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Inhibitory effects of volatile compounds released from rice callus on soybean callus growth: allelopathic evidence observed using in vitro cultures
Plant Science. 77 ( 1991 ) 103-110
103
Elsevier Scientific Publishers Ireland Ltd.
Inhibitory effects of volatile compounds released from rice callus on soybean callus growth: allelopathic evidence observed using in vitro cultures Yue-Sheng
Yang and Yuzo Futsuhara
Laboratoo' otr Theo O' ~[" Agronon O' and Plant Breeding. Faculty ~[' Agriculture, Naq'oya University, Furocho, Chil,'usaku. Nagoya 464 (Japan) (Received December 17th, 1990; revision received February 21 st, 1991: accepted March I st, 1991 )
When soybean (Glycine max Merr.) callus was cultured with rice (Oryza sativa L.) callus in the same culture bottle, an allelopathic evidence of growth inhibition on the former callus was observed. This allelopathic effect was so intense that the growth rates of the soybean callus were reduced by more than 100 fold under m a n y experimental conditions. Further studies showed that the inhibitory effect was from volatile c o m p o u n d s which were produced by rice callus. In rice cell suspension cultures, the volatile inhibitory compounds were released from rice cells and maintained in the liquid medium rather constantly. These compounds in the supernatant fluid from rice cell cultures were quite heat- and acid-stable, but their activity was reduced considerably by K M n O 4 oxidation. On the contrary, the addition of base to the supernatant fluid greatly enhanced the production of volatile inhibitory material. Limited experiments indicated that the allelopathic effect of inhibition on soybean callus growth was non-specific with respect to rice cultivars. The intensive inhibitory effect was also observed on callus growth of other legume species as well but not on members of the Solanaceae. The species-specific inhibitory effect of allelopathy on cultured cell and callus growth may be useful as a strategy for screening somatic hybrid cells from cultures of fusion-treated protoplasts.
Key words: allelopathy; in vitro culture: soybean and rice calli: inhibition on cell and callus growth: volatile inhibitory compounds
Introduction
In experiments on culturing soybean protoplasts using conditioned medium or feeder cell layer methods, we observed that rice cells or callus did not nurse the soybean protoplasts. In fact, the conditioned medium or the feeder cell layer provided by rice cultures had the opposite effect. It became evident that some substances produced by rice cells or callus had an inhibitory effect on the growth of soybean protoplasts. Allelopathy, meaning biochemical interactions among all types of plants [1], has been studied and reviewed extensively [2]. Although allelopathy is Correspondence to: Yuzo Futsuhara, Laboratory of Theory of Agronomy and Plant Breeding, Faculty of Agriculture, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.
generally a matter of ecology, its effects should exist in certain in vitro culture systems as well. For example, conditioning or nursing cells or protoplasts cultured at low densities using different plant species [3--6] was basically the application of an allelopathic effect to in vitro cultures. In the field or in greenhouses, soybean plants were observed to show allelopathic effects from various plant species [7--9]. How allelopathic chemicals affect the growth of soybean plants [10] was also reported, but whether soybean plants are affected by rice plants is not known, in in vitro cultures, Niizeki et al. [11] reported success in obtaining somatic hybrid cells from cultures of fused protoplasts of soybean and rice, but they did not mention if allelopathic effects were evident in the cultures. In this paper, we report our findings on an
0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
104
allelopathic phenomenon between soybean and rice callus in in vitro cultures. Materials and Methods
Callus induction Soybean (Glycine max Merr.) mature seeds of cultivar Kihoshu were surface-sterilized with 70% (v/v) ethanol for 30 s and then in sodium hypochlorite solution (final available chlorine at least 1%) for 15 min followed by two rinses in sterile distilled water. The aseptically treated seeds were then placed on autoclaved water-agar medium and kept at about 28°C for 4 days under dark conditions. Cotyledonary explants were obtained from the soybean seedlings and callus was induced by culturing the explants on a medium composed of Murashige and Skoog (MS) inorganic elements [12], B5 vitamins [13], 3%(w/v) sucrose, and supplemented with 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l kinetin. This medium was adjusted with 1 N N a O H or HC1 to pH 5.7, gelled with 0.8% agar (except for suspension cultures) before autoclaving for 15 min at 1.1 kg/cm -~, and was used for all the cell and callus cultures in the experiments. Rice (Oryza sativa L.) callus was induced directly from surface-sterilized dehulled mature seeds. Rice callus from the cultivar Sekiguchi-asahi was used in all the experiments and callus from the cultivar Nipponbare was used only for comparison. Calli of Glycine soja, strain Giken No. 2 and Vigna calcaratus, cultivar Takeazuki were induced from cotyledonary tissue of surface-sterilized immature seeds, while calli of G. tamentella, strain Inverell and G. tabacina, strain Miyakojimatsurumame were induced from cotyledonary tissue of seedlings grown from aseptically treated mature seeds. Calli of two species from the Solanaceae Atropa belladonna and Datura stramonium (strain names were unknown) were induced from explants of primary leaves of aseptically grown seedlings. Callus cultures on agar-solidified medium were maintained under continuous light of about 20 #E -'Y - I m - s provided by daylight fluorescent lamps at 28 ± 1 °C. All the callus lines had been maintained by subculture at 25--30-day intervals for at least 6 months before the experiments.
Init&tion and maintenance of soybean and rice suspension cell cultures Soybean and rice cell suspension cultures were initiated by transferring about 1.5 g fresh wt. of vigorously proliferating callus to 100-ml Erlenmeyer flasks containing about 33 ml of liquid medium kept in constant reciprocal agitation at 90 cycles/min under continuous dim light of about 5 ~E m -2 s -I at 24 ± 2°C. Established suspension cultures were maintained by subculture. For soybean, subculture was at l-week interval by transferring 3--5 ml suspension cultures to another culture bottle containing 30 ml of fresh medium, and for rice, subculture was performed at 2-week interval by transferring about 1.5 g (fresh wt.) cell clumps to another culture bottle containing fresh medium. Isolation and treatment o[ supernatant .[luid ./?ore rice cell cultures (SFRCC) Rice cell cultures for providing supernatant fluid were wrapped with three layers of kitchen foil (Aluminium foil. Alfamic Co., Ltd., Nakamachi 1-6-12, Kodoku, Tokyo, Japan) and sealed with Parafilm " M ''® (American National Can, Greenwich, CT). Liquid medium was collected from well sealed rice cell cultures 2 weeks after subculture and S F R C C was obtained by centrifugation at 1000 rev./min for 10 min. When investigating the growth inhibition of soybean cell suspension cultures by addition of SFRCC, supernatant fluid was added directly without any treatments to the Erlenmeyer flasks containing inoculum soybean suspension cells in liquid culture medium. Several methods of physical treatments of S F R C C were tested. When S F R C C was treated by autoclaving, the Erlenmeyer flasks containing the supernatant fluid were wrapped with layers of kitchen foil and autoclaved in the same manner as sterilization of culture medium. When S F R C C was treated by evaporation, the flasks containing the supernatant fluid were placed over a gas burner, or inside a vacuum evaporator until the fluid was reduced to 80% of the original volume and was then made to the original volume with distilled water. S F R C C was also chemically treated. Tenmilliliter aliquots of fluid were mixed with 2 ml of
105 0.3 M solutions of HCi, K O H or K M n O 4 prior to culture.
Results
Growth inhibition of soybean callus by rice callus cultured in the same bottle
Experimental culture methods Cylindrical culture bottles of 50 × 100 m m were used in most of the experiments, each of which received 30- or 35-ml culture m e d i u m ; four pieces of callus were inoculated on to the solidified medium. The weight of a n i n o c u l u m o f rice callus was approx. 60 mg fresh wt. while that o f soybean a n d the other plant species was a b o u t 10 mg fresh wt., since the growth of the rice callus was slow. Small glass bottles of 20 m m × 38 mm, each receiving 8 ml of m e d i u m a n d inoculated with one piece of soybean callus, were used in the assembly within the cylindrical culture bottles. Even smaller bottles of 17 × 35 mm, each receiving 6 ml of m e d i u m a n d inoculated with one piece of s o y b e a n callus, were assembled within test tubes of 20 × 200 mm. The cylindrical culture bottles or test tubes, in which the small bottles were assembled, also received culture m e d i u m or various testing solutions. Erlenmeyer flasks (100-mi) were used for suspension cultures. Cultures were w r a p p e d with three layers o f kitchen foil a n d sealed with Parafilm " M ''® or wrapped with three layers of Saran W r a p ~ (Sheet of polyvinylidene chlorite for food wrapping, Asahi Kasei K o g y o Co., Ltd., Y u r a k u m a c h i 1-1-2, C h i y o d a k u , T o k y o , J a p a n ) a n d were b a n d e d with a r u b b e r b a n d to be airtight d u r i n g the culture period.
The s o y b e a n callus grew very fast on culture med i u m when cultured alone. The growth of rice callus was c o n s i d e r a b l y slower in c o m p a r i s o n to the growth o f soybean callus. The former increased by a b o u t 12-fold of its original fresh weight while the latter increased in fresh weight by more than 160-fold within the culture period. W h e n both of them were cultured together in the same bottle, the growth of soybean callus was greatly inhibited. The degree o f i n h i b i t i o n did not differ much using various culture m e t h o d s (Table l). The i n h i b i t i o n was so strong that soybean callus could only proliferate very slowly in the presence of rice callus and had a proliferation rate of a b o u t five, which was only one thirtieth o f the c o n t r o l cultured in the absence of rice callus. The colour of the inhibited soybean callus was deepened a n d was very similar to that of s o y b e a n callus whose growth was inhibited by a m e d i u m c o n t a i n i n g antibiotics, for example, k a n a m y c i n at c o n c e n t r a t i o n s of 2 0 1 5 0 mg/I ( u n p u b l i s h e d results). The growth of the rice callus was practically unaffected by culture with soybean callus.
Growth &hibition of soybean suspension cells by the addition of SFRCC A d d i n g S F R C C greatly inhibited the growth of suspension soybean cells (Table ll). M e d i u m con-
Table I. Comparison of various culture methods on the growth inhibition of soybean callus by rice callus. Results were evaluated after a 30-day culture period and are presented as mean fresh wt. (mgl ± S.D. from eight pieces of callus. Culture methodsa
Rice (R) Soybean (S) (R + S)/2
Mixed
MF
St
Control
Enb En 412 ± 87
865 ± 183 56 ± 9 511 ± 96
780 .4- 162 59 ± 8 420 ± 85
778 ± 188 1679 ± 294 1229 + 241
aMixed, R and S call± were mixed in a ratio of one part of soybean callus with six parts (w/w) of rice callus prior to culture, four pieces of mixed call± were cultured in a bottle; MF, R and S call± were cultured very close to each other but were separated with a piece of membrane filter (cellulose nitrate, pore size 1.0 #m), the two pieces of soybean callus and two pieces of rice callus were cultured in a bottle; St, R and S call± were cultured at a distance of about 30 mm, the two pieces of soybean callus and two pieces of rice callus were cultured in a bottle; Control, R and S call± were cultured separately in different bottles. bEvaluation was not performed due to the difficulty in isolating the R and S call± from each other.
106 Iio Growth inhibition of soybean suspension cells by addition of SFRCC. Soybean suspension cells for inoculation were obtained from suspensions at the exponential stage. Results were evaluated 12 days after inoculation of the culture and are presented as mean fresh wt. 4- S.D. from 5 trials. Table
SFRCC (ml)
Culture medium (ml)
Original fresh wt. (rag)
Final fresh wt. (mg)
% inhibition a
1 3 6 0
30 28 25 31
450 450 450 450
4640 770 430 7230
30 96 100 0
4. 810 4. 160 490 ± 1240
a % inhibition = [1 - (treatment final fresh wt. - original fresh wt.) + (control final fresh wt. - original fresh wt.)] x 100.
taining 3% (v/v) SFRCC inhibited the cell proliferation by 30"/,, and 20% supernatant fluid could totally stop the proliferation of soybean suspension cells. It was clear that the inhibitory effect did not depend on the presence of living rice cells, and the inhibitory compounds were present constantly in the culture medium under the usual culture conditions.
Growth inhibition of soybean callus by the d([]itsion of volatile compounds from rice callus The growth of soybean callus in small bottles, was inhibited by rice callus cultured on medium outside the small bottles sharing the same air space
(Table III). In this experimental system, the influence by diffusion through culture medium was prevented so that the inhibition could only be from volatile compounds. The degree of inhibition became more pronounced as the amount of rice callus was increased. It was obvious that the concentration of the inhibitory volatile compounds inside a bottle with more rice callus was higher than a bottle with less rice callus, and therefore, produced a greater inhibition of the soybean callus growth. Generally, the inhibition caused by volatile diffusion was so pronounced that the proliferation of the soybean callus was reduced by 13--27 times.
Table III. Growth inhibition of soybean callus by volatile substances from rice callus. In each culture bottle 1, 2 or 3 small bottles were assembled, and each small bottle as well as the culture bottle assembled with the small bottlels) recieved culture medium. Soybean callus was inoculated into the small bottles while rice callus, 1, 2 or 3 pieces, was inoculated on medium outside the small bottle(s). The results were evaluated 28 days after the initiation of the culture and are given as mean fresh wt. ::e S D , from 6 to 9 pieces of callus. Rice No. of callus pieces I
Soybean Fresh wt.(mg)/bottle
843 4- 218
No. of callus pieces 1
2 3 2
1584 4. 312
Fresh wt.lnrgypiece
152 4- 32 132 + 27 134 4- 24
2 3
87 4- 21 98 4. 28 77 4- 23
I
3
2234 4- 438
1 2 3
6 6 4 - 15 55 4- 12 52 + 18
0
0 4- 0
3
1786 4- 437
107 IV. Comparison of the volatile inhibitory effect of various physically-treated SFRCC on soybean callus growth. Soybean callus was inoculated into 3 small bottles which contained medium and were placed inside a big culture bottle. Outside the small bottles 30 ml of various physically-treated solutions were introduced. Results were evaluated 28 days later and are presented as mean fresh wt. 4- S.D. from 4 replicates with 3 pieces of soybean callus per replicate.
Table
1. Treatmentof SFRCC by autoclaving Materials added SFRCC
Autoclave treatment
Callus weight (mg)
+
183 4- 32 1 9 2 4- 37 1790 4- 456
-
Water (control)
+
2. Treatmentof SFRCC by evaporation Materials added
Evaporation method
Callus wt. (mg)
SFRCC
Boiling Vacuum None None
1277 1358 105 1583
Water (control)
m
± 444-
320 314 15 354
ahree layers of Saran Wrap(~) and a rubber band Callus Medium
RUw kb. l.
T a b l e I l l also shows that the i n h i b i t o r y effect was not r e d u c e d n o t i c e a b l y by an increase in the i n o c u l u m a m o u n t s o f s o y b e a n callus within a culture bottle. T h e i n h i b i t i o n caused by including 2 pieces o f rice callus o r 3 pieces did not differ very much. These two o b s e r v a t i o n s indicated that the s o y b e a n callus c o n s u m e d or d e c o m p o s e d very little if any o f the volatile i n h i b i t o r y c o m p o u n d s , a n d the i n h i b i t o r y effect seemed to a p p r o a c h saturation when the q u a n t i t y o f rice callus derived from two rice inoculi was present.
Growth inhibition of soybean callus by the d~ffusion of volatile compounds from various physicallytreated SFRCC S F R C C also inhibited the s o y b e a n callus g r o w t h by serving as a source o f i n h i b i t o r y volatile c o m p o u n d s , a n d the volatile i n h i b i t o r y effect o f the fluid was not reduced o r w e a k e n e d significantly after a u t o c l a v e t r e a t m e n t s (Table IV). T r e a t m e n t o f the s u p e r n a t a n t fluid by e v a p o r a t i o n , however, effectively r e d u c e d the i n h i b i t o r y effect on the g r o w t h o f the s o y b e a n callus (Table IV). T h e i n h i b i t o r y effect was also greatly decreased by using a c t i v a t e d c h a r c o a l as an a b s o r b e n t e x p o s e d to the air space a r o u n d the cultures ( d a t a not shown). These results further c o n f i r m e d the volatile n a t u r e o f the i n h i b i t o r y c o m p o u n d s .
A piece of glass
Growth inhibition of soybean callus by the diffusion of volatile compounds .[rom various chemicallytreated SFRCC
2.
3,
4.
H
Fluid
Fig. I. A diagram of one stack of bottles in a test tube of the experiment on comparison of the volatile inhibitory effect of chemically treated supernatant fluid from rice cell cultures on soybean callus growth (Table V). The test tubes with cultures were kept straight up and stable during the 21-day culture period.
In o r d e r to d e t e r m i n e some o f the chemical features o f the volatile c o m p o u n d s , S F R C C was treated with s o l u t i o n s o f acid (HCI), a l k a l i n e ( K O H ) a n d o x i d a t i v e ( K M n O 4 ) reagents. Illustration o f the e x p e r i m e n t a l design is shown in Fig. 1 a n d the results o f the e x p e r i m e n t are given in T a b l e V. T h e t r e a t m e n t o f S F R C C with acid did not have a n o t i c e a b l e effect on the g r o w t h i n h i b i t i o n o f s o y b e a n callus, while t r e a t m e n t with K M n O 4 significantly decreased the i n h i b i t o r y effect. A n unexpected result was o b t a i n e d when the s u p e r n a tant fluid was treated with alkali, 0.05 M final conc e n t r a t i o n o f K O H greatly increased the i n h i b i t o r y effect. The i n h i b i t o r y effect o f the a l k a l i - t r e a t e d s u p e r n a t a n t fluid was so great that the s o y b e a n callus receiving the volatile influence by diffusion
108 Table V. Comparison of the volatile inhibitory effect of various chemically-treated SFRCC on soybean callus growth a. The results were evaluated after a 21-day culture period and each datum is the mean fresh wt. (mg) from 3 replicates. Treatment No.
1
2
3
4
5
6
7
8
Row Row Row Row
428 481 634 644
146 84 44 28
397 497 613 632
162 164 51 33
517 622 556 564
8 7 6 8
593 563 572 548
626 470 82 46
DW,12
SFRCC,10 DW,2
DW,10 H+,2
SFRCC,10 H+,2
DW,10 OH-,2
SFRCC,10 OH-.2
DW,10 OX.,2
SFRCC,10 OX.,2
No. No. No. No.
I (Top) 2 3 4
Fluid composition b (ml)
aRefer to Fig. 1 for details of experimental design. bDW, distilled water; H +, HCI at 0.3 M; O H - , KOH at 0.3 M; OX., KMnO 4 at 0.3 M. Solutions supplying H +, O H - and OX. were added to SFRCC, delivered with pipettes into each test tube just prior to assembling the small bottles filled with medium and inoculated with soybean callus into the test tube.
could not recover to grow even after being transferred to fresh medium in absence of volatile material, while call± from other treatments could recover their vigor gradually.
Investigations oj'allelopathic e['fects between call± o.]" rice and various plant species Call± of four species from the Leguminosae and two from the Solanaceae were compared with soybean callus for the susceptibility to the allelopathic influence of call± of two rice cultivars (Table VI). Rice call± of both cultivars inhibited the callus growth of all the legume species but did not influence the growth of call± of the two species from the Solanaceae. The inhibitory effect on callus growth of legume species with the call± of the two
rice cultivars was almost the same, and there was little difference in the susceptibility to the volatile allelopathic influence among the legume species. The allelopathic effect between two rice cultivars was not pronounced, neither was that between species from the same family of Leguminosae or Solanaceae (data not shown). Discussion The experiments demonstrate that a strong allelopathic effect existed in in vitro cultures between rice and soybean call±, and indicate that the effects are due to volatile compounds, which diffuse from rice callus and inhibit the growth of soybean cells and callus.
Table VI. Influence on callus growth of various plant species by culturing with rice callus. Two pieces of rice callus were cultured with two pieces of callus of various plant species in each culture bottle. Data were obtained after a I-month culture period and presented as mean fresh wt. (mg) .4- S.D. from 10 pieces of callus. Plant species
Glycine max, Kihishu Glycine soja, Giken No. 2 Glycine tabacina, Miyakojimatsurumame Glycine tamentella, Inuerell Vigna cah'¢tratus. Takeazuki Atropa belladonna a Datura slrtlmonium a Rice
+ Rice callus
-Rice callus
Sekiguchi-asahi
Nipponbare
52 45 55 44 33 1296 1422
49 46 51 51 41 1132 1582
.4- 8 46 .4- 7 .4- 6 .4- 5 -a- 121 4- 188
788 + 132 b
.4- 9 .4- 6 + 7 .4- 5 .4- 5 ± 186 4- 183
622 .4- 144 b
aStrain name unknown. bEvaluated from all the treatments of the rice cultivar cultured with various plant species.
(control) 1464 1254 1267 1552 1490 1214 1493
.4± .4.44.4+
335 302 122 246 278 209 221
109
Culturing cells from different plant species in the same culture chamber has been reported in some in vitro cultures of protoplasts. Vardi and Daveh [3] reported cross-feeder nursing cultures between tobacco and orange, while Shneyour et al. [4] reported that tobacco mesophyll protoplasts were well-nursed using petunia feeder cells. Additionally, Yamakawa et al. [5] reported that tobacco cells nursed the red-colored grape protoplasts. In these nursing culture systems, the feeder cells were assumed to be non-specific with respect to species. Very recently, differences in nursing effects among different plant species were shown by Thomas and Earle [6] including rice, which did not nurse the leaf protoplasts of Brassica oleracea. It is obvious that our results are similar to Thomas and Earle's, though at present, whether rice callus can inhibit the protoplast and callus growth of B. oleracea as strongly as soybean is unknown. Culturing protoplasts of rice and soybean after fusion treatment was performed by Niizeki et al. [11]. They reported success in obtaining somatic hybrid calli from the cultures, but did not mention any allelopathic effects between the two species. The reason why there was no evident inhibition of soybean colony formation is not clear, but might be due to the very low population of rice protoplasts in the cultures. It was observed that ethylene which is a volatile phytohormone is produced by cell cultures of Rosa sp., wheat, sweet clover, Haplopappus gracilis Nutt., rue and soybean, but the amounts of ethylene produced by the cultures did not affect growth [14]. In fact, soybean cells produced more ethylene than rice, and both of them were poor ethylene producers [15]. Moreover, an enhancing effect of ethylene on soybean cell suspension cultures has also been reported [16]. Based on the above observations, it is very probable that the inhibition of soybean callus growth is not due to the ethylene produced by rice callus, but to other allelopathic compounds. Though some of the inhibitory characteristics of the SFRCC were studied (Tables IV and V), further analysis using a gas chromatograph in combination with an in vitro bioassay are necessary to identify this soybean callus growth inhibitor. Allelopathy was observed among many plant
species, but among the large numbers of allelopathic compounds, volatile ones were rarely found [17,18]. Since plant cell and tissue culture techniques are now widely used ill a variety of basic research programs [19], our results indicate that it is very reasonable that the use of in vitro cultures could lead to the discovery of raore allelopathic effects among plant species as well as finding more allelopathic compounds, and should greatly facilitate research on the mechanisms of various allelopathic effects. Two characteristics of the volatile allelopathic effects of the SFRCC were considered to be important. One of these was the species-specific inhibition on the callus growth of soybean and other legumes and lack of effect on callus from species of the Solanaceae. The other was that the volatile inhibitory material could be increased by adding base to the fluid. These two characteristics suggested the possibility of using the allelopathic effects as a screening method to select somatic hybrid cells from protoplast fusions between legumes and the plants which are resistant to the allelopathic effects of rice callus like those of the Solanaceae, provided that the protoplasts of the resistant plants are inactivated before fusion [20], or are provided with a genetic marker.
Acknowledgements This research was partially supported by funds from The National Education Committee of China. We thank Mrs. Armenia Mendoza for critical reading of the manuscript.
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H. Molisch, Der Einfluss einer Pflanze a u f dic AndereAllelopathie. Gustav Fischer Verlag, Jena. 1937 (cited in Ref. 7). E.L. Rice, Allelopathy-an update. Bot. Rev., 45 {1979) 15 109. A. Vardi and D. Raveh, Cross-feeder experiments between tobacco and orange protoplasts. Z. Pflanzenphysiol., 78(1976) 350 359. Y. Shneyour, A. Zelcer, S. Izhar and J.S. Beckmann, A simple feeder-layer technique for the plating of plant cells and protoplasts at low density. Plant Sci. Lett., 33 {1984) 293 302. T. Yamakawa, K. Onomichi, T. Kodama and Y. Minoda.
110 Application of feeder layer method for improved colony formation of grape cells and protoplasts at low cell density, Agric. Biol. Chem.. 49 (1985) 3583--3585. 6 W.W. Thomas and E.D. Earle, A simple, versatile feeder layer system for Brossica oh, racea protoplast culture. Plant Cell Rep.. 9 (1990) 316~319. 7 D.C. Drost and J.D. Doll. The allelopathic effect of yellow nutsedge ( C)'perus esculentus) on corn (Zea mays) and soybeans (Glycine max). Weed Sci., 28 (1980) 229 233. 8 S.M. Irons and O.C. Burnside, Competitive and allelopathic effects of sunflower (Helianthus annuus). Weed Sci., 30 (1982~ 372--377. 9 M.A.B. Mallik and K. Tesfai, Allelopathic effect of common weeds on soybean growth and soybeanBradyrhizobium symbiosis. Plant and Soil, 112 (1988) 177--182. 10 D.T. Patterson, Effects of allelopathic chemicals on growth and physiological responses of soybean (Glycine max). Weed Sci., 29 (1981) 53 59. I 1 M. Niizeki, M. Tanaka, S. Akata, A. Hirai and K. Saito, Callus formation of somatic hybrid of rice and soybean and characteristics of the hybrid callus. Jpn. J. Genet., 60 (1985) 81--92. 12 T. Murashige and F. Skoog, A revised medium for rapid
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growth and bioassays with tobacco tissue cultures. Physiol. Plant, 15 (1962) 4 7 3 4 9 7 . O.L. Gamborg, R.A. Miller and K. Ojima, Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res., 50 (1968) 151 158. T.A.G. LaRue and O.L. Gamborg, Ethylene production by plant cell cultures. Plant Physiol., 48 (1971] 394--398. O.L. Gamborg and T.A.G. LaRue, Ethylene produced by plant cells in suspension cultures. Nature, 220 (1968) 604~605. F. Constable, W.G.W. Kurz, K.B. Chatson and J.W. Kirkpatrick, Partial synchrony in soybean cell suspcnsion cultures induced by ethylene. Exp. Cell Res., 105 (1977) 263--268. E.L. Rice, Allelopathy, Academic Press, New York, 1974. C.H. Muller, Volatile growth inhibitors produccd by Salvia species. Bull. Torrey Bot. Club, 91 tl964) 327---330. I.K. Vasil, Cell Culture and Somatic Cell Genetics of Plants, Academic Press, Inc., 1986. V.A. Sidorov, L. Mcnczel, F. Nagy and P. Maliga, Chloroplast transfer in Nicotiana based on metabolic complementation between irradiated and iodoacctatc treated protoplasts. Planta, 152 (1981) 341 345.
103
Elsevier Scientific Publishers Ireland Ltd.
Inhibitory effects of volatile compounds released from rice callus on soybean callus growth: allelopathic evidence observed using in vitro cultures Yue-Sheng
Yang and Yuzo Futsuhara
Laboratoo' otr Theo O' ~[" Agronon O' and Plant Breeding. Faculty ~[' Agriculture, Naq'oya University, Furocho, Chil,'usaku. Nagoya 464 (Japan) (Received December 17th, 1990; revision received February 21 st, 1991: accepted March I st, 1991 )
When soybean (Glycine max Merr.) callus was cultured with rice (Oryza sativa L.) callus in the same culture bottle, an allelopathic evidence of growth inhibition on the former callus was observed. This allelopathic effect was so intense that the growth rates of the soybean callus were reduced by more than 100 fold under m a n y experimental conditions. Further studies showed that the inhibitory effect was from volatile c o m p o u n d s which were produced by rice callus. In rice cell suspension cultures, the volatile inhibitory compounds were released from rice cells and maintained in the liquid medium rather constantly. These compounds in the supernatant fluid from rice cell cultures were quite heat- and acid-stable, but their activity was reduced considerably by K M n O 4 oxidation. On the contrary, the addition of base to the supernatant fluid greatly enhanced the production of volatile inhibitory material. Limited experiments indicated that the allelopathic effect of inhibition on soybean callus growth was non-specific with respect to rice cultivars. The intensive inhibitory effect was also observed on callus growth of other legume species as well but not on members of the Solanaceae. The species-specific inhibitory effect of allelopathy on cultured cell and callus growth may be useful as a strategy for screening somatic hybrid cells from cultures of fusion-treated protoplasts.
Key words: allelopathy; in vitro culture: soybean and rice calli: inhibition on cell and callus growth: volatile inhibitory compounds
Introduction
In experiments on culturing soybean protoplasts using conditioned medium or feeder cell layer methods, we observed that rice cells or callus did not nurse the soybean protoplasts. In fact, the conditioned medium or the feeder cell layer provided by rice cultures had the opposite effect. It became evident that some substances produced by rice cells or callus had an inhibitory effect on the growth of soybean protoplasts. Allelopathy, meaning biochemical interactions among all types of plants [1], has been studied and reviewed extensively [2]. Although allelopathy is Correspondence to: Yuzo Futsuhara, Laboratory of Theory of Agronomy and Plant Breeding, Faculty of Agriculture, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.
generally a matter of ecology, its effects should exist in certain in vitro culture systems as well. For example, conditioning or nursing cells or protoplasts cultured at low densities using different plant species [3--6] was basically the application of an allelopathic effect to in vitro cultures. In the field or in greenhouses, soybean plants were observed to show allelopathic effects from various plant species [7--9]. How allelopathic chemicals affect the growth of soybean plants [10] was also reported, but whether soybean plants are affected by rice plants is not known, in in vitro cultures, Niizeki et al. [11] reported success in obtaining somatic hybrid cells from cultures of fused protoplasts of soybean and rice, but they did not mention if allelopathic effects were evident in the cultures. In this paper, we report our findings on an
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104
allelopathic phenomenon between soybean and rice callus in in vitro cultures. Materials and Methods
Callus induction Soybean (Glycine max Merr.) mature seeds of cultivar Kihoshu were surface-sterilized with 70% (v/v) ethanol for 30 s and then in sodium hypochlorite solution (final available chlorine at least 1%) for 15 min followed by two rinses in sterile distilled water. The aseptically treated seeds were then placed on autoclaved water-agar medium and kept at about 28°C for 4 days under dark conditions. Cotyledonary explants were obtained from the soybean seedlings and callus was induced by culturing the explants on a medium composed of Murashige and Skoog (MS) inorganic elements [12], B5 vitamins [13], 3%(w/v) sucrose, and supplemented with 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l kinetin. This medium was adjusted with 1 N N a O H or HC1 to pH 5.7, gelled with 0.8% agar (except for suspension cultures) before autoclaving for 15 min at 1.1 kg/cm -~, and was used for all the cell and callus cultures in the experiments. Rice (Oryza sativa L.) callus was induced directly from surface-sterilized dehulled mature seeds. Rice callus from the cultivar Sekiguchi-asahi was used in all the experiments and callus from the cultivar Nipponbare was used only for comparison. Calli of Glycine soja, strain Giken No. 2 and Vigna calcaratus, cultivar Takeazuki were induced from cotyledonary tissue of surface-sterilized immature seeds, while calli of G. tamentella, strain Inverell and G. tabacina, strain Miyakojimatsurumame were induced from cotyledonary tissue of seedlings grown from aseptically treated mature seeds. Calli of two species from the Solanaceae Atropa belladonna and Datura stramonium (strain names were unknown) were induced from explants of primary leaves of aseptically grown seedlings. Callus cultures on agar-solidified medium were maintained under continuous light of about 20 #E -'Y - I m - s provided by daylight fluorescent lamps at 28 ± 1 °C. All the callus lines had been maintained by subculture at 25--30-day intervals for at least 6 months before the experiments.
Init&tion and maintenance of soybean and rice suspension cell cultures Soybean and rice cell suspension cultures were initiated by transferring about 1.5 g fresh wt. of vigorously proliferating callus to 100-ml Erlenmeyer flasks containing about 33 ml of liquid medium kept in constant reciprocal agitation at 90 cycles/min under continuous dim light of about 5 ~E m -2 s -I at 24 ± 2°C. Established suspension cultures were maintained by subculture. For soybean, subculture was at l-week interval by transferring 3--5 ml suspension cultures to another culture bottle containing 30 ml of fresh medium, and for rice, subculture was performed at 2-week interval by transferring about 1.5 g (fresh wt.) cell clumps to another culture bottle containing fresh medium. Isolation and treatment o[ supernatant .[luid ./?ore rice cell cultures (SFRCC) Rice cell cultures for providing supernatant fluid were wrapped with three layers of kitchen foil (Aluminium foil. Alfamic Co., Ltd., Nakamachi 1-6-12, Kodoku, Tokyo, Japan) and sealed with Parafilm " M ''® (American National Can, Greenwich, CT). Liquid medium was collected from well sealed rice cell cultures 2 weeks after subculture and S F R C C was obtained by centrifugation at 1000 rev./min for 10 min. When investigating the growth inhibition of soybean cell suspension cultures by addition of SFRCC, supernatant fluid was added directly without any treatments to the Erlenmeyer flasks containing inoculum soybean suspension cells in liquid culture medium. Several methods of physical treatments of S F R C C were tested. When S F R C C was treated by autoclaving, the Erlenmeyer flasks containing the supernatant fluid were wrapped with layers of kitchen foil and autoclaved in the same manner as sterilization of culture medium. When S F R C C was treated by evaporation, the flasks containing the supernatant fluid were placed over a gas burner, or inside a vacuum evaporator until the fluid was reduced to 80% of the original volume and was then made to the original volume with distilled water. S F R C C was also chemically treated. Tenmilliliter aliquots of fluid were mixed with 2 ml of
105 0.3 M solutions of HCi, K O H or K M n O 4 prior to culture.
Results
Growth inhibition of soybean callus by rice callus cultured in the same bottle
Experimental culture methods Cylindrical culture bottles of 50 × 100 m m were used in most of the experiments, each of which received 30- or 35-ml culture m e d i u m ; four pieces of callus were inoculated on to the solidified medium. The weight of a n i n o c u l u m o f rice callus was approx. 60 mg fresh wt. while that o f soybean a n d the other plant species was a b o u t 10 mg fresh wt., since the growth of the rice callus was slow. Small glass bottles of 20 m m × 38 mm, each receiving 8 ml of m e d i u m a n d inoculated with one piece of soybean callus, were used in the assembly within the cylindrical culture bottles. Even smaller bottles of 17 × 35 mm, each receiving 6 ml of m e d i u m a n d inoculated with one piece of s o y b e a n callus, were assembled within test tubes of 20 × 200 mm. The cylindrical culture bottles or test tubes, in which the small bottles were assembled, also received culture m e d i u m or various testing solutions. Erlenmeyer flasks (100-mi) were used for suspension cultures. Cultures were w r a p p e d with three layers o f kitchen foil a n d sealed with Parafilm " M ''® or wrapped with three layers of Saran W r a p ~ (Sheet of polyvinylidene chlorite for food wrapping, Asahi Kasei K o g y o Co., Ltd., Y u r a k u m a c h i 1-1-2, C h i y o d a k u , T o k y o , J a p a n ) a n d were b a n d e d with a r u b b e r b a n d to be airtight d u r i n g the culture period.
The s o y b e a n callus grew very fast on culture med i u m when cultured alone. The growth of rice callus was c o n s i d e r a b l y slower in c o m p a r i s o n to the growth o f soybean callus. The former increased by a b o u t 12-fold of its original fresh weight while the latter increased in fresh weight by more than 160-fold within the culture period. W h e n both of them were cultured together in the same bottle, the growth of soybean callus was greatly inhibited. The degree o f i n h i b i t i o n did not differ much using various culture m e t h o d s (Table l). The i n h i b i t i o n was so strong that soybean callus could only proliferate very slowly in the presence of rice callus and had a proliferation rate of a b o u t five, which was only one thirtieth o f the c o n t r o l cultured in the absence of rice callus. The colour of the inhibited soybean callus was deepened a n d was very similar to that of s o y b e a n callus whose growth was inhibited by a m e d i u m c o n t a i n i n g antibiotics, for example, k a n a m y c i n at c o n c e n t r a t i o n s of 2 0 1 5 0 mg/I ( u n p u b l i s h e d results). The growth of the rice callus was practically unaffected by culture with soybean callus.
Growth &hibition of soybean suspension cells by the addition of SFRCC A d d i n g S F R C C greatly inhibited the growth of suspension soybean cells (Table ll). M e d i u m con-
Table I. Comparison of various culture methods on the growth inhibition of soybean callus by rice callus. Results were evaluated after a 30-day culture period and are presented as mean fresh wt. (mgl ± S.D. from eight pieces of callus. Culture methodsa
Rice (R) Soybean (S) (R + S)/2
Mixed
MF
St
Control
Enb En 412 ± 87
865 ± 183 56 ± 9 511 ± 96
780 .4- 162 59 ± 8 420 ± 85
778 ± 188 1679 ± 294 1229 + 241
aMixed, R and S call± were mixed in a ratio of one part of soybean callus with six parts (w/w) of rice callus prior to culture, four pieces of mixed call± were cultured in a bottle; MF, R and S call± were cultured very close to each other but were separated with a piece of membrane filter (cellulose nitrate, pore size 1.0 #m), the two pieces of soybean callus and two pieces of rice callus were cultured in a bottle; St, R and S call± were cultured at a distance of about 30 mm, the two pieces of soybean callus and two pieces of rice callus were cultured in a bottle; Control, R and S call± were cultured separately in different bottles. bEvaluation was not performed due to the difficulty in isolating the R and S call± from each other.
106 Iio Growth inhibition of soybean suspension cells by addition of SFRCC. Soybean suspension cells for inoculation were obtained from suspensions at the exponential stage. Results were evaluated 12 days after inoculation of the culture and are presented as mean fresh wt. 4- S.D. from 5 trials. Table
SFRCC (ml)
Culture medium (ml)
Original fresh wt. (rag)
Final fresh wt. (mg)
% inhibition a
1 3 6 0
30 28 25 31
450 450 450 450
4640 770 430 7230
30 96 100 0
4. 810 4. 160 490 ± 1240
a % inhibition = [1 - (treatment final fresh wt. - original fresh wt.) + (control final fresh wt. - original fresh wt.)] x 100.
taining 3% (v/v) SFRCC inhibited the cell proliferation by 30"/,, and 20% supernatant fluid could totally stop the proliferation of soybean suspension cells. It was clear that the inhibitory effect did not depend on the presence of living rice cells, and the inhibitory compounds were present constantly in the culture medium under the usual culture conditions.
Growth inhibition of soybean callus by the d([]itsion of volatile compounds from rice callus The growth of soybean callus in small bottles, was inhibited by rice callus cultured on medium outside the small bottles sharing the same air space
(Table III). In this experimental system, the influence by diffusion through culture medium was prevented so that the inhibition could only be from volatile compounds. The degree of inhibition became more pronounced as the amount of rice callus was increased. It was obvious that the concentration of the inhibitory volatile compounds inside a bottle with more rice callus was higher than a bottle with less rice callus, and therefore, produced a greater inhibition of the soybean callus growth. Generally, the inhibition caused by volatile diffusion was so pronounced that the proliferation of the soybean callus was reduced by 13--27 times.
Table III. Growth inhibition of soybean callus by volatile substances from rice callus. In each culture bottle 1, 2 or 3 small bottles were assembled, and each small bottle as well as the culture bottle assembled with the small bottlels) recieved culture medium. Soybean callus was inoculated into the small bottles while rice callus, 1, 2 or 3 pieces, was inoculated on medium outside the small bottle(s). The results were evaluated 28 days after the initiation of the culture and are given as mean fresh wt. ::e S D , from 6 to 9 pieces of callus. Rice No. of callus pieces I
Soybean Fresh wt.(mg)/bottle
843 4- 218
No. of callus pieces 1
2 3 2
1584 4. 312
Fresh wt.lnrgypiece
152 4- 32 132 + 27 134 4- 24
2 3
87 4- 21 98 4. 28 77 4- 23
I
3
2234 4- 438
1 2 3
6 6 4 - 15 55 4- 12 52 + 18
0
0 4- 0
3
1786 4- 437
107 IV. Comparison of the volatile inhibitory effect of various physically-treated SFRCC on soybean callus growth. Soybean callus was inoculated into 3 small bottles which contained medium and were placed inside a big culture bottle. Outside the small bottles 30 ml of various physically-treated solutions were introduced. Results were evaluated 28 days later and are presented as mean fresh wt. 4- S.D. from 4 replicates with 3 pieces of soybean callus per replicate.
Table
1. Treatmentof SFRCC by autoclaving Materials added SFRCC
Autoclave treatment
Callus weight (mg)
+
183 4- 32 1 9 2 4- 37 1790 4- 456
-
Water (control)
+
2. Treatmentof SFRCC by evaporation Materials added
Evaporation method
Callus wt. (mg)
SFRCC
Boiling Vacuum None None
1277 1358 105 1583
Water (control)
m
± 444-
320 314 15 354
ahree layers of Saran Wrap(~) and a rubber band Callus Medium
RUw kb. l.
T a b l e I l l also shows that the i n h i b i t o r y effect was not r e d u c e d n o t i c e a b l y by an increase in the i n o c u l u m a m o u n t s o f s o y b e a n callus within a culture bottle. T h e i n h i b i t i o n caused by including 2 pieces o f rice callus o r 3 pieces did not differ very much. These two o b s e r v a t i o n s indicated that the s o y b e a n callus c o n s u m e d or d e c o m p o s e d very little if any o f the volatile i n h i b i t o r y c o m p o u n d s , a n d the i n h i b i t o r y effect seemed to a p p r o a c h saturation when the q u a n t i t y o f rice callus derived from two rice inoculi was present.
Growth inhibition of soybean callus by the d~ffusion of volatile compounds from various physicallytreated SFRCC S F R C C also inhibited the s o y b e a n callus g r o w t h by serving as a source o f i n h i b i t o r y volatile c o m p o u n d s , a n d the volatile i n h i b i t o r y effect o f the fluid was not reduced o r w e a k e n e d significantly after a u t o c l a v e t r e a t m e n t s (Table IV). T r e a t m e n t o f the s u p e r n a t a n t fluid by e v a p o r a t i o n , however, effectively r e d u c e d the i n h i b i t o r y effect on the g r o w t h o f the s o y b e a n callus (Table IV). T h e i n h i b i t o r y effect was also greatly decreased by using a c t i v a t e d c h a r c o a l as an a b s o r b e n t e x p o s e d to the air space a r o u n d the cultures ( d a t a not shown). These results further c o n f i r m e d the volatile n a t u r e o f the i n h i b i t o r y c o m p o u n d s .
A piece of glass
Growth inhibition of soybean callus by the diffusion of volatile compounds .[rom various chemicallytreated SFRCC
2.
3,
4.
H
Fluid
Fig. I. A diagram of one stack of bottles in a test tube of the experiment on comparison of the volatile inhibitory effect of chemically treated supernatant fluid from rice cell cultures on soybean callus growth (Table V). The test tubes with cultures were kept straight up and stable during the 21-day culture period.
In o r d e r to d e t e r m i n e some o f the chemical features o f the volatile c o m p o u n d s , S F R C C was treated with s o l u t i o n s o f acid (HCI), a l k a l i n e ( K O H ) a n d o x i d a t i v e ( K M n O 4 ) reagents. Illustration o f the e x p e r i m e n t a l design is shown in Fig. 1 a n d the results o f the e x p e r i m e n t are given in T a b l e V. T h e t r e a t m e n t o f S F R C C with acid did not have a n o t i c e a b l e effect on the g r o w t h i n h i b i t i o n o f s o y b e a n callus, while t r e a t m e n t with K M n O 4 significantly decreased the i n h i b i t o r y effect. A n unexpected result was o b t a i n e d when the s u p e r n a tant fluid was treated with alkali, 0.05 M final conc e n t r a t i o n o f K O H greatly increased the i n h i b i t o r y effect. The i n h i b i t o r y effect o f the a l k a l i - t r e a t e d s u p e r n a t a n t fluid was so great that the s o y b e a n callus receiving the volatile influence by diffusion
108 Table V. Comparison of the volatile inhibitory effect of various chemically-treated SFRCC on soybean callus growth a. The results were evaluated after a 21-day culture period and each datum is the mean fresh wt. (mg) from 3 replicates. Treatment No.
1
2
3
4
5
6
7
8
Row Row Row Row
428 481 634 644
146 84 44 28
397 497 613 632
162 164 51 33
517 622 556 564
8 7 6 8
593 563 572 548
626 470 82 46
DW,12
SFRCC,10 DW,2
DW,10 H+,2
SFRCC,10 H+,2
DW,10 OH-,2
SFRCC,10 OH-.2
DW,10 OX.,2
SFRCC,10 OX.,2
No. No. No. No.
I (Top) 2 3 4
Fluid composition b (ml)
aRefer to Fig. 1 for details of experimental design. bDW, distilled water; H +, HCI at 0.3 M; O H - , KOH at 0.3 M; OX., KMnO 4 at 0.3 M. Solutions supplying H +, O H - and OX. were added to SFRCC, delivered with pipettes into each test tube just prior to assembling the small bottles filled with medium and inoculated with soybean callus into the test tube.
could not recover to grow even after being transferred to fresh medium in absence of volatile material, while call± from other treatments could recover their vigor gradually.
Investigations oj'allelopathic e['fects between call± o.]" rice and various plant species Call± of four species from the Leguminosae and two from the Solanaceae were compared with soybean callus for the susceptibility to the allelopathic influence of call± of two rice cultivars (Table VI). Rice call± of both cultivars inhibited the callus growth of all the legume species but did not influence the growth of call± of the two species from the Solanaceae. The inhibitory effect on callus growth of legume species with the call± of the two
rice cultivars was almost the same, and there was little difference in the susceptibility to the volatile allelopathic influence among the legume species. The allelopathic effect between two rice cultivars was not pronounced, neither was that between species from the same family of Leguminosae or Solanaceae (data not shown). Discussion The experiments demonstrate that a strong allelopathic effect existed in in vitro cultures between rice and soybean call±, and indicate that the effects are due to volatile compounds, which diffuse from rice callus and inhibit the growth of soybean cells and callus.
Table VI. Influence on callus growth of various plant species by culturing with rice callus. Two pieces of rice callus were cultured with two pieces of callus of various plant species in each culture bottle. Data were obtained after a I-month culture period and presented as mean fresh wt. (mg) .4- S.D. from 10 pieces of callus. Plant species
Glycine max, Kihishu Glycine soja, Giken No. 2 Glycine tabacina, Miyakojimatsurumame Glycine tamentella, Inuerell Vigna cah'¢tratus. Takeazuki Atropa belladonna a Datura slrtlmonium a Rice
+ Rice callus
-Rice callus
Sekiguchi-asahi
Nipponbare
52 45 55 44 33 1296 1422
49 46 51 51 41 1132 1582
.4- 8 46 .4- 7 .4- 6 .4- 5 -a- 121 4- 188
788 + 132 b
.4- 9 .4- 6 + 7 .4- 5 .4- 5 ± 186 4- 183
622 .4- 144 b
aStrain name unknown. bEvaluated from all the treatments of the rice cultivar cultured with various plant species.
(control) 1464 1254 1267 1552 1490 1214 1493
.4± .4.44.4+
335 302 122 246 278 209 221
109
Culturing cells from different plant species in the same culture chamber has been reported in some in vitro cultures of protoplasts. Vardi and Daveh [3] reported cross-feeder nursing cultures between tobacco and orange, while Shneyour et al. [4] reported that tobacco mesophyll protoplasts were well-nursed using petunia feeder cells. Additionally, Yamakawa et al. [5] reported that tobacco cells nursed the red-colored grape protoplasts. In these nursing culture systems, the feeder cells were assumed to be non-specific with respect to species. Very recently, differences in nursing effects among different plant species were shown by Thomas and Earle [6] including rice, which did not nurse the leaf protoplasts of Brassica oleracea. It is obvious that our results are similar to Thomas and Earle's, though at present, whether rice callus can inhibit the protoplast and callus growth of B. oleracea as strongly as soybean is unknown. Culturing protoplasts of rice and soybean after fusion treatment was performed by Niizeki et al. [11]. They reported success in obtaining somatic hybrid calli from the cultures, but did not mention any allelopathic effects between the two species. The reason why there was no evident inhibition of soybean colony formation is not clear, but might be due to the very low population of rice protoplasts in the cultures. It was observed that ethylene which is a volatile phytohormone is produced by cell cultures of Rosa sp., wheat, sweet clover, Haplopappus gracilis Nutt., rue and soybean, but the amounts of ethylene produced by the cultures did not affect growth [14]. In fact, soybean cells produced more ethylene than rice, and both of them were poor ethylene producers [15]. Moreover, an enhancing effect of ethylene on soybean cell suspension cultures has also been reported [16]. Based on the above observations, it is very probable that the inhibition of soybean callus growth is not due to the ethylene produced by rice callus, but to other allelopathic compounds. Though some of the inhibitory characteristics of the SFRCC were studied (Tables IV and V), further analysis using a gas chromatograph in combination with an in vitro bioassay are necessary to identify this soybean callus growth inhibitor. Allelopathy was observed among many plant
species, but among the large numbers of allelopathic compounds, volatile ones were rarely found [17,18]. Since plant cell and tissue culture techniques are now widely used ill a variety of basic research programs [19], our results indicate that it is very reasonable that the use of in vitro cultures could lead to the discovery of raore allelopathic effects among plant species as well as finding more allelopathic compounds, and should greatly facilitate research on the mechanisms of various allelopathic effects. Two characteristics of the volatile allelopathic effects of the SFRCC were considered to be important. One of these was the species-specific inhibition on the callus growth of soybean and other legumes and lack of effect on callus from species of the Solanaceae. The other was that the volatile inhibitory material could be increased by adding base to the fluid. These two characteristics suggested the possibility of using the allelopathic effects as a screening method to select somatic hybrid cells from protoplast fusions between legumes and the plants which are resistant to the allelopathic effects of rice callus like those of the Solanaceae, provided that the protoplasts of the resistant plants are inactivated before fusion [20], or are provided with a genetic marker.
Acknowledgements This research was partially supported by funds from The National Education Committee of China. We thank Mrs. Armenia Mendoza for critical reading of the manuscript.
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