. Tech. 8, 239-244. Gray, D.J., Compton, M.E., Harrell, R.C., Cantliffe, D.J., 1995. Somatic embryogenesis and the technology of synthetic seeds, In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry, Vol. 30. Somatic Embryogenesis and Synthetic Seed I. Springer, Berlin, pp. 126-151. Hausman, J.F., Neys, 0.. Kevers, C., Caspar, T., 1994. Effect of in vitro storage at 4°C on survival and proliferation of poplar shoots. Plant Cell Tissue Organ Cult. 38, 65-67. Janeiro, L.V., 1996. Almacenamiento en frio de especies leiiosas propagadas in vitro. Aplicaci6n de las tecnologias de semilla artificial y crioconservackkr en el genera Camellia. Doctoral thesis, University of Santiago de Compostela, Spain.
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Janeiro, L.V., Ballester, A., Vieitez, A.M., 1995a. Effect of cold storage on somatic embryogenesis systems of Camellia. J. Hortic. Sci. 70, 665-672. Janeiro, L.V., Vieitez, A.M., Ballester, A., 1995b. Cold storage of in vitro cultures of wild cherry, chestnut and oak. Ann. Sci. For. 52, 287-293. Janeiro, L.V., Vieitez, A.M., Ballester, A., 1996. Cryopreservation of somatic embryos and embryonic axes of Camellia japonica L. Plant Cell Rep. 15, 699-703. Jouve, L., Fouche, J.G., Gaspar, T., 1995. Early biochemical changes during acclimation of poplar to low temperature. J. Plant Physiol. 147, 247-250. Kinoshita, I., Saito, A., 1990. Propagation of japanese white birch by encapsulated axillaty buds 1. Regeneration of plantlets under aseptic conditions. J. Jpn. For. Sot. 72, 166-170. Monette, P.L., 1987. Organogenesis and plantlet regeneration following in vitro cold storage of kiwifruit shoot tip cultures. Sci. Hortic. 31, 101-106. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473-497. Orlikowska, T., 1992. Effect of in vitro storage at 4°C on surviving and proliferation of two apple rootstocks. Plant Cell Tissue Organ Cult. 3 1, l-7. Pattnaik, S.K., Sahoo, Y., Chand, P.K., 1995. Efficient plant retrieval from alginate-encapsulated vegetative buds of mature mulberry trees. Sci. Hortic. 61, 227-239. Piccioni, E., Standardi, A., 1995. Encapsulation of micropropagated buds of six woody species. Plant Cell Tissue Organ Cult. 42, 221-226. Redenbaugh, K., Fujii, J.A.A., Slade, D., 1993. Hydrated coatings for synthetic seeds. In: Redenbaugh, K. (Ed.), Synseeds: Application of Synthetic Seeds to Crop Improvement. CRC Press, Boca Raton, pp. 35-46. Sanchez, M.C., Vieitez, A.M., 1991. In vitro morphogenetic competence of basal sprouts and crown branches of mature chestnut. Tree Physiol. 8, 59-70. San-Jose, M.C., Vieitez, A.M., 1993. Regeneration of Camellia plantlets from leaf explant cultures by embryogenesis and caulogenesis. Sci. Hortic. 54, 303-315. Sharma, T.R., Singh, B.M., Chanhan, R.S., 1994. Production of disease-free encapsulated buds of Zingiber oficinale Rose. Plant Cell Rep. 13, 300-302. Sokal, R.R., Rohlf, F.J., 1981. Biometry: The Principles and Practice of Statistics and Biological Research, 2nd edn. Freeman, New York, 859 pp. Son, S.H., Chun, Y.W., Hall, R.B., 1991. Cold storage of in vitro cultures of hybrid poplar shoots (Populus alba X P. gandidentata Michx.). Plant Cell Tissue Organ Cult. 27, 161-168. Vieitez, A.M., 1995. Somatic embryogenesis in Camellia spp. In: Jain, S.M., Gupta, P.K., Newton, R.J. (Eds.), Somatic Embryogenesis in Woody Plants, Vol. 2. Angiosperms. Kluwer Academic Publ., Dordrecht, pp. 235-276. Vieitez, A.M., Ballester, A., Vieitez, M.L., Vieitez, E., 1992. Micropropagation of Camellia spp. In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry, Vol. 19. High-Tech and Micropropagation III. Springer, Berlin, pp. 361-387.
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Cold storage of shoot cultures and alginate encapsulation of shoot tips of CumeZZiajuponica L. and Camellia reticulata Lindley A. Ballester
*,
L.V. Janeiro, A.M. Vieitez
Instituto de Inuestigaciones Agrobiokigicas de Galicia, CSIC, Apartado 122, 15080 Santiago de Compostela, Spain Accepted 25 April 1997
Abstract In vitro shoot cultures of 8 clones or cultivars of Camellia japonica L. and Camellia reticulata Lindley were stored at 2-4°C for up to 12 months. Survival frequencies near 100% were obtained during the first or second subcultures after cold storage in seven of the eight clones assayed, but the other clone could not be profitably stored for more than 6 months. When shoot tips and nodal explants of C. japonica L. (clone 2 and cv. Alba Plena) were encapsulated in alginate beads, the regrowth of shoot tips depended on the components of the gel matrix but that of nodal explants was very poor regardless of the encapsulation conditions. The best morphogenetic response of the shoot tips was obtained when the encapsulating gel was supplemented with the minerals and vitamins of Murashige and Skoog’s medium, 3% sucrose and clone-specific growth regulators. The size of the shoot tips used and the concentration of sucrose added to the gel matrix also influenced the capacity for regrowth. Encapsulated shoot tips of C. japonica L. stored at 2-4°C or 18-20°C survived for shorter periods of time than unencapsulated shoot tips stored at low temperature: after 75 days of storage, only 10% of encapsulated shoot tips survived at 2-4°C and 7% at 18-20°C. 0 1997 Elsevier Science B.V. Keywords: Camellia japonica L.; Camellia reticulata Lindley; Cold: Encapsulation;
In vitro culture; Storage
acid; IBA, indole-3Abbreviations: BA, 6benzyladenine; GA s , g ibberellic acid; IAA, indole-3-acetic butyric acid; Zip, 6-dimethylallylamino-purine; MS, Murashige and Skoog medium * Corresponding author. Tel.: +34 81 590 958; fax: f34 81 592 504; e-mail: [email protected]. 0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(97)00074-5
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1. Introduction CumeZlia (Theales, Theaceae) is a genus of tropical and subtropical trees and shrubs native to Eastern Asia. Chang and Bartolomew (1984) have carried out a major taxonomic revision, grouping some 200 recognized species in four subgenera and 20 sections. Increasing interest in ornamental camellias (mainly Camellia juponicu L., Camellia reticuluta Lindley, Camellia susanqua and Camellia suluenensis) has led to continuous efforts by researchers, growers and breeders to produce new varieties and hybrids with improved floral and growth characteristics, and to develop more efficient methods for their propagation. Protocols for micropropagation from shoot tips and nodal explants of both juvenile and mature ornamental camellias have been reported (Vieitez et al., 1992) as has the generation of adventitious shoots from leaves of C. reticulutu Lindley (San-Jose and Vieitez, 1993). The induction and development of somatic embryos of several Camellia species has recently been reviewed (Vieitez, 1995). Furthermore, such protocols are in principle applicable for ornamental camellias and tea plants not only to clonal propagation but also to artificial seed production. This has dramatically increased in recent years (Redenbaugh et al., 1993; Gray et al., 1995) leading to the establishment of gene banks for medium- or long-term storage. The low-temperature storage of germplasm produced in vitro is now a well documented alternative for the preservation of a variety of plant species (Monette, 1987; Orlikowska, 1992; Janeiro et al., 1995b). The major advantages of this approach are reduced labour and space requirements, the elimination of pathogen-related problems and the reduction of genetic erosion if optimal storage conditions are achieved (Engelmann, 1991). I n Cumellia, the effect of cold storage on embryogenic cultures of C. japonicu L. and C. reticulutu Lindley has been reported (Janeiro et al., 1995a), as have the results of attempts at long-term cryopreservation of both somatic embryos and embryonic axes of C. japonicu L. (Janeiro et al., 1996). There has been little research on the effect of encapsulation (the technology of coating somatic embryos or shoot tips in alginate beads) on the response of plant material to cold storage (Bapat et al., 1987; Bapat and Rao, 1988). Encapsulation is an aid for storage (smaller space is needed in relation to the storage of conventional in vitro cultures), but also it is important for handling and exchange of in vitro material. There is no information on the possibility of medium-term storage of either encapsulated or unencapsulated shoot tips of C. juponica L. and C. reticduta Lindley at low temperature. The objectives of this study were: (1) to study the survival and proliferation of several clones of C. japonica L. and C. reticulutu Lindley after storage of unencapsulated shoot cultures at 2-4°C for several months; (2) to study the factors affecting the encapsulation and subsequent development of shoot tips and nodal segments isolated from in vitro shoot cultures of C. juponicu L.; (3) the effect of storage on encapsulated material.
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2. Materials and methods
2.1. Cold storage of shoot cultures Shoot cultures from the species, clones or cultivars listed below were multiplied by subculturing shoot tips and nodal segments (1 cm in length) at 8-week intervals (for specific multiplication media see Janeiro, 1996). Thirty days after the last subculture or 10 days for clone 2 of C. japonica L., the 500 ml glass jars (with glass lids fixed with plastic film) containing the shoot cultures were transferred from standard conditions to cold storage. They were kept at 2-4°C in cool cabinets (340-l Sanyo Medico01 MPR 311D) with 16 h day-’ of dim light (8 pmol me2 s- I> for 3, 6, 9 or 12 months (C. japonica L. clone 2 and cv. Alba Plena), 10 months (C. japonica L. clone 1) or 12 months (C. japonica L. cv. Fimbriata Alba and C. reticulata Lindley cv. Mouchang clones 1, 3, 10 and 222). After this period, the cultures were transferred to fresh multiplication medium and kept in a growth chamber under standard growth conditions together with controls (0 month in the cold). After 8 weeks, these shoots (subculture 1) were evaluated as described below and were subcultured to produce subculture 2 which was evaluated after a further 8 weeks. For both subcultures the following parameters were evaluated: survival rate (the percentage of cultures capable of proliferating), the number of new shoots per responsive explant, the length of the longest shoot, and the multiplication coefficient, defined as the product of the proportion of explants surviving with shoot development and the mean number of 8-10 mm segments per explant (Sgnchez and Vieitez, 1991). Twelve replicate jars (containing 80 ml of multiplication medium), with 10 explants per jar, were used for each treatment. Growth conditions were provided by a growth chamber with 30 pmol rnp2 s-’ of light from Osram L4OW cool white fluorescent lamps for 16 h day-‘, with day and night temperatures of 24°C and 2O”C, respectively. 2.2. Encapsulation Shoot tips and nodal segments 5-6 mm in length were isolated at the end of a multiplication cycle from in vitro shoot cultures of C. japonica L. clone 2 and cv. Alba Plena. Tips and nodes were individually encapsulated in alginate beads by transferring them one by one with a Pasteur pipette from a 3% solution of sodium alginate in distilled water to one of the following solutions: (a) 0.1 M CaCl, .2H,O (calcium solution); (b) calcium solution with Ca-free MS medium (Murashige and Skoog, 19621, including MS vitamins; (c) calcium solution with Ca-free MS medium and 3% sucrose; (d) calcium solution with Ca-free MS medium and 3% sucrose and the growth regulators of the multiplication medium (4.40 PM BA + 0.57 FM IAA for clone 2, or 8.90 PM BA + 9.12 PM Zeatin + 9.80 /IM 2 ip + 0.049 PM IBA for cv. Alba Plena). The beads were left in the calcium solution for half an hour and then inoculated in multiplication medium, where, in accordance with conventional protocol for the multiplication of these Camellia clones, they spent 4 weeks followed by a further 4 weeks in fresh multiplication medium. At the end of this time, the survival percentage, the number of shoots per
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responsive explant and the length of the longest shoot per explant were recorded. Unencapsulated explants were used as controls. In other experiments, the effects of the size of explant were investigated using shoot tips of C. japonica L. clone 2 sized l-2 mm, 5-6 mm or lo- 11 mm, encapsulated in solution (d); and the effect of the concentration of sucrose in the encapsulating matrix were studied using 5-6 mm shoot tips of the same clone encapsulating by means of Ca solutions identical to solution (d) except that sucrose concentrations of 1.5%, 3% and 5% were employed. Twelve replicate jars (300 ml, containing 50 ml of multiplication medium), with 10 beads per jar, were used for each treatment. 2.3. Storage of encapsulated
shoot tips
Shoot tips of C. japonica L. clone 2 and cv. Alba Plena were encapsulated in beads containing 3% calcium alginate + MS + 5% sucrose + growth regulators (specific for each clone, see Section 2.2) and were then kept in sterile Petri dishes, 20 to a dish, and stored at 2-4°C or 18-20°C for 30, 60 and 75 days (clone 2) or 30 and 60 days (cv. Alba Plena). After storage, the beads were inoculated in 300 ml glass jars containing 50 ml of multiplication medium and transferred to fresh medium after 4 weeks. Unstored encapsulated explants were used as controls. At the end of the multiplication stage, the same parameters as in the experiments on the storage of unencapsulated material were recorded. Twelve replicate jars with 10 beads per jar were used for each treatment. 2.4. Statistical analysis In all experiments, treatments were arranged in a completely randomized design with subsampling and the data were statistically analyzed by one- or two-way analysis of variance (percentage data were first subjected to arcsine square root transformation), using the least significant difference (LSD) or the Tukey HSD test (Sokal and Rohlf, 1981) at the P = 0.05 level to compare means.
3. Results and discussion 3.1. Cold storage of shoot cultures In vitro shoot cultures of C. juponicu L. clone 2 were less resistant to the cold than those of cv. Alba Plena (Table 1) after more than 6 months at 2-4°C. Whereas Alba Plena had a 100% survival and unaltered morphogenetic parameters (the number of shoots produced and the length of the longest shoot were affected by subculture number, but without any clear trend), the survival percentage, number of shoots per explant and longest shoot length of clone 2 decreased significantly to very low levels. In particular, the damage caused in clone 2 by 9 months or more in the cold failed to form multiple shoots in subculture 2. In this clone necrosis was observed to spread down from the shoot tip as time elapsed.
Control 1 2 1 2 1 2 1 2
0
“b’cSignificant
F-test 104.9’ 14.lb 5.5b
LSD
13.8 8.7 6.1
F-test
156.8’ 12.2s 5.5b
P < 0.0001, respectively;
N
1.3 11.1 0.6
LSD
2.6f0.7 2.8+ 1.4 3.7+ 1.2 2.7kO.3 2.4 + 0.4 2.8kO.l 2.650.5 3.6k 1.1 3.1*0.9
50.3b 0.8b 2.lns
F-test
L
cv. Alba Plena
ns = not significant.
7.9k2.1 5.5 * 0.9 8.4 * 2.5 1.5f0.2 3.3* 1.1 1.0+0.0 1.0*0.0 1.0+0.0 1.0+0.0
Survival
Clone 2
100 100 100 100 100 100 100 100 100
cv. Alba Plena
Clone 2
100 90.6 100 68.8 39.6 29.1 1.2 14.1 1.5
(o/o)
10.5 11.1 _
LSD
37.9 + 5.9 48.1 + 3.4 58.6 + 3.2 28.9k6.1 30.3 + 1.9 20.1 k4.5 10.2* 1.3 10.2 + 2.3 10.1 +2.4
Clone 2
2.9” 0.9ns 1.3ns
F-test
N
cv. Alba Plena
68.9 i 5.4 58.9)7.3 51.3i5.4 63.2+9.1 63.5 +4.3 58.6 f 3.5 49.2 f 2.9 51.4k6.2 57.8 rf- 8.3
cv. Alba Plena
Length of longest shoot k standard error (mm)
-
_
0.7
LSD
7.9 4.9 8.4 0.8 1.3 0.3 0.07 0.2 0.07
Clone 2
9.1 -
_
LSD 4. lb l.lns 2.4ns
2.6 2.8 3.7 2.7 2.4 2.8 2.6 3.6 3.1
cv. Alba Plena
subcultures
F-test
L
Multiplication coefficient
of C. japonicu L. clone 2 and cv. Alba Plena in two successive
Clone 2
parameters Number of shoots per explant f standard error
on survival and morphogenetic
Survival
at P < 0.01, P < 0.001,
Subculture (A) Cold storage (B) AXB
Source of variation
Analvsis of Variance
12
9
6
3
Subculture
Months in cold
Table 1 Effect of cold storage (2-4°C)
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In a similar experiment in which other clones and cultivars of C. japonicu L. and C. reticulutu Lindley spent 12 months in cold storage (10 months in the case of C. juponicu L. clone l), survival percentage in subculture 1 ranged from 100% in cv. Fimbriata Alba and C. reticulutu Lindley clones 1 and 3 to 16% in C. juponicu L. clone 1. In subculture 2, however, all clones had 100% survival except C. juponicu L. clone 1, and even this clone had a survival percentage of 80% (Fig. 1). Moreover, the number of shoots per explant after 2 subcultures was close to that of control cultures (data not shown). These results show that the detrimental effects of cold storage were transitory in these clones. In all clones, surviving cultures of subculture 2 exhibited no carry-over effect of cold storage, multiplying and rooting at levels similar to those of controls in subsequent subcultures (data not shown). The results obtained in these experiments indicate that in vitro cultures of ornamental camellias are tolerant to cold storage but the degree of tolerance varies between clone or cultivar. In a germplasm bank, no general protocol can be applied, C. juponicu L. clone 2 would have to be subcultured at least every 6 months, while other clones could be left in the cold without subculture for a year or more. The causes of this differential behaviour are not known, but one may be the consistency of the shoots: clone 2 leaves were thinner and apparently less cutinized than those of other clones. One of the most important factors that can influence the success of cold storage (4°C) is the physiological state of the explants at the time of being placed in the cold
% Survival
100 90 80
70 60 50 40 30
SUbcUll
10
ture 2
culture
0 CjF
Cjl
03
CrlO Crl
Cr222
Fig. 1. Survival percentage of different Camellia clones in two successive subcultures after a prior 12.month period in cold storage. CjF, C. japonica L. cv. Fimbriata Alba; Cjl, C. japonica L. clone 1; Cr3, CrlO, Crl and Cr222, C. reticulata Lindley clones 3, 10, 1 and 222, respectively. Bars with the same letter are not significantly different at the 5% level according to the Tukey HSD test.
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(Orlikowska, 1992). The need for explants to recover from cold storage prior to the stress brought about by subculture has been shown for hybrid Populus (Son et al., 1991), Custunea and Quercus (Janeiro et al., 1995b) and Ulmus (Dorion et al., 1993), all these genera, like Camellia, also exhibit clonal variability in their response to cold storage. A possible exception to this rule is that of Populus tremulu (L.) X Populus tremuloides (L.) cv. Muhs 1, shoot tips stored at 10°C immediately after isolation had a 100% survival after 3 months storage (Hausman et al., 1994). On the basis of our previous experience, in this work Camellia cultures spent some time under standard culture conditions between the last subculture and the start of cold storage (10 or 30 days, depending on clone). As regards the biochemical basis of resistance to cold storage, Jouve et al. (1995) have suggested that an important role in cell protection and acclimatization may be played by a three-fold increase in endogenous spermidine content that they observed in wild cherry shoots stored at 10°C for 15 days. 3.2. Encapsulation In preliminary work, the optimum concentration of sodium alginate to be used for preparation of the explant beads was found to be 3%. Complexing 3% sodium alginate with 0.1 M of CaCl, . H,O afforded firm, clear, isodiametric beads suitable for handling (Fig. 2). The experiments showed that the survival percentage and development of encapsulated 5-6 mm shoot tips of C. japonica L. clone 2 improved with increasing the complexity of the matrix giving a similar survival percentage or significantly better propagating rate than those of unencapsulated controls (Table 2). Similar trends were observed with cv. Alba Plena (Fig. 3). In the experiment to evaluate the effect of gel matrix sucrose concentration on the explants, the highest concentration (5%) gave best growth in terms of the number of
Fig. 2. Camellia shoot tips encapsulated CaCl,.H,O. Scale bar = 5 mm.
in 3% sodium
alginate
matrix
after complexation
with 0.1 M
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Table 2 Effect of matrix composition Treatment
Control Alg Alg + MS Alg + MS + Sue Alg+MS+Suc+GR
on subsequent Survival
Horticulturae 71 (1997) 67-78
growth of encapsulated
shoot tips of C. japonica
L. clone 2
(%o)
Number of shoots per explant + standard error
Length of longest shoot f standard error (mm)
1OOb 55.9” 61.1” 72.1a 91.6b ***
2.6+0.2b 1.2+0.2a 1.1 +O.la 2.5 +0.5a.b 4.1 +o.3c ***
15.9 f 10.3 f 14.6& 17.0* 23.8 f ***
1.V.b 0.3” 1.2”,b 1.7b 1.5’
Alg, 3% alginate; Alg + MS, alginate + MS medium; Alg + MS + Sue, alginate + MS medium + 3% sucrose; Alg + MS + Sue + GR: alginate + MS medium + 3% sucrose + growth regulators (4.40 /*M BA + 0.57 PM IAA). Control: unencapsulated shoot tips. Within each column, values followed by the same superscript are not significantly different at the 5% level according to the Tukey HSD test. * * *F-test: significant at P < 0.0001.
shoots developed and the length of the longest shoot, and did not significantly reduce survival frequency (data not shown). The beneficial effects of including adjuvants in encapsulation matrices have also been reported for other species. Pattnaik et al. (1995) achieved efficient plant retrieval when MS mineral salts, 3% sucrose, myo-inositol, BA and GA, were present in the encapsulating axillary buds of three indigenous varieties of mulberry. A complex matrix has also been used in the production of disease-free encapsulated buds of Zingiber oficinale (Sharma et al., 1994). The presence of 5-39% sucrose in the capsule is of great importance for the emergence of shoots from encapsulated axillary buds of Bet&a
Fig. 3. Development of encapsulated 5-6 mm shoot tips of C. japonica L. cv. Alba Plena after 10 weeks in culture medium. The alginate matrix was supplemented with MS medium + 3% sucrose + 8.90 PM BA + 9.12 PM Zeatin + 9.80 PM 2 i.p. + 0.049 PM IBA. Arrows indicate encapsulation beads. Scale bar = 5 mm.
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15
platyphylla (Kinoshita and Saito, 1990). Absence of sucrose in the matrix inhibited the development of axillary buds of mulberry (Bapat and Rao, 1990). Encapsulated axillary buds of some species such as mulberry (Bapat and Rao, 1990; Pattnaik et al., 1995) can sprout and root when sown directly in soil. Although this would be the most desirable procedure, most published reports on encapsulated material of woody species refer only to growth on an appropriate culture medium, as it occurs in Camellia. Again, attempts to grow encapsulated explants of Japanese white birch (Kinoshita and Saito, 1990), ginger (Sharma et al., 1994) and other woody species (Piccioni and Standardi, 1995) on agar only failed in comparison with the regrowth on basal medium with growth regulators. In accordance with these results and those of the present study, we recommend encapsulating plant material in beads containing a proper basal medium, sucrose and growth regulators. When nodal explants of C. japonica L. were used instead of shoot tips, the results were very poor regardless of clone, cultivar or gel composition. Only 3.4% of encapsulated clone 2 nodes survived, and only 1.4% of encapsulated cv. Alba Plena nodes. These results indicate that only shoot tips should be encapsulated when working with C.
Fig. 4. Influence of the size of encapsulated shoot tips (C. japonica L. clone 2) on subsequent development after . 10 weeks in culture. A, shoot tips l-2 mm; B, shoot tips 5-6 mm. In both cases the alginate matrix was s”Pr llemented with MS medium + 3% sucrose + 4.40 FM BA + 0.57 PM IAA. Scale bar = 5 mm.
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Horticulturae 71 (1997) 67-78
juponica L. The reason for such a difference in response is not clear, but it may be related to mitotic activity being greater in the meristem of the shoot tips than in lateral buds, which are subject to apical dominance. As pointed out by Piccioni and Standardi (19951, axillary buds are quiescent. Another parameter to take into consideration, especially when working with slow growing woody species such as C. juponica L., is the size of explant to be encapsulated. In this work, clone 2 sized l-2 mm had survival rates of about 8 1% as against rates of nearly 100% for 5-6 or lo-11 mm tips regardless of whether they were encapsulated. These shoot tips also responded better than the l-2 mm tips as regards the number of new shoots produced and the length of the longest shoot (P < 0.02 and 0.0002, respectively) (Fig. 4A,B). In most work, no special study of the effect of explant size on regrowth after encapsulation has been carried out. Shoot tips as small as 2-4 mm have been used in cardamon (Ganapathi et al., 1994) and 3 mm for Japanese white birch (Kinoshita and Saito, 1990). The size of the explant is of great importance if the encapsulated material is to be used in cryopreservation programmes (Janeiro et al., 1996). In this case, the smallest explants possible should be used, yet, as we observed in Camellia, a certain minimum size is required for attainment of the highest percentages of explant regrowth. 3.3. Storage of encapsulated
shoot tips
In a preliminary experiment, in which shoot tips of C. juponicu L. clone 2 encapsulated in the most complex matrix were stored for up to 6 months at 2-4”C, only 1% of tips survived for 4 months. In view of these previous results, another experiment was carried out in which encapsulated clone 2 shoot tips were stored at either 2-4°C and 18-20°C for up to 75 days. Survival rate decreased significantly with increasing storage time regardless of storage temperature (Fig. 5). By day 75, only 10% of shoots survived at 2-4°C and 7% at 18-20°C. The number of shoots per explant and the length of the % Survival 100 80 60
30 Days of storage
0
m
Control
&Q
2-4 OC
m
60
15
Room temperature
Fig. 5. Survival rate of encapsulated shoot tips of C. japonica L. clone 2, after 8 weeks of culture in multiplication medium following different periods of storage at 2-4°C and room temperature. Unstored encapsulated shoot tips were used as controls. Bars with the same letter are not significantly different at the 5% level according to the Tukey HSD test.
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longest shoot also decreased, although not to values significantly different from those of the controls (unstored encapsulated tips). Shoot tips of cv. Alba Plena stored in the cold for up to 60 days showed similar trends: after 30 days of cold storage survival had decreased by 50%, and after 60 days only 39.5% of encapsulated shoots survived. The results on cold storage of encapsulated Camellia shoot tips contrast with those for unencapsulated shoots, which survived at 2-4°C for about 6 months (clone 2) or for at least a year (cv. Alba Plena). A similar reduction in allowable storage time for encapsulated explants has been reported for several varieties of mulberry (maximum storage time at 4°C 90 days, Pattnaik et al., 1995) and white birch (40 days, Kinoshita and Saito, 1990). The cause of the reduction in survival time may be that encapsulation gels reduce the availability of oxygen. The simple procedure outlined for in vitro conservation of Camellia at 2-4°C described in this paper appears to be a suitable method for maintaining a repository of selected genotypes, giving rise to an alternative system for germplasm conservation. To reduce labour costs and risks of handling errors, storage for long periods is preferable to storage under normal growth conditions, as storing under dim light will be less energy and space consuming than storing under light.
4. Conclusion Although encapsulated shoot tips of Camellia can be stored at 2-4°C for shorter periods of time than unencapsulated ones, the encapsulation techniques described in Section 2.2 allow new possibilities for handling, transportation and delivery of in vitro tissue cultures of these species.
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