Original Paper Environ. Control in Biol., 42(3), 177-183, 2004

Water Status and Growth of Callus in Sturt's Desert Pea ( formosa) as Affected by Media Condition

Takashi IKEDA,1 Acram TAJI,2 Yukihiro FUJIME3 and Masaki NoGucHI4 1 National Agricultural Research Center for Western Region , Ayabe, Kyoto 623-0035, Japan 2 School of Rural Science and Agriculture , University of New England, Armidale, NSW, 2351, 3 Faculty of Agriculture, Kyoto Prefectural University, Kyoto 606-8522 , Japan4 National Institute of Vegetable and Tea Science, Taketoyo, Aichi 470-2351, Japan

(Received December 9, 2003)

This work was undertaken to investigate response of callus and the water status of Sturt's desert pea () in vitro under various environmental stress condi- tions. Water potential of culture media ranged from -0 .27 MPa to -1.05 MPa so that salt and osmotic stress and different concentration of sucrose could be applied to Sturt's desert pea calli grown in the tissue culture media. The percent increase in calli was highest in standard MS medium with 30 g L-1 sucrose but was not so when the water potential of medium was adjusted to the same water potential using standard MS plus mannitol . The water potential of callus tissue was almost the same as that of culture media at any range of MS and sucrose concentrations. Turgor of callus tissue did not alter significantly under any stress conditions. It is concluded that the optimum concentration of salt , sucrose and water status of medium could be estimated by this method and that standard MS concentra- tion and its water status are suitable for the formation of Sturt's desert pea calli .

Keywords : isopiestic psychrometer, mannitol , osmoregulation

INTRODUCTION

Sturt's desert pea (Swainsona formosa) a legume native to Australia and the of , is one of Australia's most admired wild flowers. It is a member of subfamily Papilionoideae and has a trailing habit with glaucous , pinnate leaves on stem spreading to 2 m. Its large flag shaped flowers, up to 7.5 cm long , are suspended on upright peduncles in clusters. The flowers have brilliant red wings and keel with a shiny black boss incorporated into the standard petal. Sturt's desert pea has great commercial potential as cut flower or as flowering pot . In vitro propagation of Sturt's desert pea has been achieved by callus culture generated from seedlings (McIntyre and Whitehorne, 1974) or direct shoot formation on hypocotyl and cotyledon segments (Taji and Williams, 1989) and via somatic embryogenesis (Tapingkae , 1997). However, in all these cases the productivity of cultures were small and that premature decline of cultures has been the cause of suspending the in vitro propagation of this as means of commercial clonal production. Thus, the objective of the work reported here was to investigate the optimum culture medium conditions for the development and further

Corresponding author : Takashi Ikeda, fax : +81-773-42-7161 , e-mail: [email protected]

Vol. 42, No. 3 (2004) (17)177 maintenance of Sturt's desert pea callus under various concentrations of MS (Murashige and Skoog, 1962) salts and addition of sucrose. It is important to note that physical conditions of tissue culture medium and its impact on callus induction and growth has not been investigated frequently in the past. A uniform water status of culture media for different concentrations of MS salt or sucrose, used in the experiments that follow, was established using mannitol. Methods of measuring the water status of tissue-cultured plants and its media, which may have wider application, are discussed.

MATERIALS AND METHODS

Plant materials and culture conditions. Seeds of Sturt's desert pea (Swainsona formosa) were surface sterilized by successive immersion in 70% ethanol for 30 s ; 1% (v v-1) sodium hypochlorite (NaOCI) solution for 10 min ; followed by two rinses in sterile water. Seeds were germinated aseptically on 50 % of MS salt containing 10 g L-1 sucrose and 8 g L-1 agar. After germination, the excised 5 mm hypocotyl segments were transferred to 100% MS medium containing 30 g L-1 sucrose, 8 g L-1 agar, 3 mg L-1 indole-3-acetic acid (IAA) and 3 mg L-1 benzylaminopurine (BA). Callus formed on this medium within 2 weeks. The induced callus was further subcultured onto the same medium in order to increase the quantity of callus tissues for further experiments. The first experiment was conducted using a range of MS salts from 10, 50, 100, 150, 200 or 300% concentrations all containing 8 g L-1 agar, 30 g L-i sucrose, 3 mg L-1 IAA and 3 mg L-1 BA. In the second experiment sucrose at 5, 30, 45, 60 or 90 g L-1 was used. The MS salt concentration used in all these media was set at 100%. All media contained 8 g L-1 agar, 3 mg L-i IAA, and 3 mg L-1 BA.

Experiments with mannitol were commenced after establishing the optimum MS and

sucrose concentration (100% and 30 g L-i respectively) for the calli. The Sturt's desert pea

calli were subcultured onto a medium containing 10% MS salt+95 mM D-mannitol (mannitol-

molecular weight 182.17) or 50% MS salt+44 mM mannitol. The water potential of both

media was -0.48 MPa, almost the same as that for 100% MS salt+0 mM mannitol. Then, a

medium containing 100% MS + 53 mM mannitol, with a similar water potential to 150% MS

salt without mannitol, was used. Agar, sucrose, IAA and BA concentrations were kept at 8

g L-1, 30 g L-1, 3 mg L-1 and 3 mg L-1, respectively, for all ranges of concentrations of MS salt and mannitol. Additionally, culture media containing 130 mM sucrose (45 g L-1) and 0 mM

mannitol, or 88 mM sucrose (30 g L-1) and 82 mM mannitol were used for callus growth. MS

salts, agar, IAA and BA concentrations were kept at 100%, 8 g L-1, 3 mg L-1 and 3 mg L-1,

respectively, for all concentrations of sucrose and mannitol. The pH of all the media was

adjusted to 5.8 using 1 N NaOH. Ten milliliter medium was dispensed into each test tube (22

mm the inner diameter and 100 mm height, TEST-F 25-100, Iwaki Glass Co., Ltd., Chiba,

Japan), and then autoclaved for 15 min at 103 kPa and 121•Ž.

Sturt's desert pea calli were cultured at 23•}1•Ž with 90 pmol m-2 s-1 of photosynthetical

ly active photon flux density and a 16-h photoperiod for 15 days. Sixteen to 20 cultures were

used for each treatment. Callus volume was estimated as a cone after measuring the height

and width of the callus with a ruler, and then the percent increase was calculated by dividing

the value at 0 day by that at 15 days after culture. Callus volume was also estimated by water

displacement methods. Both techniques resulted in similar values for callus volume measure-

ments, however, method one proved easier and as such this method was used in callus

measurements throughout this paper. Water status measurement with isopiestic psychrometers. The water status of tissues and culture medium was determined using isopiestic psychrometers (Model-3, Isopiestic

178(18) Environ. Control in Biol. Psychrometry, Ltd., DE, USA). The thermocouple chambers were first coated with melted

and resolidified petrolatum (Boyer, 1995) and then loaded with callus tissue or culture

medium. After water potential was measured, the osmotic potential of the tissue in the

thermocouple chamber was determined in the same tissue immediately after freezing at -30•Ž

and thawing at 25•Ž (Ehlig, 1962). The turgor was calculated by subtracting the osmotic

potential from the water potential. All callus tissue in an individual vessel was taken and loaded into a thermocouple chamber for one measurement of water status. Measurements of

water status were duplicated 3-8 times for each treatment. Statistical deviations in the water

status measurement with the psychrometer were evaluated by calculating the standard error .

RESULTS

Figure 1 shows that callus growth and its water status are affected by the concentration of sucrose and MS salt. The growth of callus is largest at -0.48 MPa obtained with 100% MS and 30 g L-1 sucrose (Fig. 1, C and F). Increasing or decreasing MS concentration reduces callus size (growth rate, Fig. 1C). Similarly increasing or decreasing sucrose concentration reduces callus growth (Fig. 1F). The water potentials of the callus tissue were similar to those of the medium (Fig. 1, A and D). Water and osmotic potentials of callus increased as concentration of MS or sugar concentration decreased (Fig. 1, A and D). There was no correlation between increase in callus size and the size of turgor (Fig . 1, B and C, E and F). In order to investigate the influence of water status of culture media and concentration of

Fig. 1 Water (•›) and osmotic (•œ) potentials of the callus tissue, and water potential of tissue culture medium (dashed line) (A, D), turgor (B, E) and callus increase (C, F) for Sturt's desert pea grown in tissue culture media having different concentrations of MS salt (A, B, C) or sucrose (D, E, F). Vertical bars indicate the standard error. Each point is the mean of at least 3 determinations.

Vol. 42, No. 3 (2004) (19)179 MS salt and sucrose on growth of Sturt's desert pea callus, mannitol in different amount for different concentration of MS was added to the media (so that the water potential of the media was the same as mannitol-free medium). Results show that the water potential of callus tissue was almost the same as that of the callus grown in mannitol-free medium. That is -0.49 MPa (Fig. 2A) and -0.60 MPa (Fig. 2D). The size of callus decreased as MS concentration decreased (Fig. 2, C and F). However turgor did not correspond to the increase when calli were grown under these conditions for either treatment (Fig. 2, B and E). The growth of Sturt's desert pea callus was not altered significantly whether or not the media contained mannitol (Fig. 3C).

DISCUSSION

In this study we demonstrated that the optimum concentration for growth of Sturt's desert pea callus is 100% MS (Fig. l C). For induction of rice (Chen, 1977) and red fescue (Torello et al., 1984) callus, 50% of MS salts were the optimum concentration. Based on these reports it appears that optimum concentration of MS salt for callus induction and growth is species specific. Retardation of growth in tissue-cultured grown plants exposed to high and low sucrose concentrations has been reported in several species both monocotyledons and dicotyledons

Fig. 2 Water (•›) and osmotic (•œ) potentials of the callus tissue, and water potential of tissue culture medium (dashed line) (A, D), turgor (B, E), and callus increase (C, F) for Sturt's desert pea grown in tissue culture media having -0.48 MPa (A-C) (100% MS salt+0 mM mannitol, 50% MS salt+ 44 mM mannitol and 10% MS salt+95 mM mannitol) and -0.60 MPa (D-F) (150% MS salt+0 mM mannitol, 100% MS+53 mM mannitol). Vertical bars indicate the standard error. Each

point is the mean of at least 3 determinations.

180(20) Environ. Control in Biol. Fig. 3 Water (•›) and osmotic (•œ) potentials of the callus tissue, and water potential of tissue culture medium (dashed line) (A), turgor (B), and callus increase (C) for Sturt's desert pea grown in tissue culture media having 100% MS salt+82 mM mannitol+88 mM (30 g L-1) sucrose or 100% MS salt+0 mM mannitol+ 130 mM (45 g L-1) sucrose. Vertical bars indicate the standard error. Each point is the mean of at least 3 determinations.

(e.g. tobacco, eggplant, maize and soybean) (Brown et al., 1979; Brown and Thorpe, 1980; Myers et al., 1992 ; Ikeda et al., 1999c, 2000). In this study, the growth of Sturt's desert pea calli was also inhibited in media with high sucrose concentration. Although, the media of 300% MS with 30 g L-1 sucrose and 100% MS with 90 g L-1 sucrose had almost the same water potential, the increase in Sturt's desert pea calli was different (Fig. 1, A, C, D and F). Why such a difference occurs? One explanation is that Sturt's desert pea calli has different sensitivity to salt stress versus that of high sucrose concentration. High salt concentration is injurious to plant cell. Excess salt may cause problems with membrane, enzyme inhibition, or general metabolic dysfunction. In particular high salt concentration can affect membrane fluidity (Nilsen and Orcutt, 1996). Such a damage could account for growth differences in Sturt's desert pea callus. In Fig. 1C we demonstrated that the optimum concentration of MS for Sturt's desert pea callus development was 100% and MS concentration was critical for regulation of callus development. However when MS concentration is set at 100% using mannitol (Fig. 2F) callus did not develop as well as those grown on 150% MS medium with no mannitol, eventhough the concentration of MS salt based on our findings from Fig. l C was optimum for callus growth. If the salt concentration has regulated callus development in Sturt's desert pea then better callus growth should have been achieved on 150% MS whether or not mannitol was added. In Fig. 2F, although the concentration of MS was set at 100%, the callus growth developed less than 150% of MS when mannitol was added. Also, this difference was not caused by osmotic stress because water status of culture media is the same (Fig. 2D). Mannitol is widely used as an osmoticum which induced osmotic stress in plants (Pritchard et al., 1990; Ranathunge et al., 2003). Generally speaking, mannitol can not be utilized by plants (Thompson et al., 1986; Ramage and Leung, 1996). Thus, it appears that callus growth in Sturt's desert pea may not be controlled only by the MS concentration. Now why

Vol. 42, No. 3 (2004) (21)181 MS of 100% concentration in Fig. 1C was the best? One possibility is that the water status of culture media is also important in controlling growth of Sturt's desert pea callus. In Fig. 1F, the optimum condition was also -0.48 MPa when concentration of sucrose was altered. Additionally, when concentration of sucrose was changed while MS concentration remained the same (100%) (Fig. 3A), growth of the callus on the medium with optimum sucrose condition showed no advantages (Fig. 3C), although the development of callus on 30 g L-1 sucrose concentration was better than 45 g L-1 of it (Fig. l F). This difference was not caused by osmotic stress because water status of culture media was almost the same (Fig. 3A). Thus, it may appear that callus growth in Sturt's desert pea was not only associated with sucrose and MS salt concentration but water status of the culture medium. Ikeda et al. (1999a, b) observed that the water potentials in calli of soybean or carnation plants were adapted to that of the culture medium, indicating that osmoregulation occurred. In the present study, the water potentials of Sturt's desert pea calli were always similar to those of the medium when the water potentials of media were modified by salt or sugar concentra tions or by addition of mannitol (Figs. lA, 1D, 2A and 3A). This is evidence of osmoregula tion in Sturt's desert pea callus for maintaining turgor under conditions of salt stress and nutrient deficiency, high and low concentration of carbon source or osmotic stress induced by mannitol. However, there were no correlation between the size of turgor and increase in size of callus under all the treatments in this study (Figs. 1B and l C, 1E and l F, 2B and 2C, 2E and 2F, 3B and 3C). Similarly in some other instances Meyer and Boyer (1972), Brown and Thorpe (1980) and Ikeda et al. (1999a, b, 2000), found that the size of turgor was unrelated to plant growth indicating that the sizes of turgor are not factors involved in callus growth in Sturt's desert pea. If plants are not grown under the optimum water status for plant growth, water stress may yield a significant growth difference in tissue-cultured plants. Thus, we suggest that measure ment of the water potential of tissue culture medium could provide information about the optimum condition for tissue-cultured plants. From the viewpoint of water relation in calli of Sturt's desert pea, the optimum condition was found in this study to be the standard MS concentration with 30 g L-1 sucrose and that the water status of such medium is suitable for the formation of Sturt's desert pea calli. Further investigations should aim at finding optimum conditions such as light intensity, culture temperature, C02 concentration, etc. in obtaining and maintaining sustained callus multiplication for subsequent plant regeneration in Sturt's desert pea.

We wish to thank Prof. Dr. R. R. Williams (University of , Gatton, Australia) for critical reading of our manuscript.

REFERENCES

Boyer, J. S. 1995. Measuring the Water Status of Plants and Soils. Academic Press, San Diego. Brown, D. C. W., Leung, D. W. M., Thorpe, T. A. 1979. Osmotic requirement for shoot formation in tobacco callus. Physiol. Plant. 46:36-41. Brown, D. C. W., Thorpe, T. A. 1980. Changes in water potential and its components during shoot formation in tobacco callus. Physiol. Plant. 49:83-87. Chen, C. H. 1977. In vitro development of plants from microspores of rice. In Vitro 13:484-489. Ehlig, C. F. 1962. Measurement of energy status of water in plants with a thermocouple psychrometer. Plant Physiol. 37:288-290. Ikeda, T., Iwaya-Inoue, M., Fukuyama, T., Nonami, H. 2000. Trehalose changes hydraulic conductance of tissue-cultured soybean embryos. Plant Biotechnol. 17:119-125.

182(22) Environ. Control in Biol. Ikeda, T., Nonami, H., Fukuyama, T., Hashimoto, Y. 1999a. Hydraulic contribution in cell elongation of tissue-cultured plants : growth retardation induced by osmotic and temperature stresses and addition of 2,4-dichlorophenoxyacetic acid and benzylaminopurine. Plant Cell Environ. 22:899-912. Ikeda, T., Nonami, H., Fukuyama, T., Hashimoto, Y. 1999b. Water potential associated with cell elongation and cell division of tissue-cultured carnation plants. Plant Biotechnol. 16: 115-121 (Erratum Plant Biotechnol. 16:251). Ikeda, T., Yakushiji, H., Oda, M., Taji, A., Imada S. 1999c. Growth dependence of ovaries of facultatively parthenocarpic eggplant in vitro on indole-3-acetic acid content. Sci. Hortic. 79:143- 150. McIntyre, D. K., Whitehorne, G. J. 1974. Tissue culture in the propagation of Australian plants. Proc. International Plant Propagators Soc. 24:262-265. Meyer, R. F., Boyer, J. S. 1972. Sensitivity of cell division and cell elongation to low water potentials in soybean hypocotyls. Planta 108:77-87. Murashige, T., Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497. Myers, P. N., Setter, T. L., Madison, J. T., Thompson, J. F. 1992. Endosperm cell division in maize kernels cultured at three levels of water potential. Plant Physiol. 99:1051-1056. Nilsen, E. T., Orcutt, D. M. 1996. Physiology of Plants under Salt Stress. Wiley and Sons, Inc., New York. Pritchard, J., Wyn Jones, R. G., Tomos, A. D. 1990. Measurement of yield threshold and cell wall extensibility of intact wheat roots under different ionic, osmotic and temperature treatments. J. Exp. Bot. 41:669-675. Ramage, C. M., Leung, W. M. 1996. Influence of BA and sucrose on the competence and determination of pepper (Capsicum annuum L. var. Sweet banana) hypocotyl cultures during shoot formation. Plant Cell Rep. 15:974-979. Ranathunge, K., Steudle, E., Lafitte, R. 2003. Control of water uptake by rice (Olyza sativa L.): role of the outer part of the root. Planta 217:193-205. Taji, A., Williams, R. R. 1989. In vitro propagation of formosus (Sturt's desert pea) an Australian native legume. Plant Cell Tissue Organ Cult. 16:61-66. Tapingkae, T. 1997. Light quality and quantity : their effects on in vitro growth and development of three Australian native plant species. MSc Thesis, University of New England, Australia. Thompson, M. R., Douglas, T. J., Obata-Sasamoto, H., Thorp, T. A. 1986. Mannitol metabolism in cultured plant cells. Physiol. Plant. 67:365-369. Torello, W. A., Symington, A. G., Rufner, R. 1984. Callus initiation, plant regeneration, and evidence of somatic embryogenesis in red fescue. Crop Sci. 24:1037-1040.

<和文抄録>

培 地 条 件 が ス タ ー ツデ ザ ー トピー(Swainsona formosa)カ ル ス の 水 分状 態 と増 殖 に与 え る影 響

池 田 敬1・ ア ク ラ ム タ ジ2・ 藤 目 幸 擴3・ 野口 正 樹4

1近 畿 中 国 四 国 農 業 研 究 セ ン タ ー 野 菜 部 ・2ニ ュ ー イ ン グ ラ ン ド大 学 ・3京都 府 立 大 学 農 学 部 ・4野菜

茶 業 研 究 所 果 菜 研 究 部

ス タ ー ツ デ ザ ー トピ ー(Swainsona formosa)カ ル ス を塩 お よ び糖 濃 度,ま た 浸 透 圧 の 異 な る培 地 で 培 養 し,水 分 状 態 と増 殖 の 関 係 に つ い て 研 究 した.カ ル ス を 水 ポ テ ン シ ャ ル が-0.27MPaか ら-1.05MPaの 培 地 で 培 養 し た.カ ル ス 増 殖 率 は,標 準 濃 度 のMS塩 と30gL-1シ ョ糖 で 最 も高 か っ た が,マ ン ニ トー ル 添 加 に よ り培 地 の 水 分 状 態 を 同 じ 条 件 に し た 培 地 で は 低 く な っ た.こ の こ と か ら,ス タ ー ツ デ ザ ー トピ ー カ ル ス 増 殖 に は培 地 の塩,糖 濃 度 の み で な く,水 分 状 態 も重 要 な 要 因 で あ る こ とが わ か っ た.

Vol.42,No.3(2004) (23)183