235

Short Communication Biotechnology, 16 (3), 235- 238 (1999)

Effects of Macro- COmponents and Sucrose in the Medium on in vitro Red- COlor Pigmentation ill Dionaea muscipula Ellis and spathulata Labill.

Sayuri ICHIISHl* Toshiharu NAGAMITSU*, Yusuke Tsukasa IWASHINA* * , KONDO* , Katsuhiko KONDO* * * ,t and Norikazu TAGASHIRA* * *

*Plant Bio- technology Section, Sanmei Electric Co., Ltd., 5- 7, Sumiyoshi- Cho. Handa City 475- 0862. Japan **Tsukuba Botanical Garden, National Science Museum, 1. City O005. 4- I- Amakubo, Tsukuba 305- Japan ***Laboratory of Plant Chromosome and Gene Stock, Faculty of Science, Hiroshima University, City 739-8526, 1- 4-3 Kagamiyama. Higashi-Hiroshima Japan E- mail :[email protected] u.ac.jp Received 21 september 1998; accepted 15 March 1999

Abstract

in (1/2 red Effects of the five macro - components and sucrose half strength MS MS) agar medium on in the plant bodies of muscipula spathulata - color pigmentation were studied Dionaea and Drosera generated from multiple shoots in vitro. In 1/2 MS agar medium modified with 10.31 mM NH4N03 and 9.40 mM KN03 and supplemented with 0.75 or O% sucrose the subcultured continuously proliferated by multiple shoots and generated large, green - colored plants, while with dilution of those nitrogen components and increase of sucrose to 1.5% the red - oolor anthocyanin pigmentation spread from the glands or glandular hairs to the entire leaves and the plant sizes and dry weight decreased in inverse proportion to the depth of red color. The anthocyanin pigments of Dionaea muscipula consisted glucoside (chrysan- of delphinidin 3- O- glucoside which was new to the species and cyanidin 3- O= themin), and those of Drosera spathulata, studied here for the first time, consisted of cyanidin 3,5 - di - galactoside (idaein), cyanidin glucoside, pelargonidin O- glucoside (cyanin), cyanidin 3- O- 3- O- 3- O- galactoside, and pelargonidin 3- O- glucoside (callistephin).

Dionaea muscipula Ellis and Drosera spathulata few species of Drosera other than D, spathulata Labill. in the are ornamental, carniv- (Gascoigne et al., 1948; Paris and Denis, 1957; orous plants (Kondo and Kondo, 1983). The plant Paris and Delaveau, 1959; Bienenfeld and Katz- bodies of the two species vary in coloration from meister, 1966; Bendz and Lindberg, 1968; Bendz green to red. However, plants with stable red color and Lindberg, 1970; Ayuga et al., 1985). are horticulturally more desirable than green ones. Dionaea muscipula and Drosera spathulata, The commercialized cultivars of 'Red Dragon', which are autotrophic plants, commonly occupy 'Red Giant', 'Red Purple', and 'Royal Red' of relatively closed ecosystems where the soil is poor Dionaea muscipula are artificially selected to bring in nutrient substances, wet and acid. They take in out a stable, deep red- color pigment in whole plant and absorb nutrients directly from small animal body when cultivated in vivo. resources by way of carnivorous leaves (Lloyd, Although anthocyanin pigmentation in leaves of a 1942). Darwin (1875) and some other workers few species of Dionaea and Drosera is a common (Kellermann and Raumer, 1878; Thum, 1988, 1989; phenomenon, it has been poorly characterized with Gibson, 1991) stated that the plants of some Dros- limited references; cyanidin -3 -glucoside in Dio- era species fed small animals artificially through naea muscipula (Di Gregorio and Di Palma, 1961; their leaves increased the number of flowers, total Jay and Lebreton, 1972), cyanidin-glycoside, mal- weight of seeds and vegetative organs. In contrast, vidin- glycosides, pelargonidin- glycoside, quercetin the plants of Dionaea muscipula and some Drosera galactoside, qercetin- digalactoside, in species fed mineral nutrients through their under- - 3- and 3- a ground roots in vivo grew poorly (Robert and t addressed. To whorn correspondence should be Oosting, 1958; Juniper et al., 1989). A tissue culture 236 of another , Utricularia praelonga color pigmentation in the inner surface of the trap St. Hil., (Idei and Kondo, 1998) showed different 10be in Dionaea muscipula and in the glandular hair organogenesis, micropropagation, growth forms, in Drosera spathulata and in the whole leaves of the and so on by adjusting KN03 concentrations be- both species after four months culture. However, well they plant In 1/2 tween 24.73 and 3 mM as as BAP (N6 - made growth worse. contrast, MS benzylaminopurine) concentrations in B5 (Gamborg media with more to complete macro -components et al., 1968) Iiquid medium. However, studies on promoted deeper green color in the whole plant the correlation between nutrients and anthocyanin bodies and larger growth and more proliferation in pigmentation in Dionaea miscipula and Drosera the both species. spathulata as well as other carnivorous plants are Moreover, the modified 1/2 MS media with no very much lacking. NH4N03 resulted in some red colored glands, glan- Seeds of Dionaea muscipula and Drosera spathu- dular tissues and sensitive hairs but green color in lata Kanto type collected in cultivation were sur- the other parts of the leaves in Dionaea muscipula face-sterilized with 0.1% (w/v) benzalkonium after four months culture and the modified 1/2 MS chloride solution for 5 min, 1% (w/v) sodium medium with no NH4N03 and no KN03 resulted red hypochlorite solution for 5 min, 70% (v/v) ethanol coloration in the inner surface of the trap lobe and for 30 s and were rinsed three times with sterile, relatively red color in the whole leaves and reduc- distilled water before they were sown on half tion of dry weight (Fig. 1). Thus, NH~N03 among strength of MS (1/2 MS; Murashige and Skoog, the macro -components of the 1/2 MS media could 1962) medium supplemented with O.8% sucrose. be the major nitrogen source for Dionaea muscipula They germinated 30 to 60 days after they were to giving thin red -color pigmentation and more sown. green color and to increasing plant growth. The Individual leaves of Dionaea muscipula used as natural habitat of Dionaea muscipula in North explants were planted on 1/2 MS supplernented with Carolina, U.S.A, has low contents of NH4' (2 1.5% sucrose. After 5 months they propagated an mg/Kg dry weight), P04 (less than 2 mg/Kg), K (2 average of 6plants per explant by adventitious buds mg/Kg) and Mg (1 mglKg) and no N03~, Ca and and multiple shoots. Each plant averaged 2 cm in Mn (Robert and Oosting, 1958). diameter, 0.12 ~ 0.04 g fresh weight, and 0.02 d:: Similarly, the lack of NH4N03 and KN03 among 0.00 g dry weight was used for the present experi- the macro -components of the 1/2 MS medium ment. deepened red- color pigmentation in glandular hairs Individual plants of Drosera spathulata averaging and the other parts of the leaves and reduced plant 2.5 cm in diameter each were used as explants. growth and dry weight in Drosera spathulata. On planted and subcultured at intervals of the other hand, the lack They were of NH4N03 and MgS0.1 ' 20 days on 1/2 MS supplemented with 1.5% su- 7H20 among the macro -components of the 1/2 MS crose. After 5 months they propagated numerous medium exhibited healthy -looking plant bodies plants per explant by multiple shoots. Plants aver- without any dead leaf but no plant growth perhaps aging 2.5 cm diameter, 0.14 ~: 0.03 g fresh weight, due to the balanced combination of N, P, K, and Ca. and 0.02 i 0.00 g dry weight were used for the In contrast, when the 1/2 MS medium lacked CaC12, present experiment. MgS04 ' 7H20 and KH2P04 the leaf and shoot tips All cultures were planted on 50 ml medium died perhaps due to an unbalanced combination of supplemented with no growth regulator at pH 5.5 in less Ca against more N and K. Thus, NH4N03 and cylindrical- shaped, culture vials 80 mm diameter X KN03 among the macro-components of the 1/2 MS 129.5 mm high, 450 ml capacity, air-tight with a media could be the major nitrogen sources for transparent, clear lid at 25 ~C under 3500 Iux contin- Drosera spathulata creating thin red- color pigmen- uous illumination. tation and more green color and increasing plant 0.2 g fresh weight of plant bodies, especially growth and even propagation. leaves, per sample was utilized to extract anthoc- The anthocyanin pigments of Dionaea muscipula yanin pigments with I ml MeOH -HCI mixture consisted of delphinidin 3-O -glucoside and cya- (methanol:hydrochloric acid=1000:1) for 3 h to nidin 3-0-glucoside (chrysanthemin), and those of overnight and filtrated by Toyopak ODS M (Tosoh) Drosera spathulata consisted of cyanidin 3,5- di- O- Maisyoridisc O.45 and H - 13-5 ,Im (Tosoh) pre - glucoside (cyanin), cyanidin 3-O-glucoside, pelar- cartridge. The composition of plant extracts was gonidin 3-O-glucoside (callistephin). Delphinidin the of (1996). determined by methods lwashina 3- O- glucoside was reported here in Dionaea musci- Modified 1/2 MS media with less or no macro pula for the first time, while the other one was components and with more sucrose induced red - already known in the species (Di Gregorio and Di I

l*~~ l F:ffecrs oT t.he mas:rL)-components (Nll,¥_ O;, KNaE, C~C]., lvlgh.S0.･7H2O ancl Kll2P0.1) iTl ~ hillf ~rol;vth and coloration uf clonal Etrength of )IS agar medium SLlpplemented T~~*1rh 1 ~:~;o s]J(~r(Ise f] n

Dion(2ea tn~i ~~ipstla and Drasen7 spath~!tatr, in ~.'itrr, after 4months tFeatment - A-It: Di~?sa.a ,,ts~~c~pulp> G_. L: Droseru spath#tlata, A= D, G ~tnd J: L~r~e-siz~ plants ~rown in the medium with the complete nlacro-cQmponents* IS, E, Il and K: TYTedium-si;,e planrs grown In the medium with 9 4C mM KNO: ~nd O 75m~,1 IYlgSO*･7H20. C, F, Iand L: Slnall-si2;e plants grown in the medium wi th no macro-coT1lponents Rars= Iem 237

0.3 i,4 0.25 0.8

07 v'= I.2 o Z5 -u o.2 c~'!)c~) >u~ 0,6 l cr) ~~ o.Z ~xc(~ ~ ~~E 0.5 ~ o. 15 .g) o '~'-o0.8 v";~ e' 0.4 0.1 5 ~ q,L~~ ~ ~v'Lo 0.6 J:, o. ~ ~'1 1 a' 0.3 ~ 0.1 0.4 o.Z v, J: 0.05 v, :L OOS = 0.2 CL s o. 1 c~ o) c;, a, * = cv :i o o o O co de ad abcd abcdee cde ce none bcde be acdeabcde none Macro-components Macro-components

in red color pigmentations (line graphs) Fig. 2 Effects of macro - components of 1/2 MS medium combination on and growth in dry weight (bar graphs) in Dionaea muscipula (A) and Drosera spathulata (B). e- e =delphinidin 3- galactoside. =cyanidin 3,5 di- glucoside. =cyanidin O- O- glucoside. I- I =cyanidin 3- O- V-V - O- * - * 3- JL =pelargonidin galactoside. ~F =pelargonidin glucoside. glucoside. A - 3- O- ~- 3- O- sec) at the wavelength of by Relative peak areas of the anthocyanins (absorption maxima ' were measured 510 nm b: 1.50 CaC12; d: 0.75 HPLC. Macro-component a: 10.31 mM NH4N03; 9.40 mM KN03; c: mM mM MgS04 ' 7H20 and e: O.62 KH2P04 ; mM .

Palma, 1961; Jay and Lebreton, 1972). All of the a root consumption of llutrients to survive, grow and anthocyanins found here in Drosera spathulata have propagate. The anthocyanin pigmentation in the two been reported in other species of Drosera (Gas- species may make it possible to be biosensitive to coigne et al., 1948; Paris and Denis, 1957; Paris and nitrogen consumption uptake. Delaveau, 1959; Bienenfeld and Katzmeister, 1966; Since this phenomenon is observed throughout Bendz and Lindberg, 1968, 1970; Ayuga et al., our tissue culture experience in Drosera adelae F. 1985). Muell., D. anglica Huds., D. binata Labill., D. Thus, dilution of NH4N03 and KN03 and increase burkeana Planch., D. capensis L., D, peltata Sm, ex of sucrose up to 1.5% mainly promoted and could Willd., D. petiolaris R. Br. ex DC., and D. rotun- be in relatively inverse proportion to depths of red- difolia L. in the Droseraceae (whole plant bodies), color anthocyanin pigmentation spread from glands Nepenthes mirabilis Druce in the Nepenthaceae especially peristomes), follic- or glandular hairs to entire leaves in the both species (pitchers Cephalotus (Fig. 2- A, B). Insects contain total nutrients of N ularis Labill. in the Cephalotaceae (whole pitchers), (99- 121 g/kg dry weight), P (6- 14.7 g/kg), K (1.5- and Sarracenia flava L, and S. purpurea L. in the 31.8 g/kg), Ca (22.5 g/kg) and Mg (0.94 glkg) Sarraceaniaceae, this methodology may be gener- (Reichle et al., 1969; Dixon et al., 1980; Watson et alized to the majority of the red -color pigmented aL, 1982) that are somewhat smilar to the medium carnivorous plants. requirements studied here. Prey would be more References attracted to and captured by red -colored plants of Drosera species than by green-colored ones. Gen- Adamec, L., 1997. Mineral nutrition of carnivorous plants: A erally, carnivorous plants might have adaptation review. Bot. Rev., 63: 273- 299. C., E., P., 1985. Contribucion al strategies to barren, wet and low pH soil conditions Ayuga, Carretero, Bermejo, estudie de flavonoides rotundlfolia (A by interaction between leaf carnivory and low root en Drosera the study of flavonoids in D_ consumption of nutrients (Adamec, 1997). The contribution to rotun- dlfolia). Ann. Real Acad Farm., 51: 321 - 326. present study suggests that the two species would Bendz, G., Lindberg, G., 1968. Naphthoquinones and antho- turn red color when they became deficient in nitro- cyanins from two Drosera species. Acta Chem. Scand., themselves attractive to gen compounds to make 22: 2722- 2723. prey and would catch more prey if they had too low 238

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