Frost sensitiveness of chicory roots
(
Cichorium intybus
L.)
V. Neefs
*, S. Leuridan, N. Van Stallen,
M. De Meulemeester, M.P. De Proft
Faculty of Agricultural and Applied Biological Sciences, Department of Applied Plant Sciences, Laboratory for Plant Culture, Katholieke Universiteit Leuven, Willem de Croylaan 42,
B-3001 Heverlee, Belgium
Accepted 16 February 2000
Abstract
This study was conducted to determine if chicory roots (Cichorium intybusL. var.foliosum) can be stored at temperatures belowÿ18C. Roots from cultivar Focus, produced in hydroponics on two different arti®cial substrates, were stored atÿ1, ÿ2.5,ÿ5 andÿ78C. After storage at sub-zero temperatures, roots with highest dry weight percentage showed highest chicon production and highest organogenesis capacity. Moreover, the electrical resistance of those roots never decreased signi®cantly during storage, while roots with a lower dry weight percentage had reduced electrical resistance after storage atÿ78C. Decrease in electrical resistance was detected before frost damage became visible. Thus, measuring the electrical resistance of root tissue can be used to predict frost damage. The most distinct frost damage symptoms were `water soaking' and browning of the vascular bundles. According to these results, it can be concluded that storage temperatures belowÿ18C, but aboveÿ78C, are not necessarily fatal for chicory roots: roots with relatively high dry weight percentage are best able to withstand low storage temperatures.#2000 Elsevier Science B.V. All rights reserved.
Keywords: Chicory roots; Frost damage; Dry weight percentage; Plant electrical resistance; Storage
1. Introduction
Chicory,Cichorium intybusL. var.foliosum, is a biennial plant. After the ®rst growing season chicory roots are harvested, cold stored and forced in the dark.
Scientia Horticulturae 86 (2000) 185±195
*
Corresponding author. Tel.:32-1632-2390; fax:32-1632-2966. E-mail address: veerle.neefs@agr.kuleuven.ac.be (V. Neefs).
Root reserves are remobilised during the forcing process to support the growth of an etiolated head, the chicon, which is marketed. To ensure a continuous supply on the market, cold storage of chicory roots is a requirement. For long storage, chicory roots are kept at ÿ0.5 to ÿ18C (Scheer, 1997). Nevertheless, lower
storage temperatures would improve storage duration, if frost damage can be avoided. In the present work, storage of chicory roots at four different sub-zero temperatures has been examined to determine when frost damage occurs.
2. Materials and methods
2.1. Plant material: growth and storage conditions
Chicory seeds (Cichorium intybus L. var Foliosum cv. `Focus') were sown in hydroponics (May 1997) on two different arti®cial substrates. One substrate (50/ 50 substrate) was a mixture of 50% hydrophilic (Grodan type 012/519) and 50% hydrophobic rock wool (Grodan BU 20 granulate). The other substrate (100/ Argex substrate) was pure hydrophilic rock wool with clay granules (Argex, 0.5± 1 cm diameter) underneath to avoid root waterlogging. Plants were fertigated three times a day (at 9.00, 12.00 and 16.00 hours) with 100 ml nutrient solution per root. A solution with 50 mval/l ionic strength (De Rijck et al., 1993) was used for the ®rst 6 weeks, thereafter a solution with 16 mval/l ionic strength (solution number 4 (De Rijck et al., 1993)). Because plants can be made more tolerant to sub-zero temperatures by stress treatments (Biddington and Dearman, 1988; Gusta et al., 1996), plants received only 100 ml nutrient solution once in a day, at 9.00 hours the last 6 weeks before harvest. Roots were harvested at the beginning of October 1997 and stored at18C for 20 days. Afterwards, they were placed in
four different storage rooms where root temperatures of, respectively, ÿ1.06, ÿ2.59, ÿ5.00 and ÿ6.898C were reached within 14 days. Temperatures were
continuously recorded (Escort Junior, VEL) over a 76 day experimental storage period.
2.2. Dry weight percentage
Dry weight percentage (% DW) of root tissue was determined by drying fresh root tissue for 1 week at 808C.
2.3. Evaluation of stored roots
After 34 and 76 days storage 30 roots of each substrate were taken from each temperature treatment for analysis. Because 1 week thawing at a low positive temperature is advised (Scheer, 1997), roots were placed at 28C. Afterwards,
roots were conditioned at room temperature for 24 h and evaluated. For the evaluation, roots were divided into two parts by a transverse cut 3 cm below the apex. The upper part was used for chicon production, the lower part for all other evaluations.
2.3.1. Chicon production
After disinfection with benomyl (0.5 g/l), the upper parts of the roots were forced on white sand and tap water (dark, 168C). After 14 days chicory heads (chicons) were harvested. Weight and core length of the chicons were measured. Chicons produced on control roots (kept 20 days at18C) were evaluated the same way.
2.3.2. Plant electrical resistance
A plant electrical resistance meter (Resamet RM-90, Nieuwkoop B.V., Aalsmeer) was used to measure the electrical resistance (ER) of central parenchyma, vascular bundles and cortical parenchyma on the transverse section of the lower root parts. Frost damage of plant tissues is associated with a decrease in ER (Zhang and Willison, 1990, 1992a,b; Prive and Zhang, 1996).
2.3.3. Organogenesis capacity
A 1 cm thick root disc was taken from each root 5 cm below the apex. After disinfection with benomyl (0.5 g/l), these root discs were placed on sterile sand and tap water in miniature greenhouses (45 cm30 cm15 cm) under long day
conditions (16 h light, 228C). After 18 days, organogenesis was evaluated by counting the number of shoots and weighing the proliferated mass of the cambium ring.
2.4. Statistical analysis
Each time all measurements were done on 30 roots. The in¯uence of the combined effect of substrate, storage duration and storage temperature was tested on all evaluation parameters except for the parameters expressed in percentage. To ®nd signi®cant differences in ER the Duncan's Multiple Range Test was used. In the case of chicon evaluation, the number of roots that produced a chicon determined the sample size of the other evaluation parameters (chicon weight, core length). Thus, the sample size itself depends on the treatment. Because the differences in sample size were large, it was necessary to use the Tukey's Studentised Range Test to determine the in¯uence of the combined effect on chicon weight and core length. The same remark is valid for the evaluation of root disc organogenesis capacity. In this case the proportion of root discs that produced shoots determined the sample size. For parameters expressed in percentage, the in¯uences of substrate, storage duration and storage temperature were tested separately with the Chi-square-test. Only signi®cant in¯uences with a
strong correlation (uncertainty coef®cient symmetric, UCS>0.1) are mentioned. For each statistical analysis a signi®cance level of 5% was maintained.
3. Results
3.1. Temperature progress during cooling
Harvested roots were ®rst kept at 18C for 20 days. Afterwards they were
placed in four different storage rooms. It took about 2 weeks before the roots reached their ®nal temperatures ofÿ1.06,ÿ2.59,ÿ5.00 andÿ6.898C, respectively.
3.2. Characterisation of the roots
Roots cultivated on the 50/50 substrate were smaller (mean diameter: 33 mm, mean weight: 133 g) than roots from the 100/Argex substrate (mean diameter: 37 mm, mean weight: 157 g). Reducing the amount of hydrophilic rock wool (50/ 50 substrate) resulted in roots with a signi®cantly (Duncan's Multiple Range Test,
a0.05) lower dry weight percentage (28.3% DW) than those that were grown on
100% hydrophilic rock wool (100/Argex substrate, 30.4% DW).
3.3. Evaluation of stored roots
3.3.1. Chicon production
The in¯uence of storage temperature and duration on the number of roots still producing a chicon was signi®cant with a strong correlation (UCS, respectively, 0.179 and 0.103). Cold storage atÿ18C and lower reduced the number of roots
which are capable to form a chicon (Table 1). Roots grown on the 50/50 substrate did not produce any chicons after 34 days atÿ78C and 76 days atÿ5 orÿ78C.
On the other hand, roots cultivated on the 100/Argex substrate still produced chicons in all cases, but the number of roots with a chicon also decreased with decreasing temperature and increasing storage duration. Roots with higher % DW were more resistant to low temperature treatments.
The weight of chicons produced on control roots (20 days stored at18C) grown
on the 50/50 substrate was signi®cantly higher than the weight of all chicons produced on roots of both substrates after 76 days cold storage. The weight of all chicons produced on roots from the 50/50 substrate after 34 days cold storage was signi®cantly higher than the weight of all chicons produced on roots from the same substrate but after 76 days cold storage. The weight of chicons produced on roots, grown on the 100/Argex substrate, after 34 days storage at, ÿ1, ÿ5 and ÿ78C,
respectively, was signi®cantly higher than the weight of chicons grown on roots from the same substrate, but after 76 days storage at the same temperatures.
The chicon length was in¯uenced by the cold treatment: frost sensitive roots produced smaller chicons (not exactly measured), which had relatively smaller cores. To use the core length as a frost damage parameter it is necessary to express it as a ratio to the chicon length. Because chicon length was not measured, core lengths could not be compared.
Neither substrate, nor storage duration, nor storage temperature had a signi®cant in¯uence with a strong correlation on percentage white chicons, percentage closed chicons or percentage chicons with white core (data not shown). Nevertheless, there was a weak trend (UCS<0.05) that the proportion of white chicons and chicons with white core was lower after 76 days than after 34 days cold storage.
3.3.2. Plant electrical resistance
Roots grown on the 50/50 substrate reached signi®cantly lowest ER after storage at the lowest temperature (ÿ78C), with exception of the ER of vascular
bundles after 34 days cold storage (Table 2). After both the storage times, roots grown on the 100/Argex substrate did not show any signi®cant difference in ER between different storage temperatures. This means, those roots did not show a distinct decrease in ER, not even after storage atÿ78C. The ER of the different
Table 1
Evaluation of chicon production on roots from cv. Focus, after 20 days at18C followed by a cold treatment of 0, 34 and 76 days at different temperatures
Root
Weight of chicon (g) Core length (mm)
Mean Tukey Mean Tukey
Table 2
ER of different tissues of roots from cv. Focus, after 20 days at18C followed by a cold treatment of 34 and 76 days at different temperatures
Root
Central parenchyma Vascular bundles Cortical parenchyma
Mean Duncan Mean Duncan Mean Duncan
Table 3
Evaluation of shoot growth on root discs from cv. Focus, after 20 days at18C followed by a cold treatment of 34 and 76 days at different temperatures
Root
No. of shoots/root disc Weight of proliferated mass (g)/root disc
root tissues, from roots grown on substrate 50/50, after 34 and 76 days, respectively, with storage temperatureÿ78C were signi®cantly lower than those
from roots grown on the 100/Argex substrate after the same storage conditions. There was no clear distinction between the ER of the different root tissues.
3.3.3. Organogenesis capacity
The in¯uence of storage temperature on shoot growth was signi®cant with a strong correlation (UCS0.113). After 34 days cold storage at least 83% of all
root discs, with the exception of those from roots grown on the 50/50 substrate and stored atÿ78C, still produced shoots (Table 3). The mean number of shoots
and mean weight of proliferated mass were the same for roots stored at all temperatures (after 34 days). After 76 days at ÿ5 and ÿ78C roots from both
substrates yielded poor results. Nevertheless, at least 57% of root discs from roots cultivated on the 100/Argex substrate and kept at any temperature showed shoot growth.
The number of shoots was directly proportional to the expanded cambium: the higher the weight of proliferated mass, the more was the number of shoots. Because of this, the ratio of number of shoots to weight of proliferated cambium is more or less constant and not adequate to express the activity of the root discs.
4. Discussion
All biological living tissues have limits, like the lowest temperature at which they still have a potential to survive and recover. Chicory plants in the ®eld sometimes are exposed to very low temperatures (ÿ108C) for several days or
weeks. The next season some of the roots will form in¯orescences indicating they survived those winter ®eld conditions. On the other hand, roots kept in commercial cold storage for several months almost never survive ÿ38C. The
question is whether those roots have got the chance to adapt to low temperatures. It is important that roots are hardened enough to become more capable to withstand different kinds of stresses. For this research, two lots of chicory roots, with signi®cantly different dry weight, were used to study the in¯uence of percentage dry weight on frost sensitiveness. Roots with the highest dry weight (30.36%) still produced chicons after cold treatments, whereas roots with a lower dry weight (28.27%) did not. Their root discs also showed more retention of organogenesis capacity than those from roots with lower dry weight. Moreover, the ERs of the different root tissues with higher dry weight never showed a signi®cant decrease with decreasing storage temperature or increasing storage duration, while those of roots with a lower dry weight did. Therefore it can be concluded that roots with a higher dry weight are more frost tolerant: they can survive a low temperature exposure (above ÿ78C) for some weeks.
Because of the slow root cooling, ice formation is typically initiated in the extracellular medium (plant cell walls should be considered as part of the extracellular medium) while the cells themselves remain unfrozen (Taylor, 1987; Thomashov, 1998). A gradient in water potential is established which causes the water to ¯ow from the cell to the exterior where it subsequently freezes in an attempt to achieve an osmotic equilibrium (Mazur, 1969; Grout and Morris, 1987). If cooling is slow enough to permit this process to be prolonged, the cells could become so extensively dehydrated that no intracellular freezing would take place. Intracellular freezing is widely believed to be lethal to cells (Taylor, 1987). A decline in osmotic potential in the cells, per se, might enhance freezing resistance (Biddington and Dearman, 1988). Because a higher dry weight is related to higher solute concentration (lower osmotic potential) roots with a higher dry weight can withstand longer the intracellular freezing.
Because the roots were thawed very slowly (1 week at28C followed by 24 h
at room temperature), it must also be remarked that recrystallisation is very harmful to cells (Mazur, 1969; Taylor, 1987). Smaller ice crystals become unstable, and, as they melt, their molecules are transferred to large crystals, which increases the risk of mechanically induced cell membrane damage. This kind of damage became visible by water soaking and brown colouring of the vascular bundles (Figs. 1B and 2B). Severe damage resulted in totally water soaked roots
Fig. 1. Root discs from cv. Focus, after 76 days storage atÿ78C. A: totally water soaked root tissue; B: brown vascular bundles.
(Fig. 1A). Moline (1987) also established that ¯eshy roots often show no discoloration except for the vascular tissues.
By measuring plant ERs frost damage in chicory roots can be demonstrated clearly while visible symptoms are not convincing. Thus, measuring ER can be used to predict frost damage. As a result of enhanced membrane permeability, electrolyte leakage to extracellular space causes an increase in extracellular electrical conductivity (i.e. a decrease in resistance). Chicory roots with lower dry weight reached their signi®cantly lowest ER after storage at the lowest temperature.
It must be emphasised that the lower chicon production on roots, which were stored longer and at lower temperatures, was not always a direct consequence of destruction of the apex. Mostly, the apex appeared to be still intact (normal green colour) (Fig. 2A) but did not develop into a chicon. This observation excludes water loss at the apex as the cause of growth inhibition. Infections coming out of frozen foliage, which could not be prevented by benomyl treatment, must be considered as a possible negative effect on chicon development.
Acknowledgements
This research was funded by the IWT (Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie) Ph.D. scholarship of the ®rst author and by the DG-6 project (5820A) of the Ministerie van Middenstand en Landbouw.
Fig. 2. Roots from cv. Focus, after 34 days storage at ÿ58C. A: green intact apex; B: brown vascular bundles.
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