1021-4437/03/5004- $25.00 © 2003

MAIK “Nauka

/Interperiodica”0527

Russian Journal of Plant Physiology, Vol. 50, No. 4, 2003, pp. 527–531. Translated from Fiziologiya Rastenii, Vol. 50, No. 4, 2003, pp. 593–597.Original Russian Text Copyright © 2003 by Kovaleva, Tikhomirov, Dolgushev.

INTRODUCTION

The prospects to control the processes of plantgrowth and the synthesis of secondary metabolites arecurrently considered mostly in the cell culture context[1, 2]. The specific features and interrelations of theseprocesses in the intact plants have not been studied suf-ficiently. Photocultured plants seem to present the bestmodel for approaching this goal [3, 4]. By manipulatingthe irradiation factor under the completely controlledenvironment, one can assess the characteristic patternsof growth and development as related to the biosynthe-sis of secondary metabolites [3]. Photoculture wouldprobably help maintain biodiversity and propagation ofthe valuable and endangered species of medicinalplants. Among the latter, we find golden root (

Rhodiolarosea

L.), with its natural habitat in the alpine and sub-alpine mountain belts. A phenolic glycoside salidrosideis the most attractive among secondary metabolites [5].

The effects of the rate and duration of artificial irra-diation on the growth and development of golden rootplants have not been studied in sufficient detail. Khein-talu [6] and Kuznetsova

et al.

[7] used the sources ofadditional irradiation when culturing golden root plantsin a glasshouse, however, the levels of PAR in theseexperiments, 15 to 30 W/m

2

, were low when comparedto 70–140 W/m

2

in the natural habitats of this plant spe-cies [8].

The present study aimed at the comparative analysisof

R. rosea

growth and development under photocul-ture, in plant natural habitat, and following introductionto field culture. In addition, we planned to relate thepatterns of salidroside synthesis to the rates of growthprocesses.

MATERIALS AND METHODS

To grow

Rhodiola rosea

plants under photocultureconditions, we used growth cabinets with controlledtemperature [4]. The level of PAR in our experimentswas 100

±

10 W/m

2

and corresponded to the averageirradiation level in the natural plant habitat [8]. Thelight source was a high-pressure xenon arch tubularlamp DKsTV-6000 with water-jacket cooling (Russia).To cut down the input of infrared irradiation, the lightsource was arranged over 35–50-mm-thick layer ofrunning water [4]. The photoperiod was 16 h.

To start culturing golden root plants under artificialconditions, we germinated the stratified seeds [9] onmoistened filter paper in petri dishes at 22–23

°

C. Metalboxes were filled with the layers of sand and keramsite(1 : 3) separated with a moisture-penetrable tissue.Seedlings were planted in the sand layer and transferredfrom the laboratory conditions into the photoculturecabinets.

For watering, plants were flooded every six hourswith the Knop solution plus micronutrients. Day/night

Specific Characteristics of

Rhodiola rosea

Growth and Development under the Photoculture Conditions

N. P. Kovaleva, A. A. Tikhomirov, and V. A. Dolgushev

Institute of Biophysics, Siberian Division, Russian Academy of Sciences, Akademgorodok, Krasnoyarsk, 660036 Russia;fax: 7 (3912) 43-3400; e-mail: ubflab@ibp.ru

Received December 7, 2001

Abstract

—Growth and development of

Rhodiola rosea

L. plants (the family Crassulaceae) were compared intheir natural habitat, field stands, and in photoculture. By the indices of growth and development, plants grownfor 135–137 days under the intensive photoculture were shown to exceed the 3-year-old plants developed in thenatural habitats and 1–1.5-year-old plants grown in the field stands. Under the photoculture, 35% of all theplants under study started flowering at the day 75–77 after seed germination. The content of salidroside in therhizomes of the 135–137-day-old plants was 0.4–0.6% per dry weight. Following photoculturing for 245 days,rhizome weight increased 4.5-fold as compared to the 135–137-day-old plants, and the salidroside concentra-tion reached 1.2%, the level corresponding to the maximum content of this glycoside in the plants growing intheir natural habitat and exceeding by 1.5–3 times the levels observed in the plants grown in the field stands.Under the photoculture conditions, plants of

R. rosea

were shown to grow without the dormancy period. Severalfactors apparently raised the salidroside concentration in the 245-day-old plants under the photoculture condi-tions, including enhanced growth, absence of the dormancy period and the period of lowered temperatures; asa whole, these factors promoted the detoxification, storage, and/or transport of the primary metabolic products.

Key words: Rhodiola rosea - plant growth and development - photoculture - salidroside

Abbreviation

: PAR—photosynthetically active radiation.

528

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et al

.

air temperature was 24

±

1/14

±

1

°

C. After first trueleaves appeared, the seedlings were pricked out intokeramsite (0.5–1.5-cm-fraction), with the nutrientacquisition area of about 80 cm

2

per plant.

Salidroside content was determined by the spectro-photometric method [10] in golden root rhizomes sam-pled from the plants grown under the photoculture con-ditions for 135 and 245 days.

The data in the table and text are means and theirstandard errors from two independent experiments,each in three replicates.

RESULTS AND DISCUSSION

To assess most objectively our data for golden rootplants under the photoculture conditions, we must com-pare them to the evidence reported by other researchersfor the natural plant habitats and field stands. Regretta-bly, when defining the development periods of

R. rosea

,the authors [11, 12] are of varied opinions, and there-fore we rely on the generally accepted classification ofphenotypical developmental stages put forward byRabotnov [13].

In the Table 1, we compared the indices of goldenroot growth and development in its natural habitat, thealpine belt of the Altai Mountains [11], the field standsin the Moscow oblast [12], and under the photocultureconditions.

In the latter case, the duration of the seedling phasewas 18–20 days, that is, by 7–22 days shorter thanunder the field conditions (Table 1). Within this phaseof golden root development, hypocotyls progressivelythickened. During the next interval of 15–17 days, thatis between 18–20 and 35–37 day following germinationunder the photoculture conditions, hypocotyls became0.4–0.5-cm thick, and plants developed a rosette of 6–8true leaves (the juvenile phase). Under the photocultureconditions, the first assimilating shoots developed on

days 35–37 after seed germination, that is, by 12–17days earlier than in the field stands (Table 1).

In the natural habitat, golden root plants produceone generation of assimilating shoots per vegetativeperiod, one–two generations in the field stands [11, 12],whereas in the photoculture, we observed the formationof the third and subsequent shoot generations. It fol-lows that in the latter case, golden root behaves as aconstantly growing plant. The timing of the develop-ment of the second- and third-generation shoots wasnot set definitely: under the photoculture conditions,the second-generation shoots developed 26–30 dayslater than the first-generation shoots, and the third-gen-eration shoots appeared 70–74 days later. The data pre-sented in Table 1 demonstrate that the 135–137-day-oldgolden root plants grown under the photoculture condi-tions are comparable to the 3-year-old plants in the nat-ural habitat and the 1–1.5-year-old plants in the fieldstands. When comparing these data, one should keep inmind that the duration of gold root vegetation wasabout 60 days under high-mountain conditions and150–160 days in the Moscow oblast.

By their morphological indices, the 135–137-day-old plants grown under the photoculture conditions leftbehind the 3-year-old plants developed in the high-mountain habitat and 1-year-old plants grown in thefield stand in Gorno-Altaisk (Table 2).

These observations are supported by the datareported by other authors [12] who introduced goldenroot plants in the Moscow oblast. The latter reportstated that in the first year of growth in the field stand,plants developed one–two and less frequently, three–four shoots, with their height under 4–15 cm, and by theend of this period, hypocotyls were 0.2–2.2-cm-thick.Under the photoculture conditions, two to four shootsdeveloped, as high as 26 cm, and the hypocotyls diam-eters of the 135–137-day-old plants were 2.2–2.8 cm.

Depending on the weather conditions, the shootgrowth rates were 0.18 to 0.37 cm/day in the second

Table 1.

Comparative characteristics of growth and development rates of

Rhodiola rosea

under various cultivation condi-tions

Developmental phase

Cultivation conditions

Altai high mountains [5, 11] Moscow oblast,field stands [12]

photoculture,an irradiance of 100 W/m

2

Seedlings

The first-year vegetative period up to 25–40 days up to 18–20 days

Juvenile phase

,formation of the leaf rosette

At the end of the first vegetative period or absent

from 25–40 daysup to 40–57 days

from 18–20 daysup to 35–37 days

Development of the first-generation shoots

Starting from the secondvegetative period

by the day 40–57 by the day 35–37

Development of the second-generation shoots

Starting from the thirdvegetative period

in August of the first growingperiod or May of the second growing period

by the day 63–65

Development of the third-generation shoots

Starting from the fourthvegetative period

in May or August of the second growing period

by the day 135–137

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GROWTH 529

year of the field cultivation and 0.33 to 0.46 cm/day inthe third year; this index was maintained in the subse-quent years of field cultivation [11]. Under the photoc-ulture conditions (PAR of 100 W/m

2

), this index was ashigh as 0.40–0.44 cm/day already in the first-genera-tion shoots.

It follows that by the duration of the developmentalphases and plant morphological and production indi-ces, the 135–137-day-old golden root plants grownunder the photoculture conditions are comparable tothe 1–1.5- and sometimes 2-year-old field-grownplants.

The height of plants grown in the field and photoc-ulture varied inconsiderably within 23.5 to 26 cm(Table 2). A dramatic 4.5-fold increase in the rhizomeweight in the 245-day-old plants as compared to the135–137-day-old plants was apparently caused by planttransit from the lag phase to the early exponentialgrowth. Numerous authors reported slow growth ofgolden root plants in the postembryonic period [5, 12].Even at the optimum photoculture conditions, the ini-tial growth is as slow as in the field (Table 1). Startingfrom the moment when the third-generation shootsdevelop, the rhizome formation considerably acceler-ates. In the field stands, the fresh weight of rhizomes inthe 3-year-old plants is already 52 g, and in the 4–5-year-old plants, 150 to 300 g [5]. To obtain such rhi-zomes under the photoculture conditions, we appar-ently need longer growing periods than in the presentstudy. However, taking into an account the growth pat-tern of golden root plants under the photoculture condi-tions, such period would be considerably shorter than inthe field stands and especially under the high-mountainconditions.

Under the high-mountain conditions, golden rootplants flower and bear fruit at the year 7 to 40, and mostoften, 12 to 20. In the field stand of the introducedplants of one and the same age, the number of plants inflower increased gradually, from single plants at theyear 1 to 53.3–86.7% at the year 5 [5, 12]. Under thephotoculture conditions, the first-generation shoot pro-duced flowers at day 75–77 after seed germination. Thenumber of plants in flower was 35%, and the periods of

flower buds, flowering, and fruit bearing were 7–9, 12–15, and 20–22 days.

The morphological pattern of golden root plantflowering observed in our experiments matched thedescription reported previously [12]. Following theartificial pollination, we obtained viable seeds. Theaverage duration of the whole seed-to-seed cycle was120 days. We were first to obtain

R. rosea

seeds underthe artificial growth conditions.

The photoperiodic demands of

R. rosea

are notknown. The photoperiodic conditions in the plant natu-ral habitat and at the site of its introduction are ratherdifferent, with the day length of 14–18 h within the veg-etative period. Under the photoculture conditions, thefirst- and second-generation shoots went to flowerunder the constant 16-h photoperiod. We therefore con-clude that

R. rosea

does not belong to the short-dayplant species.

The third-generation and subsequent shoots did notdevelop flowers under the photoculture conditions, andwe presume that to resume flowering, plants must beexposed to the period of lowered temperature charac-teristic of the dormancy period. In a special study of theeffect of lowered temperatures on the golden rootgrowth rhythms [16], plants were kept through theautumn and winter in a glasshouse and under the fieldconditions. The author also suggested that for their nor-mal growth and development, golden root plants mustbe exposed to a period of lowered temperatures; how-ever, this study did not account for the possible effectsof the light factor.

Table 3 lists the data on the contents of glycosidesalidroside, a biologically active component of

R. rosea

,in the rhizome. As compared to the natural habitats,plants in the field stands are more productive (Tables 1,2); however, the salidroside content in the latter case isdramatically lower than in the former. The salidrosidecontent in the 135–137-day-old plants under the photo-culture conditions was the same as in the 1–3-year-oldplants in the field stands (0.4–0.6%).

The salidroside content in the 245-day-old plantsreached 1.2%, the level corresponding to the maximumconcentration observed in the natural

R. rosea

habitats

Table 2.

Growth patterns in

Rhodiola rosea

plants grown under various cultivation conditions

Cultivation conditions Plant age Plant height, cm The numberof renewal buds Rhizome weight, g

Natural habitat[5, 14, 15]

One year – 2.1

±

0.04 0.1

±

0.01

Two year 9.7

±

0.42 2.7

±

0.12 0.4

±

0.02

Three years 17.6

±

0.73 4.7

±

0.34 1.6

±

0.05

Field stands [5] One year 8.0

±

0.21 7.0

±

0.25 1.2

±

0.04

Two year 24.3

±

1.51 25.0

±

1.59 11.0

±

0.52

Photoculture,PAR of 100 W/m

2

135–137 days 25.9

±

1.53 7.1

±

0.70 2.3

±

0.40

245 days 23.5

±

4.37 No data 10.5

±

2.70

530

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and exceeding the index characteristic of the field-grown plants by 1.5–3 times. Such biochemical plastic-ity of golden root plants in relation to the cultivationconditions apparently depends on both environmentalconditions and the changes in the pattern of plantgrowth and development. When the accumulation ofsecondary metabolites is viewed from the point of theirmultiple functions [17], one presumes that in a particu-lar plant species, various functions of the secondarymetabolites are manifested under diverse cultivationconditions. In the natural habitats, under adverse andrapidly changing climate conditions, the major functionof the secondary metabolites is to protect plants againstabiotic stress agents. When this plant species is intro-duced into milder climate conditions, the biologicalneed for high-level salidroside synthesis diminishes,and plants grow and develop at higher rates. We alsopresume that as the content of salidroside decreases, itsrole as growth inhibitor diminishes [18].

We also believe that the rapid promotion of plantgrowth and development under the photoculture condi-tions activates other functions of the secondary metab-olites, which some authors consider minor: detoxifica-tion of the primary metabolic products as well as depo-sition and/or transport of metabolites [17, 19]. Anincrease in the salidroside content in the 245-day-oldplant under the photoculture conditions could resultfrom enhanced growth and could be related to theabsence of the periods of dormancy and lowered tem-perature.

Summing up our experimental data, we concludethat growing of golden root plants under the photocul-ture conditions promotes their growth and developmentand also changes the pattern of secondary metabolitesyntheses. Thus, the synthesis of the phenolic glycosidesalidroside notably depended on plant growth function.Our evidence supports the hypothesis of multiple func-tions of plant secondary metabolites; they can also helpsolve the practical problems of maintaining and propa-gating

R. rosea

as an endangered species.

REFERENCES

1. Bavrina, T.V., Vorob’ev, A.S., Konstantinova, T.N., Ser-geeva, L.I., and Zal’tsman, O.O., Growth and EssentialOil Production in

Artemisia balchanorum In Vitro, Fiz-

iol. Rast.

(Moscow), 1994, vol. 41, pp. 903–906 (

Russ. J.Plant Physiol.

, Engl. Transl.).

2. Orlova, I.V., Nosov, A.M., Luksha, V.G., and Volo-din, V.V., Ecdysteroid Biosynthesis in Intact Plants andCell Cultures of

Rhaponticum carthamoides

Willd.(Iljin)),

Fiziol. Rast.

(Moscow), 1994, vol. 41, pp. 907–912 (

Russ. J. Plant Physiol.

, Engl. Transl.).

3. Protasova, N.N., Photoculture as an Approach to Revealthe Potential Productivity of Plants,

Fiziol. Rast.

(Mos-cow), 1987, vol. 34, pp. 812–822 (

Sov. Plant Physiol.

,Engl. Transl.).

4. Tikhomirov, A.A., Sharupich, V.N., and Lisovskii, G.M.,

Svetokul’tura rastenii: biofizicheskie i biotekhnologi-cheskie osnovy. Uchebnoe posobie dlya vuzov

(PlantPhotoculture: Biophysical and Biochemical Bases. Man-ual for Institutes), Novosibirsk: Sib. Otd. Ross. Akad.Nauk, 2000.

5. Saratikov, A.S. and Krasnov, E.A.,

Rodiola rozovaya

(

Rhodiola rosea

), Tomsk: Tomsk. Gos. Univ., 1987.

6. Kheintalu, A.M., Growing of

Rhodiola rosea, Mekhani-zats. Elektrifikat. Sel. Kh.

, 1986, no. 12, p. 56.

7. Kuznetsova, G.K., Geier, N.I., Golovkina, D.I.,Romanenko, V.I., Denisenkova, A.I., and Shain, S.S.,

Sostoyanie i perspektivy nauchnykh issledovanii pointroduktsii lekarstvennykh rastenii

(The State andTrends of Scientific Investigations on Medicinal PlantIntroduction), Moscow: Nauchn. Proisv. Otd. VILAR,1990.

8. Kultiasov, I.M.,

Ekologiya rastenii

(Plant Ecology),Moscow: Mosk. Gos. Univ., 1982.

9. Kovaleva, N.P., Tikhomirov, A.A., and Dolgushev, V.A.,The Pathways of Germination Increase of

Rhodiolarosea

Seeds in Photoculture,

Dokl. Ross. Akad. S.-kh.Nauk

, 1997, no. 6, pp. 16–17.

10.

Gosudarstvennaya farmakopeya SSSR. Obshchiemetody analiza. Lekarstvennoe rastitel’noe syr’e

(StatePharmacopoeia of USSR. The Standard Methods ofAnalyses. Resourses of Medicinal Plants), Moscow:Meditsina, 1990, no. 2.

11. Polozhii, A.V., Revyakina, N.V., Kim, E.F., and Sviri-dova, T.P.,

Rhodiola rosea

L.,

Biologiya rastenii Sibiri,nuzhdayushchikhsya v okhrane

(Biology of SiberianPlants Required the Protection), Sobolevskaya, K.A.,Ed., Novosibirsk: Nauka, 1985.

12. Nukhimovskii, E.L., Yurtseva, N.S., and Yurtsev, V.N.,Biomorphological Peculiarities of

Rhodiola rosea

L.Growing (Moscow oblast),

Rastit. Resur.

, 1977, vol. 13,pp. 489–450.

Table 3.

Salidroside contents in rhizomes of

Rhodiola rosea

plants grown under various cultivation conditions

Cultivation conditions Plant age Salidroside content,% of dry wt Plant age Salidroside content,

% of dry wt

Natural habitat [5] One to three years No data Perennial plants 0.8–1.2

Field stands [5, 12] One to three years 0.4–0.6 Perennial plants 0.4–0.8

Photoculture,PAR of 100 W/m

2

135–137 days 0.4–0.6 245 days 1.2

Note: Under the photoculture conditions the standard error was 10%.

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13. Dvorakovskii, M.S.,

Ekologiya rastenii

(Plant Ecology),Moscow: Vysshaya Shkola, 1983.

14. Kim, E.F., The Practice of

Rhodiola rosea

Growing inthe Altai Low-Mountains,

Rastit. Resur.

, 1976, vol. 12,pp. 583–590.

15. Kim, E.F., Ecological and Biological Properties of

Rhod-iola rosea

and Its Introduction in the Altai Foothills,

Izv.Sib. Otd. Akad. Nauk SSSR, Ser. Biol. Nauk

, 1983,no. 15, pp. 66–71.

16. Nukhimovskii, E.L., The Early Stages of

Rhodiola rosea

L., Morphogenesis Grown in Moscow Oblast,

Rastit.Resur.

, 1976, vol. 12, pp. 348–355.

17. Nosov, A.M., Functions of Plant Secondary Metabolites

In Vivo

and

In Vitro, Fiziol. Rast.

(Moscow), 1994,vol. 41, pp. 873–878 (

Russ. J. Plant Physiol.

, Engl.Transl.).

18. Muzafarov, E.N. and Ruzieva, R.Kh., Phenolic Com-pounds as a Factor of Photosynthesis and Growth Regu-lation,

Rost rastenii i ego regulyatsiya

(Plant Growth andIts Regulation), Kefeli, V.I. and Toma, S.I., Eds., Chi-sinau: Shtiintsa, 1985.

19. Lovkova, M.Ya.,

Biosintez i metabolizm alkaloidov vrasteniyakh

(Biosynthesis and Metabolism of Alkaloidsin Plants), Moscow: Nauka, 1981.