Scientia Horticulturae, 26 (1985) 183--189 183 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

STARC, H C O N T E N T O F F R E E Z E - D R I E D A N T H E R S A N D a - A M Y L A S E A C T I V I T Y O F T H E I R E X T R A C T S AS C R I T E R I A T H A T D R Y - S T O R E D BULBS ( T U L I P A G E S N E R I A N A , L.) C U L T I V A R ' A P E L D O O R N ' H A V E BEEN E X P O S E D TO 5°C

N. GOR:[N and F.T. HEIDEMA

Sprenger Institute, P.O. Box 1 7, 6700 AA Wageningen (The Netherlands)

(Accepted for publication 18 January 1985)

ABSTRACT

Gorin, N. and Heidema, F.T., 1985. Starch content of freeze-dried anthers and a-amylase activity of their extracts as criteria that dry-stored bulbs (Tulipa gesneriana, L.) cultivar 'Apeldoorn' have been exposed to 5°C. Scientia Hortic., 26: 183--189.

Starch and sucrose were estimated selectively in freeze-dried anthers from tulip bulbs pre-cooled at 5°C for 12 weeks and from a control batch maintained at 17°C. a-Amylase activity in anther extracts was also estimated. Based on data from our own trials (1981, 1982 and 1983) and from 18 batches from typical bulb-producing regions in The Nether- lands (year 1983), sucrose was discarded as indicator. The following tentative criteria are proposed for detecting whether dry bulbs cultivar 'Apeldoorn' have been exposed to 5°C: starch content of freeze-dried anthers should exceed 10% and specific activity of a-amylase should be below 0.16 (arbitrary unit per mg protein). These criteria are not indicative of the duration of the 5°C period.

Keywords : a-amylase; anthers; pre-cooling; starch ; tulip bulbs.

INTRODUCTION

This work fo rms pa r t o f a p r o j e c t a imed at f o r m u l a t i n g c r i t e r ia fo r as- ce r t a in ing w h e t h e r tu l ip bulbs have been spec ia l ly p re -coo led , i.e. k e p t a t 5°C fo r 12 weeks (De Her togh , 1981) . Spec ia l p re -coo l ing is cr i t ica l fo r bu lbs sent to the U.S.A. to f lower for St. Va l en t i ne ' s D a y (De H e r t ogh et al., 1983) and f o r bu lbs e x p o r t e d to West G e r m a n y and Sweden .

The r e q u i r e m e n t s t h a t the c o n t e n t s o f subs tances mus t fulfi l in o r d e r to be useful as c r i t e r ia to ind ica te w h e t h e r special p re -coo l ing has t a k e n p lace were e s t ab l i shed a rb i t r a r i l y as fo l lows: ( 1 ) t h e d i f f e rence in c o n t e n t s o f sub,; tances at the 12th week o f t r e a t m e n t b e t w e e n p r e - c o o l e d bu lbs and c o n t r o l (17°C) shou ld be g rea te r t h a n 40% ( twice the m a x i m u m co- e f f i c i en t o f va r i a t ion o f 20%); (2) th is d i f f e rence shou ld pers is t dur ing the s imu la t i on o f t r a n s p o r t c o n d i t i o n s (1 week at 17°C) car r ied o u t immed i - a t e ly a f t e r the 12 th week o f the pre-cool ing .

0304-4238]85]$03.30 © 1985 Elsevier Science Publishers B.V.

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The following contents of substances do not satisfy both requirements mentioned above, and consequently could not be used as criteria: content of soluble sugars in whole bulbs (Moe and WickstrOm, 1973) or in shoots (Moe and Wickstr¢m, 1979); starch content in scales and in shoots (Haaland and WickstrCm, 1975); ethylene and carbon dioxide production of whole bulbs (Prince et al., 1982); or ethylene production of anthers (from bulbs 'Paul Richter') (N. Gorin and T. Honkoop, unpublished data, 1982).

Earlier qualitative determinations (J. Beyersbergen, unpublished data, 1965) of starch in anthers from bulbs at the end of a pre-cooling treatment of 12 weeks suggested that their carbohydrate content might indicate whether the bulbs had indeed been pre-cooled for 12 weeks. In a period of 12 weeks, we studied the changes in starch content, a-amylase activity and sucrose content, in anthers from bulbs undergoing pre-cooling and in control bulbs, to ascertain whether any of these substances, singly or in combination, could be used as an indicator of pre-cooling. Using the criteria presented in this work, we could detect that dry bulbs had been exposed to 5°C, but we would not establish the exact duration of the 5°C-treatment.

MATERIALS AND METHODS

A batch of 2500 tulip bulbs of 'Apeldoorn' was obtained from the Bulb Research Centre, Lisse, after they had reached Stage G of flower develop- ment. They were divided into 2 groups of 1250 bulbs, one stored at 5°C (pre-cooling) and the other at 17°C (control). Each group of 1250 bulbs was distributed between 5 stacked storage trays (De Hertogh, 1981) (75 × 50 × 15 cm) whose bases were of wire netting to ensure good ventilation. A 6th empty tray was kept at the bot tom of the stack so that the lowest tray with bulbs did not come into contact with the storeroom floor. Each week (starting 20 September 1982) for 12 weeks, 70 healthy bulbs were randomly sampled from each group and divided into 3 samples of 10 bulbs each for chemical analysis (triplicates for starch determination), one sample of 20 bulbs for preliminary determination of a-amylase and another sample of 20 bulbs for determination of sucrose. At the end of the cooling (12th week), the ambient temperature was raised from 5 to 17°C (to simulate conditions during transport), and during 2 weeks, 9 samplings, each of 15 bulbs, were made.

The 1983 experiments started on 13 September; they were similar to those of 1982 with the following slight differences. The batch was 800 bulbs divided into 2 groups of 400 (one kept at 5°C and the other at 17°C). Each week, 20 bulbs were removed and divided into 2 samples of 10 for biochemical analysis, so that a-amylase activity could be determined in duplicate. Conditions during transport were simulated as previously men- tioned, but the number of bulbs in the sample for chemical analysis was 20 instead of 15. In addition, 18 batches of bulbs from different producers in The Netherlands were given the cooling (5°C) t reatment or the 17°C

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control t rea tment at the Bulb Research Centre, Lisse. In the 12th week, they were brought to our Institute for fixation of material (see below).

For the 1981 experiment , which started on 21 September and was a pre- liminaE.¢ for the starch determination, we used 900 bulbs divided into 2 groups of 450 each, which were given t reatments of 5 and 17°C, respectively. Anther~ from 20 bulbs were used for chemical analysis.

Fixation o f material

Anthers were removed from the bulbs, submerged in liquid nitrogen and freeze-dried (Leybold GT-3, West Germany). The freeze-dried material was stored at - 3 0 ° C until required.

Starch analysis

Freeze-dried anthers (circa 100 mg) were extracted in a mini-Soxhlet apparatus for 6 h with 30 ml of 80% methanol (v/v). The extract was dis- carded and the last traces of methanol were removed by placing the thimbles with extracted anthers in a thermosta ted oven at 50°C under vacuum for 1 h. Tile dried methanol-insoluble residue was submitted to the enzymatic analysi~ of starch (Boehringer Mannheim, 1980; Gorin et al., 1978).

The recovery of commercial starch (BDH, art. 10271), previously ex- tracted with methanol, was 90 + 2.6% and the coefficient of variation was 3.0% (J~2 replicates). Adding estimated starch to a sample of anthers ("ad- mixture; technique") gave a recovery of 98.2%. There was a linear relation- ship between absorbance (0.20--0.50) and the amount of starch in the cuvette (20.35--40.70 pg). Using this method, a quant i ty of starch as small as 2 pg could be detected. All samples pertaining to anthers from bulbs were analysed within the limits of the linear relationship and the results expressed as mg starch per 100 mg freeze-dried anthers {i.e. dry matter).

a-Amylase activity

The principle of the method described below is that only a-amylase (not ~-amylase) acts on insoluble stained starch (Phadebas tablet) to produce soluble coloured products, whose absorbance is measured at 620 nm (Barnes and Blakeney, 1974; Wahlefeld, 1984). We found that this starch was not cleaved either by pure phosphorylase a or b (Boehringer Mannheim, articles 108553 and 108570, respectively).

Enzyme extract. -- Before use, freeze-dried anthers were ground to powder in a laboratory cross-beater mill (1982 trial) or by pestling (1983 trial). The powder (100 mg) was mixed with 2 ml of 0.1 M phosphate buffer pH 7.0, and extracted by stirring the suspension in an ice bath. After 30 rain, the mi~:ture was centrifuged at 5°C for 30 min at 45000 g. The extract ion

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was repeated twice with 2 ml of fresh buffer. The combined supernatants consti tuted the enzyme extract, which was stable for 48 h at 5°C. The en- zyme extract was used within 24 h of preparation.

I n c u b a t i o n m i x t u r e . - - The enzyme extract (0.5--2.0 ml) was diluted with double-distilled water to a volume of 4.2 ml and placed in a water bath at 37.0°C for approximately 5 min (Phadebas Amylase Test, Directions for use, Pharmacia Diagnostics AB, Uppsala, Sweden). One tablet of commercial substrate Phadebas was then added. After 30 min, 1 ml of 0.5 M NaOH solution (end-point reaction) was added and the mixture was centrifuged at 5--6°C for 30 min at 2000 g. Absorbance of the supernatant was meas- ured at 620 nm with a disposable cuvette of I cm light path (Sarstedt, art. 67741), with the cuvette-holder of the spec t rophotometer maintained at 25.0°C.

Successive dilutions of one of the anther enzyme extracts demonstrated a linear relationship between enzyme activity (absorbance of 0.152--0.600} and amount of enzyme extract. All measurements pertaining to the enzyme extracts from anthers were done in such a manner that they were within the above-mentioned linear relationship. Absorbances were arbitrary units (U) of catalytic activity and were expressed in terms of 1 ml enzyme extract.

P r o t e i n . - - Protein in the enzyme extracts was estimated according to Lowry et al. (1951).

S u c r o s e . - - Sucrose in anthers was estimated by high-performance liquid chromatography as described in Gorin and Heidema (1980), but wi thout clean up of the extract. Integrated peaks of glucose and fructose in the chromatograms were too small for accurate calculations and therefore data on these sugars are not presented. No a t tempt was made to estimate the monosaccharides with a more sensitive procedure.

RESULTS AND DISCUSSION

S t a r c h

Starch in anthers from dry bulbs stored at 5°C increased from Week 0 to Week 1 and remained at about the same level until Week 12, while those from the 17°C t rea tment decreased from Week 0 to Week 8, there- after remaining at that level until Week 12. Thus by 7--8 weeks the largest difference was found between the starch content in anthers from 5°C bulbs and that from control bulbs (Fig. 1). This large difference (higher than 40%) remained even during the transport simulation at 2 weeks (Fig. 1). Analysis of variance demonstrated that there was a significant difference (P < 1%) between bulbs kept at 5 and 17°C. Both the changes in starch content during 12 weeks and the starch contents themselves were the same as those found in the preliminary experiment of the previous year (1981).

187

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a-Amy lase activity

During the 12 weeks of the treatment, the values for specific catalytic activity (obtained by the quotient of activity units per ml and mg protein per ml} oscillated between 0.04 and 0.08 in the enzyme extracts from anthers from cooled bulbs, but in the enzyme solutions from control bulbs, specific catalytic activity was between 0.04 and 0.10 from Week 0 until Week 4, and then increased steadily until Week 12, by which time the mean value had increased to 0.40 from the initial value of 0.04 (Fig. 2). Consequently, there was a large difference (higher than 40%) in the mean values for specific catalytic activity between pre-cooled (0.08) and control bulbs (0.40) at the 12th week, and this persisted during the simulated transport period. Analysis of variance showed that there was a significant difference between bulbs kept at 5 and 17°C (P < 1%). The same results were obtained in the preliminary trial of 1982, which did not include simulation of transport. The specific catalytic activity of a-amylase within trials in different years

188

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was reproducible, whereas activity per ml enzyme extract and the protein contents were not reproducible from year to year. Thus, the only satisfactory indicator found for exposure to 5°C was specific catalytic activity, as it clearly allowed rejection of the bulbs kept at 17°C.

S u c r o s e . - - Sucrose in anthers (mg/100 mg dry matter) (1982 trial) was discarded as a possible indicator of pre-cooling because at the 12th week the sucrose content of pre-cooled bulbs (6.17 -+ 0.15) was similar to that of control bulbs (6.66 + 0.15).

The starch content in anthers from bulbs exposed at 5°C was higher than that of control bulbs, suggesting that at 5°C synthesis predominates over hydrolysis: the specific catalytic activity of a-amylase was lower in anther extracts from pre-cooled bulbs than in anther extracts from control bulbs, thereby confirming this hypothesis.

189

The same test on 18 batches o f bulbs f rom typical bulb-growing regions in The Nether lands gave the fol lowing results for Week 12: starch, 16--30% for pre-cooled bulbs and 4--8% for con t ro l bulbs; specific a-amylase activity, 0 .05- -0 .16 and 0 .32--0 .49 , respectively. On the basis o f these practical tests and of the cont ro l led trials, we p ropose the fol lowing provisional criteria. Starch should exceed 13 + 3% ( f rom coeff ic ient o f variat ion o f 20%) and specific catalyt ic activity o f a-amylase should be below 0.16 + 0.03. We are; still looking for bet ter criteria o f the dura t ion o f t r ea tment , as those presented here (5°C bulbs) did no t change linearly during the 12 weeks o f t rea tment . Also, the effects o f al terations in the storage t empera tu re need to be examined more closely.

ACKNOWLEDGEMENTS

We thank Ir. W. Klop for suggesting the use of the Phadebas Amylase tablet , Dr. J. Beyersbergen (Bulb Research Centre, Lisse) for valuable discus- sions, Hanna Lagerwerf for statistical analysis o f the data and J.C. Rigg (Centre for Agricultural Publishing and D o c u m e n t a t i o n , Wageningen) for help in draf t ing the manuscr ipt .

REFERENCES

Barnes, W.C. and Blakeney, A.B., 1974. Determination of cereal alpha amylase using a commercially available dye-labelled substrate. Staerke, 26 : 193--197.

Boehringer Mannheim GmbH, 1980. Starch analysis. In: Methods of Enzymatic Food Analysis. Mannheim, West Germany.

De Hertogh, A.A., 1981. Holland Bulb-Forcer's Guide, 2nd edn. International Flower- Bulb Centre, The Netherlands, and Netherlands Flower-Bulb Institute, New York, pp. 62--67.

De Hertogh, A.A., Aung, L.H. and Benschop, M., 1983. The tulip: botany, usage, growth and development. Hortic. Rev., 5: 45--125.

Gorin, N. and Heidema, F.T., 1980. Clean-up of methanolic extract in high-pressure liquid chromatography of fructose, glucose, and sucrose in onion powder. J. Agric. Food Chem., 28: 1340--1342.

Gorin, N., Bonisolli, F., Heidema, F.T., Klop, W. and Williams, A.A., 1978. Changes in starch content and amylase zymograms during storage of Golden Delicious and Cox's Orange Pippin apples. Z. Lebensm.-Unters.-Forsch., 166: 157--161.

Haaland, E. and Wickstr~bm, A., 1975. The effect of storage temperature on carbohydrate interconversion in tulip bulbs. Acta Hortic., 47: 371--376.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the folin-phenol reagent. J. Biol. Chem;. 193 : 265--275.

Moe, R and Wickstr~m, A., 1973. The effect of storage temperature on shoot growth, flowering, and carbohydrate metabolism in tulip bulbs. Physiol. Plant., 28: 81--87.

Moe, R and Wickstr~m, A., 1979. Effect of precooling at 5 or --I°C on shoot growth, flowering and carbohydrate metabolism in tulip bulbs. Scientia Hortic., 10: 187--201.

Prince, T.A., Herner, R.C. and De Hertogh, A.A., 1982. Increases in ethylene and carbon dioxide production by Tulipa gesneriana L. 'Prominence' after completion of the cold-requirement. Scientia Hortic., 16: 77--83.

Wahlefeld, A.W., 1984. Glycosidases. a-Amylase, Determination with coloured insoluble substrates. In: H.U. Bergmeyer (Editor-in-Chief), J. Bergmeyer and M. Grabl (Editors), Methods of Enzymatic Analysis. 3rd edn., Vol. IV. Verlag Chemic, Weinheim, Deer- field Beach, Florida, Basel, pp. 161--167.