Trees (2012) 26:1181–1192 DOI 10.1007/s00468-012-0694-7
ORIGINAL PAPER
Dormancy development during cold hardening of in vitro cultured Malus domestica Borkh. plants in relation to their frost resistance and cryotolerance Alois Bilavcˇ´ık • Jirˇ´ı Za´mecˇnı´k • Martin Grospietsch Milosˇ Faltus • Petra Jadrna´
•
Received: 17 April 2011 / Revised: 5 January 2012 / Accepted: 31 January 2012 / Published online: 14 February 2012 Ó Springer-Verlag 2012
Abstract The development of dormancy, frost resistance and cryotolerance of in vitro apple plants (Malus domestica Borkh.), cv. Greensleeves during their exposure to cold hardening was studied. In vitro cultures were cold hardened at 4°C under a short photoperiod up to 25 weeks. The dormancy status, non-structural saccharides, proline, water content and frost resistance were evaluated for optimization of cryopreservation. According to regrowth tests, in vitro cultures exhibited endogenous dormancy after the maximal frost resistance was reached. The highest regeneration ability of shoot tips after cryopreservation by encapsulation–dehydration method coincided with the period of the plant’s dormant state and maximum of frost resistance. All studied saccharides and proline exhibited the maximal values at the beginning of cold hardening and/ or the dormancy phase. Contrary to the accumulation of saccharides and proline, water content showed the inverse time behaviour. According to these results, the cold hardening-induced endodormancy, high frost resistance and accumulation of saccharides and proline are the important prerequisites for the successful cryopreservation of shoot tips of in vitro grown apple plants. Keywords Apple tree Endodormancy Frost tolerance Cryopreservation Saccharides Proline
Communicated by D. Treutter. A. Bilavcˇ´ık J. Za´mecˇnı´k (&) M. Grospietsch M. Faltus P. Jadrna´ Crop Research Institute, Drnovska´ 507, 16106 Prague 6-Ruzyneˇ, The Czech Republic e-mail:
[email protected]
Introduction Plant dormancy is a strategy of plant organisms for surviving unsuitable periods of the year, such as a winter or dry seasons, with arrested growth. Winter dormancy, common for mild climate plant species can be brought on by decreasing temperatures and shortened days, but arises each season automatically and can be broken only by a certain degree of cold hardening temperatures. So, a factitious ‘‘eternal summer’’ would not avoid automatic endodormancy and would cause lethality of plants in the dormant stage (Bewley and Black 1994). The length of the endodormancy period depends on the genotype (Palonen and Linde´n 1999). The induction of endodormancy is controlled by plant hormones and numerous integrated plant structures and functions (Simpson 1990). Winter dormancy in trees of mild and cold climates is associated with increased frost hardiness; endodormancy is physiologically the most important part of winter dormancy (Faust et al. 1995b). During endodormancy, most water becomes bound and unfreezable in tissues, which also enhances the frost tolerance (Faust et al. 1995a; Buba´n and Faust 1995). To break endodormancy and to resume a growth, the exposure of trees to low temperatures, which is connected with cold acclimation, is required; it is defined as a cold hardening requirement (Arora et al. 1997), which differs among plant genotypes according to their genetic predispositions (Hauagge and Cummins 1991). However, cold hardening for too long in controlled conditions can negatively affects further growth and the quality of the tree plantlets ex vitro (Borkowska 1986). The release of dormancy can be influenced by many environmental factors and chemicals like temperature, light, anaerobic conditions, injury, oils, chemical uncouples, toxic substances, salts, acids, organic solvents etc. (Lavee and May 1997).
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In contrast to most temperate species, for which a photoperiod was declared as one of the factors inducing dormancy (Arora et al. 2003; Allona et al. 2008; Kalcsits et al. 2009), apples and pears were proven to be non-sensitive to the photoperiod for the growth cessation and dormancy induction (Heide and Prestrud 2005). In vitro cultures were already used earlier to test the cold hardening requirements in apple at 4°C, but without mentioning the role of a photoperiod (Borkowska 1981). Barthe and Bulard (1982) proved that the break of dormancy in embryos of Malus domestica cultivated in vitro is influenced by the medium and some of its components (sucrose, agar); sucrose and solid media caused a delay in the break of dormancy. Further, it was proven that anaerobic conditions induce the release from dormancy in apple embryos (Barthe and Bulard 1983). In vitro cultures enable permanent subcultivations of temperate species thanks to the strong hormonal influences on small explants parts and controlled conditions during the cultivation. However, endodormancy and the associated frost resistance can be induced in the explants tissues by appropriate changes of cultivation conditions (temperature, day-length). Biochemical changes, which are observed in vivo during cold hardening and the winter period, can also be observed in vitro, when the cultures are exposed to low temperatures (Caswell et al. 1986; Travert et al. 1997). The aim of this study was to investigate relations between the progression of endodormancy, frost resistance, and some physiologically important compounds associated with frost resistance in plants, like non-structural saccharides and proline and water content, during long-lasting cold hardening of in vitro culture of apple cv. Greensleeves. Rohde and Bhalerao (2007) define dormancy as the inability to initiate growth from meristems (and other organs and cells with the capacity to resume growth) under favourable conditions. Endodormancy, a part of dormancy, refers to a state of buds regulated by internal factors where growth is possible only after plants have been exposed to sufficient amount of chilling (Lang et al. 1987; Welling and Palva 2006). Dormancy of in vitro plants has been frequently studied on bulb plant species (Maksimovic´ et al. 2008; Kwang-Soo Kim et al. 1994; Aguettaz et al. 1990; Delvalle´e et al. 1990). Some investigations of partial aspects related to endodormancy were made on fruit trees in vitro but there is a lack of studies for the investigation of dormancy induction and release in trees in vitro. This study aimed to investigate the development of endodormancy in apple plantlets during long-term cold culture in relation to other changes in plantlets which are closely associated with the initiation of endodormancy, such as the frost resistance and the contents of matters, whose changes are directly
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linked to the frost resistance rate. Influence of the cold hardening for successful cryopreservation of apple in vitro cultures will be evaluated.
Materials and methods For the experiments, in vitro cultures of Malus domestica Borkh. cv. Greensleeves were used. In vitro cultures were subcultivated on medium Murashige and Skoog (1962), which was slightly modified by authors Yepes and Aldwinckle (1994) and Paul T. Lynch (personal communication). Sorbitol (30 g l-1) was used instead of the normally used sucrose as carbohydrate source and phytohormones BAP (1 mg l-1), IBA (1 mg l-1) and GA3 (1 mg l-1) were added to the media at a temperature of 23 ± 0.5°C, photoperiod of 16/8 (day/night) and light intensity of 120 lE s-1 m-2 PAR. The subcultivation period was approximately 30 days. Seven days after subcultivation (after a formation of remedial tissues), plantlets cultures were transferred from standard cultivation conditions into cold hardening conditions: 4°C, short photoperiod 8/16 (day/night), light intensity of 120 lE s-1 m-2 PAR. At the beginning of cold hardening, and then at 7-day interval, samples were collected 0–1 h before the end of the dark period for the observation of the beginning and the end of endodormancy, assessment of proline content and sugars contents. Morphological changes of plants were also observed during the experiment. Two replications of all measured characteristics were done. In addition, shoot apices were cryopreserved in periods of 14 days during the second replication of the cold hardening experiment by encapsulation–dehydration protocol. Development of dormancy Shoots were collected from the cold hardening chamber every 7 days. To test the end of endodormancy, they were defoliated, and their central parts were cut into uninodal segments. Thirty uninodal segments were divided into three replications per ten segments. They were transferred onto fresh media and into standard cultivation conditions, and the number of sprouting buds, average number of leaves per each regrowing plantlet, and average sizes of newly growing shoots, were rated every 7 days during cultivation. The sprouting of chilled buds was evaluated 5 weeks after transferring the samples onto fresh media into standard cultivation conditions and the endodormancy period (that means a period, in which the rate of sprouting buds was lower than 50%) was examined. The experiment was replicated twice.
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Frost resistance estimation Each week, the cold-hardened plantlets and the control plantlets were tested for their frost resistance. They were transferred into glass tubes with MS medium without phytohormones (one plant per tube). A frost treatment was achieved by the programmed freezer Ultra Kryomat Lauda RUK 50 in a gradually cooled ethanol bath. The tubes were inserted into the bath at a temperature of -2°C. After stabilization of the temperature in the tubes (controlled by thermistor temperature sensor Ahlborn in reference tube) bases of tubes were inserted into liquid nitrogen for several seconds to initiate ice nucleation of the medium. After the initiation of nucleation, the tubes were immediately moved back into the ethanol bath and let to equilibrate again (thanks to the state change in media, a heat released during the process of nucleation, and the temperature increased up to -0.3°C). After that, the bath temperature was stepwise decreasing at 1°C min-1, and then kept for 15 min at each step. At the end of each step, one tube with a plantlet was taken out of the bath and placed into a chamber at 4°C for 24 h to thaw. After that, the plantlets were transferred from the tubes into Erlenmayer flasks, onto modified MS medium containing phytohormones, and cultivated at 23 ± 0.5°C and a photoperiod of 16/8 h day/night. Plant regeneration and regrowth were evaluated visually after 14 and 28 days. The last temperature, at which the plantlet was still able to regenerate, was considered the critical temperature for the frost resistance of the plantlet. Estimation of resistance of in vitro culture to ultralow temperatures During the second replication of dormancy assessment by in vitro cultures, 1–2 mm apices were extirpated from the in vitro culture of cv. Greensleeves every second week, and transferred onto standard MS medium with 3% w/v sucrose and cryoprotective solution (2 M sucrose with 100 mg l-1 vitamin C). After 48 h pretreatment in cryoprotective solution, apices were encapsulated in natrium alginate (3% natrium alginate in 0.75 M sucrose) which was fixed into solid state after dropping the liquid capsules containing apices into 0.1 M CaCl2 in 0.6 M sucrose (reaction natrium alginate with CaCl2) (Dereuddre et al. 1991). Encapsulated apices were slightly dried on sterile filter paper. Approximately 20 capsules were placed onto petri dishes (5 cm diameter) and then controlled dehydration followed in laminar flow box at room temperature for 4 h. Dehydration level was assessed at the end of dehydration gravimetrically by comparing the weight of the controlled dehydrated sample and the same sample after total drying in the oven at 85°C. At least five dehydrated apices were used as a control for assessment of survival after
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dehydration. Dehydrated encapsulated apices were sealed into 2 ml cryotubes (10–20 pieces/tube) and immersed into liquid nitrogen (LN) at freezing rate of approximately -200°C min-1. After at least 24 h, two sets of 20 encapsulated apices were transferred from LN and rapidly thawed by immersion into water at 40°C for 60 s. After that, the encapsulated apices were transferred onto standard MS medium (3% w/v sorbitol instead of sucrose, BAP 1 mg l-1). Survival and growth of apices cultivated in vitro in Erlenmayer flasks were visually evaluated. Sprouting and creating of new shoots were observed. Regrowth was evaluated after 4 weeks of cultivation on three-level-scale from fully sprouting shoots over 10 mm (9) through badly growing 1–10 mm (5) to non-growing (1) plants. Water content estimation Five plantlets were taken for assessing their water content every week. They were weighed immediately after removal from media, and then put into a dryer and dried out at a temperature of 105°C for at least 48 h into constant weight. The water content (WC) was calculated as the difference between the fresh and dry weight and expressed in gH2O g-1dry matter (DM). Estimation of non-structural soluble saccharides The content of sugars was assessed by the method of HPLC (High Performance/Pressure Liquid Chromatography). Approximately 100 mg of fresh material was taken from each of 5 randomly chosen in vitro plantlets every 7 days. These samples were weighed and put into a freezing box at -135°C. After that, they were lyophilized and weighed for assessing dry matter content. Further the samples were incubated in 80% methanol (1 ml of methanol, 10 min at 75°C, IncuBlock Denville, model 285), which was then evaporated in a vacuum evaporator (SpeedVac, 2–3 h). Soluble saccharides were extracted from these desiccated samples by redistilled water. After addition of redistilled water, microtubes with samples were dipped into an ultrasound pool (Julabo USR 05) for 10 min and centrifuged at a rotation speed of 233 Hz for clearing. The supernatant was filtered through membrane filters Whatman 0.45 lm, and stored at -15°C before HPLC analysis. Samples were injected in volumes of 15 ll into a mobile phase which was redistilled water at a flow speed of 0.5 ml min-1. Particular saccharides were identified by comparing their retention with standards. The identities of less common saccharides (raffinose, stachyose) were confirmed by a different system of HPLC with amperometric detection (Dionex, Sunnyvale, USA; mobile phase 100 mM NaOH). From five analysed samples three samples without the maximal and minimal values were used for
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evaluations. Changes of the levels of soluble saccharides were compared with development of frost resistance during the cold hardening. Proline estimation The plant material, 5 samples of entire shoots, was homogenized in 10 ml of 3% (v/w) water solution of sulfosalicyl acid and filtered. Two ml of filtrate, 2 ml of ninhydrine solution (prepared by boiling 1.25 g ninhydrine in 10 ml of acetic acid and 20 ml of 6 M phosphoric acid) and 2 ml of cold acetic acid were put into a testing tube. The tube was sealed and dipped into boiling water for 1 h. After removing and cooling down the tube, 4 ml of toluene was added, the tube was sealed again and shaken for 15–20 s. Then, absorbency at 520 nm of wavelength was measured in the coloured toluene solution. Proline content was assessed from the calibrated diagram and calculated for the fresh weight unit according to this formula: [(lg proline/ml 9 ml toluene)/115.5 lg/lmol]/[(g sample)/5] = lmol proline/g sample (Bates et al. 1973).
Results Development of dormancy in in vitro plantlets of apple cv. Greensleeves During the cold hardening of apple plantlets cv. Greensleeves, morphological changes were observed visually: From the second week, the stems started to be coloured by anthocyans, later the leaves started to get yellow or reddish (in the 6th week only approximately 30% of leaves were green). Later, they started to get brown and go down; after 14 weeks of cold hardening, most shoots were defoliated. The endodormancy of variously long, cold-hardened plantlets was evaluated according to regrowth of the plantlets at 23°C when less than 50% of them were able to create new sprouts. This was examined after 5 weeks of standard cultivation of cold-hardened plantlets, when all sprouted buds could already be well recognized (Fig. 1). The period of endodormancy, as detected by treated plantlets, was lasting for 4 weeks, from the 8th to 11th week after starting the cold hardening (Fig. 2a–c). Actually, some buds were always able to sprout during that period, but a distinct decrease was detected in the average number of sprouting buds below 50% (Fig. 2a), sizes of sprouted shoots (Fig. 2b) and a number of new leaves on the shoots (Fig. 2c). After the end of endodormancy, from the 13th week, 100% of buds were in a stage of ecodormancy, able to fully sprout again, and the sizes of shoots and numbers of leaves on the shoots increased to the levels
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Fig. 1 Sprouting of uninodal segments of plantlets of apple cv. Greensleeves after 14 weeks of cold hardening at 4°C at short photoperiod (8/16 h). Newly sprouted buds and new leaves were evaluated from the uninodal segments. The regrowth was evaluated as new leaves regrowth after 5 weeks of regeneration in standard (23°C) growth conditions. The plants with new leaves were considered as nondormant plants, the plants with decreased or without regrowth of new leaves were considered as dormant plants
detected in the plantlets cold hardened for less than 8 weeks, which still had not entered endodormancy. Development of frost resistance during cold hardening The frost resistance of the cold-treated in vitro plantlets increased from -4.5°C in non-chilled plantlets to -21°C in plantlets after 7 weeks of cold treatment (Fig. 3b). The plantlets were the most frost tolerant in the 7th week, 1 week before entering endodormancy (Fig. 2a). From the 8th week, the frost resistance decreased, and from the 20th week of cold hardening it was on the level of non-chilled control plants and reached -4 to -5°C (Fig. 3b). At the end of the cultivation, the frost resistance was even lower than in the control plants. Cryotolerance of in vitro cultures to ultra-low temperatures Tests of plantlet tissues tolerance to ultra-low temperatures from the beginning to 20th week of cold hardening were carried out (Fig. 3a). Apices were able to regenerate after cryopreservation in the period from the 6th to 14th week of cold hardening with a maximum of 88% regrowth in the 6th and 8th week. However, the frost resistance decreased after its maximum in the 7th week, and from the 9th week it was generally at lower levels than frost resistance detected before the 7th week, although some plantlets successfully regenerated after cryopreservation until the 14th week of cold hardening at the rate of 46% in the 14th
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Cold hardening [weeks] Fig. 2 Measured signs of endodormancy in vitro plantlets of apple cv. Greensleeves: Number of newly sprouted buds (a), sizes of newly sprouted buds (b) and number of new leaves (c) regenerated from uninodal segments after 5 weeks of regeneration in standard growth conditions. The plants were cold hardened at temperature 4°C from 1 to 18 weeks at short photoperiod (8/16 h). The values are means from three replications, vectors represent SD
week (Fig. 3a). The small number of measurements in plant regrowth after cryopreservation (Fig. 7) is due to short possible sampling time (rapid increase of frost resistance of plantlets to the highest level). The higher the plantlets were frost resistant the higher regrowth after liquid nitrogen exposure the plantlets had (Figs. 7a, 8a). Plantlets regrowth after liquid nitrogen exposure rapidly increased in the beginning of cold hardening contrary to the slow decrease after reaching the maximum cold hardiness at 7th week (Figs. 7a, 8a). Changes of water content during cold hardening Water content was relatively low between the 4th and 15th week of cultivation—3.81 to 4.37 g H2O g-1 DM; while
Fig. 3 Dormancy and regrowth after cryopreservation in liquid nitrogen (a) frost resistance treatment (b) in apple plantlets cv. Greensleeves during cold hardening at 4°C at short photoperiod (8/ 16 h) for 25 weeks. The dormancy was considered from 8th to 12th week according to the 50% stop of bud proliferation to new shoots. Columns with the same letter belong to the same homogeneous group according to Duncan’s test (a = 0.05). Vectors represent SD. Bars without SD are values from one measurement only
the water content was 5.58 g H2O g-1 DM at the beginning of cold hardening, and it was 4.89 g H2O g-1 DM in the 27th week (Fig. 4). The initial decrease in water content corresponded to the increased frost resistance which was the highest in the 5th–7th week of cultivation and then decreased (Fig. 3b). In the plantlets cold hardened for longer than 15 weeks, a gradual increase of water content in their tissues with some fluctuations was detected. The higher the plantlets were frost resistant the lower water content they had (Figs. 7b, 8b). The initial water content was slightly higher than the final one (Figs. 7b, 8b). Changes of non-structural saccharides content Analysis of non-structural saccharides contents in plantlet tissues during the cold hardening proved specific changes and trends of various sugars. The content of sucrose (Fig. 5a) was the highest at the beginning of cold hardening, 23.6 lg sucrose mg-1DM in the 1st and 25.7 lg sucrose mg-1 DM in the 2nd week. Then it decreased gradually with some variations, as the photosynthesis and
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sucrose accumulation were inhibited in plantlets due to cold conditions. Glucose content was the highest in the 3rd week—25.7 lg glucose mg-1 DM—and remained high to the 7th week of cold hardening—which corresponded to the maximal frost resistance, then decreased (Fig. 5b). Fructose content (Fig. 5c) was the highest in the 6th week of cold hardening, 18.1 lg fructose mg-1 DM, and then decreased. High levels of glucose and especially fructose contents fitted perfectly with the frost resistance which was the highest in the 5th–7th week of cold hardening, then decreased continuously (Fig. 3b). Raffinose and stachyose contents (data not shown) were apparently connected with the development of frost resistance and dormancy, although their values differed very strongly between the first and second replication. In the 1st replication, stachyose was detected in the plantlets only from the 3rd to 10th week and its highest content was in the 8th week, 1.3 lg stachyose mg-1 DM; raffinose was detected from the 1st to 11th week and its highest content was in the 3rd week, 4.2 lg raffinose mg-1 DM. In the second replication, the stachyose and raffinose contents were much higher and reached their maxima of 10.3 lg raffinose mg-1 DM in the 7th week and 19.0 lg stachyose mg-1 DM in the 9th week of cold hardening. Stachyose appeared in the tissues the first time in the 4th week and remained detectable until the end of the experiment (25 weeks); raffinose appeared in the 2nd week of cold hardening and the last time it was detected was in the 21st week. The positive correlation was found between frost resistance of the plantlets and the content of glucose, fructose, stachyose and raffinose in both periods before (Fig. 7e–h) and after maximal frost resistance (Fig. 8e–h). Only the sucrose content was the highest at the beginning of cold hardening, corresponding with low frost resistance, and decreased significantly with the decrease of frost resistance after the plantlets reached the maximum of the cold hardiness.
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Fig. 4 Changes of average water contents in plantlet tissues of in vitro culture of apple cv. Greensleeves during cold hardening at 4°C at short photoperiod (8/16 h)
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Changes of proline content Proline content values in in vitro plantlets were intensively increasing from the 5th week. In the 4th week, the proline content was 3.2 lg mg-1 DM, and the highest average value of the proline content—17.3 lg mg-1 DM—was
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reached in the 10th week of cold hardening. Then the proline content was decreasing continuously until the 21st week to the levels measured in non-cold-hardened plants (Fig. 6). So the maximum peak of the proline content was achieved in the period of endodormancy. The higher the plantlets were frost resistant the higher content of proline the plantlets had (Figs. 7c, 8c). The content of proline increased slower till the plantlets obtained the maximal hardiness in contrary to faster decrease during their cold dehardening phase, although the maximal cold-hardened plantlets did not have the highest content of proline, which was obtained in the 10th week. (Figs. 7c, 8c).
Discussion Endodormancy in plantlets of apple cv. Greensleeves occurred in the 8th–11th week of cold hardening at 4°C and at a short photoperiod. The sprouting was significantly lower than 50% during the endodormancy period, but some sprouting shoots always appeared. According to the definition, endodormancy—or sometimes called true dormancy—should be a stage of deep sleeping of buds. Some published data imply that specific processes inside the buds are not ceased definitely also during dormancy in situ. For example, it was observed in apple trees, that the growth and development of primordia occur in buds also during dormancy (Buba´n and Faust 1995). A study of dormancy in buds of Fraxinus mentions in vitro buds in a resting period as ‘‘dormant-like buds’’, and describes their histological parallels to buds dormant in nature (Nougare`de et al. 1996). For the first 7-week period as well as for the 12th–25th week period of the cold hardening at 4°C, the plantlets
regenerated in 100% similarly in vigour and number of leaves after their transfer to standard growing conditions. The endodormancy was strictly bounded by these two periods in the beginning and in the end. This is a phenomenon typical for in vitro cultures of deciduous plants kept in artificial conditions with external supplementation of plant hormones. The cold hardening was prolonged more than in other studies, but the plants were able to regrowth at favourable conditions (23°C) even without transfer into a fresh medium. Also dry matter of uninodal segments (data not shown) did not change during whole cold hardening period (25 weeks). From these findings and with taking into account the cultivation at low temperature (4°C) the plants probably did not suffer from lack of nutrients in the medium during cold hardening. During the cold hardening at 4°C, increased frost resistance of plantlets was detected until the 7th week, as well as increased amounts of non-structural saccharides in their tissues like glucose and fructose. The glucose and fructose maxima were found in the 7th week, and for raffinose family sugars—raffinose and stachyose, whose presence is typical for cold acclimated plants, the maximum was from the 7th to 9th week. After that, amounts of sugars decreased continually. This phenomenon is supported by many studies, which report the increasing contents of these sugars in association with increased cold hardiness. The enhancement of contents of total sucrose, glucose, fructose and raffinose was proven during a 5-week cold acclimation of raspberry (Palonen et al. 2000). The levels of raffinose family oligosaccharides highly correlate to low temperature exposure during acclimation and to the lowest survival temperatures; endogenous raffinose and stachyose increase as the temperature drops in early winter, and diminish as the temperature rises in spring (Cox and Stushnoff 2001). The ratio of raffinose-like saccharides to glucose, sucrose and fructose is also considered an important factor influencing the frost resistance of plants; the higher this ratio, the more cold resistant plants should be (Imanishi et al. 1998). The occurrence of raffinose or stachyose saccharides is also associated with dormancy (Keller and Loescher 1989; Jones et al. 1999). Other nonstructural saccharides, sucrose, increased in our study only at the beginning of the treatment (until the 3rd week) and then decreased continuously. This observation is supported by other studies, from which it arises that the increase of sucrose at the beginning of cold hardening is an effect of rapid adaptation of the plant growth and metabolism to cold, as proven by poplar cuttings and in vitro shoots (Hausman et al. 2000), and subsequent contents under continuing cold conditions are determined by the photosynthetic rate in the plant (Du and Nose 2002), which means that the continual decrease in sucrose content is caused by inhibited photosynthesis. In our experiments the
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0
Frost resistance [°C]
Fig. 7 Correlation between the regrowth (a) after liquid nitrogen (LN) treatment, water content (b), proline content (c), sucrose content (d), glucose content (e), fructose content (f), stachyose content (g), raffinose content (h) and the frost resistance of apple plantlets cv. Greensleeves in the period of increasing of the frost resistance, before
entering plantlets into dormancy up to the 7th week of plantlets cold hardening at 4°C at short photoperiod (8/16 h). Vectors represent SD. The data were fitted by linear relationship. Significances of the correlation coefficients are on ***p B 0.001, **p B 0.01 and *p B 0.05
plant did not suffered from lack of photosynthates because at 4°C the growth was ceased under short-day radiation. The dry matter of plants was constant during the whole
period of cold hardening—this also implies the plants did not suffer during long-lasting cold hardening. In contrast, Palonen et al. (2000) observed that mainly the sucrose
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b
100 80 60 40 20
Proline content [µg mg -1 DM]
-20
-15
20
-10
-5
y = -0,72x + 1,2786 r = 0,7418***
15 10 5
-25
-25
d
30 25
-15
-10
-5
f
30 25
Fructose content [µg mg -1 DM]
y = -0,7015x + 1,7823 r = 0,6139**
20 15 10 5
-25
-20
-15
-10
-5
y = -0,5749x- 1,4067 r = 0,8563*** 10
5
-10
-5
0
15 10 5 0
25
-20
-15
-10
-5
0
-10
-5
0
y = -0,5448x + 2,3331 r = 0,5971**
20 15 10 5 0 -25
h
-15
20
0
15
-20
y = -0,4994x + 7,2547 r = 0,4579*
-25
0
Raffinose content [µg mg -1 DM]
Glucose content [µg mg -1 DM]
-20
0
Stachyose content [µg mg -1 DM]
4
0
0
g
5
Sucrose content [µg mg -1 DM]
-25
e
y = 0,0369x + 4,6812 r= 0,5416*
3
0
c
6
r = 0,9340***
Water content [g H 2O g-1 DM]
Regrowth after LN treatment [%]
a
1189
-20
-15
10
y = -0,1745x + 0,0854 r = 0,5625 *
8 6 4 2 0
0 -25
-20
-15
-10
-5
0
Frost resistance [°C]
-25
-20
-15
-10
-5
0
Frost resistance [°C]
Fig. 8 Correlation between the regrowth (a) after liquid nitrogen (LN) treatment, water content (b), proline content (c), sucrose content (d), glucose content (e), fructose content (f), stachyose content (g), raffinose content (h) and the frost resistance of apple plantlets cv. Greensleeves in the period of decreasing of the frost resistance, from
entering plantlets into dormancy; since the 8th week of plantlets cold hardening at 4°C at short photoperiod (8/16 h). Vectors represent SD. The data were fitted by linear relationship except a, where the logistic relationship was used. Significances of the correlation coefficients are on ***p B 0.001, **p B 0.01 and *p B 0.05
content (more than that of other non-structural saccharides) increased during cold hardening and was the highest at the end of a 5-week cold acclimation of Rubus idaeus L. in vitro plantlets, which correlated with the highest frost resistance of these plantlets during the 5-week experiment. The accumulation rate of the non-structural saccharides is also dependent on the genotype and the actual frost
resistance of the plant; more hardy genotypes within one species contain higher concentrations than more tender ones (Va´gu´jfalvi et al. 1999). The period in which the cryopreservation was successful (the 6th–14th week) overlapped the period of endodormancy (the 8th–11th week) and corresponded to high contents of stachyose during the 6th–14th week. The
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1190
beginning of successful cryopreservation period also corresponded with the maximum of raffinose content and with a maximal frost resistance (-21°C, the 7th week). The frost resistance in our experiments was higher than obtained by Kushnarenko et al. (2009) whose acclimated plantlets had lethal temperature (Lt50) values from -12 to -15°C after 1–3 weeks. Sedla´k et al. (2001) cold hardened apple and pear in vitro plantlets for 4–5 weeks at 4°C with long daylengths (16 light/8 dark) and noted their cold hardiness of -13°C. In vitro apple shoots cold acclimated with short day (8 h light) at 2°C for 8 weeks, followed by a 48 h period at -3°C, reached an Lt50 of -17.5°C only (Kushnarenko et al. 2009). The in vitro apple plantlets reached cold-hardened state after shorter period (3 weeks) of the cold hardening at 5°C (Halmagyi et al. 2010). Three weeks of alternating temperature of cold hardening can be recommended as a standard protocol for Malus germplasm cryopreservation (Kushnarenko et al. 2009). Baldwin et al. (1998) obtained slightly higher frost resistance of in vitro Amelanchier cultures, down to -27°C after 6 weeks at 4°C, short day (8 h light). They also mentioned an important role of the photoperiod and hormone composition in the medium for the frost resistance. Their higher frost resistance could be obtained either due to use hormone free-medium during cold hardening or use of another species, Amelanchier, contrary to our study. Successful cryopreservation was apparently not linked to the ratio of raffinose family sugars (raffinose, stachyose) to sucrose ? glucose ? fructose, which is considered as a good indicator for frost resistance, since plantlets were able to regenerate after cryopreservation already 1 week before starting endodormancy, when the ratio was more than 0.19, while they did not regenerate after cryopreservation between the 16th and 20th week when the ratio was also more than 0.19 and the frost resistance of plantlets was similar to that from the 14th week. Preculture was critical for the survival of apple shoot tips after cryopreservation. Among four different tested sugars and sugar alcohols (glucose, sucrose, mannitol and sorbitol) the sucrose treatment for 24 h was the most efficient one (Halmagyi et al. 2010). The timing of maxima of fructose, glucose and raffinose family contents in context with frost resistance corresponded to the experience of the 14-week cold hardening of sour cherry in vitro plantlets, which gave the most vigorous plantlets (according to the genotype) after 4–6 or 6–8 weeks of cold hardening, and unsuitable weak plantlets after a longer period of cold hardening (Borkowska 1986). A study of influence of cold hardening on strawberries shows that 9 weeks of cold hardening is the best, while longer chilled plantlets also have a weak growth (Borkowska and Michalczuk 2000). Two weeks before assessing of endodormancy in the 8th week, leaves of
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plantlets started to cease (became yellowish, brownish, went down). This was apparently connected with entering the endodormant period, for which the increased production of abscisic acid (ABA) in plants, the main hormone controlling endodormancy and the associated autumn leaf fall in plants, is typical (Arora et al. 2003; Li et al. 2003; Debeaujon and Koornneef 2000). When cultivated as in vitro cultures, its ABA concentration is also significantly lower in growing shoots than in dormant ones (Piola et al. 1998). Addition of ABA into cultivation medium increased cold hardiness of Amleanchier in vitro cultures contrary to cultures grown on hormone-free medium or even ABA antagonist benzylaminopurine (BAP) rich medium (Baldwin et al. 1998). After the end of endodormancy, during the maintaining of the plantlets at 4°C, the contents of measured sugars in apple cv. Greensleeves plantlets (sucrose, glucose and fructose) decreased (Fig. 5a–c). The plantlets were not able to process photosynthesis due to the complete loss of chlorophyll from their leaves and their growth was stopped; the sugars were accumulated from medium. That could be surely the reason for the weakness of such plantlets after planting them in vivo, as mentioned in this discussion above. As dormancy and cold hardening in vitro are frequently studied in bulb plants too, it is worthy to mention that the increases or stabile levels of sucrose contents during the cold hardening are typical for bulb plants (e.g. Lilium or Frittilaria sp.) (Xu et al. 2006; Maksimovic´ et al. 2008). The increasing proline contents in tissues of hardy plants are also associated with a cold acclimation and increasing frost resistance of plants (Yelenosky 1979; Kushad and Yelenosky 1987); the proline content corresponds to the rate of frost resistance among genotypes of one species and they are higher in hardier genotypes than in tender ones (Kushad and Yelenosky 1987; Tantau et al. 2004). The increase of proline content can be induced by exposure of plant to cold conditions as well as by exposure to ABA, a plant hormone controlling the endodormancy, after which the frost resistance increases (Duncan and Widholm 1987). During the cold hardening of apple cv. Greensleeves plantlets, a decrease of water content was detected between the 4th and 13th week. There was no study focusing on changes in water content of plantlets during the cold hardening of in vitro cultures yet, but other facts show that the decrease of water content improves the frost resistance in plants and that the mild-climate plants have native mechanisms to improve their frost resistance for surviving winters through adapting the water status in their tissues. The bound water in tissues during winter dormancy enhances their frost resistance because it cannot freeze and damages the tissues (Faust et al. 1995a; Buba´n and Faust 1995). These observations suggest that the freezing of water in nonhardy tissue dried the tissue to moisture levels
Trees (2012) 26:1181–1192
at which severe dehydration damage occurred. It appears that acclimation of vegetative apple buds involves at least two processes: (1) an increase in tolerance to dehydration and (2) an increase in the level of unfreezable water (Vertucci and Stushnoff 1992). Water stress induces frost resistance and decrease of freezable water in possibly cold-hardened plant tissues (but also cold-tender plants can be cryopreserved), both of which qualities are necessary for plant survival at ultra-low temperatures. This induction of frost resistance and decrease in water content (Figs. 7b, 8b) are also a key step for all cryoprotocols. The water loss from tissues can be induced by a pre-cultivation of plantlets on media with high sucrose content or by various treatments in osmotic solutions, which enhance the osmotic potential in cells and avoid frost injuries. In apples, as deciduous mild-climate tree species, the cold hardening is proved to be satisfying for successful pre-treatment before the cryopreservation of dormant buds. In in vitro cultures, the extirpated apices need to be chilled, pretreated in osmotic solution and then dehydrated (Chang and Reed 2000). The regrowth of cryopreserved apices of apple cv. Greensleeves in our study (Fig. 3a, b) was the best during the highest frost resistance of plantlets, when they were just entering endodormancy and the amounts of all measured saccharides and proline were relatively high and water content relatively low. The last regrowth after liquid nitrogen treatment was detected (46%) in the 14th week, when the proline content was still high, although decreasing, and the water content was still low and slowly increasing. The success of cryopreservation was evidently strongly linked to the frost resistance, endodormancy and amounts of the associated crucial substances in plantlets. A high regrowth after cryopreservation despite of endodormancy can be caused by exposure to liquid nitrogen which caused bud break in normally endodormant buds (Cox and Stushnoff 2001).
Conclusions Low temperatures induced dormancy in in vitro culture of apple cv. Greensleeves. The endodormancy occurred in the 8th–11th week of cold hardening, proceeded for 4 weeks, and was characterized by distinct depression in creating new sprouts (less than 50% of plantlets created sprouts after transfer to growing conditions), their lengths and numbers of leaves. The regeneration ability of plantlets after cryopreservation and their frost resistance corresponded with the period of dormancy and a higher proline, glucose, fructose, raffinose and stachyose contents, and lower water content in plantlet tissues. Maximal regeneration ability after cryopreservation was proven in the 6th– 8th week of cold hardening and correlated with the
1191
maximal frost resistance of the plantlets and high values of all examined substances in their tissues, whereas the plantlets were able to regenerate after cryopreservation until the 14th week of cold hardening. The success of cryopreservation was fully linked to the high frost resistance of the plantlets and to the amounts of associated accumulated high energy substances in the plantlets. Acknowledgments This work was supported by 0002700604 project of the Ministry of Agriculture of the Czech Republic and the OC08062 project financed by the Ministry of Education, Youth and Sport of the Czech Republic. Authors are thankful to Nadˇa Kantnerova´, student of Czech Agricultural University, Prague who assisted in experimental part of in vitro cultures, to Dr. Helena Lipavska´ and Dr. Hana Konra´dova´, Charles University, Prague for estimations of saccharides content in plantlets and to Prof. Paul T. Lynch, University Derby, UK providing us by Greensleeves apple cultivar.
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