Regulatory factors to affect sporophytic shoot formation of Equisetum arvense induced by exogenously supplied cytokinin

JPlantPhysiol. Vol. 137.pp. 233-237(1990)

Regulatory Factors to Affect Sporophytic Shoot Formation of Equisetum arvense Induced by Exogenously Supplied Cytokinin A.

KURIYAMA*, **

and M.

TAKEUCHI

Laboratory of Plant Morphogenesis, Department of Regulation Biology, Faculty of Science, Saitama University, Urawa 338,Japan * Present address: Interdisciplinary Research Institute of Environmental Sciences, Shichihon-matsu, Itsutsuji-dori, Kamigyo-ku, Kyoto, 602 Japan ** To whom correspondence should be sent Received March 12, 1990 . Accepted July 16, 1990

Summary We examined the regulatory factors involved in the formation of the sporophytic shoot of Equisetum arvense, which is produced from the gametophyte in response to exogenously supplied cytokinin. Among the tested cytokinins (BA, BA-riboside, Kinetin, Zeatin and 4-PU), BA showed the highest effect. As the concentrations of the cytokinins except Zeatin increased to more than those optimal for shoot formation, callus was dominantly produced from the explanted gametophyte tissue instead of the sporophytic shoot. Although cytokinin played a key role in expression of the sporophytic genes in the haploid generation of E. arvense (Kuriyama et aI. 1990 a), no apogamous shoout could form on the sugar-free medium even when it was cultured with exogenous cytokinin. For the production of a number of shoots, a suitable concentration of sugar was needed with a suitable concentration of cytokinin. The necessity of a sugar supply did not depend on increasing osmotic pressure of culture medium because sucrose was more effective than glucose, and sorbitol and mannitol had no effect on shoot formation. Dlumination was not required for sporophytic shoot formation, but shoots formed in darkness were yellow or whitish and thinner or longer than those formed in the light. NAA, 2,4-D, ABA, and GA inhibited apogamous shoot formation.

Key words: Equisetum arvense, Apogamy, Growth regulators, Pteridophyte, Sporophytic shoot formation. Abbreviations: ABA = abscisic acid; BA = benzyl aminopurine; 2,4-D = 2,4-dichlorophenoxyacetic acid; GA - gibberellic acid; NAA = naphthaleneacetic acid; 4-PU = 4-pyridylurea.

Introduction In Equisetum arvense gametophyte culture, exogenously supplied BA is known to produce apogamous shoots (Kuriyama et aI. 1990 a). As previously described (Kuriyama et aI. 1990 b), this aberrantly induced sporophytic shoot differentiates directly from the surface part of gametophyte tissue. Furthermore, external morphological observation by scanning electron microscope revealed that an apical cell peculiar to sporophytes of Equisetum species is recognized on a shoot tip that is surrounded by several leaf primordia. These results suggest that an integrated capacity to form spo© 1990 by Gustav Fischer Verlag, Stuttgart

rophytic structure is possessed in a gametophytic cell or cell groups of E. arvense, and it can be expressed by exogenously supplied cytokinin. In other words, cytokinin plays a key role in the activation of sporophytic genes in the haploid generation of E. arvense although it can not be concluded that cytokinin directly triggers expression of sporophytic genes. In cell or tissue cultures of higher plants, various regulatory factors are involved in the process of morphogenesis (Tran Thanh van 1981). Also in the aberrant morphogenesis in pteridophytes, namely apogamy, different regulatory factors should interact with each other. Actually, sugar

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(Whittier and Steeve 1960, Whittier 1964), growth substances (Whittier 1966, Sulklyan and Mehra 1977), light (Duncan 1941, Whittier 1964, Whittier and Pratt 1971), and ethylene {Elmore and Whittier 1973} are known as regulatory factors involved in artificially produced apogamy in pteridophytes. However, the manner in which promotion of apogamy is brought about is still wholly unknown (Sheffield and Bell 1987). It is important for further physiological and genetic investigation to examine the regulatory factors involved in the activation of sporophytic genes in haploid cells or tissues. In this study, we examined different regulatory factors that affect sporophytic shoot formation in gametophyte culture of E. arvense.

Materials and Methods Plant materials

Gametophytes of Equisetum arvense L. derived from a single spore were used for experimental materials. Gametophytes with rapid growth in culture were established and subcultured on Murashige and Skoog's (1962) agar medium (MS medium) supplemented with 3 % sucrose under 1000 lux continuous light as reported previously (Kuriyama et al. 1989). Gametophyte tissues cultured for 12 - 18 days were cut in pieces 2 - 3 mm in length and thickness by a razor and explanted on 10 mL of agar medium in test tubes (20 mm x 150 mm).

Culture conditions

MS medium was used as a basal medium and modified in various forms by adding sugars and/or growth regulators. All media were adjust to a pH of 5.8 before autoclaving. All cultures were carried out at 26°C in light (1000 lux) or in darkness. Shoot formation rate and average number of shoots were determined after culture for 40 days as follows: shoot formation rate = number of tube that contain at least one sporophytic shoot divided by the total number of tube used for the culture and multiplied by 100, average number of shoots = total number of shoots formed under the same culture condition divided by the number of tubes that contain sporophytic shoots.

Results Effects of sugars on the apogamous shoot formation It is well known that provision of sugar to the culture medium is important for the production of artificial apogamy in pteridophytes (Bopp 1968, Miller 1968, Sheffield and Bell 1987). To examine how sugars were involved in sporophytic shoot formation induced by cytokinin, different concentrations of sucrose or glucose were added to MS medium containing 1 x 10- 7 and 1 x 1O- 6 M BA. Table 1 shows the effect of sucrose and glucose on sporophytic shoot formation induced by BA. On the medium without sugar, sporophytic structures were not induced by BA. This result indicates that sugar is a necessary factor of sporophytic shoot formation, but more than optimal concentrations of sugar decreased the

Table 1: Effect of different concentrations of sucrose or glucose on sporophytic shoot formation rate (%) in E. arvense gametophyte culture. Concentration (g VI)

BA (M) 1 X 10- 7

Sucrose Glucose 0 0 0 71 (5) 90 (6) 5 10 96 (13) 95 (15) 100 (19) 100 (7) 20 100 (19) 70 (3) 30 80 (6) 50 (3) 40 43 (2) 50 30 (2) ( ): Mean number of shoots formed per tube.

1 X 10- 6 Sucrose Glucose 0 0 13 (1) 15 (I) 63 (3) 45 (3) 100 (32) 100 (2) 100 (113) 100 (78) 100 (103) 100 (32) 100 (65) 95 (16)

Table 2: Effect of illumination on sporophytic shoot formation rate (%) in E. arvense gametophyte culture (sucrose, 30 g L -I). BA(M)

Light condition Dark Light o 0 o 1 X 10- 8 o 10 (2) 5 X 10- 8 100 (6) 60 (5) 1 X 10- 7 100 (19) 100 (19) 5 X 10- 7 100 (47) 100 (90) 1 X 10- 6 100 (21) 100 (86) 5 X 10- 6 15 (1) 47 (2) 1 x 10- 5 20 (2) 0 ( ): Mean number of shoots formed per tube. shoot formation rate and/or the number of shoots formed per tube. As a substitutable carbon source, xylose, ribose, mannose, galactose, fructose, sorbitol, maltose, lactose, raffinose, dextran, succinate, malate, and citrate were tested. Only fructose appeared to be a substitutable compound (data not shown), but the number of shoots formed on the medium containing fructose was lower than that on the medium with added sucrose.

Effect of illumination on the apogamous shoot formation Table 2 gives the shoot formation rate and the mean number of shoots per tube in different concentrations of BA in light or in darkness. A hundred percent of shoot formation rate was obtained at 1 x 10- 7,5 X 10- 7 and 1 x 10- 6 M BA in both conditions. However, shoot morphology was different: shorts that developed in darkness were yellow or whitish and thinner or longer in comparison with green shoots formed in light. At more than 1 x 10- 6 M BA, shoot formation rates and shoot numbers were decreased in both light conditions. These reductions are related to callus production as described previously (Kuriyama et al. 1990 a).

Effects of various cytokinins on the apogamous shoot formation Five different cytokinins were added at various concentrations to MS medium supplemented with 3 % sucrose, and ga-

Regulatory factors in Equisetum shoot formation SA

Kinetin

SA-riboside

235

4-PU

Zeatin

'" '0'"

50 (; 0 .c

Fig. 1: Effect of various cytokinins on sporophytic shoot formation rate (%) and mean number of shoots formed per tube in E. ar· vense gametophyte culture (sucrose, 30gL -1).

0.1

0

1x10- 8

1 x 10- 7 1 x 10- 6 2,4-D (M)

1 x 10- 8

BA (M) 1 X 10- 7

1 X 10- 6

10 (12) 15 (1) 0 0

100 (17) 90 (11) 30 (2) 0

100 (71) 100 (54) 100 (27) 0

100 (10) 100 (88) 30 (2) 100 (64) 25 (2) 80 (6) 100 (4) 0 0 0 0 0 ( ): Mean number of shoots formed per tube. Table 4: Effect of different combinations of BA and ABA on sporophytic shoot formation rate (%) in E. aruense gametophyte culture (sucrose, 30 g L -1). BA (M) 1 X 10- 7 1 X 10- 6 (6) 100 (10) 100 (98) (4) 97 (7) 100 (88) 100 (58) (1) 50 (4) 27 (2) 100 (31) 90 (14) 33 (3) of shoots formed per tube.

1 x 10- 8 0 1 x 10- 8 1 x 10- 7 1 x 10- 6 1 x 10- 5

10 17 3 0 0 ( ): Mean number

100.1

10

Table 5: Effect of different combinations of BA and GA on sporophytic shoot formation rate (%) in E. aruense gametophyte culture (sucrose, 30 g L -1). GA(M)

0 1 x 10- 8 1 x 10- 7 1 x 10- 6

ABA(M)

10500.1

E

Concentration (pM)

Table 3: Effect of different combinations of BA and NAA or 2,4-D on sporophytic shoot formation rate (%) in E. aruense gametophyte culture (sucrose, 30 g L -1). NAA(M)

10500.1

Jo

~

z"

metophyte tissues were cultured on them for 40 days in continuous light. The shoot formation rate and mean number of shoots formed per tube are shown in Fig. 1. All tested cytokinins showed a positive effects on sporophytic shoot formation, but the level of their effects differed. BA was the most effective cytokinin. Among purine-type cytokinins, Zeatin showed a poor effect, and 100 % of shoot formation rate could not attained at any concentrations (1 x 10- 7 to 1 x 10- 5 M). On the medium in which BA, BA-riboside, or Kinetin was added at 5 x 10 - 5 M, growth of explanted gametophyte tissues could barely be recognized. Optimal concentrations for maximum shoots were apparent in cultures with BA, BA-riboside, and Kinetin. At higher concentrations of these cytokinins, callus formation became dominant, with a

1 x 10- 8

BA (M) 1 X 10- 7

0 1 x 10- 8 1 x 10- 7 1 x 10- 6 1 x 10- 5

1 X 10- 6

20 (3) 100 (8) 100 20 (1) 85 (2) 100 0 10 (1) 60 0 0 20 0 0 0 ( ): Mean number of shoots formed per tube.

(64) (34) (3) (2)

decreasing number of shoots formed per tube (Kuriyama et al. 1990 a). In contrast to these cytokinins, Zeatin did not induce callus formation at any tested concentrations. A cytokinin of 4-PU-type was also tested and showed lower effectiveness than purine-type cytokinins. Callus formation was also recognized in higher concentrations of 4-PU (5 x 10- 6 and 1 x 10- 5 M). cAMP and adenine were tested between 1 x 10- 7 and 5 x 10 - 5 M as similar compounds of purine-type cytokinins and appeared to have no effect. In all tested concentrations of these compounds, responses of explanted tissues were the same as the controls.

Effect 0/ other kinds 0/growth regulators Tables 3 - 5 shows the effects of other growth regulators including NAA, 2,4-0, ABA, and GA 3. NAA and 2,4-0 showed strong inhibitory effects on shoot formation. At 1 x 10- 6 M NAA or 2,4-0, BA-induced shoot formation was completely inhibited together with the inhibition of growth of explanted tissues. ABA and GA3 showed similar effects on shoot formation.

Discussion The organization of sporophytic plants from gametophyte tissue without syngamy was induced artificially in Pteridium gametophytes by high concentrations of sugar in the culture medium (Whittier and Steeves 1960). Higher irradiance and succinic acid in place of sugar also produced sporophytic

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A. KURIYAMA and M. TAKEUCHI

plants in Pteridium (Whittier 1964). Also in several other species of ferns, sugar in the culture medium induced or hastened the formation of sporophytes (Whittier and Steeve 1962, Sulklyan and Mehra 1977). These results support the hypothesis that the formation of apogamous structures in pteridophytes depends on a sufficient supply of carbohydrate or other organic substrates (Bopp 1968). However, experiments on culture of E. arvense gametophytes showed that appropriate concentration of sucrose enhanced gametophytic growth markedly but did not induce sporophytic differentiation at any concentrations (0- 5 %) (Kuriyama et al. 1989). E. arvense required an exogenously supplied cytokinin for the production of sporophytic shoots (Kuriyama et al. 1990 a). These facts suggest that sugars cannot permit the activity of genes that are necessary for the differentiation of sporophyte in E. arvense. Nevertheless, we first considered a supportive role in induction or development of apogamous structure and chose sugar as a regulatory factor to examine. Sucrose and glucose were tested and shown to play an important role in the induction of apogamy. On the medium without sugar, no apogamous shoot could be induced even if the explanted tissues were cultured over the period of 40-100 days. A higher concentration (5 %) of sugar was also unsuitable for sporophytic shoot formation. Therefore, it is considered that both cytokinin and sugars are necessary to regulate the induction or development of apogamy in E. arvense. Besides sucrose and glucose, only fructose was effective in induction of apogamy. Other sugars or carbon sources like xylose, ribose, mannose, galactose, maltose, lactose, raffinose, dextran, mannitol, sorbitol, succinate, malate, or citrate were not supportive for the growth of explanted tissues and showed no effect on apogamy. Sucrose was more effective than glucose, which indicated that the osmotic values are not the controlling factor. Higher osmotic values alone inhibit growth and sporophyte formation as was shown in experiments with applied mannitol or sorbitol. In Doodia caudata, apogamy was induced by high irradiances (Duncan 1941). Also in Pteridium, an appropriate irradiance was required for apogamy (Whittier 1964). Many years ago, the involvement of light in fern apogamy was reported (Heim 1896, Lang 1898, 1929, Heilbronn 1910). Thus, we determined the light requirement for apogamy of E. arvense. Surprisingly, 100 % of apogamous shoot formation rate was obtained in darkness with the appropriate concentration of BA and sucrose although the mean number of shoots per tube at optimal concentration was relatively lower than that in light in the case of 5 x 10 -7 M and 1 x 10- 6 M BA. Illumination was not needed for the initiation of apogamous shoot buds of E. arvense. Both purine- and 4-PU-type cytokinins could induce shoot formation. Although the strong effect of 4-PU-type cytokinins on shoot formation was shown in culture of tobacco pith disk and callus (Isogai et al. 1976, Isogai and Kawachi 1980), apogamous shoots of E. arvense could not be produced at a higher rate than with purine-type cytokinins. Whittier (1966) investigated the influence of growth substances on the induction of apogamy in Pteridium gametophytes. The most significant difference of his work from ours is that, in Pteridium, cytokinin does not play an es-

sential role for apogamy, but NAA and GA are promotive. We examined the effect of NAA, 2,4-D, ABA, and GA on the apogamy of E. arvense but could not detect a synergistic effect with either substance. Although the mechanism of action is different among auxins, ABA, and GA, all of these growth regulators inhibited apogamy induction.

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