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The antioxidant BHT — A new factor disturbing plant morphogenesis?
Scientia Horticulturae, 26 (1985) 217--224
217
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
THE ANTIOXIDANT MORPHOGENESIS?
BHT -- A NEW FACTOR DISTURBING PLANT
MARIA J. GROCHOWSKA' and GEORGE J. BUTA
Plant Hormone and Regulator Laboratory, USDA, Beltsville Agricultural Research Center, i~eltsviUe, MD 20705 (U.S.A.) (Accepted for publication 6 February 1985)
ABSTRACT
Grochowska, M.J. and Buta, G.J., 1985. The antioxidant BHT -- a new factor disturbing plant morphogenesis? Scientia Hortic., 26: 217--224. The antioxidant BHT -- commonly used as a food stabilizer -- when applied in aqueous solution to the tips of 5-day-old pea seedlings, to young apple seedlings or to germinating lettuce seeds, produced a suden cessation of growth that, when growth was resumed, was followed by certain morphological changes. Treatments with BHT caused the formation of (1) a bunch of new shoots arising at the apex of apple seedlings, (2) 2 or 3 adventitious fast-growing shoots from the base of the pea hypocotyl, and (3) adventitious growing points in lettuce plants. This multiplication of growing points was followed, during further development, by splitting of the apple seedling stems and lettuce leaf blades into independently growing organs. Keywords: antioxidant; apple; BHT = butylated hydroxytoluene; lettuce; morphogenesis; pea.
ABBREVIATION BHT = 2,6-di-tert-4-methyl phenol, or butylated hydroxytoluene.
INTRODUCTION
BHT, also known as ionol, is an inhibitor of free radical-mediated processes and an anti-tumor agent whose activity has been ascribed to the inhibition of certain phases of mitosis (Alekperov and Shcherbakov, 1967; Askerov et al., 1973). Ionol evoked certain disturbances in metaphasic spindle functioning, resulting in polyploidisation o f Crepis capilaris (Askerov et al., 1973) and multi-polar mitosis followed by multi-nuclearity in onion cells (Alekperov et al., 1975). ~Permanent address: Research Institute of Pomology and Floriculture, 96-100 Skierniewice, Poland.
0304-4238/85/$03.30
© 1 9 8 5 Elsevier S c i e n c e Publishers B.V.
218 BHT has also been used as a food antioxidant to stabilize fats and oils, and to preserve the nutritional value of the diet of man and animals. In some reviews on safety evaluation of hindered phenolic anti-oxidants, BHT was described as a factor decreasing the activities o f plasma catalase, cholin-esterase and peroxidase (Hathway, 1966; Branen, 1975). In monkeys, BHT induced fragmentation and the presence of large nuclear fibrils in hepatic nuclei (Branen, 1975). This phenolic anti-oxidant has been recommended in plant analyses to prevent the oxidation of IAA and other hormones (Iino et al., 1980). The use of BHT during analytical preparation of apple tree tissues for gibbereUin determination b y bioassays contributed to the discovery o f some striking additional effects of this compound (Grochowska et al., 1984). The previous observations of BHT effects were concerned with the ultrastructural changes produced in plants and animals. The present results deal with macrostructural growth modifications that were provoked by BHT treatments in three plant species. MATERIAL AND METHODS Pea ( P i s u m s a t i v u m L.). - - Pea seedlings (150 plants per cultivar 'Laxton Progress No. 9', 'Alaska', 'Sugar Snap') were treated with aqueous solutions o f BHT b y drop-application to the shoot tip (plumule). An emulsion was formed b y dissolving 1, 5, 100 or 300 mg of BHT in a few drops of ethyl acetate, and vigorously shaking with 4 ml H20 plus 10 pl of Tween 80. On the 5th day of germination (the 2nd day after planting the seedling), 10 pl of the BHT emulsion was applied to the pea plumule twice within 18 h. No damage was observed in control plants treated with emulsion without BHT. Some untreated seedlings were decapitated in order to release plants from apical dominance. In an independent experiment, the activities o f 8 other phenolic compounds closely related to BHT were examined b y the pea seedling assay. Preparation of aqueous solutions of the compounds and application of 20 pl (10 pl, twice) of 3 X 10 -2, 2 X 10 .3 or 2 X 10 .4 M solution per plant were performed in the manner described above. Plant elongation was measured after 6 days further growth. The pea plants were prepared for gibberellin bioassay as described b y McComb and Carr (1958). L e t t u c e ( L a c t u c a sativa L.). - - Germination of lettuce seeds ('Grand Rapids')
in BHT aqueous solutions was carried out in the light and in the dark. BHT dissolved in ethyl acetate was applied in 6 concentrations to filterpaper discs (3 cm in diameter) in the range 10--300 pg per disc. After drying, the discs were wetted with water and put into petri dishes with 10 lettuce seeds; they were kept at 22°C for 36, 48 or 72 h, when plant elongation and morphological changes were recorded.
219
Apple seedlings (Malus domestica Borg.). -- Aqueous solutions (emulsion) of BHT were applied to 'York' seedlings at different ages. Two-, 3.5- and 7month-old seedlings were treated with BHT in 3 ways: 2 drops applied to the apex of the plant; spraying the whole plant; dipping the upper part (with 5 leaves) c,f the seedling. The emulsion was prepared b y dissolving 0.1--3 g of BHT in 2 ml of ethyl acetate, which was poured into vigorously stirred 100 ml H20 plus 1 ml Tween 80. The emulsion without BHT was applied to control plants and did not cause damage. The uppermost (youngest) node of the seedling was marked (by a spot) with a marker pen on the day of treatment. The new growth increment only (without any contribution from the elongation of internodes below), was measured 2 months later, which gave the seedling's response to the treatment. Standard deviations of the results obtained were calculated for all three species. RESULTS
Applications of BHT to dwarf pea seedlings inhibited the growth of the plants which, although still alive and w i t h o u t visible damage, did not resume growth for at least 2 weeks. Pea plants of the 3 cultivars responded to the highest a m o u n t of applied BHT (1500 ~g/plant) b y production of 2 or 3 ad-
Fig. 1. Effect of B H T on growth of pea seedlings 'Laxton Progress' No. 9. T w o drops of B H T so|ution (1500 ~g) applied to 5-day-old plant (depicted with arrow) caused the formation of vigorously growing adventitious shoots (left).Control plant, right.
220 ventitious vigorously growing shoots, which arose at the base of the hypoc o t y l ( F i g . 1 a n d T a b l e I). T h e l o w e r c o n c e n t r a t i o n o f B H T a p p l i e d t o s e e d lings affected growth to a lesser degree, but caused swelling of the upper p a r t o f t h e h y p o c o t y l s . E v e n less i n h i b i t i o n a n d a l m o s t n o s w e l l i n g w a s o b tained after applications of 5 ~g BHT. TABLE I Effect of BHT on growth of 5-day-old pea seedlings of 3 cultivars ~g/plant (in 2 applications)
1500 500 25 5
Average growth increment in % of controls -+ standard deviation 'Laxton Progress' No. 9 (dwarf)
'Alaska' (dwarf)
'Sugar Snap' (tall)
34-+ 91 65 -+ 102 78-+ 11 84_+ 13
16-+ 21 22 +- 2 30+- 7
15-+ 41 23 -+ 6: 57-+ 14 103 ± 12
1Plants with adventitious shoots. 2Plants with swelling in the upper part of the hypocotyl. TABLE II Effect of B H T applications on s t e m extension and m o r p h o l o g i c a l changes o f 2-month-old, 3.5-monthold a n d 7 - m o n t h - o l d ' Y o r k ' a p p l e s e e d l i n g s BHT concentration ( m g m1-1)
Treatment
Number of applications
Number of plants
Average growth increment ( c m -+ s t a n d a r d deviation)
New shoots springing up Number
Split shoot number
Total length
(cm) 2 m o n t h s old 10 5 1 H20
Two drops on the tip
5 5 5 5
23 18 21 21
1.1 4.2 5.4 9.0
+ 0.8 -+ 2.1 -+ 3.0 -+ 4.2
5 5 5 5
65 59 62 50
3.8 6.0 8.5 12.0
+ 2.5 + 2.8 -+ 3 . 2 -+ 4.1
8 8 8 8
50 52 48 64
1.1 1.8 2.3 7.8
-+ 0 . 5 -+ 0.7 +- 1.0 -+ 2.2
67 42 8 0
17.0 132.0 6.2 0
3 10 0 0
12 12 12 12
12 12 11 14
1.3 1.4 13.7 12.3
-+ 0.9 -+ 1.6 -+ 5.2 -+ 3.1
24 17 0 0
51.1 89.6 0 0
4 0 0 0
2 m o n t h s old 10 5 1 H20
Spraying
3.5 m o n t h s old 30 20 10 H20
Spraying
7 m o n t h s old 10 5 1 H20
Dipping of the apical part
221
Control plants, either treated with the emulsion without BHT or decapitated, did not develop adventitious shoots and did not swell. Lettuce seed germination was not disturbed by any of the BHT concentrations, neither in the light nor in the dark. When measured on the 3rd day of incubation, the primary root and the hypocotyl of the treated plants showed redttced growth; by hall in the light and by several times in the dark as compared to controls. The light-grown seedlings were swollen and greenishyellow in colour. The BHT-grown plants did not resume growth for at least 3 weeks, although they remained alive. When transplanted into soil beds, some of the treated plants developed 1 or 2 additional growing points 3 weeks later, which produced small, malformed leaves. Some of the leaf blades ~split along the central vein into 2 separate parts which continued independent growth. An aqueous solution of BHT also modified the growth of 'York' apple seedlings (Table II). Two drops of the antioxidant emulsion at a concentration of 10 mg m1-1, applied 5 times to the shoot apices, almost completely inhibited the growth of 2-month-old apple seedlings. Spraying the plants was less effective than drop-application, but it was as effective as dipping (Table II). Four to five weeks later, all seedlings resumed growth. After treatments with lower BHT concentrations (1--5 mg ml-1), the newly-grown part of the stem had longer internodes than those of controls.
Fig. 2. B u n c h o f new shoots springing f r o m the apical bud o f 3.5-month-old apple seedling treated w i t h BHT (sprayed 8 times at a c o n c e n t r a t i o n o f 30 mg m F 1) within I m o n t h after the r e s u m p t i o n of g r o w t h (left). Control plant, right.
222
Two to three times higher concentrations of BHT sprayed on 3.5-monthold seedling of 'York', as well as drastic inhibition o f elongation, also caused some disturbance in plant growth after growth was resumed. About onethird of all seedlings produced a bunch of new shoots (2--5) which arose from the very top of the plant (Table II and Fig. 2). The number of shoots per plant was proportional to the BHT concentration; a higher concentration usually provoking a larger number of new shoots. Other seedlings which at first seemed normal, exhibited splitting in the newly grown parts o f the stem a few months after treatment (Fig. 3). Similar effects were obtained with 7-month-old seedlings of the same cultivar when their apical parts were dipped into BHT solutions. When the number of applications was increased from 8 to 12 (dipping was equally effective as spraying), the concentration of BHT necessary for these effects was decreased 4 times (Table II). In comparison with 8 closely chemically related phenolic compounds, BHT showed a unique a['ility in producing growth modifications of pea seedlings 'Sugar Snap' (Table III). Only BHT induced formation of new shoots arising from the base of the pea hypocotyl. None of the BHT concentrations damaged the plants, while the highest concentration (10 ~1 of 3 X 10 -2 M) of all other phenolic compounds killed the shoot apices or the whole plant.
Fig. 3. S p l i t t i n g o f t h e n e w l y g r o w n p a r t o f t h e s t e m o f a 7 - m o n t h - o l d a p p l e seedling w h o s e apical p a r t was d i p p e d 12 t i m e s in B H T s o l u t i o n at a c o n c e n t r a t i o n o f 10 m g m1-1. T h e seedling r e s u m e d g r o w t h after 1 m o n t h o f i n h i b i t i o n and splitting o c c u r r e d 2 m o n t h s later.
223
Chloro-substitute phenols were the most toxic, since their lowest concentrat i o n (10 pl o f 2 × 10 -4 M) also severely damaged t he plants. None o f t he pea seedlings with killed shoot tips or which were decapitated produced adventitious shoots at the base of the h y p o c o t y l . This showed t h a t t he f o r m a t i o n o f adventitious shoots due to BHT t r e a t ment s did not result from t he removal of apical dominance. TABLE ]:II Effect of BHT and 8 chemically allied compounds at 3 concentrations on growth of 5-day-old 'Sugar Snap' pea seedlings Compound
2,6-di-tert-butyl-4-methyl phenol (BHT) 2,4,6-tri-methyl-phe nol 2,4,6-tri-tert-butyl phenol 2,3,5-tri-methyl phenol O-cresol 2,6-di-methyl-phenol 2,4,5-tri-chloro-phenol 2,3,5- tri-chloro-phenol 2,3,6-tri..chloro-phenol
Average growth of 30 plants (% of controls) 3× 10 -2M 2× 10 -3M
2x 10 -4M
121 63 53 73 183 173 43 43 43
65 85 90 100 82 83 582 482 63
23 522 622 302 302 183 10 3 452 43
1Adventi:tious shoots formed at the bottom of the hypocotyl. 2Damaged apex, growth continued from lateral bud. 3Plant killed.
DISCUSSION The growth disturbances induced by BHT in three species were concerned with a sudden growth stop or a drastic reduction o f shoot elongation, and with some morphological changes. The growth stop might be due to the inhibition o f certain phases o f cell mitosis and to suppression o f prot ei n biosynthesis (Alekperov and Shcherbakov, 1967; Askerov et al., 1973). Could one assume that t he multiplication o f growing points observed in pea, lettuce and apple seedlings as well as the splitting o f their stems and leaves, have something in c o m m o n with t he fragmentation o f nuclei (Branen, 1975) and multi-polar mitosis leading to multi-nuclearity (Askerov et al., 1973; Alekperov et al., 1975) t hat were recorded in ultrastructural analyses o f plant and animal tissues treated with BHT? Microscopic analyses o n t he ultrastructural level might answer this question. Although use of BHT in practice needs m ore detailed investigation, any interference in the functioning of t he nuclear apparatus seems to be undesirable.
224 ACKNOWLEDGEMENTS T h e a u t h o r s t h a n k Dr. M i k l o s F a u s t , F r u i t L a b o r a t o r y , a n d Dr. J a m e s D. A n d e r s o n , P l a n t H o r m o n e L a b o r a t o r y , Beltsville A g r i c u l t u r a l R e s e a r c h C e n t e r , U . S . D . A . , f o r d i s c u s s i o n . This w o r k w a s s u p p o r t e d b y I n t e r n a t i o n a l Communication Agency, Grant 2420, administered by the Council for International Exchange of Scholars, U.S.A.
REFERENCES Alekperov, U.K. and Shcherbakov, V.K., 1967. An incomplete metaphase block and other effects accompanying the action of ionol on mitosis. Tsitologia, 9 : 6 0 6 - - 6 0 9 (in Russian). Alekperov, U.K., Abutalybov, M.G. and Askerov, I.T., 1975. Multipolar mitosis and multinuclearity of cells in the onion arrow roots under the effect of ionol. Tsitologia, 17:101--103 (in Russian). Askerov, I.T., Abutalybov, M.G. and Alekperov, U.K., 1973. A comparative study of polyploidizing effects of colchiein and ionol. Tsitologia, 15:341--843 (in Russian). Branen, A.L., 1975. Toxicology and biochemistry of butylated hydroxyyanisole and butylated hydroxytoluene. J. Am. Oil Chem. Soc., 52: 59--63. Grochowska, M.J., Buta, G.J., Steffens, G.L. and Faust, M., 1984. Endogenous auxin and gibberellin levels in low and high vigor apple seedlings. Acta Hortic., 146: 125--134. Hathway, D.E., 1966. Metabolic fats in animals of hindered phenolic antioxidants in relation to their safety evaluation and antioxidant function. Adv. Food Res., 15: 1--56. Iino, M., Yu, R.S.T. and Carr, D.J., 1980. Improved procedure for the estimation of nanogram quantities of indole-3-acetic acid in plant extracts using the indole-~-pyrone fluorescence method. Plant Physiol., 66:1099--1105. McComb, A.J. and Carr, D.J., 1958. Evidence for a dwarf pea bioassay of naturally occurring gibberellins in the growing plant. Nature (London), 181: 1548--1549.
217
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
THE ANTIOXIDANT MORPHOGENESIS?
BHT -- A NEW FACTOR DISTURBING PLANT
MARIA J. GROCHOWSKA' and GEORGE J. BUTA
Plant Hormone and Regulator Laboratory, USDA, Beltsville Agricultural Research Center, i~eltsviUe, MD 20705 (U.S.A.) (Accepted for publication 6 February 1985)
ABSTRACT
Grochowska, M.J. and Buta, G.J., 1985. The antioxidant BHT -- a new factor disturbing plant morphogenesis? Scientia Hortic., 26: 217--224. The antioxidant BHT -- commonly used as a food stabilizer -- when applied in aqueous solution to the tips of 5-day-old pea seedlings, to young apple seedlings or to germinating lettuce seeds, produced a suden cessation of growth that, when growth was resumed, was followed by certain morphological changes. Treatments with BHT caused the formation of (1) a bunch of new shoots arising at the apex of apple seedlings, (2) 2 or 3 adventitious fast-growing shoots from the base of the pea hypocotyl, and (3) adventitious growing points in lettuce plants. This multiplication of growing points was followed, during further development, by splitting of the apple seedling stems and lettuce leaf blades into independently growing organs. Keywords: antioxidant; apple; BHT = butylated hydroxytoluene; lettuce; morphogenesis; pea.
ABBREVIATION BHT = 2,6-di-tert-4-methyl phenol, or butylated hydroxytoluene.
INTRODUCTION
BHT, also known as ionol, is an inhibitor of free radical-mediated processes and an anti-tumor agent whose activity has been ascribed to the inhibition of certain phases of mitosis (Alekperov and Shcherbakov, 1967; Askerov et al., 1973). Ionol evoked certain disturbances in metaphasic spindle functioning, resulting in polyploidisation o f Crepis capilaris (Askerov et al., 1973) and multi-polar mitosis followed by multi-nuclearity in onion cells (Alekperov et al., 1975). ~Permanent address: Research Institute of Pomology and Floriculture, 96-100 Skierniewice, Poland.
0304-4238/85/$03.30
© 1 9 8 5 Elsevier S c i e n c e Publishers B.V.
218 BHT has also been used as a food antioxidant to stabilize fats and oils, and to preserve the nutritional value of the diet of man and animals. In some reviews on safety evaluation of hindered phenolic anti-oxidants, BHT was described as a factor decreasing the activities o f plasma catalase, cholin-esterase and peroxidase (Hathway, 1966; Branen, 1975). In monkeys, BHT induced fragmentation and the presence of large nuclear fibrils in hepatic nuclei (Branen, 1975). This phenolic anti-oxidant has been recommended in plant analyses to prevent the oxidation of IAA and other hormones (Iino et al., 1980). The use of BHT during analytical preparation of apple tree tissues for gibbereUin determination b y bioassays contributed to the discovery o f some striking additional effects of this compound (Grochowska et al., 1984). The previous observations of BHT effects were concerned with the ultrastructural changes produced in plants and animals. The present results deal with macrostructural growth modifications that were provoked by BHT treatments in three plant species. MATERIAL AND METHODS Pea ( P i s u m s a t i v u m L.). - - Pea seedlings (150 plants per cultivar 'Laxton Progress No. 9', 'Alaska', 'Sugar Snap') were treated with aqueous solutions o f BHT b y drop-application to the shoot tip (plumule). An emulsion was formed b y dissolving 1, 5, 100 or 300 mg of BHT in a few drops of ethyl acetate, and vigorously shaking with 4 ml H20 plus 10 pl of Tween 80. On the 5th day of germination (the 2nd day after planting the seedling), 10 pl of the BHT emulsion was applied to the pea plumule twice within 18 h. No damage was observed in control plants treated with emulsion without BHT. Some untreated seedlings were decapitated in order to release plants from apical dominance. In an independent experiment, the activities o f 8 other phenolic compounds closely related to BHT were examined b y the pea seedling assay. Preparation of aqueous solutions of the compounds and application of 20 pl (10 pl, twice) of 3 X 10 -2, 2 X 10 .3 or 2 X 10 .4 M solution per plant were performed in the manner described above. Plant elongation was measured after 6 days further growth. The pea plants were prepared for gibberellin bioassay as described b y McComb and Carr (1958). L e t t u c e ( L a c t u c a sativa L.). - - Germination of lettuce seeds ('Grand Rapids')
in BHT aqueous solutions was carried out in the light and in the dark. BHT dissolved in ethyl acetate was applied in 6 concentrations to filterpaper discs (3 cm in diameter) in the range 10--300 pg per disc. After drying, the discs were wetted with water and put into petri dishes with 10 lettuce seeds; they were kept at 22°C for 36, 48 or 72 h, when plant elongation and morphological changes were recorded.
219
Apple seedlings (Malus domestica Borg.). -- Aqueous solutions (emulsion) of BHT were applied to 'York' seedlings at different ages. Two-, 3.5- and 7month-old seedlings were treated with BHT in 3 ways: 2 drops applied to the apex of the plant; spraying the whole plant; dipping the upper part (with 5 leaves) c,f the seedling. The emulsion was prepared b y dissolving 0.1--3 g of BHT in 2 ml of ethyl acetate, which was poured into vigorously stirred 100 ml H20 plus 1 ml Tween 80. The emulsion without BHT was applied to control plants and did not cause damage. The uppermost (youngest) node of the seedling was marked (by a spot) with a marker pen on the day of treatment. The new growth increment only (without any contribution from the elongation of internodes below), was measured 2 months later, which gave the seedling's response to the treatment. Standard deviations of the results obtained were calculated for all three species. RESULTS
Applications of BHT to dwarf pea seedlings inhibited the growth of the plants which, although still alive and w i t h o u t visible damage, did not resume growth for at least 2 weeks. Pea plants of the 3 cultivars responded to the highest a m o u n t of applied BHT (1500 ~g/plant) b y production of 2 or 3 ad-
Fig. 1. Effect of B H T on growth of pea seedlings 'Laxton Progress' No. 9. T w o drops of B H T so|ution (1500 ~g) applied to 5-day-old plant (depicted with arrow) caused the formation of vigorously growing adventitious shoots (left).Control plant, right.
220 ventitious vigorously growing shoots, which arose at the base of the hypoc o t y l ( F i g . 1 a n d T a b l e I). T h e l o w e r c o n c e n t r a t i o n o f B H T a p p l i e d t o s e e d lings affected growth to a lesser degree, but caused swelling of the upper p a r t o f t h e h y p o c o t y l s . E v e n less i n h i b i t i o n a n d a l m o s t n o s w e l l i n g w a s o b tained after applications of 5 ~g BHT. TABLE I Effect of BHT on growth of 5-day-old pea seedlings of 3 cultivars ~g/plant (in 2 applications)
1500 500 25 5
Average growth increment in % of controls -+ standard deviation 'Laxton Progress' No. 9 (dwarf)
'Alaska' (dwarf)
'Sugar Snap' (tall)
34-+ 91 65 -+ 102 78-+ 11 84_+ 13
16-+ 21 22 +- 2 30+- 7
15-+ 41 23 -+ 6: 57-+ 14 103 ± 12
1Plants with adventitious shoots. 2Plants with swelling in the upper part of the hypocotyl. TABLE II Effect of B H T applications on s t e m extension and m o r p h o l o g i c a l changes o f 2-month-old, 3.5-monthold a n d 7 - m o n t h - o l d ' Y o r k ' a p p l e s e e d l i n g s BHT concentration ( m g m1-1)
Treatment
Number of applications
Number of plants
Average growth increment ( c m -+ s t a n d a r d deviation)
New shoots springing up Number
Split shoot number
Total length
(cm) 2 m o n t h s old 10 5 1 H20
Two drops on the tip
5 5 5 5
23 18 21 21
1.1 4.2 5.4 9.0
+ 0.8 -+ 2.1 -+ 3.0 -+ 4.2
5 5 5 5
65 59 62 50
3.8 6.0 8.5 12.0
+ 2.5 + 2.8 -+ 3 . 2 -+ 4.1
8 8 8 8
50 52 48 64
1.1 1.8 2.3 7.8
-+ 0 . 5 -+ 0.7 +- 1.0 -+ 2.2
67 42 8 0
17.0 132.0 6.2 0
3 10 0 0
12 12 12 12
12 12 11 14
1.3 1.4 13.7 12.3
-+ 0.9 -+ 1.6 -+ 5.2 -+ 3.1
24 17 0 0
51.1 89.6 0 0
4 0 0 0
2 m o n t h s old 10 5 1 H20
Spraying
3.5 m o n t h s old 30 20 10 H20
Spraying
7 m o n t h s old 10 5 1 H20
Dipping of the apical part
221
Control plants, either treated with the emulsion without BHT or decapitated, did not develop adventitious shoots and did not swell. Lettuce seed germination was not disturbed by any of the BHT concentrations, neither in the light nor in the dark. When measured on the 3rd day of incubation, the primary root and the hypocotyl of the treated plants showed redttced growth; by hall in the light and by several times in the dark as compared to controls. The light-grown seedlings were swollen and greenishyellow in colour. The BHT-grown plants did not resume growth for at least 3 weeks, although they remained alive. When transplanted into soil beds, some of the treated plants developed 1 or 2 additional growing points 3 weeks later, which produced small, malformed leaves. Some of the leaf blades ~split along the central vein into 2 separate parts which continued independent growth. An aqueous solution of BHT also modified the growth of 'York' apple seedlings (Table II). Two drops of the antioxidant emulsion at a concentration of 10 mg m1-1, applied 5 times to the shoot apices, almost completely inhibited the growth of 2-month-old apple seedlings. Spraying the plants was less effective than drop-application, but it was as effective as dipping (Table II). Four to five weeks later, all seedlings resumed growth. After treatments with lower BHT concentrations (1--5 mg ml-1), the newly-grown part of the stem had longer internodes than those of controls.
Fig. 2. B u n c h o f new shoots springing f r o m the apical bud o f 3.5-month-old apple seedling treated w i t h BHT (sprayed 8 times at a c o n c e n t r a t i o n o f 30 mg m F 1) within I m o n t h after the r e s u m p t i o n of g r o w t h (left). Control plant, right.
222
Two to three times higher concentrations of BHT sprayed on 3.5-monthold seedling of 'York', as well as drastic inhibition o f elongation, also caused some disturbance in plant growth after growth was resumed. About onethird of all seedlings produced a bunch of new shoots (2--5) which arose from the very top of the plant (Table II and Fig. 2). The number of shoots per plant was proportional to the BHT concentration; a higher concentration usually provoking a larger number of new shoots. Other seedlings which at first seemed normal, exhibited splitting in the newly grown parts o f the stem a few months after treatment (Fig. 3). Similar effects were obtained with 7-month-old seedlings of the same cultivar when their apical parts were dipped into BHT solutions. When the number of applications was increased from 8 to 12 (dipping was equally effective as spraying), the concentration of BHT necessary for these effects was decreased 4 times (Table II). In comparison with 8 closely chemically related phenolic compounds, BHT showed a unique a['ility in producing growth modifications of pea seedlings 'Sugar Snap' (Table III). Only BHT induced formation of new shoots arising from the base of the pea hypocotyl. None of the BHT concentrations damaged the plants, while the highest concentration (10 ~1 of 3 X 10 -2 M) of all other phenolic compounds killed the shoot apices or the whole plant.
Fig. 3. S p l i t t i n g o f t h e n e w l y g r o w n p a r t o f t h e s t e m o f a 7 - m o n t h - o l d a p p l e seedling w h o s e apical p a r t was d i p p e d 12 t i m e s in B H T s o l u t i o n at a c o n c e n t r a t i o n o f 10 m g m1-1. T h e seedling r e s u m e d g r o w t h after 1 m o n t h o f i n h i b i t i o n and splitting o c c u r r e d 2 m o n t h s later.
223
Chloro-substitute phenols were the most toxic, since their lowest concentrat i o n (10 pl o f 2 × 10 -4 M) also severely damaged t he plants. None o f t he pea seedlings with killed shoot tips or which were decapitated produced adventitious shoots at the base of the h y p o c o t y l . This showed t h a t t he f o r m a t i o n o f adventitious shoots due to BHT t r e a t ment s did not result from t he removal of apical dominance. TABLE ]:II Effect of BHT and 8 chemically allied compounds at 3 concentrations on growth of 5-day-old 'Sugar Snap' pea seedlings Compound
2,6-di-tert-butyl-4-methyl phenol (BHT) 2,4,6-tri-methyl-phe nol 2,4,6-tri-tert-butyl phenol 2,3,5-tri-methyl phenol O-cresol 2,6-di-methyl-phenol 2,4,5-tri-chloro-phenol 2,3,5- tri-chloro-phenol 2,3,6-tri..chloro-phenol
Average growth of 30 plants (% of controls) 3× 10 -2M 2× 10 -3M
2x 10 -4M
121 63 53 73 183 173 43 43 43
65 85 90 100 82 83 582 482 63
23 522 622 302 302 183 10 3 452 43
1Adventi:tious shoots formed at the bottom of the hypocotyl. 2Damaged apex, growth continued from lateral bud. 3Plant killed.
DISCUSSION The growth disturbances induced by BHT in three species were concerned with a sudden growth stop or a drastic reduction o f shoot elongation, and with some morphological changes. The growth stop might be due to the inhibition o f certain phases o f cell mitosis and to suppression o f prot ei n biosynthesis (Alekperov and Shcherbakov, 1967; Askerov et al., 1973). Could one assume that t he multiplication o f growing points observed in pea, lettuce and apple seedlings as well as the splitting o f their stems and leaves, have something in c o m m o n with t he fragmentation o f nuclei (Branen, 1975) and multi-polar mitosis leading to multi-nuclearity (Askerov et al., 1973; Alekperov et al., 1975) t hat were recorded in ultrastructural analyses o f plant and animal tissues treated with BHT? Microscopic analyses o n t he ultrastructural level might answer this question. Although use of BHT in practice needs m ore detailed investigation, any interference in the functioning of t he nuclear apparatus seems to be undesirable.
224 ACKNOWLEDGEMENTS T h e a u t h o r s t h a n k Dr. M i k l o s F a u s t , F r u i t L a b o r a t o r y , a n d Dr. J a m e s D. A n d e r s o n , P l a n t H o r m o n e L a b o r a t o r y , Beltsville A g r i c u l t u r a l R e s e a r c h C e n t e r , U . S . D . A . , f o r d i s c u s s i o n . This w o r k w a s s u p p o r t e d b y I n t e r n a t i o n a l Communication Agency, Grant 2420, administered by the Council for International Exchange of Scholars, U.S.A.
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