Euphytica 122: 269–277, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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In vitro screening of strawberry plants for cold resistance Rytis Rugienius & Vidmantas Stanys Lithuanian Institute of Horticulture, 4335 Babtai, Kaunas district, Lithuania Received 31 August 1999; accepted 30 January 2001
Key words: cold resistance, in vitro screening, Fragaria × ananassa, strawberry
Summary Formation of embryo autonomy of strawberry, plant regeneration from embryo components, plant freezing conditions in vitro and the possibility to differentiate objectively genotypes by freezing them in vitro and in vivo were studied to create strawberry screening technology in vitro for cold resistance. It was established that autonomy of strawberry embryos manifests itself not earlier than on 14–16th day after pollination and full autonomy is reached on 20–22nd day. Plants regenerated from 26 days old embryos grew most intensively. At the highest rate strawberry plants regenerated from an isolated embryo axis on MS medium without phytohormones, and from rescued cotyledons – on the medium with 1.0 BA and 0.5 NAA. The temperature interval, at which genotypes differentiated according to cold resistance in vitro, was –8 to 12 ◦ C. Differentiation of strawberry genotypes according to this character conformed to their differentiation in vivo, provided hardening proceeded not less than 21 days. The correlation between cold resistance in vitro and in vivo reached 0.93. Domination of cold resistance manifested itself in strawberry seedlings from various crossing combinations. Abbreviations: BA – 6-benzylaminopurine; IBA – indole-3-butyric acid; NAA – 1-naphthaleneacetic acid; GA3 – gibberellic acid; MS – Murashige & Skoog medium
Introduction Insufficient winterhardiness is one of the major constrains limiting strawberry production in moderate climates. Low temperature at the beginning of winter when snow cover is unstable and frequent thawings and freezing in the middle of winter cause considerable yield losses in the following growing season. Following unfavorable winter conditions productivity may decrease by as much as 40% (Nestby, 1997; Dalman & Matala, 1997). Strawberry varieties differ in winterhardiness. As yet there is no agreement on the heritability of cold resistance and winterhardiness. Most researchers believe the character is determined by a large group of genes (Scott & Lawrence, 1975; Zubov, 1990). Filipenko (1996) asserts that the whole genome determines winterhardiness. It has been established that high winterhardiness is partially dominant and is inherited independently of other economic characters, thus allowing combination of winterhardiness
with other valuable economic properties such as productivity, berry size and quality, and disease and pest resistance. One of the most important components of winterhardiness is cold resistance. Assessment of strawberry resistance to cold in the field takes several years due to alternating overwintering conditions. In vitro screening saves time and space since only screened seedlings or their clones are transplanted into the field. It is important that the cold resistant plants selected in vitro should be resistant in vivo as well. In spite of all the advantages of screening in vitro, there have not been many successful trials on the selection of cold resistant plants in vitro. Cold resistance of various raspberry varieties and seedlings is assessed reliably in vitro only when hardened plants are used. Hardening for two weeks at +15 and +2 ◦ C lowered the temperature at which plants of a resistant variety could survive in vitro, by as much as 5 ◦ C (Zatylny et al., 1993). According to the data of Caswell et al. (1986)
270 and Dix et al. (1994) sucrose increased cold resistance of plants. Palonen & Buszard (1997) investigated cold resistance of strawberry varieties. They established the dependence of cold resistance of strawberry in vitro with phytohormones in the nutritive medium. However, they failed to report correspondence of resistance in vitro and in vivo of varieties. Hitherto conditions have not been determined to allow objective differentiation of strawberry genotypes according to cold resistance in vitro. Nor has an effective screening system in vitro been developed yet for cold resistant strawberries. The advantage of in vitro methods in breeding is supplemented by the possibility of obtaining plants from isolated, even immature, embryos or their parts (Miller et al., 1993; Stanys, 1997). After investigations of the dynamics of embryo development, the rise of autonomy and the optimization of plant regeneration, it would be possible to speed up seedling production by saving the time for seed stratification. The aim of our investigation is to establish optimal conditions for differentiation of strawberry genotypes according to cold response and to create a screening technology in vitro for cold resistant strawberry seedlings.
Materials and methods In the experiments the following varieties of strawberry (Fragaria × ananassa Duch.) were used, differing in cold resistance: Venta, Lvovskaja Raniaja, Pegasus, Holiday, Kama and Redgauntlet, their seedlings following open pollination and seedlings from 4 cross combinations: Kent × Nida, Nida × Holiday, Kent × Holiday and Holiday × Marmolada. To investigate the dynamics of development and the rise of autonomy, embryos were rescued from 30–50 free pollinated seeds of Venta and Redgauntlet varieties every second day starting on 8th and finishing on 30th day after flower pollination and cultured in tubes with Murashige & Skoog (1962) medium supplemented with 3% sucrose. Variants of this medium were supplemented with phytohormones (mg/l): 0.2 kinetin; 1.0 gibberellic acid; 1.0 BA and 1.5 IBA. The control medium was without phytohormones. For regeneration of strawberry plants, freely pollinated achenes of Venta and Redgauntlet were cut into two parts under a binocular microscope (16x) in such a way that an embryo axis would remain in one part, and cotyledons in another part. Both parts were established
in the medium. Variants of MS and White (1943) media were supplemented with hormones (mg/l) BA 0.4–3.3; IBA 0.5–1.5; NAA 0.1–0.5; gibberellic acid 1.0. For each variant, 20–60 seeds of each variety were taken. The control was produced by the conventional method of breeding: seeds stratified at 0 – +5 ◦ C for 3 months and germinated in Petri dishes for 1 month (Zubov, 1990). For in vitro investigation of cold resistance of strawberry varieties and seedlings, micro plants and proliferating structures were hardened for 7, 21 and 35 days at 2 ± 1 ◦ C. They were then frozen in vitro for 12 hours at –8, –9, –10, –11, –12, –13 ◦ C in MS medium, which was supplemented with 1.5, 3, 6, and 9% sucrose. Explants were thawed over 12 hours at +15 ◦ C. For in vivo investigation of cold response of strawberry varieties, plants of varieties Venta and Holiday and seedlings of 4 crossing combinations were frozen at –12 and –14 ◦ C for 12 hours. After freezing, boxes with plants were transferred to a greenhouse, in which the temperature was +12 – +20 ◦ C. The proportion (%) of plants remaining viable in vitro was established after 30 days and the lengths of plants and their roots were measured. Cold injuries were recorded according to the scale: 0 – without injury, 1 – injured up to 10% of plant tissues, 2 – injured 10 - 30%, 3 – injured 30–50%, 4 – more than 50% injury, 5 – dead. Data were analyzed by variation and dispersal analysis methods and grouped by the Duncan test p ≤0.05 (Tarakanovas, 1996; Volf, 1966).
Results Embryo autonomy formation During the investigation it was discovered that differentiation of a strawberry embryo started on the 10th day after pollination and stopped on the 18th day when the achene reached its maximum size (1.7–2 mm). At that time the embryo length was 0.2 mm (Figure 1). These were youngest and smallest embryos that developed in vitro. Thus, embryo autonomy begins at this moment. Rapid growth of the embryo was observed from the 16th day after pollination. Over 2 days (from 16th till 18th day after pollination) embryo length increased by more than 4 times. Later its growth slowed down. By the 26th day after flower pollination embryos had reached their maximum and stable length
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Figure 1. Dynamics of in vitro embryo growth and development rate isolated embryos of the strawberry variety Venta.
(1.5 mm). Longer embryos produced a higher percentage of developing in vitro isolated embryos. When embryos were isolated on the 18th day, 60% of them developed, on the 20th day the comparative value was 93%. From the 22nd day the maximum percentage of rescued embryos developed. Thus, embryos became fully autonomous at that time. Later than 90 days after pollination, i.e. when a seed is in dormancy, a significantly smaller proportion of embryos developed (83%). During the investigation a positive correlation was established between embryo age and its development in vitro. The later embryos were isolated the faster they developed in vitro, and plants regenerated from them grew faster and were more differentiated (Figure 2). Plants regenerated from embryos isolated no earlier than on the 26th day after pollination reached the greatest length. Plants regenerated from young embryos, rescued on the 14 and 16th days after pollination, achieved only 10–30 per cent of the maximum length and were still without rootlets after 30 days of isolation. Plants formed roots provided they were regenerated from rescued embryos that reach least 18 days old. The shoots of plants which were regenerated from embryos of dormant seeds (90 days after flower pollination), did not differ significantly from those of plants
regenerated from 26–30 days old embryos. However, in the former plants, rootlets were longer. Plant regeneration from embryo parts During cultivation of isolated embryo components in vitro, the development of an explant and the rate of plant regeneration did not coincide. An embryo axis regenerated plants at the highest rate in the medium without phytohormones (Figure 3). In that medium nearly all explants regenerated plants. Meanwhile, a greater number of embryo axes developed in the medium with 0.2 mg/l kinetin but plant regeneration rate was lower than in the control medium. Cytokinins, particularly higher concentrations, inhibited embryo development and plant regeneration. Auxins did not have a decisive effect on regeneration rate. According to the rate of the development of embryo axes and plant regeneration, the medium with 1.0 mg/l GA3 was almost as good as the control medium. GA3 addition promoted higher shooting up of epocotyl, and that effected plant viability in further subcultivations and freezing in vitro. In contrast to the cultivation of embryo axes, supplements of exogenic phytohormones were indispensable for plant regeneration from cotyledons and even then, fewer explants regenerated plants (Figure 4). The highest proportion of regenerants (53%) was obtained
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Figure 2. Effect of age of embryo isolation on growth of segments after 30 days of culture.
Figure 3. Regeneration of strawberry plants from embryo axes of open pollinated seeds of Venta depending on phytohormone composition in MS medium.
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Figure 4. Strawberry regeneration from isolated cotyledons of open pollinated seedlings from the variety Venta.
in the medium supplemented with 1.0 mg/l BA and 0.5 mg/l NAA; the higher the level of cytokinins the lower the output of regenerants. When regenerating plants from cotyledons, proliferating structures were formed, from which several plants differentiated later. Selection of conditions for screening of cold resistant plants in vitro Relative survival rates of various strawberry genotypes following in vitro freezing are shown in Figure 5. After freezing at –8 ◦ C, plants (90%) survived, and after freezing at –12 ◦ C more than 90% of plants died. Maximum differences between genotypes occurred at temperatures –9–11 ◦ C where, depending on the variety, between 20 and 100% of plants survived. It was established that cold resistance of strawberry plants depended on hardening duration (Figure 6). The longer the period of hardening the more cold resistant they became. The plants were most susceptible when hardened for only 7 days, when the survival rate was 11.1–37.5% depending on the variety. The plants were most resistant when they had been hardened for
35 days (53.3–100% survival). The effectiveness of hardening depended on the variety. Longer hardening (21 or 35 days) increased the viability of Venta plants in vitro more than in any other variety tested. Under optimal conditions for strawberry discrimination according to cold resistance (hardening 21 days, freezing at –10 ◦ C, 6% sucrose in the medium, thawing for 12 h at +15 ◦ C) the correlation between strawberry cold resistance in vitro and in vivo reached 0.93. Efficiency comparison of in vitro and in vivo screening of strawberry seedlings By establishing optimal conditions for discrimination of strawberry genotypes according to cold resistance it was possible to select cold resistant seedlings. During freezing in vitro it was observed that the proportion of seedlings that were resistant or viable after freezing depended upon the cross combination, ranging from 53.3% (Holiday × Marmolada) to 73.6% (Kent × Nida), and the proportion without cold injury was 5.0 and 13.8%, respectively. During freezing under con-
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Figure 5. Percentage of viable plants of several strawberry varieties after freezing in vitro depending on freezing temperature.
trolled conditions in vivo 90 and 65% of seedlings from these combinations survived, and 2.3 and 14.4%, respectively, were without cold injury (Figure 7). Correlation between cold resistance in vitro and in vivo reached 0.89. The winter of 1997–1998 was unfavorable for overwintering strawberries due to low temperatures and thin snow cover in December. This allowed reliable discrimination between strawberry seedlings according to their winterhardiness and enabled comparison of cold resistance in vitro with plant resistance under natural conditions. In that winter the proportion of seedlings without cold injury ranged from 5.3% (Holiday × Marmolada) to 19.6% (Kent × Nida) (Figure 7). The correlation between cold resistance in vitro and winterhardiness in situ reached 0.78.
Discussion In this work the autonomy of a strawberry embryo was apparent after termination of seed growth and at the final stage of embryo differentiation. The maximal autonomy was reached within two-thirds of the
period, from pollination to full ripening of the berries. In practical breeding it is known that strawberry seeds have highest germination levels when rescued from fully ripened berries (not sooner than 30 days after pollination). The germination rate is usually around 60% and achieved after stratification for 3–4 months (Zubov, 1990). Under in vitro conditions, 20 days after pollination, all isolated embryos develop and regenerate plants at a rate of almost 100%. Therefore, the method of isolating embryos not only reduces the term of seedling production but increases the output as well. Plants, regenerated from older embryos, grew faster. The same patterns were observed when growing black currant and actinide plants from embryos of various maturities (Stanys, 1997). Apparently this is characteristic of most plant species. Full embryo autonomy does not determine the maximum growth of a regenerant. This can be explained by the fact that after embryo differentiation, a period of synthesis starts, during which various components are synthesized in a seed (including long-term iRNA), that are indispensable for normal seed germination and plant growth (Walbot, 1978). After this period an embryo enters dormancy, which is bound up with
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Figure 6. Cold resistance of different strawberry varieties depending on duration of hardening.
accumulation of inhibitors in the coat and the embryo itself. Results showed that plants could be successfully regenerated not only from the entire embryo but from its parts as well: an embryo axis and cotyledons. There is no agreement in the literature on whether exogenic phytohormones are necessary for seed germination or for the induction of the development of isolated embryos. There are reports (Wang et al., 1984; Sayegh & Hennerty, 1989) that germination of strawberry seeds, especially of immature ones, could be induced by the supplements of gibberallin or even cytokinins and auxins. Our investigation corroborated with Erb et al. (1989) statement that phytohormone addition is not efficient for induction of embryo development and plant regeneration. Inactivity of exogenic phytohormones for regeneration of plants from embryo axes could be explained by the fact that embryo axes contain an adequate amount of growth regulators enabling explant’s development. In contrast to embryo axes, plants from cotyledons regenerated in morphogenesis de novo. For embryoid formation and growth exogenic cytokinins and auxins
were needed. Miller et al. (1992) showed that a supplement of cytokinin BA alone did not induce but inhibited plant regeneration from cotyledons and growth in further subcultivations. Apparently, BA induced excessive formation of embryoids, which adversely affected the translocation of nutrients for growth and further development. Thus, in order to obtain the maximum rate of regenerants from isolated cotyledons, different phytohormones and combinations of them should be selected for separate regeneration stages. Our investigation proves the possibility of producing plants from isolated cotyledons at the rate of viable seeds. By regenerating from cotyledons we usually obtain not one plant but a group – proliferating structure, therefore, plant propagation and clone formation are facilitated. Thus, by using isolated embryos or their components it is possible to shorten plant development period to 55 days after pollination and to achieve it at a higher rate to produce strawberry population suitable for in vitro screening for cold resistant genotypes. Cold resistance of different strawberry cultivars manifests itself objectively in a very narrow interval
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Figure 7. Cold resistance of strawberry seedlings in vitro, in vivo and winterhardiness in situ depending on cross combination. Correlation between cold resistance in vitro and in vivo reached r = 0.89, between cold resistance in vitro and winterhardiness in situ – r = 0.78.
of temperatures only when hardening duration is sufficient. Hardening increased strawberry cold resistance significantly. During the investigation it was found that strawberry varieties differ in ability to harden. The varieties Venta and Lvovskaja Raniaja under field conditions are rather hardy, nevertheless, plants of the first variety reacted more to longer hardening than the plants of the second. If the character of reacting differently to hardening is determined genetically, it may be possible to create genotypes that are not only cold resistant but winter hardy as well, since the ability to harden affects a number of winterhardiness factors. When comparing seedlings of four cross combinations according to the number of cold resistant seedlings, dominance of cold resistance was indicated. In the combinations with at least one winter hardy parent, the production of winter hardy seedlings was higher than in the combination where both parents were not winter hardy. A close correlation was established between strawberry cold resistance in vitro and in vivo, between cold resistance in vitro and winterhardiness in situ, thus proving that screening in in vitro conditions is possible and useable. Based on the results, an in vitro
screening technology was determined for cold resistant strawberry seedlings permitting the screening to be undertaken shortly after crossing and enabling a significant reduction in breeding time and field nursery space required for field assessment (Rugienius, 2000).
Conclusions 1. Strawberry embryos can become autonomous not earlier than on the 14–16th day after flower pollination and greatest autonomy (100%) is reached on the 20–22nd day. Plants regenerated from 26 day-old embryos grow best. 2. MS medium without phytohormones was the best for regenerating strawberry plants from isolated embryo axes. Regeneration from rescued cotyledons was most successful on the medium with 1.0 BA and 0.5 NAA. 3. The temperature interval, at which genotypes discriminated according to cold resistance, is –8– 12 ◦ C. Discrimination of strawberry genotypes according to this character conformed to their discrimination in vivo, provided plants were hardened
277 for at least 21 days. Close correlation between cold resistance in vitro and in vivo (0.93) proves that screening for cold resistant seedlings in vitro is effective.
References Caswell, K.L., N.J. Tyler & C. Stushnoff, 1986. Cold hardening of in vitro apple and saskatoon shoot cultures. Hort Science 1207– 1209. Dalman, P. & V. Matala, 1997. The effect of cultivation practices on the overwintering and yield of strawberry. Acta Horticulturae 439(2): 881–886. Dix, P.J., I. Finch & J.I. Burke, 1994. Genotypic differences in cold tolerance are masked by high sucrose and cytokinin in shoot cultures of sugarbeet. Plant cell, Tissue and Organ Cult 36: 285–290. Erb, P.S., A.R. Miller, J.C. Scheerens & C.K. Chandler, 1989. Enhanced strawberry seed germination by in vitro culture of embryo axis sections. Hort Sci 71. Filipenko, I.M., 1996. Hypothesis about nature and peculiarities of cold hardiness inheritance of plants. Agricultural Biology. Ser. Biology of Plants (Rus.). 3: 121–127. Miller, A.R., J.C. Scheerens, P.S. Erb & C.K. Chandler, 1992. Enhanced strawberry seed germination through in vitro culture of cut achenes. J Amer Soc Hort Sci 117(2): 313–316. Murashige, T. & F. Skoog, 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473–479. Nestby, R., 1997. Influence of wintercovers on crown temperature, tissue browning and yield of Korona strawberries. Acta Hort 439(2): 887–892.
Palonen, P. & D. Buszard, 1997. Screening strawberry cultivars for cold hardiness in vitro. Acta Hort 39(1): 217–220. Rugienius, R., 2000. Comparison of screening technologies in vitro, in vivo and in situ of cold hardy strawberry seedlings. Hort veg grow 19(2): 3–10. Sayegh, A.J. & M.J. Hennerty, 1989. Androgenesis and embryo rescue for strawberry haploid production. Acta Hort 265: 129– 135. Scott, D.H. & F.J. Lawrence, 1975. Strawberries. In: J. Janick & J.N. Moore (Eds.), Advances in Fruit Breeding, pp. 71–83. N.Y. Univ. Press, New York. Stanys, V., 1997. In vitro kultûra augal¸u selekcijoje. Kintamumas ir stabilumas, pp. 68–82, Babtai. Tarakanovas, P., 1996. Selekcini¸u – genetini¸u tyrim¸u rezultatç apdorojimo ir á¸ivertinimo sistema ‘Selekcija’, pp. 11–30, DotnuvaAkademija. Volf, V.G., 1966. Dispersional analysis of qualitative traits. Statistical treatment of experimental data. (Rus.) M. 194 pp. Walbot, V., 1978. Control mechanisms for plant embryogeny. In: M.E. Clutter (Ed.), Dormancy and Development Arrest – Experimental Analysis in Plants and Animals, pp. 114–166. N.Y. Academic Press, New York. Wang, D., W.P. Wergin & R.H. Zimmerman, 1984. Somatic embryogenesis and plant regeneration from immature embryos of strawberry. Hort Science 71. White, P.R., 1943. A handbok of Plant Tissue Culture J. Cattel, Lancaster, Pa. 277 pp. Zatylny, A.M., J.T.A. Proctor & J.A. Sullivan, 1993. Screening red raspberry for cold hardiness in vitro. HortScience 740–741. Zubov, A.A., 1990. Genetic peculiarities and breeding of strawberry. Methodical references, pp. 29–70. Michurinsk, 81 pp.
