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Occurrence of an inhibitor in wheat endosperm as revealed by embryo transplantation in irradiated seeds
Radiation Botany, 1964, Vol. 4, pp. 497 to 502. Pergamon Press Ltd. Printed in Great Britain.
OCCURRENCE OF AN INHIBITOR IN WHEAT ENDOSPERM AS REVEALED BY EMBRYO T R A N S P L A N T A T I O N IN IRRADIATED SEEDS* P. MELETT]L, C. FLORIS a n d F. D'.4,MATO
Istituto di Botanica della Universit~t, Cagliari and Istituto di Genetica delia Universit~t, Pisa, Italy
(Received 20 January 1964) Alanstract--Seeds (caryopses) of durum wheat var. Cappelli were soaked in water for 24 hr at 20°C to soften them enough for excision of embryos with scutellum. After irradiation of half of the soaked seeds (2-10 kr of X-rays), embryos were excised from both irradiated and unirradiated seeds and the following transplantations were made: (1) unirradiated embryo on irradiated endosperm, EMtu)/EN(I ) and (2)unirradiated embryo on unirradiated endosperm, EM~u)/EN (.) (homotransplant, to serve as control). In 1962 and 1963, using seeds of the crops of 1961 and 1962 respectively, it was demonstrated that endosperms of after-ripened seeds stimulated growth in the EM(u)/EN(i ) seedlings. Evidence was found that this stimulatory effect results from the destruction--or inactivation-by radiation of an inhibitor present in the endosperm of after-ripened seeds. The inhibitor is not present, in detectable amounts, in the endosperm of seeds during the total period of dormancy (first three months or more after harvest). The results are discussed in relation to the general problem of growth responses, including stimulation, in irradiated seeds. R a s u m # - - D e s graines (caryopses) de b16 durum var. Cappelli ont 6t6 immerg6es dans de l'eau pendant 24 heures/~ 20°C afin de les ramollir sutTasamment pour permettre l'excision des embryons avec leurs scutellums. Apr~s irradiation de la moiti6 du lot de graines hydrat~es (2 ~t 10 Kr de rayons X), des embryons ont 6t6 excis6s des graines irradi6es aussi bien que des graines non irradi6es et les transplantations suivantes ont 6t6 r6alis6es: (1) embryon non irradi6 sur endosperme irradi6 EM~u)/EN(i ) et (2) embryon non irradi6 sur endosperme non irradi6, EM(u)/EN(u) (homo-transplantation servant de t6moin). En 1962 et 1963, en utilisant les graines des r~coltes de 1961 et 1962 respectivement, nous avons d6montr6 que les endospermes des graines aprb.s maturation stimulaient la croissance darts les plantules EM~u)/EN(0. On a prouv6 que l'effet stimulant provient de la destruction ou de l'inactivation, par les radiations, d'un inhibiteur present dam l'endosperme des graines apr~ maturation. L'inhibiteur n'est pas prdse~nt en quantit6 ddcelable darts l'endosperme des graines pendant la p6riode totale de dormance (les 3 premieres mois ou plus apr6s la r6colte). Ces rdsultats sont discut6s dam leur relation avee le probl~me g6n6ral de la croissance y compris la stimulation des graines irradi~es. Z u s ~ m m e n f a s s u n g - - S a m e n (Karyopsen) von durum-Weizen var Cappelli wurden 24 S'tunde.fi lang bei 20 ° in Wasser angequollen, um sie for das Herauspr/iparieren von Embryo plus Skutellum gentigend zu erweichen. Die eine H~lfte der gequollenen Samen wurde *Part of the investigation was supported by a sub-contract with the Euratom-ITAL Association, Wageningen, Holland. 497 M
OCCURRENCE OF AN INHIBITOR I N W H E A T ENDOSPERM
498
bestrahlt (2-10 kr R6ntgenstrahlen). Dann wurden sowohl aus bestrahlten wie unbestrahlten Samen die Embryonen herauspr~ipariert, und die folgenden Transplantationen vorgenommen : (1) Unbestrahlter Embryo auf bestrahltes Endosperm, EM(u)/EN(0. (2) Unbestrahlter Embryo auf unbestrahltes Endosperm, EM(u)/EM(u) (Homotransplantate, die als Kontrollen dienten). In denJahren 1962 und 1963 wurde unter Benutzung von Samen der Ernten von 1961 bzw 1962 gezeigt, dass Endosperme von nachgereiften Samen das Wachstum bei EM(u}/EN(0Keimlingen stimulieren. Es wurde gefunden, dass diese stimulierende Wirkung auf der strahlenbedingten Zerst6rung oder Inaktivierung eines Inhibitors beruht, der im Endosperm yon naehgerefften Samen vorkommt. Der Inhibitor l~isst rich im Endosperm yon Samen w~ihrend der gesamten Periode der Samenruhe die ersten 3 oder mehr Monate naeh der Ernte nicht naehweisen. Die Ergebnisse werden mit Hinsicht aufdas aUgemeine Problem yon Waehstumsreaktionen, unter Einschluss yon Stimulationserseheinungen, bei bestrahltenSamen diskutiert. INTRODUCTION
IN A PREVZOUSpaperO), the results of embryo transplantation experiments in irradiated seeds (caryopses) of barley and wheat were reported. Dry seeds were irradiated with high radiation exposures of X-rays, y-rays and thermal neutrons; irradiated and unirradiated seeds were soaked in water at 16°C for 24 hr in order to have them softened enough for excision of endosperm-free embryos with scutellum. Seedling growth was compared in the following two types of transplants : (1) unirradiated embryo on irradiated endosperm, EM(,)/EN(i) and (2) unirradiated embryo on unirradiated endosperm, EM/(,) EN(,) (homotransplant). Both in barley (X-ray exposures of 50, 100, 200 and 400 kr) and in
wheat (y-ray exposures of 250, 500 and 1000 kr) it was found that X-ray (or y-ray) irradiated endosperm can depress growth in both the root and the shoot systems in the EM(,,)/EN(0 seedlings. These results have been confirmed recently by FONSTEJN(s)in embryo transplantation experiments in wheat seeds irradiated with y-rays ( 100 and 500 kr) in the dry state. In continuing our work on embryo-endosperm relations in irradiated seeds, we decided to concentrate our attention on the durum wheat variety Cappelli, a material we are using in extensive radiation biological and genetic studies. We also introduced a new experimental procedure, namely: irradiation of seeds pregerminated for 24 hr at 20°C with X-ray
Table 1. Seed germinability and seedling growth in Triticum durum vat. Cappelli at different times afteT seed harvest (seeds stored under laboratory conditions). For each test two germinations of 50 seeds each were made at 20°C in the dark
% germinated after days Crop
1961 1962
1962"
After 4 days
Days after harvest
Seed moisture °/o
2
4
6
10
14
375 2 9 16 23 69 16 69
11"0 6"0 8"3 11"0 11'3 11"3 2"6 2"6
95 0 32 36 56 68 9 39
99 3 76 80 92 88 48 88
100 35 90 90 94 90 65 90
62 92 92 94 92 76 92
74 92 92 95 94 80 92
* Kept in a dry atmosphere in a desiccator.
First leaf length mm
Root length mm
32"80
58"00
13"00 25"40 25"70
30"00 56"30 56"40
24"70
50"50
499
P. MELETTI, C. FLORIS and F. D'AMATO
Table 2. Growth of s;edlings obtainedfrom irradiated embo~os transplanted on irradiated endosperms, EM(t)/EWo) (homotransplants) and from irradiated embryos transplanted on unirradiated endosperms, EMq)]EW(u). For each radiation exposure 300 transplants were made (150 EM(i)]EN(i ) and 150 EM(i)]EW(u) ). Material: durum wheat seeds stored under laboratory conditionsfor 27 weeks Radiation exposure 4 kr
6 kr
8 kr
Transplant First leaf length mm EMq)/ENq) EM0)/EN(, )
36"20* 27"86
Root length mm 16"74++ 14"51
First leaf length mm
Root length mm
First leaf length mm
Root length mm
26"62 22"17
13"18 11"16
20"39* 15"67
10"847 9"84
Significant differences between EM(/)/EN(i) means and the corresponding EM0)/EN(u ) means are indicated with the following P values: *0"001 ; ~0"01 ; +0"05 (other differences not significant).
exposures of 2-10 kr and completion of the embryo transplantation work within one hour from the end of irradiation. Using this experimental procedure on after-ripened seeds, we tbund that EM~I)/EN~i) seedlings grew better than EM~0/EN(, ) seedlings (see Table 2). In the present paper, results are reported showing that the seemingly stimulatory effect of the irradiated wheat endosperm is due to the abolition, or reduction, by irradiation of an inhibitory condition present in that tissue.
reported here (Table 2), EMli)/EN
MATERIAL AND METHODS
RESULTS 1. Germinability and seedling growth at different times after seed harvest Observations on the percentage and rate of germination at different times after harvest were made on seeds of the crops 1961, 1962 and 1963. Fully ripened seeds (harvest time) manifested a 65-75 per cent germination and a lower germination rate as compared with after-ripened seeds (seeds stored under laboratory conditions for at least 3 months). Further test on seeds stored under laboratory conditions showed that both germination percentage and germifiation rate increased progressively with increasing distance from the harvest time; so did also the growth of the seedlings. Three months or more after harvest, the seeds had reached complete after-ripeness.
The experiments were carried out in 1962 and 1963 using seeds of the durum wheat variety Cappelli, crops 1961 and 1962, stored under laboratory conditions. Dry seeds were soaked in distilled water in petri dishes (100 seeds in 50 ml water in each dish) for 24 hr at 20°C in the dark and soon afterwards half of the seeds was irradiated with an X-ray machine operated at 210 kV, 11 mA, I000 r/n'dn, with a l-ram A1 filter. Using a sharp gouge of appropriate size, embryos with scutellum were excised from both irradiated and unirradiated seeds; the embryo transplantation work was completed within an hour from the end of irradiation. T h e transplants were sown in sterilized sand, at 2-5 cm depth, in wooden boxes in a controlled room at 20°C. I n the initial phase of the experiments, of which only one series is
5()0
OCCURRENCE OF AN INHIBITOR IN WHEAT ENDOSPERM
Table 3. Growth oj-seedlings obtainedfrom unirradiated embryos transplanted on irradiated endosperms, EM(u)[EW(t) and of seedlings from homotransplants, EM(u)[EN(u). Material: durum wheat seeds, crop 1961, stored under laboratory conditionsfor 32 weeks Transplant type
EM(u)fENiu) EM(u)fEN(~) EM(u)fEN(u) EM(u)fEM(~) EM(u)fEN(,) EM(u)fEN(I) EM(,)fEN(u) EM(u)fEN¢, ) EM(u)/EN(u) EM(u)fEN(i) E M cu)fEN (u) EM(u)fENct)
2 kr 4 kr 6 kr 8 kr 10 kr 20 kr
Transplants no.
First leaf length mm
Root length mm
160 160 180 180 180 180 100 100 100 100 100 100
35-52 51.07* 39"35 51.72* 45- 75 70.30* 27.95 39.79* 46.57 56'I1++ 40.36 40.00
21-11 32.82* 27.37 34.65* 34.33 47.56* 18.68 27"68* 22" 76 32"25 t 23.50 26"23
Significant differences between EM(ul/EN(i ) means and the corresponding EM(u)fEN(u ) means are indicated with the following P values: *0"001 ; ~0"01 ; +~0"05 (other differences not significant). I f the seeds were stored at laboratory temperature in a dry atmosphere in a desiccator, the dormancy phenomenon was slightly prolonged. The situation described above is summarized in Table 1, in which the data of a germination test performed in 1962 is reported. According to BELDEROCKO),the average duration of dormancy is the time elapsing from the fully-ripe stage (harvest time) to the time when half of the grains germinate within 3 days. We can statc that in Cappelli the average duration of dormancy is l 0-15 days, while the total period of dormancy lasts three months or more.
2. Effect of#radiated endosperm on growth of unirradiated embryo In December 1961, embryo transplantation experiments were started using seeds of the crop 1961 (harvest: June 18-20); EMli)/ENci ) and EMt,)/ENt,,) transplants were compared in order to ascertain whether unirradiated endosperm was able to improve growth in the irradiated embryo. In these experiments, very variable results were obtained: with some irradiations, EMt,)/ENt,) seedlings grew better than the E~VI(i)/ENt/) ones; with other irradiations, the
reverse was true. In no case, however, was a statistically significant difference in growth between the two types of transplants found. In an experiment performed in the second half of J a n u a r y 1962, quite unexpected results were obtained: the EM(i)/EN¢i ) transplants grew better than the EM(i)/EN¢,,) transplants in all cases and, in some radiation exposures, the differences were highly significant (Table 2). To assess further the "growth stimulating" effect of the irradiated endosperm, we carried out new experiments comparing growth in EMt,,)/ENt, ~ and EM(u)/EN,,,~ transplants. One of such experiments, made 32 weeks after seed harvest, is reported in Table 3. It is apparent that a significant growth stimulation by the irradiated endosperm is obtained throughout the irradiation series 2-10 kr, whilst no clear effect is seen with 20 kr. This last result, also met with in further experiments, seems to indicate that, in our material, 20 kr is too high a radiation exposure, probably inducing some general damage to the endosperm. In 1962-3, the embryo transplantation experiments were repeated on seeds of the crop 1962, at different times, starting from the first week
501
P. MELETTI, C. FLORIS and F. D'AMATO Table 4. Growth of seedlings obtainedfrom unirradiated embryos transplanted on irradiated endosperms, EA,I(u)[EN(o and of seedlingsfrom homotransplants, EM(u)]EN(u). Material: durum wheat seeds, crop 1962, stored under laboratory conditions Duration of seed storage after harvest Transplant type
EM(u)/EN(u) EM(u)/EN(0 EM(u)/EN(u) EM(u)/EN(0 EM(u)/EN(u) EM(u)[EN(i)
1-3 weeks Transplants no.
First leaf length mm
Root length mm
Transplants no.
First leaf length mm
Root length mm
200 200 200 200 200 200
23" 12 21.84 23" 12I" 19.79 23.12 25"46
22 "45 21 "52 22-45 20"91 22.45 24"47
100 100 100 I00 100 100
58.23 64.97 t 56.89 62.63I" 62.23 70.18 t
42.65 56.76* 42.65 42.96 42.65 52.02*
4kr 6 kr 8 kr
28-30 weeks
Significant differences between EM(u)/EN(o means and the corresponding EM(u)/EN(u) means are indicated with the following P values: *0.01; ~'0.05 (other differences not significant). after harvest. We could demonstrate that, in seeds irradiated during the total period of dormancy, the irradiated endosperm did not exert any "growth stimulating" effect; this appeared only in completely after-ripened seeds, 28-30 weeks after storage (Table 4), and became still more pronounced in the next 2 months. 3. Occurrence of an inhibitor in the endosperm The results presented in the preceding section could be interpreted by assuming either (I) a true stimulating effect of the irradiated endosperm or (2) a destruction, or inactivation, by irradiation of an inhibitor present in the endosperm. Experimental evidence for the validity of the second assumption is as follows (4) (for more detailed data, reference is made to the original paper) : (a) if endosperms excised from after-ripened seeds (presoaking in water for 24 hr at 20°C) are placed in the center of petri dishes in which 40 dry after-ripened seeds are put to germinate in 2 circles of 16 and 24 seeds (endosperms renewed every 24 hr), seedling growth is depressed as compared with controls (seed germination in the absence of excised endosperms). Moreover, growth depression is greater in the seedlings of the inner circle than in those of the outer; (b) endosperms escised from dry after-ripened seeds show a greater growth depressing effect
than those excised from presoaked seeds, thus providing further evidence that the inhibitor is a water-soluble substance, which may partly leak out during seed soaking; (c) endosperms excised with and without pericarp from after-ripened seeds show a similar growth depressing effect. If pericarps instead of endosperms are used in experiments of the type under (a), no effect is observed; (d) since the inhibitor can be extracted from after-ripened seeds in water at 2°C, it seems very likely that it occurs in the dry seeds and is not the result of the metabolic activity associated with germination (Prof. E. MarrY, personal communication) ; (e) the inhibitor is not present, in detectable amounts at least, in the endosperm during the period of dormancy (experiments of type a) ; (f) using the method reported under (a), it can be shown that, in after-ripened material, endosperms excised from seeds irradiated after presoaking in water have lost their inhibitory activity. DISCUSSION The present investigation shows that in Cappelli seeds, stored under laboratory conditions, the average duration of dormancy (1) is rather short, whilst the total period of dormancy lasts at least three months. During dormancy, no
502
OCCURRENCE OF AN INHIBITOR IN WHEAT ENDOSPERM
inhibitor is found in the Cappelli endosperm; this situation, already known for wheat and other cereal speciesC1), demonstrates that the factors governing dormancy in cereals are not localized in the endospermteL When Cappelli seeds have attained complete after-ripeness, an inhibitory condition occurs in the endosperm that lasts rather long: using water extraction and germination tests in presence of excised endosperms, the presence of the inhibitor in the 1961 seeds could still be ascertained in October 1963. As shown by this paper, the detection of an inhibitor in after-ripened seeds of durum wheat (a phenomenon so far unknown in the wheat literature) was made possible by the embryo transplantation technique, which allows a dissociation of the effects of radiation on the embryo from those on the endosperm. This confirms our previous statement that the embryo transplantation technique may prove useful in studying embryo-endosperm relations in irradiated seedsl 5). For the general problem of seed radiobiology, our results are of significance in that they show the striking physiological differences occurring in wheat seeds at different times after harvest. Dormancy and after-ripening, being phenomena of rather common occurrence in seed physiologyt~), must be regarded among the important factors governing seed response to ionizing radiation. It seems probable that the increased growth of seedlings observed on several occasions after seed irradiationt 7) may result from the destruction of an inhibitor present in the seed or from other changes, which could be expected to occur only in some special physiological condition of the seed at the time of irradiation. In
our opinion, inadequacy of knowledge on the physiology of seeds used in radiobiological experiments and inability to control the factors responsible for seed maturation offer a reasonable explanation of the irregularity and unreproducibility of results met with in studies on growth responses, including stimulation, in irradiated seeds. Work on the problems raised by this investigation is being continued, partly in collaboration with the Laboratory of Plant Physiology of the University of Milan, Italy (Director: Prof. E. MarrY). Acknowledgements--We are indebted to our colleagues, Drs. M. BUIATTIand P. G. AReARA, for help in the statistical analysis.
REFERENCES . I. BELDEROKB. (1961) Studies on dormancy in wheat. Proe. Seed Test. Ass. 26, 697-760. 2. CROCKERW. and BARTONL. V. (1953) Physiology ofseeds. Chronica Botanica, Waltham, p. 267. 3. FONS'rBJNL. M. (1961) Data on the influence of the irradiation of wheat endosperm on growth and development of plants. Radiobiologia 1, 446-451. 4. MELETTIP. (1964) Nuove prospettive nello studio dei fattori che controllano la germinazionc dci semi. Giorn. But. Ital. (to be printed). 5. MELE'I"rlP. and D'AMATOF. (1961) The embryo transplantation technique in the study of embryoendosperm relations in irradiated seeds. Effects of ionizing radiations on seeds, IAEA, Vienna, 47-54. 6. MOORMANNB.(1942) Untersuchungeniiber Keimruhe bei Hafer und Gerste. Kiihn-Arch. 56, 41-79. 7. Shx K. (1963) The stimulation of plant growth by ionizing radiation. Radiation Botany 3, 179-186.
OCCURRENCE OF AN INHIBITOR IN WHEAT ENDOSPERM AS REVEALED BY EMBRYO T R A N S P L A N T A T I O N IN IRRADIATED SEEDS* P. MELETT]L, C. FLORIS a n d F. D'.4,MATO
Istituto di Botanica della Universit~t, Cagliari and Istituto di Genetica delia Universit~t, Pisa, Italy
(Received 20 January 1964) Alanstract--Seeds (caryopses) of durum wheat var. Cappelli were soaked in water for 24 hr at 20°C to soften them enough for excision of embryos with scutellum. After irradiation of half of the soaked seeds (2-10 kr of X-rays), embryos were excised from both irradiated and unirradiated seeds and the following transplantations were made: (1) unirradiated embryo on irradiated endosperm, EMtu)/EN(I ) and (2)unirradiated embryo on unirradiated endosperm, EM~u)/EN (.) (homotransplant, to serve as control). In 1962 and 1963, using seeds of the crops of 1961 and 1962 respectively, it was demonstrated that endosperms of after-ripened seeds stimulated growth in the EM(u)/EN(i ) seedlings. Evidence was found that this stimulatory effect results from the destruction--or inactivation-by radiation of an inhibitor present in the endosperm of after-ripened seeds. The inhibitor is not present, in detectable amounts, in the endosperm of seeds during the total period of dormancy (first three months or more after harvest). The results are discussed in relation to the general problem of growth responses, including stimulation, in irradiated seeds. R a s u m # - - D e s graines (caryopses) de b16 durum var. Cappelli ont 6t6 immerg6es dans de l'eau pendant 24 heures/~ 20°C afin de les ramollir sutTasamment pour permettre l'excision des embryons avec leurs scutellums. Apr~s irradiation de la moiti6 du lot de graines hydrat~es (2 ~t 10 Kr de rayons X), des embryons ont 6t6 excis6s des graines irradi6es aussi bien que des graines non irradi6es et les transplantations suivantes ont 6t6 r6alis6es: (1) embryon non irradi6 sur endosperme irradi6 EM~u)/EN(i ) et (2) embryon non irradi6 sur endosperme non irradi6, EM(u)/EN(u) (homo-transplantation servant de t6moin). En 1962 et 1963, en utilisant les graines des r~coltes de 1961 et 1962 respectivement, nous avons d6montr6 que les endospermes des graines aprb.s maturation stimulaient la croissance darts les plantules EM~u)/EN(0. On a prouv6 que l'effet stimulant provient de la destruction ou de l'inactivation, par les radiations, d'un inhibiteur present dam l'endosperme des graines apr~ maturation. L'inhibiteur n'est pas prdse~nt en quantit6 ddcelable darts l'endosperme des graines pendant la p6riode totale de dormance (les 3 premieres mois ou plus apr6s la r6colte). Ces rdsultats sont discut6s dam leur relation avee le probl~me g6n6ral de la croissance y compris la stimulation des graines irradi~es. Z u s ~ m m e n f a s s u n g - - S a m e n (Karyopsen) von durum-Weizen var Cappelli wurden 24 S'tunde.fi lang bei 20 ° in Wasser angequollen, um sie for das Herauspr/iparieren von Embryo plus Skutellum gentigend zu erweichen. Die eine H~lfte der gequollenen Samen wurde *Part of the investigation was supported by a sub-contract with the Euratom-ITAL Association, Wageningen, Holland. 497 M
OCCURRENCE OF AN INHIBITOR I N W H E A T ENDOSPERM
498
bestrahlt (2-10 kr R6ntgenstrahlen). Dann wurden sowohl aus bestrahlten wie unbestrahlten Samen die Embryonen herauspr~ipariert, und die folgenden Transplantationen vorgenommen : (1) Unbestrahlter Embryo auf bestrahltes Endosperm, EM(u)/EN(0. (2) Unbestrahlter Embryo auf unbestrahltes Endosperm, EM(u)/EM(u) (Homotransplantate, die als Kontrollen dienten). In denJahren 1962 und 1963 wurde unter Benutzung von Samen der Ernten von 1961 bzw 1962 gezeigt, dass Endosperme von nachgereiften Samen das Wachstum bei EM(u}/EN(0Keimlingen stimulieren. Es wurde gefunden, dass diese stimulierende Wirkung auf der strahlenbedingten Zerst6rung oder Inaktivierung eines Inhibitors beruht, der im Endosperm yon naehgerefften Samen vorkommt. Der Inhibitor l~isst rich im Endosperm yon Samen w~ihrend der gesamten Periode der Samenruhe die ersten 3 oder mehr Monate naeh der Ernte nicht naehweisen. Die Ergebnisse werden mit Hinsicht aufdas aUgemeine Problem yon Waehstumsreaktionen, unter Einschluss yon Stimulationserseheinungen, bei bestrahltenSamen diskutiert. INTRODUCTION
IN A PREVZOUSpaperO), the results of embryo transplantation experiments in irradiated seeds (caryopses) of barley and wheat were reported. Dry seeds were irradiated with high radiation exposures of X-rays, y-rays and thermal neutrons; irradiated and unirradiated seeds were soaked in water at 16°C for 24 hr in order to have them softened enough for excision of endosperm-free embryos with scutellum. Seedling growth was compared in the following two types of transplants : (1) unirradiated embryo on irradiated endosperm, EM(,)/EN(i) and (2) unirradiated embryo on unirradiated endosperm, EM/(,) EN(,) (homotransplant). Both in barley (X-ray exposures of 50, 100, 200 and 400 kr) and in
wheat (y-ray exposures of 250, 500 and 1000 kr) it was found that X-ray (or y-ray) irradiated endosperm can depress growth in both the root and the shoot systems in the EM(,,)/EN(0 seedlings. These results have been confirmed recently by FONSTEJN(s)in embryo transplantation experiments in wheat seeds irradiated with y-rays ( 100 and 500 kr) in the dry state. In continuing our work on embryo-endosperm relations in irradiated seeds, we decided to concentrate our attention on the durum wheat variety Cappelli, a material we are using in extensive radiation biological and genetic studies. We also introduced a new experimental procedure, namely: irradiation of seeds pregerminated for 24 hr at 20°C with X-ray
Table 1. Seed germinability and seedling growth in Triticum durum vat. Cappelli at different times afteT seed harvest (seeds stored under laboratory conditions). For each test two germinations of 50 seeds each were made at 20°C in the dark
% germinated after days Crop
1961 1962
1962"
After 4 days
Days after harvest
Seed moisture °/o
2
4
6
10
14
375 2 9 16 23 69 16 69
11"0 6"0 8"3 11"0 11'3 11"3 2"6 2"6
95 0 32 36 56 68 9 39
99 3 76 80 92 88 48 88
100 35 90 90 94 90 65 90
62 92 92 94 92 76 92
74 92 92 95 94 80 92
* Kept in a dry atmosphere in a desiccator.
First leaf length mm
Root length mm
32"80
58"00
13"00 25"40 25"70
30"00 56"30 56"40
24"70
50"50
499
P. MELETTI, C. FLORIS and F. D'AMATO
Table 2. Growth of s;edlings obtainedfrom irradiated embo~os transplanted on irradiated endosperms, EM(t)/EWo) (homotransplants) and from irradiated embryos transplanted on unirradiated endosperms, EMq)]EW(u). For each radiation exposure 300 transplants were made (150 EM(i)]EN(i ) and 150 EM(i)]EW(u) ). Material: durum wheat seeds stored under laboratory conditionsfor 27 weeks Radiation exposure 4 kr
6 kr
8 kr
Transplant First leaf length mm EMq)/ENq) EM0)/EN(, )
36"20* 27"86
Root length mm 16"74++ 14"51
First leaf length mm
Root length mm
First leaf length mm
Root length mm
26"62 22"17
13"18 11"16
20"39* 15"67
10"847 9"84
Significant differences between EM(/)/EN(i) means and the corresponding EM0)/EN(u ) means are indicated with the following P values: *0"001 ; ~0"01 ; +0"05 (other differences not significant).
exposures of 2-10 kr and completion of the embryo transplantation work within one hour from the end of irradiation. Using this experimental procedure on after-ripened seeds, we tbund that EM~I)/EN~i) seedlings grew better than EM~0/EN(, ) seedlings (see Table 2). In the present paper, results are reported showing that the seemingly stimulatory effect of the irradiated wheat endosperm is due to the abolition, or reduction, by irradiation of an inhibitory condition present in that tissue.
reported here (Table 2), EMli)/EN
MATERIAL AND METHODS
RESULTS 1. Germinability and seedling growth at different times after seed harvest Observations on the percentage and rate of germination at different times after harvest were made on seeds of the crops 1961, 1962 and 1963. Fully ripened seeds (harvest time) manifested a 65-75 per cent germination and a lower germination rate as compared with after-ripened seeds (seeds stored under laboratory conditions for at least 3 months). Further test on seeds stored under laboratory conditions showed that both germination percentage and germifiation rate increased progressively with increasing distance from the harvest time; so did also the growth of the seedlings. Three months or more after harvest, the seeds had reached complete after-ripeness.
The experiments were carried out in 1962 and 1963 using seeds of the durum wheat variety Cappelli, crops 1961 and 1962, stored under laboratory conditions. Dry seeds were soaked in distilled water in petri dishes (100 seeds in 50 ml water in each dish) for 24 hr at 20°C in the dark and soon afterwards half of the seeds was irradiated with an X-ray machine operated at 210 kV, 11 mA, I000 r/n'dn, with a l-ram A1 filter. Using a sharp gouge of appropriate size, embryos with scutellum were excised from both irradiated and unirradiated seeds; the embryo transplantation work was completed within an hour from the end of irradiation. T h e transplants were sown in sterilized sand, at 2-5 cm depth, in wooden boxes in a controlled room at 20°C. I n the initial phase of the experiments, of which only one series is
5()0
OCCURRENCE OF AN INHIBITOR IN WHEAT ENDOSPERM
Table 3. Growth oj-seedlings obtainedfrom unirradiated embryos transplanted on irradiated endosperms, EM(u)[EW(t) and of seedlings from homotransplants, EM(u)[EN(u). Material: durum wheat seeds, crop 1961, stored under laboratory conditionsfor 32 weeks Transplant type
EM(u)fENiu) EM(u)fEN(~) EM(u)fEN(u) EM(u)fEM(~) EM(u)fEN(,) EM(u)fEN(I) EM(,)fEN(u) EM(u)fEN¢, ) EM(u)/EN(u) EM(u)fEN(i) E M cu)fEN (u) EM(u)fENct)
2 kr 4 kr 6 kr 8 kr 10 kr 20 kr
Transplants no.
First leaf length mm
Root length mm
160 160 180 180 180 180 100 100 100 100 100 100
35-52 51.07* 39"35 51.72* 45- 75 70.30* 27.95 39.79* 46.57 56'I1++ 40.36 40.00
21-11 32.82* 27.37 34.65* 34.33 47.56* 18.68 27"68* 22" 76 32"25 t 23.50 26"23
Significant differences between EM(ul/EN(i ) means and the corresponding EM(u)fEN(u ) means are indicated with the following P values: *0"001 ; ~0"01 ; +~0"05 (other differences not significant). I f the seeds were stored at laboratory temperature in a dry atmosphere in a desiccator, the dormancy phenomenon was slightly prolonged. The situation described above is summarized in Table 1, in which the data of a germination test performed in 1962 is reported. According to BELDEROCKO),the average duration of dormancy is the time elapsing from the fully-ripe stage (harvest time) to the time when half of the grains germinate within 3 days. We can statc that in Cappelli the average duration of dormancy is l 0-15 days, while the total period of dormancy lasts three months or more.
2. Effect of#radiated endosperm on growth of unirradiated embryo In December 1961, embryo transplantation experiments were started using seeds of the crop 1961 (harvest: June 18-20); EMli)/ENci ) and EMt,)/ENt,,) transplants were compared in order to ascertain whether unirradiated endosperm was able to improve growth in the irradiated embryo. In these experiments, very variable results were obtained: with some irradiations, EMt,)/ENt,) seedlings grew better than the E~VI(i)/ENt/) ones; with other irradiations, the
reverse was true. In no case, however, was a statistically significant difference in growth between the two types of transplants found. In an experiment performed in the second half of J a n u a r y 1962, quite unexpected results were obtained: the EM(i)/EN¢i ) transplants grew better than the EM(i)/EN¢,,) transplants in all cases and, in some radiation exposures, the differences were highly significant (Table 2). To assess further the "growth stimulating" effect of the irradiated endosperm, we carried out new experiments comparing growth in EMt,,)/ENt, ~ and EM(u)/EN,,,~ transplants. One of such experiments, made 32 weeks after seed harvest, is reported in Table 3. It is apparent that a significant growth stimulation by the irradiated endosperm is obtained throughout the irradiation series 2-10 kr, whilst no clear effect is seen with 20 kr. This last result, also met with in further experiments, seems to indicate that, in our material, 20 kr is too high a radiation exposure, probably inducing some general damage to the endosperm. In 1962-3, the embryo transplantation experiments were repeated on seeds of the crop 1962, at different times, starting from the first week
501
P. MELETTI, C. FLORIS and F. D'AMATO Table 4. Growth of seedlings obtainedfrom unirradiated embryos transplanted on irradiated endosperms, EA,I(u)[EN(o and of seedlingsfrom homotransplants, EM(u)]EN(u). Material: durum wheat seeds, crop 1962, stored under laboratory conditions Duration of seed storage after harvest Transplant type
EM(u)/EN(u) EM(u)/EN(0 EM(u)/EN(u) EM(u)/EN(0 EM(u)/EN(u) EM(u)[EN(i)
1-3 weeks Transplants no.
First leaf length mm
Root length mm
Transplants no.
First leaf length mm
Root length mm
200 200 200 200 200 200
23" 12 21.84 23" 12I" 19.79 23.12 25"46
22 "45 21 "52 22-45 20"91 22.45 24"47
100 100 100 I00 100 100
58.23 64.97 t 56.89 62.63I" 62.23 70.18 t
42.65 56.76* 42.65 42.96 42.65 52.02*
4kr 6 kr 8 kr
28-30 weeks
Significant differences between EM(u)/EN(o means and the corresponding EM(u)/EN(u) means are indicated with the following P values: *0.01; ~'0.05 (other differences not significant). after harvest. We could demonstrate that, in seeds irradiated during the total period of dormancy, the irradiated endosperm did not exert any "growth stimulating" effect; this appeared only in completely after-ripened seeds, 28-30 weeks after storage (Table 4), and became still more pronounced in the next 2 months. 3. Occurrence of an inhibitor in the endosperm The results presented in the preceding section could be interpreted by assuming either (I) a true stimulating effect of the irradiated endosperm or (2) a destruction, or inactivation, by irradiation of an inhibitor present in the endosperm. Experimental evidence for the validity of the second assumption is as follows (4) (for more detailed data, reference is made to the original paper) : (a) if endosperms excised from after-ripened seeds (presoaking in water for 24 hr at 20°C) are placed in the center of petri dishes in which 40 dry after-ripened seeds are put to germinate in 2 circles of 16 and 24 seeds (endosperms renewed every 24 hr), seedling growth is depressed as compared with controls (seed germination in the absence of excised endosperms). Moreover, growth depression is greater in the seedlings of the inner circle than in those of the outer; (b) endosperms escised from dry after-ripened seeds show a greater growth depressing effect
than those excised from presoaked seeds, thus providing further evidence that the inhibitor is a water-soluble substance, which may partly leak out during seed soaking; (c) endosperms excised with and without pericarp from after-ripened seeds show a similar growth depressing effect. If pericarps instead of endosperms are used in experiments of the type under (a), no effect is observed; (d) since the inhibitor can be extracted from after-ripened seeds in water at 2°C, it seems very likely that it occurs in the dry seeds and is not the result of the metabolic activity associated with germination (Prof. E. MarrY, personal communication) ; (e) the inhibitor is not present, in detectable amounts at least, in the endosperm during the period of dormancy (experiments of type a) ; (f) using the method reported under (a), it can be shown that, in after-ripened material, endosperms excised from seeds irradiated after presoaking in water have lost their inhibitory activity. DISCUSSION The present investigation shows that in Cappelli seeds, stored under laboratory conditions, the average duration of dormancy (1) is rather short, whilst the total period of dormancy lasts at least three months. During dormancy, no
502
OCCURRENCE OF AN INHIBITOR IN WHEAT ENDOSPERM
inhibitor is found in the Cappelli endosperm; this situation, already known for wheat and other cereal speciesC1), demonstrates that the factors governing dormancy in cereals are not localized in the endospermteL When Cappelli seeds have attained complete after-ripeness, an inhibitory condition occurs in the endosperm that lasts rather long: using water extraction and germination tests in presence of excised endosperms, the presence of the inhibitor in the 1961 seeds could still be ascertained in October 1963. As shown by this paper, the detection of an inhibitor in after-ripened seeds of durum wheat (a phenomenon so far unknown in the wheat literature) was made possible by the embryo transplantation technique, which allows a dissociation of the effects of radiation on the embryo from those on the endosperm. This confirms our previous statement that the embryo transplantation technique may prove useful in studying embryo-endosperm relations in irradiated seedsl 5). For the general problem of seed radiobiology, our results are of significance in that they show the striking physiological differences occurring in wheat seeds at different times after harvest. Dormancy and after-ripening, being phenomena of rather common occurrence in seed physiologyt~), must be regarded among the important factors governing seed response to ionizing radiation. It seems probable that the increased growth of seedlings observed on several occasions after seed irradiationt 7) may result from the destruction of an inhibitor present in the seed or from other changes, which could be expected to occur only in some special physiological condition of the seed at the time of irradiation. In
our opinion, inadequacy of knowledge on the physiology of seeds used in radiobiological experiments and inability to control the factors responsible for seed maturation offer a reasonable explanation of the irregularity and unreproducibility of results met with in studies on growth responses, including stimulation, in irradiated seeds. Work on the problems raised by this investigation is being continued, partly in collaboration with the Laboratory of Plant Physiology of the University of Milan, Italy (Director: Prof. E. MarrY). Acknowledgements--We are indebted to our colleagues, Drs. M. BUIATTIand P. G. AReARA, for help in the statistical analysis.
REFERENCES . I. BELDEROKB. (1961) Studies on dormancy in wheat. Proe. Seed Test. Ass. 26, 697-760. 2. CROCKERW. and BARTONL. V. (1953) Physiology ofseeds. Chronica Botanica, Waltham, p. 267. 3. FONS'rBJNL. M. (1961) Data on the influence of the irradiation of wheat endosperm on growth and development of plants. Radiobiologia 1, 446-451. 4. MELETTIP. (1964) Nuove prospettive nello studio dei fattori che controllano la germinazionc dci semi. Giorn. But. Ital. (to be printed). 5. MELE'I"rlP. and D'AMATOF. (1961) The embryo transplantation technique in the study of embryoendosperm relations in irradiated seeds. Effects of ionizing radiations on seeds, IAEA, Vienna, 47-54. 6. MOORMANNB.(1942) Untersuchungeniiber Keimruhe bei Hafer und Gerste. Kiihn-Arch. 56, 41-79. 7. Shx K. (1963) The stimulation of plant growth by ionizing radiation. Radiation Botany 3, 179-186.