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Germination Inhibitors in Developing Seeds of Phaseol1ls vulgaris L.
Department of Biology, The University, Southampton, England
Germination Inhibitors in Developing Seeds of Phaseolus vulgaris L. D. A.
MORRIS
With 5 figures Received October 10, 1977 . Accepted November 2, 1977
Summary The acidic ether-soluble fraction of extracts of immature French bean seeds (Phaseolus vulgaris L. cv Masterpiece) contained appreciable amounts of a growth regulator with
chromatographic and physiological properties similar to abscisic acid. The compound was absent from 15-d old seed and increased in concentration with seed age until shortly before pod maturity (35 to 40 d after pollination) when its level fell abruptly. The changes in concentration of the inhibitor were closely correlated with the ability of the isolated embryos to germinate precociously on an agar-based medium containing sucrose, minerals and vitamins but lacking growth substances. Embryos isolated from 20 to 30 d after pollination remained dormant on this medium but germinated normally if removed and leached in water; the leachate was found to contain the inhibitor. Younger (15-d old) and mature (35- to 40-d old) embryos germinated readily on the basal medium. The addition of 5 mg . 1-1 kinetin to the medium reversed the effects of the inhibitor on germination of isolated bean embryos. Kinetin was also found to reverse the inhibition of lettuce seed germination by the partially purified bean inhibitor. It is suggested that the inhibitor is part of the natural hormonal system regulating embryo development and functions to prevent premature embryo axis elongation during the later stages of embryo growth when the cotyledonary storage tissues are being laid down. Key words: Phaseolus vulgaris, germination inhibitors, embryo development.
Introduction During an investigation involving the aseptic culture of isolated embryos of P. vulgaris cv Masterpiece it was found that although young (12- to 15-d old) embryos grew readily on a simple nutrient medium lacking growth substances, older (20- to 30-d old) embryos failed to grow unless the medium was supplemented with cytokinins. This result was unexpected in view of previous observations which had shown that endogenous cytokinin levels in the seed were highest shortly after fertilization and fell to an undetectable level in 20-d old seed, suggesting a decreasing dependence on cytokinins for growth as the embryo aged (NESLING, 1975). The Z. Pflanzenphysiol. Bd. 86. S. 433-441. 1978.
434
D. A. MORRIS
disappearance of cytokinins from extracts of whole seeds coincided with a falling growth rate of the embryo axis and the onset of the phase of rapid cotyledonary expanSlOn. The growth exhibited by isolated embryos in culture was typical of precocious germination rather than of continued embryonic development, and it seemed possible that the failure of older embryos to germinate precociously on the basal medium alone might have resulted from the presence in them of a germination inhibitor, the effects on embryo axis growth of which could be reversed by cytokinins. The work reported here was carried out to test this possibility and to investigate in more detail the influence of medium composition on the growth behaviour of isolated P. vulgaris embryos in culture. Materials and Methods Embryo culture
Isolated P. vulgaris embryos were cultured on a modified White's medium contammg sucrose, minerals and vitamins with or without the addition of 5.0 mg .1-1 6-furfurylaminopurine (kinetin) (TEGLEY et aI., 1971). Freshly-harvested pods, previously tagged at anthesis, were surface-sterilized by swabbing with ethanol and the developing ovules were transferred aseptically to sterile Petri dishes. The testas were removed and the isolated embryos were placed singly in plugged glass boiling tubes (145 mm X 23 mm) containing 15 ml sterile nutrient medium stiffened with 1.1 Ofo agar. The embryos were cultured in racks in a growth room at 20°C under continuous illumination from Philips' Warm White fluorescent tubes (ca 10 klux). Each treatment was replicated 10 times. Extraction of plant material
Seeds of the required age were removed from freshly-harvested pods, quickly frozen on solid CO 2 and freeze-dried. A known number of dried seed was homogenized in 80 0/0 aqueous ethanol and extracted for 48 h in a large excess of 80 % ethanol at 2 °C in darkness. The supernatant was decanted and the extraction of the residue was repeated in a further volume of 80 % ethanol. Extracts and washings were combined and reduced to the aqueous fraction by rotary evaporation under reduced pressure at 35°C. The aqueous solution was adjusted to pH 8.5 by the addition of a saturated solution of NaHC0 3 and was partitioned three times against equal volumes of freshly re-distilled ethyl ether. The ether extracts were bulked and retained. The aqueous residue was re-adjusted to pH 3.0 by the addition of HCI and again partitioned against three changes of ethyl ether. The basic and acidic ether-soluble fractions were concentrated by evaporation at room temperature and purified by ascending paper chromatography on Whatman 1 CHR paper in isopropanol: ammonia: water (10: 1 : 1, v/v). Extract equivalent to 25 seeds was spotted on each chromatogram. Bioassay procedures Lettuce seed germination assay: Developed chromatograms were routinely assayed using a simple lettuce seed germination test (WEBB and WAREING, 1972). Chromatograms were cut into 10 or 15 equal sections and each was placed on two 55 mm diameter Whatman No. t filter paper disks contained in a 90 mm glass Petri dish and moistened with 2.0 ml distilled water. Approximately 75 lettuce seed (Lactuca sativa L. cv Arctic King) were evenly spread over the surface of the chromatogram and filter paper, the dishes were covered
Z. Pflanzenphysiol. Bd. 86. S. 433-441. 1978.
Germination inhibitors in Phaseolus
435
and the seeds were allowed to germinate in daylight at room temperature (ca 21 DC) for 36-48 h. Controls were prepared in the same way using sections of solvent-washed chromatography paper after initial checks had shown that the extraction procedure and materials themselves did not cause inhibition of lettuce seed germination. Each assay was replicated three times. At the end of the assay period the percentage germination in each dish was determined. Where required the inhibitor content of extracts was quantified in abscisic acid (ABA) equivalents by reference to standard curves constructed from lettuce seed germination assays of known concentrations of authentic ABA. Wheat coleoptile straight-growth assay: The results of some lettuce seed germination assays were checked by means of the wheat coleoptile section straight-growth assay of NITSCH and NITSCH (1956).
Results
Growth of isolated embryos in culture In one experiment the effect of kinetin additions to the basal medium on the growth behaviour of isolated 12-, 15- and 22-d old embryos was studied (Table 1). Twelve-day old embryos germinated precociously in the absence of kinetin and by 25 d after isolation had developed a small but vigorous shoot system. Fifteen-day old embryos germinated more slowly and embryos isolated 22 d after pollination exhibited no growth at all during the course of the experiment. The addition of 5.0 mg .1- 1 kinetin to the basal medium greatly stimulated the growth of the embryos of all three ages, promoting rapid shoot elongation, expansion of the primary leaves and the development of a vigorous, branched root system. Kinetin also increased the growth of callus tissue on the surface of the cotyledons, the amount of callus tissue formed decreasing with embryo age at isolation.
Table 1: Influence of kinetin on the growth of isolated P. vulgaris embryos in sterile culture. Measurements were made 25 d after isolation and each value is the mean of 10 replications. Age of embryos (d) Kinetin (mg .1- 1 ) Mean shoot length (mm) Mean no. of lateral roots Mean length of primary root (mm) Callus score"-)
12
15
a
5
6.8
18.8 3.2 10.6 5.0
a
1.6 2.8
22
a
5
3.8
30.3 10.5 22.4 3.9
a
4.9 1.5
a a a a a
5 91.0 12.9 74.3 1.9
".) The development of callus tissue on the surface of the cotyledons was scored on a scale: a = no callus growth to 5 = complete coverage of cotyledon surface by callus.
In a second experiment the influence of kinetin on the growth of isolated 15- to 40-d old embryos was investigated (Figs. 1 and 5 b). Approximately 60 0J0 of the 15-d old embryos germinated precociously on the basal medium alone. However, Z. Pjlanzenphysiol. Bd. 86. S. 433-441.1978.
436
D. A.
MORRIS
Fig. 1: Response of isolated P. vulgaris embryos to the incorporation of 5.0 mg .1- 1 kinetin in the basal nutrient medium. Top left to bottom right: 20-, 25-, 30-, 35- and 40-d old embryos. In each pair the left-hand embryo received kinetin and the right-hand embryo was grown on the basal medium alone. except for very slight elongation of the radicle in a small proportion of those examined, 20-, 25- and 30-d old embryos failed to grow on the basal medium. Older (35- and 40-d old) embryos isolated from mature pods nearing dehiscence exhibited vigorous growth typical of normal germination. The inclusion of kinetin in the basal medium again resulted in the vigorous growth of embryos of all ages tested between 15 and 40 d after pollination, and completely overcame the failure of the 20- to 30-d old embryos to grow on the basal medium alone.
Germination inhibitors The observed response of 20- to 30-d old embryos to the inclusion of kinetin in the nutrient medium suggested that their failure to grow on the basal medium alone may have been caused by the presence in the embryos of a germination inhibitor. In an initial attempt to check this possibility the embryos which had failed to grow in culture were removed and soaked for 16 h in a large volume of aerated distilled water in an attempt to leach out possible inhibitory compounds. The embryos were then placed on moist filter paper in an illuminated incubator at 20 °C and within 48 h all had germinated. The leach water itself was found to contain appreciable quantities of a potent germination inhibitor (Fig. 2). A detailed examination was then
Z. Pflanzenphysiol. Bd. 86. S. 433-441.1978.
Germination inhibitors in Phaseolus
437
100
Fig. 2: Lettuce seed germination assay of acidic ether extracts of the leach water from 25-d old embryos which had failed to germinate on the basal nutrient medium.
40
o
0·5
10
Rf
] 50 f15 d
Of-
J
c
o
o
50
c
.
20d
E ~
a 00 1 l5 a f-
T
t
25d
40d
0I
a
0,5
o
1·0
0·5
1·0
Rf
Fig. 3: Lettuce seed germination assays of the acidic, ether-soluble fraction of extracts of 15- to 4o-d old P. vulgaris seeds. Each assay is the mean of three replications of 25 seed equivalents per chromatogram. Z. Pjlanzenphysiol. Bd. 86. S. 433-441.1978.
438
D. A.
MORRIS
ABA
120
t----<
ec 100 0 u
'0 ~
c:
0
0
80 60
01
c:
0
40
Fig. 4: Wheat coleoptile section straight growth assay of the acidic, ether-soluble fraction of extracts of 25-d old P. vulgaris seeds. (Mean of three replications of 25 seed equivalents per chromatogram.) The horizontal bar indicates the position of a spot of authentic ABA co-chromatogrammed with the extracts. Broken lines indcate 5 % confidence limits for control mean.
IlJ
20
0
iii
400
>
"
CT III
« m « 200 01
..s L
0 .Q
:2
c:
0 100
..o! 3
g-
50
""oen
III
o
o ~"
"
III
01
.§ 400
:c01
"C; ~
~
c
200 0 15
20
25
30
35
40
Days after pollination
Z. Pflanzenphysiol. Bd. 86. S. 433-441.1978.
Fig. 5: Changes in the level of the main acidic inhibitor with age of immature P. vulgaris seed(a); germination of isolated embryos in sterile culture in the presence (e) or absence (0) of kinetin (b); and increase in dry weight with age of P. vulgaris embryos (c).
Germination inhibitors in Phaseolus
439
undertaken of the changes in the occurrence and levels of the inhibitor in 15- to 40-d old seed by means of extraction and bioassay. Chromatograms of the acidic ether-soluble fraction of 80 % ethanol extracts of freeze-dried seeds revealed the presence of two inhibitory compounds. In extracts of 15-d old seed an inhibitor was present at Rf 0.2-0.4 on chromatograms developed in isopropanol: ammonia: water. However, the level of this compound rapidly fell as the seeds aged and by day 25 it had disappeared (Fig. 3). A second inhibitor (Rf 0.5-0.7) appeared in extracts of 20-d old seeds and this increased in amount until day 30, thereafter declining until by day 40 no further significant inhibitory activity was detected in the extracts (Figs. 3 and 5 a). This second inhibitor corresponded in Rf value to samples of authentic ABA. Chromatograms of the 25-d seed extracts were also assayed in the wheat coleoptile segment straight growth assay (Fig. 4). A powerful inhibitor of coleoptile elongation was detected at an Rf value corresponding to that of the seed germination inhibitor and to authentic ABA. None of the basic ether-soluble fractions were active in the lettuce seed germination assay.
Table 2: Influence of kinetin on the germination of lettuce seed Phaseolus inhibitor. Age of embryos extracted (d)
Mean germination (Ufo) Water Kinetin
25 30
19.2 15.5 98.1
Controls
III
the presence of the
91.7 95.4
Lettuce seed were placed on sections cut from the main inhibitory zone (Rf 0.5-0.7) of chromatograms of the acidic ether-soluble fraction (25 seed equivalents per assay) and moistened with either 2.0 ml distilled water or 2.0 ml kinetin solution (10 mg .1-1). Each value given is the mean of three replications of ca 100 lettuce seed. Control = solventwashed chromatography paper moistened with distilled water. The changes in the levels of the main seed germination inhibitor detected in the lettuce seed germination assay were closely correlated with the ability of isolated P. vulgaris embryos to germinate precociously on the basal medium alone (Fig. 5). Since the growth of the embryos in culture was premature germination rather than continued normal embryo development it seemed possible that the ability of kinetin to stimulate the growth of the isolated embryos might result from an ability to counteract the effect of the inhibitor. This was tested by studying the effect of kinetin on the germination of lettuce seed in the presence of the partially purified Phaseoius inhibitor. The results presented in Table 2 indicate a complete reversal by kinetin of the effect of the inhibitor on lettuce seed germination.
z. Pjlanzenphysiol. Bd. 86. S. 433-441.1978.
440
D. A. MORRIS
Discussion The observed changes with age in the levels of the main acidic inhibitor in the seed help to explain the in vitro growth behaviour of the isolated embryos. Embryos transferred to nutrient media before the build-up of the inhibitor (up to about 15 days after pollination) grew readily, although slowly, on the basal medium alone. Those transferred after the inhibitor began to accumulate in the seed only grew if the medium was supplemented with kinetin, or if they were leached in water - a treatment demonstrated to remove the inhibitor. These results contrast with those of WALBOT et al. (1972) who found that embryos of P. vulgaris isolated at any age between about 10 and 35 d after fertilization would germinate precociously in sterile nutrient media. However, this apparent discrepancy may be explained by differences in the techniques used to isolate and culture the embryos. The method used by WALBOT et al. (1972) involved surface sterilization of the isolated embryos themselves in 70 % aqueous ethanol or in 0.5 Ufo sodium hypochlorite solution followed by rinsing in distilled water. It is possible that these procedures may have led to leaching of the very water-soluble inhibitory compounds. Furthermore this effect may have been exaggerated by the use of a «wet» agar-based medium (0.8 Ufo agar compared with 1.1 Ufo in the present experiments), and the use of liquid media in some of their experiments. In all cases growth of the embryos in culture was typically precocious germination rather than continued embryonic development - that is, growth of the embryonic axis occurred without further growth and development of the cotyledonary tissues. From their studies of embryo development in P. vulgaris WALBOT et al. (1972) concluded that developmental controls must operate in the seed to prevent precocious germination in the pod and to permit the continuation of embryogeny. The main acidic inhibitor detected in the present experiments probably forms part of such a control mechanism. It also seems probable that those mature seed dormancy mechanisms which involve germination inhibitors may have evolved by selection for mechanisms which delay the loss of the inhibitors necessary to prevent precocious germination before seed maturity and dispersal. Although not positively identified the main inhibitor isolated in the present experiments exhibited chromatographic and physiological properties characteristic of ABA. This compound is a known potent inhibitor of seed germination and of coleoptile elongation and has been identified as the major endogenous inhibitor present in the dormant seed of many species (see reviews by WAREING and SAUNDERS, 1971; MILBORROW, 1974; TAYLORSON and HENDRICKS, 1977). Several workers have previously detected ABA or physiologically similar compounds in mature P. vulgaris seed (SIEGEL, 1950; TILLBERG, 1975), and in immature P. lunatus seed (BARNES and LIGHT, 1969). The ability of cytokinins to counteract the effects of ABA on both bud and seed dormancy are well documented [see literature reviewed by WAREING and SAUNDERS (1971) and HUBER and SANKHLA (1974)]. Z. Pjlanzenphysiol. Bd. 86. S. 433-441. 1978.
Germination inhibitors in Phaseolus
441
In spite of its wide-ranging inhibitory effects on metabolism and growth, the presence of ABA in developing seeds appears not to interfere with cotyledon development while at the same time it effectively retards embryo axis growth. Indeed the inhibitor detected in the present work increased to its maximum level in the seed during the period when rapid cotyledonary growth was taking place and when the seed was undergoing its major increase in dry weight (Fig. 5 c). This differential effect of the inhibitor on the growth of tissues of the same genetic origin warrants further investigation as a system which might provide valuable information on the mechanism of action of ABA. Furthermore, as W ALBOT et al. (1972) have suggested, it would be of interest to investigate the long-term response of embryos isolated at different stages during their development to the inclusion of such inhibitors in the culture medium. Such inclusions might well provide the key to the successful maintenance of an embryonic pattern of development in the cultured embryos of a wider range of species than is at present possible. Acknowledgements
I thank Mrs. R. P. BELL for assistance, and Dr. helpful discussions.
J.
SMARTT and Dr. F. A. V. NESLING for
References BARNES, M. F. and E. N. LIGHT: Planta (Bed.) 89,30'3-308 (1969). HUBER, W. and N. SANKHLA: Z. Pflanzenphysiol. 73, 160-166 (1974). MILBORROW, B. V.: Ann. Rev. Plant Physiol. 25, 259-307 (1974). NESLING, F. A. V.: Some physiological aspects of embryo development 111 the genus Phaseolus. Ph. D. thesis, Univ. Southampton (1975). NITSCH, J. P. and C. NITSCH: Plant Physiol. 31, 94-111 (1956). SIEGEL, S. M.: Bot. Gaz. 111, 353-356 (1950). TAYLORSON, R. B. and S. B. HENDRICKS: Ann. Rev. Plant Physiol. 28, 331-354 (1977). TEGLEY, J. R., F. H. WITHAM, and M. KRASNUK: Plant Physiol. 47, 581-585 (1971). TILLBERG, E.: Physiol. Plant. 34, 192-195 (1975). WALBOT, V., M. CLUTTER, and 1. M. SUSSEX: Phytomorphology 22, 59-68 (1972). WAREING, P. F. and P. F. SAUNDERS: Ann. Rev. Plant Physiol. 22, 261-288 (1971). WEBB, D. P. and P. F. WAREING: Planta (Bed.) 104, 115-125 (1972).
Dr. D. A. MORRIS, Department of Biology, Building 44, The University, Southampton, 509 5NH, England.
Z. Pjlanzenphysiol. Bd. 86. S. 433-441.1978.
Germination Inhibitors in Developing Seeds of Phaseolus vulgaris L. D. A.
MORRIS
With 5 figures Received October 10, 1977 . Accepted November 2, 1977
Summary The acidic ether-soluble fraction of extracts of immature French bean seeds (Phaseolus vulgaris L. cv Masterpiece) contained appreciable amounts of a growth regulator with
chromatographic and physiological properties similar to abscisic acid. The compound was absent from 15-d old seed and increased in concentration with seed age until shortly before pod maturity (35 to 40 d after pollination) when its level fell abruptly. The changes in concentration of the inhibitor were closely correlated with the ability of the isolated embryos to germinate precociously on an agar-based medium containing sucrose, minerals and vitamins but lacking growth substances. Embryos isolated from 20 to 30 d after pollination remained dormant on this medium but germinated normally if removed and leached in water; the leachate was found to contain the inhibitor. Younger (15-d old) and mature (35- to 40-d old) embryos germinated readily on the basal medium. The addition of 5 mg . 1-1 kinetin to the medium reversed the effects of the inhibitor on germination of isolated bean embryos. Kinetin was also found to reverse the inhibition of lettuce seed germination by the partially purified bean inhibitor. It is suggested that the inhibitor is part of the natural hormonal system regulating embryo development and functions to prevent premature embryo axis elongation during the later stages of embryo growth when the cotyledonary storage tissues are being laid down. Key words: Phaseolus vulgaris, germination inhibitors, embryo development.
Introduction During an investigation involving the aseptic culture of isolated embryos of P. vulgaris cv Masterpiece it was found that although young (12- to 15-d old) embryos grew readily on a simple nutrient medium lacking growth substances, older (20- to 30-d old) embryos failed to grow unless the medium was supplemented with cytokinins. This result was unexpected in view of previous observations which had shown that endogenous cytokinin levels in the seed were highest shortly after fertilization and fell to an undetectable level in 20-d old seed, suggesting a decreasing dependence on cytokinins for growth as the embryo aged (NESLING, 1975). The Z. Pflanzenphysiol. Bd. 86. S. 433-441. 1978.
434
D. A. MORRIS
disappearance of cytokinins from extracts of whole seeds coincided with a falling growth rate of the embryo axis and the onset of the phase of rapid cotyledonary expanSlOn. The growth exhibited by isolated embryos in culture was typical of precocious germination rather than of continued embryonic development, and it seemed possible that the failure of older embryos to germinate precociously on the basal medium alone might have resulted from the presence in them of a germination inhibitor, the effects on embryo axis growth of which could be reversed by cytokinins. The work reported here was carried out to test this possibility and to investigate in more detail the influence of medium composition on the growth behaviour of isolated P. vulgaris embryos in culture. Materials and Methods Embryo culture
Isolated P. vulgaris embryos were cultured on a modified White's medium contammg sucrose, minerals and vitamins with or without the addition of 5.0 mg .1-1 6-furfurylaminopurine (kinetin) (TEGLEY et aI., 1971). Freshly-harvested pods, previously tagged at anthesis, were surface-sterilized by swabbing with ethanol and the developing ovules were transferred aseptically to sterile Petri dishes. The testas were removed and the isolated embryos were placed singly in plugged glass boiling tubes (145 mm X 23 mm) containing 15 ml sterile nutrient medium stiffened with 1.1 Ofo agar. The embryos were cultured in racks in a growth room at 20°C under continuous illumination from Philips' Warm White fluorescent tubes (ca 10 klux). Each treatment was replicated 10 times. Extraction of plant material
Seeds of the required age were removed from freshly-harvested pods, quickly frozen on solid CO 2 and freeze-dried. A known number of dried seed was homogenized in 80 0/0 aqueous ethanol and extracted for 48 h in a large excess of 80 % ethanol at 2 °C in darkness. The supernatant was decanted and the extraction of the residue was repeated in a further volume of 80 % ethanol. Extracts and washings were combined and reduced to the aqueous fraction by rotary evaporation under reduced pressure at 35°C. The aqueous solution was adjusted to pH 8.5 by the addition of a saturated solution of NaHC0 3 and was partitioned three times against equal volumes of freshly re-distilled ethyl ether. The ether extracts were bulked and retained. The aqueous residue was re-adjusted to pH 3.0 by the addition of HCI and again partitioned against three changes of ethyl ether. The basic and acidic ether-soluble fractions were concentrated by evaporation at room temperature and purified by ascending paper chromatography on Whatman 1 CHR paper in isopropanol: ammonia: water (10: 1 : 1, v/v). Extract equivalent to 25 seeds was spotted on each chromatogram. Bioassay procedures Lettuce seed germination assay: Developed chromatograms were routinely assayed using a simple lettuce seed germination test (WEBB and WAREING, 1972). Chromatograms were cut into 10 or 15 equal sections and each was placed on two 55 mm diameter Whatman No. t filter paper disks contained in a 90 mm glass Petri dish and moistened with 2.0 ml distilled water. Approximately 75 lettuce seed (Lactuca sativa L. cv Arctic King) were evenly spread over the surface of the chromatogram and filter paper, the dishes were covered
Z. Pflanzenphysiol. Bd. 86. S. 433-441. 1978.
Germination inhibitors in Phaseolus
435
and the seeds were allowed to germinate in daylight at room temperature (ca 21 DC) for 36-48 h. Controls were prepared in the same way using sections of solvent-washed chromatography paper after initial checks had shown that the extraction procedure and materials themselves did not cause inhibition of lettuce seed germination. Each assay was replicated three times. At the end of the assay period the percentage germination in each dish was determined. Where required the inhibitor content of extracts was quantified in abscisic acid (ABA) equivalents by reference to standard curves constructed from lettuce seed germination assays of known concentrations of authentic ABA. Wheat coleoptile straight-growth assay: The results of some lettuce seed germination assays were checked by means of the wheat coleoptile section straight-growth assay of NITSCH and NITSCH (1956).
Results
Growth of isolated embryos in culture In one experiment the effect of kinetin additions to the basal medium on the growth behaviour of isolated 12-, 15- and 22-d old embryos was studied (Table 1). Twelve-day old embryos germinated precociously in the absence of kinetin and by 25 d after isolation had developed a small but vigorous shoot system. Fifteen-day old embryos germinated more slowly and embryos isolated 22 d after pollination exhibited no growth at all during the course of the experiment. The addition of 5.0 mg .1- 1 kinetin to the basal medium greatly stimulated the growth of the embryos of all three ages, promoting rapid shoot elongation, expansion of the primary leaves and the development of a vigorous, branched root system. Kinetin also increased the growth of callus tissue on the surface of the cotyledons, the amount of callus tissue formed decreasing with embryo age at isolation.
Table 1: Influence of kinetin on the growth of isolated P. vulgaris embryos in sterile culture. Measurements were made 25 d after isolation and each value is the mean of 10 replications. Age of embryos (d) Kinetin (mg .1- 1 ) Mean shoot length (mm) Mean no. of lateral roots Mean length of primary root (mm) Callus score"-)
12
15
a
5
6.8
18.8 3.2 10.6 5.0
a
1.6 2.8
22
a
5
3.8
30.3 10.5 22.4 3.9
a
4.9 1.5
a a a a a
5 91.0 12.9 74.3 1.9
".) The development of callus tissue on the surface of the cotyledons was scored on a scale: a = no callus growth to 5 = complete coverage of cotyledon surface by callus.
In a second experiment the influence of kinetin on the growth of isolated 15- to 40-d old embryos was investigated (Figs. 1 and 5 b). Approximately 60 0J0 of the 15-d old embryos germinated precociously on the basal medium alone. However, Z. Pjlanzenphysiol. Bd. 86. S. 433-441.1978.
436
D. A.
MORRIS
Fig. 1: Response of isolated P. vulgaris embryos to the incorporation of 5.0 mg .1- 1 kinetin in the basal nutrient medium. Top left to bottom right: 20-, 25-, 30-, 35- and 40-d old embryos. In each pair the left-hand embryo received kinetin and the right-hand embryo was grown on the basal medium alone. except for very slight elongation of the radicle in a small proportion of those examined, 20-, 25- and 30-d old embryos failed to grow on the basal medium. Older (35- and 40-d old) embryos isolated from mature pods nearing dehiscence exhibited vigorous growth typical of normal germination. The inclusion of kinetin in the basal medium again resulted in the vigorous growth of embryos of all ages tested between 15 and 40 d after pollination, and completely overcame the failure of the 20- to 30-d old embryos to grow on the basal medium alone.
Germination inhibitors The observed response of 20- to 30-d old embryos to the inclusion of kinetin in the nutrient medium suggested that their failure to grow on the basal medium alone may have been caused by the presence in the embryos of a germination inhibitor. In an initial attempt to check this possibility the embryos which had failed to grow in culture were removed and soaked for 16 h in a large volume of aerated distilled water in an attempt to leach out possible inhibitory compounds. The embryos were then placed on moist filter paper in an illuminated incubator at 20 °C and within 48 h all had germinated. The leach water itself was found to contain appreciable quantities of a potent germination inhibitor (Fig. 2). A detailed examination was then
Z. Pflanzenphysiol. Bd. 86. S. 433-441.1978.
Germination inhibitors in Phaseolus
437
100
Fig. 2: Lettuce seed germination assay of acidic ether extracts of the leach water from 25-d old embryos which had failed to germinate on the basal nutrient medium.
40
o
0·5
10
Rf
] 50 f15 d
Of-
J
c
o
o
50
c
.
20d
E ~
a 00 1 l5 a f-
T
t
25d
40d
0I
a
0,5
o
1·0
0·5
1·0
Rf
Fig. 3: Lettuce seed germination assays of the acidic, ether-soluble fraction of extracts of 15- to 4o-d old P. vulgaris seeds. Each assay is the mean of three replications of 25 seed equivalents per chromatogram. Z. Pjlanzenphysiol. Bd. 86. S. 433-441.1978.
438
D. A.
MORRIS
ABA
120
t----<
ec 100 0 u
'0 ~
c:
0
0
80 60
01
c:
0
40
Fig. 4: Wheat coleoptile section straight growth assay of the acidic, ether-soluble fraction of extracts of 25-d old P. vulgaris seeds. (Mean of three replications of 25 seed equivalents per chromatogram.) The horizontal bar indicates the position of a spot of authentic ABA co-chromatogrammed with the extracts. Broken lines indcate 5 % confidence limits for control mean.
IlJ
20
0
iii
400
>
"
CT III
« m « 200 01
..s L
0 .Q
:2
c:
0 100
..o! 3
g-
50
""oen
III
o
o ~"
"
III
01
.§ 400
:c01
"C; ~
~
c
200 0 15
20
25
30
35
40
Days after pollination
Z. Pflanzenphysiol. Bd. 86. S. 433-441.1978.
Fig. 5: Changes in the level of the main acidic inhibitor with age of immature P. vulgaris seed(a); germination of isolated embryos in sterile culture in the presence (e) or absence (0) of kinetin (b); and increase in dry weight with age of P. vulgaris embryos (c).
Germination inhibitors in Phaseolus
439
undertaken of the changes in the occurrence and levels of the inhibitor in 15- to 40-d old seed by means of extraction and bioassay. Chromatograms of the acidic ether-soluble fraction of 80 % ethanol extracts of freeze-dried seeds revealed the presence of two inhibitory compounds. In extracts of 15-d old seed an inhibitor was present at Rf 0.2-0.4 on chromatograms developed in isopropanol: ammonia: water. However, the level of this compound rapidly fell as the seeds aged and by day 25 it had disappeared (Fig. 3). A second inhibitor (Rf 0.5-0.7) appeared in extracts of 20-d old seeds and this increased in amount until day 30, thereafter declining until by day 40 no further significant inhibitory activity was detected in the extracts (Figs. 3 and 5 a). This second inhibitor corresponded in Rf value to samples of authentic ABA. Chromatograms of the 25-d seed extracts were also assayed in the wheat coleoptile segment straight growth assay (Fig. 4). A powerful inhibitor of coleoptile elongation was detected at an Rf value corresponding to that of the seed germination inhibitor and to authentic ABA. None of the basic ether-soluble fractions were active in the lettuce seed germination assay.
Table 2: Influence of kinetin on the germination of lettuce seed Phaseolus inhibitor. Age of embryos extracted (d)
Mean germination (Ufo) Water Kinetin
25 30
19.2 15.5 98.1
Controls
III
the presence of the
91.7 95.4
Lettuce seed were placed on sections cut from the main inhibitory zone (Rf 0.5-0.7) of chromatograms of the acidic ether-soluble fraction (25 seed equivalents per assay) and moistened with either 2.0 ml distilled water or 2.0 ml kinetin solution (10 mg .1-1). Each value given is the mean of three replications of ca 100 lettuce seed. Control = solventwashed chromatography paper moistened with distilled water. The changes in the levels of the main seed germination inhibitor detected in the lettuce seed germination assay were closely correlated with the ability of isolated P. vulgaris embryos to germinate precociously on the basal medium alone (Fig. 5). Since the growth of the embryos in culture was premature germination rather than continued normal embryo development it seemed possible that the ability of kinetin to stimulate the growth of the isolated embryos might result from an ability to counteract the effect of the inhibitor. This was tested by studying the effect of kinetin on the germination of lettuce seed in the presence of the partially purified Phaseoius inhibitor. The results presented in Table 2 indicate a complete reversal by kinetin of the effect of the inhibitor on lettuce seed germination.
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D. A. MORRIS
Discussion The observed changes with age in the levels of the main acidic inhibitor in the seed help to explain the in vitro growth behaviour of the isolated embryos. Embryos transferred to nutrient media before the build-up of the inhibitor (up to about 15 days after pollination) grew readily, although slowly, on the basal medium alone. Those transferred after the inhibitor began to accumulate in the seed only grew if the medium was supplemented with kinetin, or if they were leached in water - a treatment demonstrated to remove the inhibitor. These results contrast with those of WALBOT et al. (1972) who found that embryos of P. vulgaris isolated at any age between about 10 and 35 d after fertilization would germinate precociously in sterile nutrient media. However, this apparent discrepancy may be explained by differences in the techniques used to isolate and culture the embryos. The method used by WALBOT et al. (1972) involved surface sterilization of the isolated embryos themselves in 70 % aqueous ethanol or in 0.5 Ufo sodium hypochlorite solution followed by rinsing in distilled water. It is possible that these procedures may have led to leaching of the very water-soluble inhibitory compounds. Furthermore this effect may have been exaggerated by the use of a «wet» agar-based medium (0.8 Ufo agar compared with 1.1 Ufo in the present experiments), and the use of liquid media in some of their experiments. In all cases growth of the embryos in culture was typically precocious germination rather than continued embryonic development - that is, growth of the embryonic axis occurred without further growth and development of the cotyledonary tissues. From their studies of embryo development in P. vulgaris WALBOT et al. (1972) concluded that developmental controls must operate in the seed to prevent precocious germination in the pod and to permit the continuation of embryogeny. The main acidic inhibitor detected in the present experiments probably forms part of such a control mechanism. It also seems probable that those mature seed dormancy mechanisms which involve germination inhibitors may have evolved by selection for mechanisms which delay the loss of the inhibitors necessary to prevent precocious germination before seed maturity and dispersal. Although not positively identified the main inhibitor isolated in the present experiments exhibited chromatographic and physiological properties characteristic of ABA. This compound is a known potent inhibitor of seed germination and of coleoptile elongation and has been identified as the major endogenous inhibitor present in the dormant seed of many species (see reviews by WAREING and SAUNDERS, 1971; MILBORROW, 1974; TAYLORSON and HENDRICKS, 1977). Several workers have previously detected ABA or physiologically similar compounds in mature P. vulgaris seed (SIEGEL, 1950; TILLBERG, 1975), and in immature P. lunatus seed (BARNES and LIGHT, 1969). The ability of cytokinins to counteract the effects of ABA on both bud and seed dormancy are well documented [see literature reviewed by WAREING and SAUNDERS (1971) and HUBER and SANKHLA (1974)]. Z. Pjlanzenphysiol. Bd. 86. S. 433-441. 1978.
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In spite of its wide-ranging inhibitory effects on metabolism and growth, the presence of ABA in developing seeds appears not to interfere with cotyledon development while at the same time it effectively retards embryo axis growth. Indeed the inhibitor detected in the present work increased to its maximum level in the seed during the period when rapid cotyledonary growth was taking place and when the seed was undergoing its major increase in dry weight (Fig. 5 c). This differential effect of the inhibitor on the growth of tissues of the same genetic origin warrants further investigation as a system which might provide valuable information on the mechanism of action of ABA. Furthermore, as W ALBOT et al. (1972) have suggested, it would be of interest to investigate the long-term response of embryos isolated at different stages during their development to the inclusion of such inhibitors in the culture medium. Such inclusions might well provide the key to the successful maintenance of an embryonic pattern of development in the cultured embryos of a wider range of species than is at present possible. Acknowledgements
I thank Mrs. R. P. BELL for assistance, and Dr. helpful discussions.
J.
SMARTT and Dr. F. A. V. NESLING for
References BARNES, M. F. and E. N. LIGHT: Planta (Bed.) 89,30'3-308 (1969). HUBER, W. and N. SANKHLA: Z. Pflanzenphysiol. 73, 160-166 (1974). MILBORROW, B. V.: Ann. Rev. Plant Physiol. 25, 259-307 (1974). NESLING, F. A. V.: Some physiological aspects of embryo development 111 the genus Phaseolus. Ph. D. thesis, Univ. Southampton (1975). NITSCH, J. P. and C. NITSCH: Plant Physiol. 31, 94-111 (1956). SIEGEL, S. M.: Bot. Gaz. 111, 353-356 (1950). TAYLORSON, R. B. and S. B. HENDRICKS: Ann. Rev. Plant Physiol. 28, 331-354 (1977). TEGLEY, J. R., F. H. WITHAM, and M. KRASNUK: Plant Physiol. 47, 581-585 (1971). TILLBERG, E.: Physiol. Plant. 34, 192-195 (1975). WALBOT, V., M. CLUTTER, and 1. M. SUSSEX: Phytomorphology 22, 59-68 (1972). WAREING, P. F. and P. F. SAUNDERS: Ann. Rev. Plant Physiol. 22, 261-288 (1971). WEBB, D. P. and P. F. WAREING: Planta (Bed.) 104, 115-125 (1972).
Dr. D. A. MORRIS, Department of Biology, Building 44, The University, Southampton, 509 5NH, England.
Z. Pjlanzenphysiol. Bd. 86. S. 433-441.1978.