Uptake and translocation of 45Ca2+ in sour cherry shoots cultivated in vitro

Scientia Horticulturae, 40 (1989) 35-41 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

35

U p t a k e and T r a n s l o c a t i o n of 4 5 C a 2 + in Sour Cherry Shoots Cultivated in Vitro BOZENA BORKOWSKA and LECH MICHALCZUK Institute of Pomology and Floriculture, 96-100 Skierniewice (Poland) (Accepted for publication 29 November 1988)

ABSTRACT Borkowska, B. and Michalczuk, L., 1989. Uptake and translocation of 4~Ca2÷ in sour cherry shoots cultivated in vitro. Scientia Hortic., 40: 35-41. Uptake of radioactive Ca from the medium and its distribution in stems and leaves of sour cherry cultures during the course of their development was investigated. Single shoots of Prunus cerasus (L.) cultivar 'Schattenmorelle'were placed on a rooting medium supplemented with 4~CaCl~. They were cultured for 5 weeks and the radioactivity taken up was followed during this period. Radioactivity of cultured shoots was detected as early as 2 days after excision. Total radioactivity rose continuously, reaching a high value at the end of the experiment. Specific activity was negatively correlated with intensity of growth. Ca absorption was improved by the formation of roots. The ratio of leaf to stem activity increased with the period of culturing. The content of Ca in the lower part of the stems was higher than in the upper parts. Similarly, the higher the position of the leaves, the lower their activity. Leaves with necrotic spots and vitrified leaves showed lower specific activity than healthy ones. Keywords: calcium; necrosis; Prunus cerasus; roots; tissue culture; vitrification. Abbreviations: IBA-NH4 = ammonium salt of indole-3-butyricacid; MS = Murashige and Skoog's medium (1962).

INTRODUCTION

Physiological disorders such as necrosis and vitrification frequently affect plant material cultured in vitro (Debergh, 1983). Apex and leaf necrosis seem to be analogous to physiological disorders of apple and tomato fruits or leaves of lettuce, caused by a low concentration of Ca in tissue. A comparison of nutrient levels in apple explants with those of leaves from apple trees indicated that Ca, Mg and Cu values in cultured material were below the normal range (Shear and Faust, 1980). The Ca concentrations in callus cultures of carrot and other plants are also lower than in intact plants (Walkinshaw et al., 1972).

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36 Since some abnormalities observed in cultures are suspected to be caused by Ca deficiency, we investigated the uptake and distribution in stems and leaves of radioactive Ca during the development of sour cherry cultures. MATERIALSAND METHODS Single shoots of P r u n u s cerasus (L.) cultivar 'Schattenmorelle' about 1.5 cm long were separated from proliferated cultures and placed on a rooting medium containing 1/2 MS major salts (Murashige and Skoog, 1962 ), full strength of microelements and vitamins with 2 mg l-1 IBA (Serva) dissolved in ammonium (IBA-NH4). The medium was solidified with Bacto Difco agar, 6.5 g l - 1 and pH was adjusted to 5.3 before autoclaving. 45CAC12 (specific activity 80 MBq mg-1 Ca), 40 MBq in total, was added to 0.5 1 of the rooting medium. Final concentration of Ca was 220 mg l - 1. Each test tube contained 10 ml of the medium. The shoots were cultured at 24 ° C and 16 h of fluorescent light at 80 W m -2. They were analyzed after 2, 4, 7, 9, 16, 25 and 32 days. From the entire population, the plantlets growing fast (the largest) and those with weak growth (the smallest) were chosen for analysis. At each sampling time, 10 vigorous and 10 weak plantlets were selected. Each plantlet was analyzed separately. The parts growing above the agar surface were divided into stem (upper and lower part separately) and individual leaves. Precautions were taken to avoid contamination by the medium. Each leaf and piece of stem was weighed separately and after solubilisation in 1 N HC1 at 80°C for 10 h, 10 ml of a scintillation cocktail (7 g PPO, 100 mg PoPoP in 1 1 of dioxane) was added. The radioactivity was measured in an Intertechnique NBAC SL 40 liquid scintillation counter. Radioactivity was calculated per mg of fresh weight (specific activity) or presented as total. The experiment was repeated twice. The results were subjected to statistical analysis. RESULTS Characterization of shoot development. - During the first week of culturing, no signs of shoot development were seen. During the second and third weeks, the preexisting leaves enlarged. Active growth of the apex, formation of new leaves and the first signs of rhizogenesis occurred about 3 weeks after starting the cultures. One week later the growth of roots was fast, but elongation of shoots was slow, even though new leaves were formed. The increase in plantlet weight was mainly owing to a pronounced enlargement of leaves. The weight of plantlets with low rate of growth increased slowly, and finally reached only 40-50 mg. The weight of those with fast rate of growth rose rapidly, reaching about 500 mg (Figs. 1 and 2 ). After the entire period of cultivation the average number of leaves per shoot increased from 3.7 to 5.9. The length of the shoots

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increased only 2-4 m m . Differences in i n t e n s i t y of leaf a n d s h o o t g r o w t h were n o t i n f l u e n c e d b y r o o t f o r m a t i o n . T h e r e were some s h o o t s with signs of l e a f / apex necrosis a n d a few s h o o t s w i t h vitrified leaves.

Absorption of Ca byplantlets. - R a d i o a c t i v i t y was d e t e c t e d in p l a n t l e t s as early as 2 days a f t e r excision. C o m b i n e d t o t a l activity in b o t h vigorous a n d weak

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Fig. 3. Increase of total radioactivityof the plantlets during the growth period. TABLE 1 The effect of roots on the specific radioactivity of 4~Ca2+ (Bq mg 1fresh weight) in leaves and stems of sour cherry Leaves

Stems

With roots

Without roots

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Values followedby the same superscript do not differ at P ~<0.05. plantlets rose continuously and reached a high value at the end of the experiment (Fig. 3 ). Specific activity per mg of fresh weight depended on the rate of growth. For slow growing plantlets, the specific activity constantly increased. Whereas in fast growing ones the specific activity decreased with time (see Figs. 1 and 2). Ca absorption was improved by roots, the presence of which caused higher accumulation of 45Ca2 + in leaves (Table 1). Distribution of Ca in plantlets. - From the beginning of the culture period ra-

dioactivity was detected in both stems and leaves. However, during the first 4 days of culturing, most of the activity (over 80% ) was found in the stems. Later, the leaf: stem activity ratio rose, and after 16 days relatively more radioactivity could be detected in the leaves t h a n in the stems (Fig. 4 ). Ca content in the lower part of the stems was higher and its level increased faster t h a n in the upper part, including the apex, although not significantly.

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Fig. 5. Specific radioactivity of lower and upper parts of stem separated from plantlets during the growth period. X - - X =upper part of stem; X--- X =lowerpart of stem. The radioactivity i n t h e upper part of stems stopped increasing after 16 days, whereas in the lower part it rose to the end of the experiment (Fig. 5 ). After 2 days of culture, the radioactivity in leaves was low and was similar for each leaf position from the base to the apex. Later, the radioactivity of all leaves increased and a distinct gradient was observed. The higher the leaf position, the lower the activity (Fig. 6).

Activity of plantlets with necrotic and vitrified leaves~apices. - Specific activity

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Fig. 6. Specific radioactivity of individual leaves, numbered from base (1) to apex (6) and measured after 2, 9, 16 and 25 days of culture. × - - × =2 days; O - - - O --9 days; [3"" F7= 16 days; • . . . . • = 25 days. TABLE 2 Specific radioactivity of 45Ca2+ (Bq mg -1 fresh weight) of apices and leaves of sour cherry in vitro, showing healthy, necrotic and vitrified appearance. Not statistically analyzed Apices

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of h e a l t h y apices was similar to t h a t of necrotic ones. L e a v e s with n e c r o t i c spots a n d especially vitrified leaves s h o w e d a t e n d e n c y to lower specific activity t h a n h e a l t h y ones ( T a b l e 2). DISCUSSION Calcium a c c u m u l a t i o n , e x p r e s s e d b y i n c r e a s e d r a d i o a c t i v i t y of t h e tissue, was d e t e c t e d 2 days a f t e r s t a r t i n g t h e s h o o t culture. T h i s is in good a g r e e m e n t with d a t a of D e b e r g h ( 1983 ), who f o u n d a decrease o f Ca c o n c e n t r a t i o n in the m e d i u m o n t h e s e c o n d day of culture. A f u r t h e r increase of Ca c o n t e n t dep e n d e d m a i n l y on t h e g r o w t h i n t e n s i t y of t h e e x p l a n t s a n d root f o r m a t i o n . In fast developing p l a n t l e t s t h e rate of g r o w t h was h i g h e r t h a n t h e rate o f Ca

41 accumulation, thus the relative Ca content was lower t h a n in shoots with slower growth rate. Formation of roots increased significantly the accumulation rate of Ca in plantlets. Faust and Shear (1973) have reported t h a t in intact apple seedlings the calcium was transported mainly by phloem, whereas in de-rooted seedlings it was transported mainly by xylem. Since sour cherry shoot explants should behave similarily to de-rooted seedlings, the xylem pathway of Ca translocation should also be expected. Further, since the xylem transport depends mostly on the transpiration stream, in in vitro culture very small translocation rates may be expected. The root formation changes the translocation pathway of Ca to phloem, which occurs mostly at the expense of metabolic energy and is independent of the transpiration rate - thus more effective in in vitro conditions. Calcium deficiency is suspected to cause so called "physiological disorders" as tipburn of lettuce and brown-heart in brassicas, and also several disorders of apple and tomato fruits (Ferguson, 1984). A lower level of Ca in leaves with necrosis t h a n in healthy ones, as well as the occurrence of this disorder at the apex, where Ca concentration is the lowest, could suggest t h a t the mechanism of these disorders is also calcium-related. However, to confirm this hypothesis, more detailed study would be required. ACKNOWLEDGEMENTS We t h a n k K. Galer and D. Salamonczyk for technical assistance.

REFERENCES Debergh, P.C., 1983. Effects of agar brand and concentrationon the tissue culture medium. Plant Physiol., 59: 270-276. Faust, M. and Shear, C.B., 1973. Calciumtranslocationpattern in apples. The 3rd Symposiumon accumulation and translocation of nutrients and regulators in plant organisms, Warszawa, Jablonna, Skierniewice,Brzezna, Krakow,14-18 May, pp. 423-436. Ferguson, I.B., 1984. Calciumin plant senescence and fruit ripening. Plant Cell Environ., 7: 477489. Murashige,T. and Skoog,F., 1962. A revisedmediumfor rapid growthand bioassayswith tobacco tissue cultures. Physiol. Plant., 15: 473-497. Shear, C.B.and Faust, M., 1980. Nutritional rangesin deciduoustree fruits and nuts. Hortic. Rev., 2: 142-163. Walkinshaw,C.W.,Johnson, P.W. and Venketeswaran,V., 1972. Elemental abundances in callus tissues of carrot, pine, rice, soybeanand tobacco. In Vitro, 7: 391-396.