- Email: [email protected]
The effect of gibberellins and growth retardants on in vitro flowering of the vegetative apex of Japanese pear
Scientia Horticulturae, 41 (1990) 223-232 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
223
The Effect of Gibberellins and G r o w t h R e t a r d a n t s on in vitro F l o w e r i n g of the V e g e t a t i v e A p e x of Japanese Pear
YOSHIHIRO HIGASHIUCHI1, KEISUKE KIDO 2, TAKAO ICHII3, TETSU NAKANISHI3, YOSHITAKA KAWAI8 and TAKESHI OZAKIz
1Nippon Zoki Pharmaceutical (Co.), Yashirocho, Katogun, Hyogo (Japan) 2Mochida Pharmaceutical (Co.), 1-7 Yotsuya, Shinjikuku, Tokyo (Japan) 3Department of Agriculture, Kobe University, Kobe (Japan) (Accepted for publication 19 May 1989 )
ABSTRACT Higashiuchi, Y., Kido, K., Ichii, T., Nakanishi, T., Kawai, Y. and Ozaki, T., 1990. The effect of gibberellins and growth retardants on in vitro flowering of the vegetative apex of Japanese pear. Scientia Hortic., 41: 223-232. Effects of gibberellins GA3 and GA4 and of growth retardants succinic acid-2,2-dimethylhydrazide(B-9), (2-chloroethyl)trimethylammoniumchloride (CCC)and (E)-l-(4-chlorophenyl)-4,4dimethyl-2- ( 1,2,4-triazol-1-yl) - 1-penten-3-oi (S-3307) on flowering of Japanese pear cultivars 'Kosui' and 'Chojuro' were investigated. Explants were prepared from the shoot apex or the axillary bud. The presence of GA4 but not GAs in the culture medium tended to decrease flowering of 'Chojuro' explants, although inhibition was less clear in 'Kosui' explants which showed low flowering percentages. S-3307 effectively promoted flowering, particularly for 'Kosui' explants. The promotive effect of CCC was slight or none, and B-9 reduced flowering. GA4 (but not GA~) or the growth retardants had a tendency to increase or decrease shoot and internode length of the explants; these morphological changes were not always associated with flowering behavior. The growth regulators had little or no effect on node number of the explants, which was closely related to flowering. Successive subculture of the vegetative apex in the adult form resulted in a rapid loss of flowering ability with an increase in shoot or internode length in the resulting explants, shifting to juvenile characteristics. Keywords: flowering; gibberellin; growth retardant; in vitro; pear. Abbreviations: BA=benzyladenine; B-9=succinic acid-2,2-dimethylhydrazide; CCC= (2-chloroethyl )trimethylammoniumchloride; G = gibberellin; GA8-- gibberellic acid; S-3307 = (E) - 1(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-l-yl)-l-penten-3-ol.
0304-4238/90/$03.50
© 1990 Elsevier Science Publishers B.V.
224
Y. H I G A S H I U C H I E T AL.
INTRODUCTION It is well known that growth regulators, particularly gibberellin (G) and growth retardants such as B-9 and CCC play important roles in flower formation in certain herbaceous and woody plants including a wide variety of fruit trees (Weaver, 1972; Hoad, 1984). In general, G inhibits flowering, while growth retardants, which block G biosynthesis or action, promote it (Luckwill, 1970). Since there is a well-known antagonism between vegetative and reproductive growth, it remains unclear whether these growth regulators exert their effects on flowering directly (Hoad, 1979), or indirectly by influencing vegetative growth (Sachs, 1977; Sachs and Hackett, 1977). In our previous paper (Tsujikawa et al., 1989), we reported on an isolated vegetative apex culture of Japanese pear ( P y r u s serotina Rehd.) cultivars, in which the explants could form flowers in the bract axils of their main shoot. The results indicated that the factors related to flowering capacity are inherent in the apex structure itself, and that GA4+ 7 can directly inhibit flowering. The present study was undertaken to define further how different kinds of G and growth retardants can influence flowering in cultures of Japanese pear cultivats, 'Kosui' and 'Chojuro'. MATERIALSAND METHODS Axillary leaf buds on very long shoots were collected in winter in 1985-1986 from mature trees of Japanese pear cultivars 'Kosui' and 'Chojuro', which were planted in the orchard of Kobe University at Kasai. During May-June in 19851987 vigorously growing shoots were collected. Explants were prepared from axillary buds or shoot apices as described previously (Tsujikawa et al., 1989). The culture medium was the same as those used previously (Tsujikawa et al., 1989). All the culture media contained 1 mg 1-1 benzyladenine (BA) as a growth promotor because it is necessary for the growth of explants (Yotsuya et al., 1980). Other growth regulators used were GA3 and GA4 (Kyowa Hakko Kogyo Co.), B-9 (Nippon Soda Co.), CCC (Nakarai Chemicals Co.) and S3307 (Sumitomo Chemicals Co.). The pH was adjusted to 5.7 with 0,1 N KOH before autoclaving a t 120°C for 20 min. The cultures were kept at 25-27°C and a 14-h photoperiod under 2000-3000 lx (52.2-88.2 #mol m -2 s - 1; 400-700 n m ) from 40 W white fluorescent tubes, unless otherwise mentioned. At the end of the culture period, length and number of leaves and n u m b e r of flowers on the main shoot of explants, and explant fresh weight were determined. The number of flowers a n d / o r leaves, counted under a binocular microscope, is referred to as "node number of the explants". E x p e r i m e n t 1. - Explants were prepared from axillary buds sampled in January
1985. After incubation on the medium with I m g 1-1 GA4 for 30 days, the
GA3, GA4AND GROWTH RETARDANTS ON JAPANESE PEAR
225
explants were transferred to the medium with or without 1 mg 1-1 GA4, and subcultured for 45 days. At the start of subculture, the explants in each group were divided into three subgroups, to which three droplets of GA4 or BA each at 100 mg l - 1 or none were applied, respectively, through the apex with microfilter (0.45 ttm in diameter). 2 . - Explants were prepared from shoot apices sampled in June 1987. After incubation on the medium with 1 mg 1-1 GA4 for 40 days, the explants were transferred to the media containing GA3 or GA4 each at 0, 0.1, 0.5 or 1.0 mg 1-1 and subcultured for 55 days. S-3307 at 1 mg 1-1 in 'Kosui' and 0.1 mg 1-1 in 'Chojuro' was added to the subculture medium.
Experiment
3. - Explants were prepared from axillary buds sampled in January 1985. After incubation on the medium with I mg 1-1 GA4 for 25 or 35 days, the explants in each group were transferred to four different media which contained B-9 or CCC each at 10 mg 1-1, S-3307 at i mg 1-1, or none, and subcultured for 50 days in the former group and 45 days in the latter. Experiment
4. - Explants were prepared from shoot apices sampled in M a y June 1985. After incubation on the medium with 1 mg 1-1 GAt for 40 days, the explants were transferred to the media containing CCC at 1, 5 or 10 mg 1-1 and S-3307 at 0.1, 0.5 or 1.0 mg 1-1, and subcultured for 55-60 days. A medium containing no growth retardant was regarded as control. Explants were also prepared from axillary buds sampled in February 1986, and treated similarly, except that they were subcultured only on the medium with S-3307 for 45 days. Experiment
5. - Axillary buds were sampled in November 1986 and January 1987, and shoot apices were sampled in June 1987. Explants were prepared immediately from the second sample, and after the tenth and fourth subcultures from the first and third samples, respectively. In each successive subculture, shoot apices only were cut and grown on the medium with I mg 1-1 GAt for 35 days. Juvenile materials were obtained from 4-5-month-old seedlings from the seeds of 'Kosui' and 'Chojuro'. Their shoot apices were similarly proliferated by successive subcultures before explants were prepared. After incubation on the medium with I mg 1-1 for 35 days, the explants from the juvenile and adult material were transferred to the medium without GAI but with I mg 1-1 S-3307 for 'Kosui' and 0.1 mg 1-1 for 'Chojuro' and grown for 45 days in a growth chamber at 25 + 2°C and a 10-h photoperiod under 3000 lx (76.5/tmol m -2 s - l ; 400-700 nm) from 40 W white fluorescent tubes. Experiment
226
Y. HIGASHIUCHI ET AL.
RESULTS
Effect of growth promoters. - GA4 applied to the medium or the shoot apex
promoted shoot growth of explants in both cultivars, except when applied to the shoot apex in 'Kosui' (Table 1). GA4 had no significant effect on node number of the explants. The presence of GA4 in the medium reduced the flowering percentages only in 'Chojuro'. GA4 applied to the shoot apex had no significant effect on flowering. This is equally true of BA applied to the apex. GA3 had scarcely any effect on growth and flowering of the explants in both cultivars (Table 2). On the other hand, in both cultivars, increasing GA4 concentration tended to decrease fresh weight of explants but to increase their shoot length (Table 2). Thus internode length increased from 0.50 to 0.86 m m in 'Kosui' and from 0.72 to 1.07 m m in 'Chojuro'. The flowering percentage in 'Chojuro' explants decreased with increasing GA4 concentration (Table 2). However, this tendency was not clear in 'Kosui' explants which showed an extremely low flowering percentage or none at all despite the addition of S3307 to the medium which would be expected to increase flowering (see Table 4). Effect of growth retardants. - Although the effects of B-9, CCC and S-3307 on
growth and flowering were tested for two groups of explants, incubated on the TABLE1 M a i n effects o f GA4 a p p l i e d to t h e m e d i u m , a n d o f GA4 or B A a p p l i e d to t h e a p e x o f e x p l a n t s o n g r o w t h a n d f l o w e r i n g of e x p l a n t s ( E x p e r i m e n t 1 ) Treatment
Medium GA4 Control GA4 Control Shoot apex GA4 BA Control GA4 BA Control
Cultivar
Number of explants
Shoot length (mm)
Node number
Percent of flowering
'Kosui'
121 123 151 152
33.5 ~1 31.15 28.5 a 23.4 b
28.6 al 29.4 a 32.2 ~ 31.3 a
16.6 ~ 21.9 a 9.9 a 19.7 b
54 70 120 79 102 122
28.7 a 30.0 a 35.35 29.6 ~ 25.05 24.35
28.7 ab 27.3 a 30.25 32.1 ~ 31.0 a 32.2 a
11.1 a 28.6 a 17.6 ~ 15.2 a 16.7 ~ 13.1 ~
'Chojuro'
'Kosui'
'Chojuro'
1Values w i t h i n c o l u m n p e r c u l t i v a r followed b y t h e s a m e s u p e r s c r i p t a r e n o t s i g n i f i c a n t l y d i f f e r e n t a t t h e 5% level ( D u n c a n ' s m u l t i p l e r a n g e t e s t ) . 2Significance as a b o v e u s i n g )/2 t e s t .
GAa, GA4 AND GROWTH RETARDANTS ON JAPANESE PEAR
227
TABLE 2 E f f e c t o f d i f f e r e n t c o n c e n t r a t i o n s o f GA3 a n d GA4 o n g r o w t h a n d f l o w e r i n g o f e x p l a n t s ( E x p e r i ment 2 ) Cultivar
Treatment
Number of explants
Fresh w e i g h t (g)
Shoot length (mm)
Node number
Percent of flowering
GA3 ( m g l - 1) 'Kosui' 0 0.1 0.5 1.0 'Chojuro' 0 0.1 0.5 1.0
37 35 39 31 31 33 35 27
0.47 ~1 0.44 a 0.49 ~ 0.45 ~ 0.38 ~ 0.38 ~ 0.40 ~ 0.48"
21.7 al 21.2 ~ 20.9 a 17.35 20.9 ~ 19.3 a 18.6 ~ ~ 18.4"
20.0 ~1 20.2" 19.2" 17.15 23.1 ~ 22.0 a 24.4 ~ 23.1 ~
8.1 ~2 0~ 5.1" 6.4" 48.3 "5 63.6 ~ 31.45 59.2 ~
GA4 ( m g 1-1) 'Kosui' 0 0.1 0.5 1.0 'Chojuro' 0 0.1 0.5 1.0
24 25 20 17 30 29 32 20
0.63" 0.345 0.235 0.245 0.48 a 0.51 ~ 0.325 0.265
12.0 a 12.3" 12.6" 16.45 15.7" 19.2 a5 18.4 ~5 22.6 b
24.1 a 22.7 a 20.05 19.15 21.8" 21.5 a 18.85 21.0 a
4.1 a 0a 0" 0" 66.7" 31.05 25.05 20.05
1Values w i t h i n c o l u m n p e r c u l t i v a r followed b y t h e s a m e s u p e r s c r i p t are n o t s i g n i f i c a n t l y d i f f e r e n t a t t h e 5% level ( D u n c a n ' s m u l t i p l e r a n g e t e s t ) . 2Significance as a b o v e u s i n g X2 t e s t .
medium with GA4 for 25 or 35 days, essentially similar results were obtained. Therefore, the average values of the two groups are presented in Table 3. All growth retardants decreased shoot length of the explants in both cultivars except CCC with 'Chojuro'. In both cultivars S-3307 slightly decreased node number. Both S-3307 and CCC increased the flowering percentage in 'Chojuro' but not in 'Kosui'. On the other hand, B-9 decreased it in both cultivars. In the experiment with shoot apices in 1985, S-3307 did not significantly decrease shoot length in both cultivars except when the concentration of 1 mg 1-1 was applied to 'Chojuro' explants (Table 4). In 'Kosui', S-3307 slightly increased node number. Although there was no significant effect on flowering in 'Chojuro' explants, S-3307 markedly increased flowering in 'Kosui'. CCC at 1-10 mg 1-1 did not affect either growth or flowering in both cultivars (data not shown). In the experiment with axillary buds in 1986, increasing S-3307 concentration significantly decreased shoot length, thus internode length, in both cultivars. In 'Kosui' explants S-3307 at 0.5-1.0 mg 1-1 slightly increased node number. Although S-3307 produced variable results on shoot growth of
228
Y. HIGASHIUCHI ET AL.
TABLE 3 Effect of B-9, CCC a n d S-3307 o n g r o w t h a n d flowering of e x p l a n t s ( E x p e r i m e n t 3 ) Cultivar
Growth retardant
N u m b e r of explants
Shoot length (m m )
Node number
P e r c e n t of flowering
'Kosui'
Cont. B-9 CCC S-3307 Cont. B-9 CCC S-3307
99 75 68 73 122 73 76 79
29.3 ~1 16.7 c 23.1 b 18.4 c 20.35 16.8 ~ 23.4 a 16.1 ¢
29.3 a51 30.2 a 28.25~ 27.2 ~ 30.55 33.7 ~ 30.55 28.3 c
23.5 4 5.35 23.5 ~ 17.3 ~ 21.35 13.7 c 48.7 ~ 63.3 ~
'Chojuro'
~Values w i t h i n c o l u m n p e r cultivar followed b y t h e s a m e s u p e r s c r i p t are n o t significantly different at t h e 5% level ( D u n c a n ' s m u l t i p l e range t e s t ) . 2Significance as above u s i n g Z 2 test.
TABLE 4 Effect of different c o n c e n t r a t i o n s of S-3307 o n g r o w t h a n d flowering of e x p l a n t s ( E x p e r i m e n t 4 ) Cultivar
Concentration (mg l- t )
N u m b e r of explants
Shoot length (mm)
apex) 32 29 27 34 16 8 14 15
23.1 al 24.7 ~ 21.9 ~ 25.1 ~ 17.3 a 15.6 a5 15.1 a5 12.9 b
S a m p l e d in F e b r u a r y 1986 (axillary b u d ) 'Kosui' 0 58 0.1 59 0.5 60 1.0 58 'Chojuro' 0 56 0.1 58 0:5 57 1.0 58
29.1 a 26.9 ab 24.35c 21.2 c 20.0 a 16.45 15.95 14.95
S a m p l e d in M a y - J u n e 1985 ( s h o o t 'Kosui' 0 0.1 0.5 1.0 'Chojuro' 0 0.1 0.5 1.0
Node number
P e r c e n t of flowering
22.5 al
28.05
21.9 ab2 6.9 a
25.2 a5 26.45 19.7 ~ 22.8 ~ 20.1 ~ 18.9 ~
73.5 c 50.0 ~ 87.5 a 71.4 a 66.7 ~
25.2 a 25.3 a 28.5 b 27.55 22.7 ~ 23.0 a 23.6 a 23.7 ~
8.6 a 16.98 36.65 37.95 71.4 a 68.9 a 71.9 a 63.8 ~
52.05c
1Values w i t h i n c o l u m n p e r cultivar followed by t h e s a m e s u p e r s c r i p t are n o t significantly different at t h e 5% level ( D u n c a n ' s m u l t i p l e range t e s t ) . 2Significance as above u s i n g Z 2 test.
GA3,GA4AND GROWTH RETARDANTS ON JAPANESE PEAR
229
TABLE 5 G r o w t h a n d flowering of explants from adult cultivars w i t h different n u m b e r s of subculture a n d from juvenile seedlings ( E x p e r i m e n t 5) Cultivar
N u m b e r of subculture
N u m b e r of explants
Fresh weight (g)
Shoot length (mm)
Node number
Internode length (mm)
Percent of flowering
0 4
'Seedling'
-
17 11 44
0.65 0.52 0.52
15.8 18.5 26.8
23.4 17.3 20.2
0.65 1.07 1.32
17.6 al 0a 0a
'Chojuro'
0 4 10
42 17 21 47
0.50 0.38 0.67 0.47
14.3 28.7 35.4 23.9
21.6 22.6 26.7 19.4
0.66 1.26 1.32 1.23
71.8 a 17.65 0b 05
'Kosui'
'Seedling'
1Values w i t h i n column followed by the same superscript are n o t significantly different at the 5% level (X2 test).
the explants in the two experiments, it gave almost identical results on flowering behavior; it effectively promoted flowering of the explants from 'Kosui', a cultivar having low flowering capacity. Effect of number of subculture. - With the number of subculture, shoot and
internode length tended to increase, and the flowering percentage rapidly decreased in both cultivars (Table 5). The juvenile seedlings had larger shoot and internode length than their adult materials without subculture, and did not flower. Unfortunately, however, we have no data for the juvenile seedlings without subculture since they have been proliferated by successive subculture. DISCUSSION
Flower formation in most fruit trees is known to be under competitive control by shoot growth and fruit development. Since there is usually an antagonism between shoot growth and flowering, G or growth retardants, which stimulate or inhibit shoot growth, respectively, have been thought to act on flowering indirectly through influencing shoot growth (Sachs, 1977; Sachs and Hackett, 1977). On the other hand, there is evidence that vegetative and reproductive growth are sometimes independent. For example, G inhibited flowering of apple (Wertheim, 1973) and B-9 promoted it (Tromp, 1972) in the absence of any growth effect. These lines of evidence proposed the idea that growth regulators directly regulate flowering. Further evidence which supports this view comes from the biennial bearing in apple and pear, in which inhibition of flowering is suggested to be due to G produced by seeds (Hoad, 1979).
230
Y. HIGASHIUCHIET AL.
In an effort to clarify the direct effect of these regulators on flowering, independent of the influence of the rest of the tree as mentioned above, the present vegetative apex culture was carried out. The GA4 reduced flowering of the explants, while the growth retardant S-3307 promoted it, supporting the idea of the direct role of growth regulators in flowering (Hoad, 1979). The effect of GA4 on flowering was sometimes not clear. This may result partly from the interaction with BA in the medium, which has been reported to cancel GA4induced inhibition of flowering (McLaughlin and Greene, 1984). It is known that the different types of G produce different effects on flowering (Tromp, 1982; Looney et al., 1985). In contrast with GA4, GA3 was inactive in either flowering or growth of the explants in the present study. Gibberellin is suggested to increase the activity of the subapical meristem of the vegetative apex (Sachs et al., 1959). Thus the activity can be reduced by growth retardants which inhibit G biosynthesis (Hackett, 1976). On the other hand, it is suggested that node production is a function of the activity of apical meristem, and internode length and height are functions of that of subapical meristem (Stein and Fosket, 1969; Zimmerman, 1973). In agreement with these suggestions, GA4 or S-3307, an inhibitor of G biosynthesis (Izumi et al., 1985 ), increased or decreased shoot and internode length, respectively, but they had little or no effect on node production, indicating that they exert their effects through modifying the subapical meristematic activity. However, in accord with our previous study (Tsujikawa et al., 1989), these morphological changes were not always associated with flowering behavior. When the average flowering percentage for each experimental plot used in the present study is plotted against the average node number or shoot length, the percentage is more closely related to node number. There is a significant curvilinear relationship between the flowering percentage and node number; quadratic correlation coefficients (d.f.--29) were 0.725 and 0.470, and the node numbers which gave rise to a maximum flowering were 23.5 and 25.6 for 'Chojuro' and 'Kosui', respectively. Thus the mode of action of these growth regulators in flowering and the abovementioned morphological changes seem to be independent. Stein and Fosket (1969) indicated that juvenile Hedera helix has a much more active subapical meristem t h a n the adult form, resulting in more rapid shoot elongation. Multiple subcultures of the vegetative apex in the adult form have been reported to tend to result in juvenile characteristics (Mullins et al., 1979; Gupta et al., 1981; Lyrene, 1981). Successive subculture of vegetative apices of Japanese pear cultivars produced resulting explants with a longer shoot and internode length. Associated with these morphological changes, there was a rapid loss of the flowering capacity of the explants. The opposite is true for the rooting ability as reported elsewhere (Satsu et al., 1988). This suggests the possibility that the successive subcultures induce rejuvenation in the vegetative apex of the adult form. Unfortunately, we have no data available for juvenile explants without subculture. In the present study, vegetative apices
GA3, GA4 AND GROWTH RETARDANTS ON JAPANESE PEAR
231
were successively subcultured on the medium with GA4. Gibberellin is known to have an important role in rejuvenation of adult H. helix (Rogler and Hackett, 1975). Further, mature leaves are suggested to aid in stabilizing the adult form by producing some substances antagonistic to the G effect (Hackett, 1976). An alternative possibility that the vegetative apex in the adult form has some factors capable of inducing flowering, which disappear temporarily with successive subculture, is not excluded.
REFERENCES Gupta, P.K., Mascarenhas, A.F. and Jagannathan, V., 1981. Tissue culture of forest trees: clonal propagation of mature trees of Eucalyptus citriodora by tissue culture. Plant Sci. Lett., 20: 195-201. Hackett, W.P., 1976. Control of phase change in woody plants. Acta Hortic., 56: 143-154. Hoad, G.V., 1979. Growth regulators, endogenous hormones and flower initiation in apple. Annu. Rep. Long Ashton Res. Stn., pp. 199-206. Hoad, G.V., 1984. Hormonal regulation of fruit-bud formation in fruit trees.. Acta Hortic., 149: 13-23. Izumi, K., Kamiya, Y., Sakurai, A., Oshio, H. and Takahashi, H., 1985. Studies of sites of action of a new plant growth retardant (E) - 1- (4-chlorophenyl) -4,4-dimethyl-2- (1,2,4-triazol-1-yl) 1-penten-3-ol (S-3307) and comparative effects of its stereoisomers in a cell-free system from Cucurbita maxima. Plant Cell Physiol., 26: 821-827. Looney, N.E., Pharis, R.P. and Noma, M., 1985. Promotion of flowering in apple trees with gibberellin A4 and C-3 epi-gibberellin A4. Planta, 165: 292-294. Luckwill, L.C., 1970. The control of growth and fruitfullness of apple trees. In: L.C. Luckwill and C.V. Cutting (Editors), Physiology of Tree Crops. Academic Press, London, pp. 237-254. Lyrene, P.M., 1981. Juvenility and production of fast-rooting cuttings from blueberry shoot cultures. J. Am. Soc. Hortic. Sci., 106: 396-398. McLaughlin, J.M. and Greene, D.W., 1984. Effects of BA, GA4+7, and daminozide on fruit set, fruit quality, vegetative growth, flower initiation, and flower quality of 'Golden Delicious' apple. J. Am. Soc. Hortic. Sci., 109: 34-39. Mullins, M.G., Nair, Y. and Sampet, P., 1979. Rejuvenation in vitro: Induction of juvenile characters in an adult clone of Vitis vinifera L. Ann. Bot., 44: 623-627. Rogler, C.R. and Hackett, W.P., 1975. Phase change in Hedera helix: Induction of the mature to juvenile change by gibberellin A3. Physiol. Plant., 34: 148-152. Sachs, R.M., 1977. Nutrient diversion: An hypothesis to explain the chemical control of flowering. HortScience, 12: 220-222. Sachs, R.M. and Hackett, W.P., 1977. Chemical control of flowering. Acta Hortic., 68: 29-49. Sachs, R.M., Bretz, C.F. and Lang, A., 1959. Shoot histogenesis: the early effects of gibberellin upon stem elongation in two rosette plants. Am. J. Bot., 46: 376-384. Satsu, T., Ichii, T., Nakanishi, T. and Kawai, Y., 1988. In vitro propagation of Japanese pear (Pyrus serotina). Sci. Rep. Fac. Agric. Kobe Univ., 18:1-7 (in Japanese). Stein, O.L. and Fosket, E.B., 1969. Comparative developmental anatomy of shoots of juvenile and adult Hedera helix. Am. J. Bot., 54: 546-551. Tromp, J., 1972. Effects of growth-regulating substances and tree orientation on growth and flowerbud formation in apple. J. Hortic. Sci., 47: 525-533. Tromp, J., 1982. Flower-bud formation in apple as affected by various gibberellins. J. Hortic. Sci., 57: 277-282.
232
Y. HIGASHIUCHI ET AL.
Tsujikawa, T., Ichii, T., Nakanishi, T., Ozaki, T. and Kawai, Y., 1989. In vitro flowering of Japanese pear and the effect of GA4+7. Scientia Hortic., 41: 233-245. Weaver, R.J., 1972. Flowering. In: R.J. Weaver (Editor), Plant Growth Substance in Agriculture. W.H. Freeman, San Francisco, CA, pp. 176-221. Wertheim, S.J., 1973. Fruit set and June drop in Cox's Orange Pippin apple as affected by pollination and treatment with a mixture of gibberellins A4 and AT. Scientia Hortic., 1: 85-105. Yotsuya, T., Ichii, T., Sawano, M. and Nakanishi, T., 1980. Studies on growth cycle of Japanese pear buds: Effects of GA4+7 and BA on the in vitro growth of Kosui leaf buds. Sci. Rep. Fac. Agric. Kobe Univ., 14:45-49 (in Japanese). Zimmerman, R.H., 1973. Juvenility and flowering in woody plants. Acta Hortic., 34: 139-142.
223
The Effect of Gibberellins and G r o w t h R e t a r d a n t s on in vitro F l o w e r i n g of the V e g e t a t i v e A p e x of Japanese Pear
YOSHIHIRO HIGASHIUCHI1, KEISUKE KIDO 2, TAKAO ICHII3, TETSU NAKANISHI3, YOSHITAKA KAWAI8 and TAKESHI OZAKIz
1Nippon Zoki Pharmaceutical (Co.), Yashirocho, Katogun, Hyogo (Japan) 2Mochida Pharmaceutical (Co.), 1-7 Yotsuya, Shinjikuku, Tokyo (Japan) 3Department of Agriculture, Kobe University, Kobe (Japan) (Accepted for publication 19 May 1989 )
ABSTRACT Higashiuchi, Y., Kido, K., Ichii, T., Nakanishi, T., Kawai, Y. and Ozaki, T., 1990. The effect of gibberellins and growth retardants on in vitro flowering of the vegetative apex of Japanese pear. Scientia Hortic., 41: 223-232. Effects of gibberellins GA3 and GA4 and of growth retardants succinic acid-2,2-dimethylhydrazide(B-9), (2-chloroethyl)trimethylammoniumchloride (CCC)and (E)-l-(4-chlorophenyl)-4,4dimethyl-2- ( 1,2,4-triazol-1-yl) - 1-penten-3-oi (S-3307) on flowering of Japanese pear cultivars 'Kosui' and 'Chojuro' were investigated. Explants were prepared from the shoot apex or the axillary bud. The presence of GA4 but not GAs in the culture medium tended to decrease flowering of 'Chojuro' explants, although inhibition was less clear in 'Kosui' explants which showed low flowering percentages. S-3307 effectively promoted flowering, particularly for 'Kosui' explants. The promotive effect of CCC was slight or none, and B-9 reduced flowering. GA4 (but not GA~) or the growth retardants had a tendency to increase or decrease shoot and internode length of the explants; these morphological changes were not always associated with flowering behavior. The growth regulators had little or no effect on node number of the explants, which was closely related to flowering. Successive subculture of the vegetative apex in the adult form resulted in a rapid loss of flowering ability with an increase in shoot or internode length in the resulting explants, shifting to juvenile characteristics. Keywords: flowering; gibberellin; growth retardant; in vitro; pear. Abbreviations: BA=benzyladenine; B-9=succinic acid-2,2-dimethylhydrazide; CCC= (2-chloroethyl )trimethylammoniumchloride; G = gibberellin; GA8-- gibberellic acid; S-3307 = (E) - 1(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-l-yl)-l-penten-3-ol.
0304-4238/90/$03.50
© 1990 Elsevier Science Publishers B.V.
224
Y. H I G A S H I U C H I E T AL.
INTRODUCTION It is well known that growth regulators, particularly gibberellin (G) and growth retardants such as B-9 and CCC play important roles in flower formation in certain herbaceous and woody plants including a wide variety of fruit trees (Weaver, 1972; Hoad, 1984). In general, G inhibits flowering, while growth retardants, which block G biosynthesis or action, promote it (Luckwill, 1970). Since there is a well-known antagonism between vegetative and reproductive growth, it remains unclear whether these growth regulators exert their effects on flowering directly (Hoad, 1979), or indirectly by influencing vegetative growth (Sachs, 1977; Sachs and Hackett, 1977). In our previous paper (Tsujikawa et al., 1989), we reported on an isolated vegetative apex culture of Japanese pear ( P y r u s serotina Rehd.) cultivars, in which the explants could form flowers in the bract axils of their main shoot. The results indicated that the factors related to flowering capacity are inherent in the apex structure itself, and that GA4+ 7 can directly inhibit flowering. The present study was undertaken to define further how different kinds of G and growth retardants can influence flowering in cultures of Japanese pear cultivats, 'Kosui' and 'Chojuro'. MATERIALSAND METHODS Axillary leaf buds on very long shoots were collected in winter in 1985-1986 from mature trees of Japanese pear cultivars 'Kosui' and 'Chojuro', which were planted in the orchard of Kobe University at Kasai. During May-June in 19851987 vigorously growing shoots were collected. Explants were prepared from axillary buds or shoot apices as described previously (Tsujikawa et al., 1989). The culture medium was the same as those used previously (Tsujikawa et al., 1989). All the culture media contained 1 mg 1-1 benzyladenine (BA) as a growth promotor because it is necessary for the growth of explants (Yotsuya et al., 1980). Other growth regulators used were GA3 and GA4 (Kyowa Hakko Kogyo Co.), B-9 (Nippon Soda Co.), CCC (Nakarai Chemicals Co.) and S3307 (Sumitomo Chemicals Co.). The pH was adjusted to 5.7 with 0,1 N KOH before autoclaving a t 120°C for 20 min. The cultures were kept at 25-27°C and a 14-h photoperiod under 2000-3000 lx (52.2-88.2 #mol m -2 s - 1; 400-700 n m ) from 40 W white fluorescent tubes, unless otherwise mentioned. At the end of the culture period, length and number of leaves and n u m b e r of flowers on the main shoot of explants, and explant fresh weight were determined. The number of flowers a n d / o r leaves, counted under a binocular microscope, is referred to as "node number of the explants". E x p e r i m e n t 1. - Explants were prepared from axillary buds sampled in January
1985. After incubation on the medium with I m g 1-1 GA4 for 30 days, the
GA3, GA4AND GROWTH RETARDANTS ON JAPANESE PEAR
225
explants were transferred to the medium with or without 1 mg 1-1 GA4, and subcultured for 45 days. At the start of subculture, the explants in each group were divided into three subgroups, to which three droplets of GA4 or BA each at 100 mg l - 1 or none were applied, respectively, through the apex with microfilter (0.45 ttm in diameter). 2 . - Explants were prepared from shoot apices sampled in June 1987. After incubation on the medium with 1 mg 1-1 GA4 for 40 days, the explants were transferred to the media containing GA3 or GA4 each at 0, 0.1, 0.5 or 1.0 mg 1-1 and subcultured for 55 days. S-3307 at 1 mg 1-1 in 'Kosui' and 0.1 mg 1-1 in 'Chojuro' was added to the subculture medium.
Experiment
3. - Explants were prepared from axillary buds sampled in January 1985. After incubation on the medium with I mg 1-1 GA4 for 25 or 35 days, the explants in each group were transferred to four different media which contained B-9 or CCC each at 10 mg 1-1, S-3307 at i mg 1-1, or none, and subcultured for 50 days in the former group and 45 days in the latter. Experiment
4. - Explants were prepared from shoot apices sampled in M a y June 1985. After incubation on the medium with 1 mg 1-1 GAt for 40 days, the explants were transferred to the media containing CCC at 1, 5 or 10 mg 1-1 and S-3307 at 0.1, 0.5 or 1.0 mg 1-1, and subcultured for 55-60 days. A medium containing no growth retardant was regarded as control. Explants were also prepared from axillary buds sampled in February 1986, and treated similarly, except that they were subcultured only on the medium with S-3307 for 45 days. Experiment
5. - Axillary buds were sampled in November 1986 and January 1987, and shoot apices were sampled in June 1987. Explants were prepared immediately from the second sample, and after the tenth and fourth subcultures from the first and third samples, respectively. In each successive subculture, shoot apices only were cut and grown on the medium with I mg 1-1 GAt for 35 days. Juvenile materials were obtained from 4-5-month-old seedlings from the seeds of 'Kosui' and 'Chojuro'. Their shoot apices were similarly proliferated by successive subcultures before explants were prepared. After incubation on the medium with I mg 1-1 for 35 days, the explants from the juvenile and adult material were transferred to the medium without GAI but with I mg 1-1 S-3307 for 'Kosui' and 0.1 mg 1-1 for 'Chojuro' and grown for 45 days in a growth chamber at 25 + 2°C and a 10-h photoperiod under 3000 lx (76.5/tmol m -2 s - l ; 400-700 nm) from 40 W white fluorescent tubes. Experiment
226
Y. HIGASHIUCHI ET AL.
RESULTS
Effect of growth promoters. - GA4 applied to the medium or the shoot apex
promoted shoot growth of explants in both cultivars, except when applied to the shoot apex in 'Kosui' (Table 1). GA4 had no significant effect on node number of the explants. The presence of GA4 in the medium reduced the flowering percentages only in 'Chojuro'. GA4 applied to the shoot apex had no significant effect on flowering. This is equally true of BA applied to the apex. GA3 had scarcely any effect on growth and flowering of the explants in both cultivars (Table 2). On the other hand, in both cultivars, increasing GA4 concentration tended to decrease fresh weight of explants but to increase their shoot length (Table 2). Thus internode length increased from 0.50 to 0.86 m m in 'Kosui' and from 0.72 to 1.07 m m in 'Chojuro'. The flowering percentage in 'Chojuro' explants decreased with increasing GA4 concentration (Table 2). However, this tendency was not clear in 'Kosui' explants which showed an extremely low flowering percentage or none at all despite the addition of S3307 to the medium which would be expected to increase flowering (see Table 4). Effect of growth retardants. - Although the effects of B-9, CCC and S-3307 on
growth and flowering were tested for two groups of explants, incubated on the TABLE1 M a i n effects o f GA4 a p p l i e d to t h e m e d i u m , a n d o f GA4 or B A a p p l i e d to t h e a p e x o f e x p l a n t s o n g r o w t h a n d f l o w e r i n g of e x p l a n t s ( E x p e r i m e n t 1 ) Treatment
Medium GA4 Control GA4 Control Shoot apex GA4 BA Control GA4 BA Control
Cultivar
Number of explants
Shoot length (mm)
Node number
Percent of flowering
'Kosui'
121 123 151 152
33.5 ~1 31.15 28.5 a 23.4 b
28.6 al 29.4 a 32.2 ~ 31.3 a
16.6 ~ 21.9 a 9.9 a 19.7 b
54 70 120 79 102 122
28.7 a 30.0 a 35.35 29.6 ~ 25.05 24.35
28.7 ab 27.3 a 30.25 32.1 ~ 31.0 a 32.2 a
11.1 a 28.6 a 17.6 ~ 15.2 a 16.7 ~ 13.1 ~
'Chojuro'
'Kosui'
'Chojuro'
1Values w i t h i n c o l u m n p e r c u l t i v a r followed b y t h e s a m e s u p e r s c r i p t a r e n o t s i g n i f i c a n t l y d i f f e r e n t a t t h e 5% level ( D u n c a n ' s m u l t i p l e r a n g e t e s t ) . 2Significance as a b o v e u s i n g )/2 t e s t .
GAa, GA4 AND GROWTH RETARDANTS ON JAPANESE PEAR
227
TABLE 2 E f f e c t o f d i f f e r e n t c o n c e n t r a t i o n s o f GA3 a n d GA4 o n g r o w t h a n d f l o w e r i n g o f e x p l a n t s ( E x p e r i ment 2 ) Cultivar
Treatment
Number of explants
Fresh w e i g h t (g)
Shoot length (mm)
Node number
Percent of flowering
GA3 ( m g l - 1) 'Kosui' 0 0.1 0.5 1.0 'Chojuro' 0 0.1 0.5 1.0
37 35 39 31 31 33 35 27
0.47 ~1 0.44 a 0.49 ~ 0.45 ~ 0.38 ~ 0.38 ~ 0.40 ~ 0.48"
21.7 al 21.2 ~ 20.9 a 17.35 20.9 ~ 19.3 a 18.6 ~ ~ 18.4"
20.0 ~1 20.2" 19.2" 17.15 23.1 ~ 22.0 a 24.4 ~ 23.1 ~
8.1 ~2 0~ 5.1" 6.4" 48.3 "5 63.6 ~ 31.45 59.2 ~
GA4 ( m g 1-1) 'Kosui' 0 0.1 0.5 1.0 'Chojuro' 0 0.1 0.5 1.0
24 25 20 17 30 29 32 20
0.63" 0.345 0.235 0.245 0.48 a 0.51 ~ 0.325 0.265
12.0 a 12.3" 12.6" 16.45 15.7" 19.2 a5 18.4 ~5 22.6 b
24.1 a 22.7 a 20.05 19.15 21.8" 21.5 a 18.85 21.0 a
4.1 a 0a 0" 0" 66.7" 31.05 25.05 20.05
1Values w i t h i n c o l u m n p e r c u l t i v a r followed b y t h e s a m e s u p e r s c r i p t are n o t s i g n i f i c a n t l y d i f f e r e n t a t t h e 5% level ( D u n c a n ' s m u l t i p l e r a n g e t e s t ) . 2Significance as a b o v e u s i n g X2 t e s t .
medium with GA4 for 25 or 35 days, essentially similar results were obtained. Therefore, the average values of the two groups are presented in Table 3. All growth retardants decreased shoot length of the explants in both cultivars except CCC with 'Chojuro'. In both cultivars S-3307 slightly decreased node number. Both S-3307 and CCC increased the flowering percentage in 'Chojuro' but not in 'Kosui'. On the other hand, B-9 decreased it in both cultivars. In the experiment with shoot apices in 1985, S-3307 did not significantly decrease shoot length in both cultivars except when the concentration of 1 mg 1-1 was applied to 'Chojuro' explants (Table 4). In 'Kosui', S-3307 slightly increased node number. Although there was no significant effect on flowering in 'Chojuro' explants, S-3307 markedly increased flowering in 'Kosui'. CCC at 1-10 mg 1-1 did not affect either growth or flowering in both cultivars (data not shown). In the experiment with axillary buds in 1986, increasing S-3307 concentration significantly decreased shoot length, thus internode length, in both cultivars. In 'Kosui' explants S-3307 at 0.5-1.0 mg 1-1 slightly increased node number. Although S-3307 produced variable results on shoot growth of
228
Y. HIGASHIUCHI ET AL.
TABLE 3 Effect of B-9, CCC a n d S-3307 o n g r o w t h a n d flowering of e x p l a n t s ( E x p e r i m e n t 3 ) Cultivar
Growth retardant
N u m b e r of explants
Shoot length (m m )
Node number
P e r c e n t of flowering
'Kosui'
Cont. B-9 CCC S-3307 Cont. B-9 CCC S-3307
99 75 68 73 122 73 76 79
29.3 ~1 16.7 c 23.1 b 18.4 c 20.35 16.8 ~ 23.4 a 16.1 ¢
29.3 a51 30.2 a 28.25~ 27.2 ~ 30.55 33.7 ~ 30.55 28.3 c
23.5 4 5.35 23.5 ~ 17.3 ~ 21.35 13.7 c 48.7 ~ 63.3 ~
'Chojuro'
~Values w i t h i n c o l u m n p e r cultivar followed b y t h e s a m e s u p e r s c r i p t are n o t significantly different at t h e 5% level ( D u n c a n ' s m u l t i p l e range t e s t ) . 2Significance as above u s i n g Z 2 test.
TABLE 4 Effect of different c o n c e n t r a t i o n s of S-3307 o n g r o w t h a n d flowering of e x p l a n t s ( E x p e r i m e n t 4 ) Cultivar
Concentration (mg l- t )
N u m b e r of explants
Shoot length (mm)
apex) 32 29 27 34 16 8 14 15
23.1 al 24.7 ~ 21.9 ~ 25.1 ~ 17.3 a 15.6 a5 15.1 a5 12.9 b
S a m p l e d in F e b r u a r y 1986 (axillary b u d ) 'Kosui' 0 58 0.1 59 0.5 60 1.0 58 'Chojuro' 0 56 0.1 58 0:5 57 1.0 58
29.1 a 26.9 ab 24.35c 21.2 c 20.0 a 16.45 15.95 14.95
S a m p l e d in M a y - J u n e 1985 ( s h o o t 'Kosui' 0 0.1 0.5 1.0 'Chojuro' 0 0.1 0.5 1.0
Node number
P e r c e n t of flowering
22.5 al
28.05
21.9 ab2 6.9 a
25.2 a5 26.45 19.7 ~ 22.8 ~ 20.1 ~ 18.9 ~
73.5 c 50.0 ~ 87.5 a 71.4 a 66.7 ~
25.2 a 25.3 a 28.5 b 27.55 22.7 ~ 23.0 a 23.6 a 23.7 ~
8.6 a 16.98 36.65 37.95 71.4 a 68.9 a 71.9 a 63.8 ~
52.05c
1Values w i t h i n c o l u m n p e r cultivar followed by t h e s a m e s u p e r s c r i p t are n o t significantly different at t h e 5% level ( D u n c a n ' s m u l t i p l e range t e s t ) . 2Significance as above u s i n g Z 2 test.
GA3,GA4AND GROWTH RETARDANTS ON JAPANESE PEAR
229
TABLE 5 G r o w t h a n d flowering of explants from adult cultivars w i t h different n u m b e r s of subculture a n d from juvenile seedlings ( E x p e r i m e n t 5) Cultivar
N u m b e r of subculture
N u m b e r of explants
Fresh weight (g)
Shoot length (mm)
Node number
Internode length (mm)
Percent of flowering
0 4
'Seedling'
-
17 11 44
0.65 0.52 0.52
15.8 18.5 26.8
23.4 17.3 20.2
0.65 1.07 1.32
17.6 al 0a 0a
'Chojuro'
0 4 10
42 17 21 47
0.50 0.38 0.67 0.47
14.3 28.7 35.4 23.9
21.6 22.6 26.7 19.4
0.66 1.26 1.32 1.23
71.8 a 17.65 0b 05
'Kosui'
'Seedling'
1Values w i t h i n column followed by the same superscript are n o t significantly different at the 5% level (X2 test).
the explants in the two experiments, it gave almost identical results on flowering behavior; it effectively promoted flowering of the explants from 'Kosui', a cultivar having low flowering capacity. Effect of number of subculture. - With the number of subculture, shoot and
internode length tended to increase, and the flowering percentage rapidly decreased in both cultivars (Table 5). The juvenile seedlings had larger shoot and internode length than their adult materials without subculture, and did not flower. Unfortunately, however, we have no data for the juvenile seedlings without subculture since they have been proliferated by successive subculture. DISCUSSION
Flower formation in most fruit trees is known to be under competitive control by shoot growth and fruit development. Since there is usually an antagonism between shoot growth and flowering, G or growth retardants, which stimulate or inhibit shoot growth, respectively, have been thought to act on flowering indirectly through influencing shoot growth (Sachs, 1977; Sachs and Hackett, 1977). On the other hand, there is evidence that vegetative and reproductive growth are sometimes independent. For example, G inhibited flowering of apple (Wertheim, 1973) and B-9 promoted it (Tromp, 1972) in the absence of any growth effect. These lines of evidence proposed the idea that growth regulators directly regulate flowering. Further evidence which supports this view comes from the biennial bearing in apple and pear, in which inhibition of flowering is suggested to be due to G produced by seeds (Hoad, 1979).
230
Y. HIGASHIUCHIET AL.
In an effort to clarify the direct effect of these regulators on flowering, independent of the influence of the rest of the tree as mentioned above, the present vegetative apex culture was carried out. The GA4 reduced flowering of the explants, while the growth retardant S-3307 promoted it, supporting the idea of the direct role of growth regulators in flowering (Hoad, 1979). The effect of GA4 on flowering was sometimes not clear. This may result partly from the interaction with BA in the medium, which has been reported to cancel GA4induced inhibition of flowering (McLaughlin and Greene, 1984). It is known that the different types of G produce different effects on flowering (Tromp, 1982; Looney et al., 1985). In contrast with GA4, GA3 was inactive in either flowering or growth of the explants in the present study. Gibberellin is suggested to increase the activity of the subapical meristem of the vegetative apex (Sachs et al., 1959). Thus the activity can be reduced by growth retardants which inhibit G biosynthesis (Hackett, 1976). On the other hand, it is suggested that node production is a function of the activity of apical meristem, and internode length and height are functions of that of subapical meristem (Stein and Fosket, 1969; Zimmerman, 1973). In agreement with these suggestions, GA4 or S-3307, an inhibitor of G biosynthesis (Izumi et al., 1985 ), increased or decreased shoot and internode length, respectively, but they had little or no effect on node production, indicating that they exert their effects through modifying the subapical meristematic activity. However, in accord with our previous study (Tsujikawa et al., 1989), these morphological changes were not always associated with flowering behavior. When the average flowering percentage for each experimental plot used in the present study is plotted against the average node number or shoot length, the percentage is more closely related to node number. There is a significant curvilinear relationship between the flowering percentage and node number; quadratic correlation coefficients (d.f.--29) were 0.725 and 0.470, and the node numbers which gave rise to a maximum flowering were 23.5 and 25.6 for 'Chojuro' and 'Kosui', respectively. Thus the mode of action of these growth regulators in flowering and the abovementioned morphological changes seem to be independent. Stein and Fosket (1969) indicated that juvenile Hedera helix has a much more active subapical meristem t h a n the adult form, resulting in more rapid shoot elongation. Multiple subcultures of the vegetative apex in the adult form have been reported to tend to result in juvenile characteristics (Mullins et al., 1979; Gupta et al., 1981; Lyrene, 1981). Successive subculture of vegetative apices of Japanese pear cultivars produced resulting explants with a longer shoot and internode length. Associated with these morphological changes, there was a rapid loss of the flowering capacity of the explants. The opposite is true for the rooting ability as reported elsewhere (Satsu et al., 1988). This suggests the possibility that the successive subcultures induce rejuvenation in the vegetative apex of the adult form. Unfortunately, we have no data available for juvenile explants without subculture. In the present study, vegetative apices
GA3, GA4 AND GROWTH RETARDANTS ON JAPANESE PEAR
231
were successively subcultured on the medium with GA4. Gibberellin is known to have an important role in rejuvenation of adult H. helix (Rogler and Hackett, 1975). Further, mature leaves are suggested to aid in stabilizing the adult form by producing some substances antagonistic to the G effect (Hackett, 1976). An alternative possibility that the vegetative apex in the adult form has some factors capable of inducing flowering, which disappear temporarily with successive subculture, is not excluded.
REFERENCES Gupta, P.K., Mascarenhas, A.F. and Jagannathan, V., 1981. Tissue culture of forest trees: clonal propagation of mature trees of Eucalyptus citriodora by tissue culture. Plant Sci. Lett., 20: 195-201. Hackett, W.P., 1976. Control of phase change in woody plants. Acta Hortic., 56: 143-154. Hoad, G.V., 1979. Growth regulators, endogenous hormones and flower initiation in apple. Annu. Rep. Long Ashton Res. Stn., pp. 199-206. Hoad, G.V., 1984. Hormonal regulation of fruit-bud formation in fruit trees.. Acta Hortic., 149: 13-23. Izumi, K., Kamiya, Y., Sakurai, A., Oshio, H. and Takahashi, H., 1985. Studies of sites of action of a new plant growth retardant (E) - 1- (4-chlorophenyl) -4,4-dimethyl-2- (1,2,4-triazol-1-yl) 1-penten-3-ol (S-3307) and comparative effects of its stereoisomers in a cell-free system from Cucurbita maxima. Plant Cell Physiol., 26: 821-827. Looney, N.E., Pharis, R.P. and Noma, M., 1985. Promotion of flowering in apple trees with gibberellin A4 and C-3 epi-gibberellin A4. Planta, 165: 292-294. Luckwill, L.C., 1970. The control of growth and fruitfullness of apple trees. In: L.C. Luckwill and C.V. Cutting (Editors), Physiology of Tree Crops. Academic Press, London, pp. 237-254. Lyrene, P.M., 1981. Juvenility and production of fast-rooting cuttings from blueberry shoot cultures. J. Am. Soc. Hortic. Sci., 106: 396-398. McLaughlin, J.M. and Greene, D.W., 1984. Effects of BA, GA4+7, and daminozide on fruit set, fruit quality, vegetative growth, flower initiation, and flower quality of 'Golden Delicious' apple. J. Am. Soc. Hortic. Sci., 109: 34-39. Mullins, M.G., Nair, Y. and Sampet, P., 1979. Rejuvenation in vitro: Induction of juvenile characters in an adult clone of Vitis vinifera L. Ann. Bot., 44: 623-627. Rogler, C.R. and Hackett, W.P., 1975. Phase change in Hedera helix: Induction of the mature to juvenile change by gibberellin A3. Physiol. Plant., 34: 148-152. Sachs, R.M., 1977. Nutrient diversion: An hypothesis to explain the chemical control of flowering. HortScience, 12: 220-222. Sachs, R.M. and Hackett, W.P., 1977. Chemical control of flowering. Acta Hortic., 68: 29-49. Sachs, R.M., Bretz, C.F. and Lang, A., 1959. Shoot histogenesis: the early effects of gibberellin upon stem elongation in two rosette plants. Am. J. Bot., 46: 376-384. Satsu, T., Ichii, T., Nakanishi, T. and Kawai, Y., 1988. In vitro propagation of Japanese pear (Pyrus serotina). Sci. Rep. Fac. Agric. Kobe Univ., 18:1-7 (in Japanese). Stein, O.L. and Fosket, E.B., 1969. Comparative developmental anatomy of shoots of juvenile and adult Hedera helix. Am. J. Bot., 54: 546-551. Tromp, J., 1972. Effects of growth-regulating substances and tree orientation on growth and flowerbud formation in apple. J. Hortic. Sci., 47: 525-533. Tromp, J., 1982. Flower-bud formation in apple as affected by various gibberellins. J. Hortic. Sci., 57: 277-282.
232
Y. HIGASHIUCHI ET AL.
Tsujikawa, T., Ichii, T., Nakanishi, T., Ozaki, T. and Kawai, Y., 1989. In vitro flowering of Japanese pear and the effect of GA4+7. Scientia Hortic., 41: 233-245. Weaver, R.J., 1972. Flowering. In: R.J. Weaver (Editor), Plant Growth Substance in Agriculture. W.H. Freeman, San Francisco, CA, pp. 176-221. Wertheim, S.J., 1973. Fruit set and June drop in Cox's Orange Pippin apple as affected by pollination and treatment with a mixture of gibberellins A4 and AT. Scientia Hortic., 1: 85-105. Yotsuya, T., Ichii, T., Sawano, M. and Nakanishi, T., 1980. Studies on growth cycle of Japanese pear buds: Effects of GA4+7 and BA on the in vitro growth of Kosui leaf buds. Sci. Rep. Fac. Agric. Kobe Univ., 14:45-49 (in Japanese). Zimmerman, R.H., 1973. Juvenility and flowering in woody plants. Acta Hortic., 34: 139-142.