spore germination and viability and with protein diffusion

Cytologia 41: 387-393, 1976

The Effect of Freezing and Organic Solvents on

Viability of Chlorophyllous Fern Spores

Yukio Kato

Biological Laboratory, Fukui University, Fukui, 910 Japan

Received November 10, 1974

Chlorophyllous spores, which occur in the genus Equisetum and Osmunda, are

particularly suitable as experimental material in the area of cell differentiation and morphogenesis (cf. Miller 1968). Chlorophyll-bearing spores, however, rapidly lose germinability at room temperature. It is desirable to obtain, therefore, viable spores throughout the year.

Germinating pollens of a number of plants release protein and other chemical

substances to an agar culture medium, on the stigma or in the style (Stanley and Linskens 1965, Stanley and Search 1971). In pear pollen, successive elution reduces

greatly germinability by the loss of materials (e. g. enzymes) within the pollen. The

present paper is concerned with the influence of freezing and organic solvents on spore germination and viability and with protein diffusion from stored spores.

Materials and methods

Cones of Equisetum arvense were collected in and around the campus of Fukui

University. Strobili were spread on paper and allowed to dry. Spores were stuffed

in glass tubes with plastic cap (2.5 cm in diameter and 5 cm in length) placed into

dark box. They are divided into two lots, and stored at plus 5-6•Ž and minus 20•Ž,

respectively, before use. Pteris vittata as a species bearing non-green spores and

Osmunda japonica bearing green spores were also used for comparison.

Chlorophyllous and non-chlorophyllous spores were soaked in various kinds

of organic solvents and stored at 5-6•Ž for 2 or 3 days. After the treatment, spores

were sown and cultured on a modified Moore's agar medium supplemented with

Nitsch's trace element solution (Kato 1969). The cultures were grown under con

tinuous illumination at a light intensity between 1,500 and 2,000 lux. Temperature

in the culture room varied from 23 to 25•Ž.

Germination percentage was scored by counting 100-150 individuals. The

appearance of the primary rhizoid or protonemal cell or both was used as an indi

cation of germination.

Results

1. Effects of freezing on viability and protein diffusion from spores

When spores were stored at room temperatur ranging 19-23•Ž, percentage of

the viable spores becomes progressively lower with the time. A typical result is

388 Y. Kato Cytologia 41

shown in Table 1. The length of viability under room temerature varied from 10

to 20 days from year to year.

The effect of various temperature conditions upon spore viability is shown in

Table 2. After storage at minus 20•Ž for about one year, the viability ranged

Table 1. Germination percentage of Equisetum arvence spores

stored under room temperature (19-23•Ž)

Table 2. Effect of various temperature conditions upon

Equisetum spore viability

Table 3. Summary of the results of some colour-reaction tests of

the supernatants from fresh and aged Equisetum spores

* Degree of colour-reaction: -; negative; •}, weak; +, strong reaction.

16-64%, whereas at plus 5•Ž germinability decreased greatly even for a short period

of 50 or 70 days. Spores stored for one year germinated readily, grew and formed

mature prothalli on agar medium.

When 0.5 g of dried spores were soaked in 10 ml of distilled water or a nutrient

1976 The Effect of Freezing and Organic Solvents on Viability 389

medium, left at room temperature (22•Ž) for 5 minutes and then centrifuged, the

supernatants frequently coloured brownish yellow, whereas those from fresh spores

milky white. These facts suggested that the quantity or quality of chemical moieties

excreted from stored spores is different to those of fresh ones, and that one of the

Figs. I and 2. Germination of Equisetum arvense spores. P, protonemal cell; r, primary rhizoid;

vs, viable (chlorophyllous) spore; ds, ungerminated (dead) spore; gs, germinating spore. 1, fresh

spores. •~150. 2, spores stored for 380 days at minus 20•Ž. •~125.

Table 4. Relative amount of phenol reacting compounds (method of Lowry et al. 1951) diffusing from Equisetum spores *

* 0 .5 g spores were suspended in 20 ml distilled water. The Folin-phenol reagent was added

to 0.5 ml of the supernatant and then measured by Hitachi-Model 101 spectrophotometer.

cause of low viability in Equisetum arvense may be due to loss of substances required

to germinate. The results of some chemical tests for substances diffused out of fresh and stored spores are summarized in Table 3. Urine sugar analysis paper, "Tes -Tape", was used for detecting the presence of sugar (Takayanagi and Murakami

1968).It is apparent from these colour-reaction that protein is present in the super


natant from stored spores but not from fresh ones. The supernatant was also

analyzed with a Hitachi-Model 356 double beam spectrophotometer using 1 cm

quartz cells, as shown in Fig. 3. The Equisetum spores excreted the ultraviolet

Fig. 3. Absorption spectra of supernatants incubated with Equisetum spores in distilled water for

10 minutes. Each curve is adjusted to show the similar levels. A, a supernatant from spores

stored for 7 days at room temperature (23•Ž). The absorption peak is observed at 320mƒÊ. B,

a supernatant after the fifth water elution of the same material as Fig. 3A. Beside 320mƒÊ, two

absorption peaks, 245mƒÊ and 285mƒÊ, are apparent. C, a supernatant from stored for about one

year at minus 20•Ž. D, a supernatant after the fifth water elution of the same material as Fig. 3C.

Large peaks at 320mƒÊ and 245mƒÊ and a small peak at 285mƒÊ are observed.

absorbing in distilled water. The extracts from spores stored for 7 days at 23•Ž

or for about one year at minus 20•Ž showed a maximum absorption peak at about

320mƒÊ (Fig. 3A). By the third water elution of these spores secondary absorption


peak of about 285mƒÊ and 245mƒÊ was apparent (Fig. 3B and 3D).

The relative amount of Lowry-reacting material diffusing from spores under

various storage conditions are summarized in Table 4. There is a correlation be

tween the spore germinability (%) and the relative amounts of Lowry-reacting com

pounds transmitting at 520mƒÊ. Germination percentage of stored spores reduced

more or less after successive washing with distilled water, whereas that of fresh spores

did not reduced by the same treatment (Table 5). It seems to probable that a con

siderable amount of materials required for germination releases from stored spores

during washing.

Table 5. Effect of water elution on spore germination of

Equisetum arvense

* The fifth water elution

Table 6. Germination rate of Equisetum arvense, Osmunda japonica and Pteris vittata

spores which had been soaked in various organic solvents for 3 days at about 5•Ž

2. The viability of fern spores in organic solventsIwanami (1972a) and Iwanami and Nakamura (1972b) reported that Camellia

japonica and. Lilium pollens retained their viability in various organic solvents. In the present study, green and non-green spores were treated with some organic solvents

to find suitable solvents for spore storage. About 0.5 g of the spores immersed in


10 ml of each organic solvents and then refrigerated at 5-6•Ž. After 3 days, these

solvents were discarded and spores were dried with a vacuum pump. Thus treated

spores were sown and grown on an agar medium. A typical result is shown in Table

6. Osmunda and Pteris spores which has been soaked in organic solvents retained

their viability, but Equisetum spores did not.

Discussion

Green spores are characterized by rapid germination after sowing and have

short period of viability. The most rapid germination in fern species is observable

in Equisetum, but under usual storage condition at room temperature the ability to

germinate loses within 10 days or near (Table 1). Castle (1953) stored Equisetum

arvense spores for period of three months at 5•Ž and obtained 7% viability. Hauke

(1969) stored wet spores of Equisetum myriochaetum, E. giantetum and E. bogotense

at 8•Ž for 18-48 days; germinability varied from 1 to 90%. Jones and Hook (1970)

reported spores of E. telmateia retained high viability after two years of storage in

glycerine at minus 10•Ž. On the other hand, the length of viability of Osmunda

japonica spores is 23-43 days (Okada 1929) and can be lengthened for more than 6

months by cold storage of 5-6•Ž (Kato unpub.)

It is well known that relatively short viability in chlorophyllous spores is related

to 1) the lack of protection of spore contents from desiccation because of the absence

of a resistant cell wall (Wollersheim 1957), 2) high rate of respiration (Hauke 1963),

3) the lack of dormancy (Lloyd and Klekowski 1970), and 4) high water content

(Okada 1929, Gullvag 1968, 1969). These are true in Equisetum arvense which is

absent a resisting spore wall. The fact that Equisetum spores soaked in organic

solvents lose their germinability (Table 6) supports the above described assumption.

Generally speaking, there are increases in cell permeability as plants approach

senescence (cf. Varner 1961). The membrane deterioration may occur during spore

storage and as a result spores may be apted to release protein and other cell consti

tuents in the medium. An increasing leakage of substance (e. g. enzymes) from spores

with age can account for low viability, as suggested by Table 4 and 5. Spore

membrane of Osmunda japonica with visible chloroplasts may alike to that Pteris

vittata with non-green spores.

Flavonoid compounds such as gossypitrin are very common in fern spores (Na

kazawa, personal communication). These compounds are so water-soluble that

some of absorption peaks found in Fig. 3 are more probably the result of flavonoid

type compounds than protein.

Summary

Green spores of Equisetum arvence retained 16-64% viability after about one

year of storage at minus 20•Ž. Judging from ultraviolet absorption and various

chemical tests, release of proteineous substances from spores in the medium incre

ases correlatively with the length of storage. Fresh spores do not diffuse protein,

dissimilar with germinating pollens of higher plants. It is concluded from the present


and other results that spore membrane deterioration is one of the causes of low viabi

lity. Osmunda japonica bearing chlorophyllous spores and Pteris vittata bearing non-chlorophyllous spores retained high vitability in various organic solvents , but E

quisetum arvence did not.

Acknowledgement

The author wishes to express his sincere thanks to Prof . S. Nakazawa, Yamagata University, for giving valuable suggestions regarding gossypitrin

, and to Dr. H. Ookawa, Nagoya University, and Mrs. K. Takeshita for technical assistance .

References

Castle, H. 1953. Notes on the development of the gametophytes of Equisetum arvense in sterile media. Bot. Gaz. 114: 323-328.

Gullvag, B. M. 1968. On the fine structure of the spores of Equisetum fluviatile var . verticillatum studied in the quieacent, germinated and non-viable state. Grana Palynol . 8: 23-69.

- 1969. Primary storage products of some pteridophyte spores-A fine structural study . Phytomorphology 19: 82-92.

Hauke, R. L. 1969. Gametophyte development in Latin American horsetails. Bull. Torrey Bot. Club 96: 568-577.

Iwanami, Y. 1972. Retaining the viability of Camellia japonica pollen in various organic solvents. Plant and Cell Physiol. 13: 1139-1141.

- and Nakamura, N. 1972. Storage in an organic solvent as a means for preserving viability of

pollen grains. Stain Technol. 47: 137-139.Kato, Y. 1969. Physiological and morphogenetic studies of fern gametophytes and sporophytes

in aseptic culture VII. Experimental modifications of dimensional growth in gameto

phytes of Pteris vittata. Phytomorphology 19: 114-121.Lloyd, R. M. and Klekowski, E. J. Jr. 1970. Spore germination and viability in Pteridophyta:

Evolutionary significance of chlorophyllous spores. Biotropica 2: 129-137.Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. 1951. Protein measurement with

folin-phenol reagent. Jour. Bio. Chem. 193: 265-275.Miller, J. H. 1968. Fern gametophytes as experimental material. Bot. Rev. 34: 361-440.Okada, Y. 1929. Notes on the germination of the spores of some pteridophytes with special regard

to their viability. Sci. Rep. Tohoku Imp. Univ. Ser. 4. Biol. 4: 127-182.Stanley, R. G. and Linskens, H. F. 1965. Protein diffusion from germinating pollen. Physiol.

Plant. 18: 47-53.- and Search, R. W. 1971. Pollen diffusates. Pollen: 174-176. Ed. by J. Helsop-Harrison,

Butterworths, London.Takayanagi, K. and Murakami, K. 1968. Rapid germinability test with exudates from seed.

Nature 218: 493-494.Varner, J. E. 1961. Biochemistry of senescence. Ann. Rev. Plant Physiol. 12: 245-264.Wollersheim, M. 1957. Untersuchungen uber die Keimungsphysiologie der Sporen von Equisetum

arvense and Equisetum limosum. Zeit. Bot. 45: 145-159.

spore germination and viability and with protein diffusion

Documents