Planta (Bdrl.) 122, 105--107 (1975) 9 by Springer-Verlag 1975

Short Communication

Cold-induced Sweetening of Tissue Cultures of Solanum tuberosum L. C. J. Pollock* and T. ap g e e s Botany School, University of Cambridge, Downing Street, Cambridge CB2 3EA, U.K. R,eceived 13 November; accepted 18 November, 1974

Summary. Callus cultures derived from tubers of Solarium tuberosum L. cv. ':Record" were transferred from a complete medium to a maintenance medium that lacked sugar and were then incubated at 2 ~ or 25 ~ Incubation at 2 ~ for 5 days led to an accumulation of sucrose. This accumulation did not occur at the expense of stored reducing sugars and was not found at 25~ Such cultures could be used for the study of cold-induced sweetening in potatoes. P o t a t o t u b e r s a c c u m u l a t e sucrose and hexose if t h e y are stored at 2-8 ~ Much is k n o w n of the economic i m p o r t a n c e and t h e general physiological characteristics of this sweetening, b u t little is k n o w n about t h e m e c h a n i s m s i n v o l v e d (Isherwood, 1973). This ignorance is due, in part, to the difficulties i n v o l v e d in using labelled s u b s t r a t e s t o follow, in vivo, t h e changes t h a t t a k e place during sweetening. U n i f o r m and reproducible labelling of i n t a c t tubers u n d e r physiological conditions has not been reported. T h e p r o f o u n d effects t h a t slicing has on t h e m e t a b o l i s m of t u b e r tissue preclude the use of disks or other small pieces of tissue as experim e n t a l m a t e r i a l (Laties, 1963; L a t i e s e t a l . , 1972). If the p h e n o m e n o n of sweetening could be m a d e to occur in tissue cultures of p o t a t o t u b e r s t h e n such cultures could be used for s t u d y i n g t h e effects of cold on t h e m e t a b o l i s m of labelled substrates during sweetening. W e wish to report t h a t we h a v e m a d e p o t a t o tissue cultures a c c u m u l a t e sucrose at low t e m p e r a t u r e s . Callus cultures were obtained from explants of mature tubers of Solanum tuberosum L. ev. "Record ". Calli were grown in boiling tubes in the dark at 25 ~ on 25 ml of the solid medium described by Ingram and Robertson (1965). For the experimental treatment each culture was transferred to a filter-paper raft supported in a boiling tube by 25 ml of a liquid, sugarfree, maintenance medium that had the composition of the standard salts medium described by Heller (1953). Sugar analyses were carried out on individual cultures. Five replicate cultures were analysed for each treatment. At the end of the incubation each culture was harvested, washed successively with 3 lots of 1OOvolumes of 20 mM Ktt2PO 4 (pH 5.5), blotted dry, weighed, and then killed with boiling 80% (v/v) aqueous ethanol. The methods used to extract and assay sucrose and reducing sugars have been described (Lyne and ap Rees, 1971). W e used cultures of t h e v a r i e t y " t ~ e e o r d " because we h a v e m e a s m e d the e x t e n t and t i m e course of the appreciable sweetening which occurs w h e n tubers of this v a r i e t y of p o t a t o are stored at 2 ~ (Pollock and ap Rees, 1975). I n each

* Present address: Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth Su U.K.

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C.J. Pollock and T. ap l~ees Table l. Sugar contents of tissue cultures maintained at 2~ or at 25~ Each value is the mean • standard error of estimates from 5 different cultures. Incubation temperature (~

-

-

25 25 25 25 2 2 2 2

Days after transfer to maintenance medium

Sugar content (mg/g F.wt.) Sucrose

Reducing sugars

0 3 5 7 9 3 5 7 9

0.63 -4-0.10 0.39 ::k0.06 0.29 j= 0.07 0.27 • 0.08 0.23 ::k 0.05 0.35 =[:0.08 1.20 =~0.13 0.66 :~ 0.22 0.40 j : 0.17

3.31 :=k0.14 1.26 ~: 0.24 0.85 =]=0.08 0.62 • 0.I1 0.25 =i=0.03 2.13 • 0.16 2.10 • 0.]6 1.44 =E0.10 1.14 • 0.19

e x p e r i m e n t we set u p 50 replicate cultures on the full growth m e d i u m a n d grew t h e m for 8 weeks. D u r i n g this time t h e fresh weight of t h e cultures increased from 0~2 to 1.0 g. W e t h e n transferred the cultures to m a i n t e n a n c e m e d i u m a n d i n c u b a t e d half of t h e m at 2 ~ a n d half at 25 ~ in the dark. We followed the changes in the c o n t e n t s of sucrose a n d reducing sugars over the n e x t 9 days. We chose to s t u d y the effects of cold on cultures on sugar-free m e d i u m i n order to avoid complications t h a t m i g h t arise from a n y differential effect of cold on the relative rates of u p t a k e a n d m e t a b o l i s m of sugars. The changes t h a t occurred in the sugar c o n t e n t s of the cultures are shown i n Table 1. The cultures i n c u b a t e d at 25 ~ serve as a control. I n these cultures the sucrose c o n t e n t fell steadily t h r o u g h o u t the 9 days. There was a sharper a n d more noticeable fall in the c o n t e n t of reducing sugars. I t is probable t h a t these changes represent the c o n s u m p t i o n of storage sugar caused b y the transfer of the cultures to the m a i n t e n a n c e medium. Over the 9-day e x p e r i m e n t a l period as a whole, there was also a fall in the sugar c o n t e n t s of the cultures kept at 2 ~. This fall was less t h a n t h a t observed at 25 ~. This would be expected from the lower rate of m e t a b o l i s m of the colder cultures. The cardinal feature of the results i n T a b l e 1 is t h a t i n c u b a t i o n at 2 ~ led to a 3 4 fold increase in sucrose c o n t e n t b e t w e e n days 3 a n d 5. After 5 days on the m a i n t e n a n c e m e d i u m at 2 ~, the sucrose c o n t e n t of the cultures was double t h a t f o u n d at the time of transfer to the m a i n t e n a n c e m e d i u m . This cold-induced n e t synthesis of sucrose coincided with a cessation in the decline in reducing sugars. T h u s t h e increase i n sucrose did n o t occur at the expense of pre-existing storage reducing sugars. Therefore the sucrose synthesis resulted in a n e t increase in the t o t a l c o n t e n t of sugars b e t w e e n the t h i r d a n d fifth d a y after transfer to 2 ~. The percentage increase in sucrose c o n t e n t t h a t occurred in the cold cultures is comparable to t h a t f o u n d w h e n t u b e r s of this v a r i e t y are stored at 2 ~. I t is also of interest to note t h a t appreciable a c c u m u l a t i o n of sucrose is first detected 5 days after transfer of the t u b e r s to 2 ~ (Pollock a n d ap Rees, 1975). Thus the general characteristics of the cold-induced a c c u m u l a t i o n of sucrose i n the tissue cultures are similar to those described for the sweetening of whole tubers. We

Cold-induced Sweetening

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have f o u n d t h a t labelled hexose is readily a n d reproducibly metabolized b y our cultures at 25 ~ a n d at 2 ~. Our results suggest t h a t the difficulties i n using feeding e x p e r i m e n t s in the s t u d y of, at least the initial, phase of sweetening could be overcome b y the use of the above tissue cultures. Our results also suggest t h a t sweetening is due to some f u n d a m e n t a l p r o p e r t y of potato t u b e r cells r a t h e r t h a n to a n y p r o p e r t y of the i n t a c t tuber. We thank Walkers Crisps for their advice and their gifts of potatoes. C. J. P. thanks the Managers of the Broodbank Fund for a fellowship.

References Heller, 1%.: Recherches sur la nutrition min~rale des tissus v~g~taux cultiv~s in vitro. Arm. Sci. Nat. Bot. 14, 1-223 (1953) Ingram, D. S., Robertson, N.F.: Interaction between Phytophthora in/estans and tissue cultures of Solanum tuberosum. J. gem Microbiol. 40, 431-437 (1965) Isherwood, F. A. : Starch-sugar interconversion in Solanum tuberosum. Phytoehemistry 12, 2579-2591 (1973) Laties, G. G. : Control of respiratory quality and magnitude during development. In: Control mechanisms in respiration and fermentation, p. 129 155, Wright, B., ed. New York: Ronald Press 1963 Laties, G. G., Hoelle, C., Jacobson, B. S.: a-Oxidation of endogenous fatty acids in fresh potato slices. Phytochemistry l l , 3403-3411 (1972) Lyne, R. L., ap Rees, T. : Invertase and sugar content during differentiation of roots of Pisum sativum. Phytochemistry 10, 2593-2599 (1971) Pollock, C. J., ap Rees, T. : Activities of enzymes o~ sugar metabolism in cold-stored tubers of Solanum tuberosum. Phytochemistry, in press (1975)

Cold-induced sweetening of tissue cultures of Solanum tuberosum L.

Callus cultures derived from tubers of Solanum tuberosum L. cv. "Record" were transferred from a complete medium to a maintenance medium that lacked s...
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