Plant Cell Reports (1984) 3:183-185

PlantCell Reports © Springer-Verlag 1984

C l o n a l p r o p a g a t i o n o f Stevia rebaudiana B e r t o n i by s t e m - t i p c u l t u r e Yukiyoshi Tamura 1 Shigeharu Nakamura 1 Hiroshi Fukui 2, and Mamoru Tabata 2 1 Research Laboratory, Maruzen Kasei Co., Ltd., Mukaihigashi, Onomichi 722, Japan 2 Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, J a p a n Received September 20, 1983/Revised version received July 13, 1984 - Communicated by J. Widholm

ABSTRACT

Clonal propagation of Stevia rebaudiana has been established by culturing stem-tips with a few leaf primordia on an agar medium supplemented with a high concentration (I0 mg/l) of kinetin. Anatomical examination has suggested that these multiple shoots originate from a number of adventitious buds formed on the margin of the leaf. Innumerable shoots can be obtained by repeating the cycle of multiple-shoot formation from a single stem-tip of Stevia. These shoots produce roots when transferred to a medium containing NAA (0.I mg/l) without kinetin. The regenerated plantlets can be transplanted %o soil.

INTRODUCTION

Stevia (Stevia rebaudiana Bertoni, Compositae), a p e r e n n i a l h e r b n a t i v e %o P a r a g u a y , i s known to a c c u m u l a t e sweet d i t e r p e n e g l u c o s i d e s such as s t e v i o s i d e and r e b a u d i o s i d e A m a i n l y i n t h e l e a v e s . Stevioside, which i s a p p r o x i m a t e l y 500 t i m e s a s sweet a s s u c r o s e , i s r e g a r d e d as a v a l u a b l e n a t u r a l s w e e t e n i n g a g e n t b e c a u s e of i t s r e l a t i v e l y good t a s t e and c h e m i c a l stability. With an increasing demand for stevioside in food industries, Stevia also has been cultivated in Asian countries. However, the Stevia plants propagated by seeds generally show a wide variation in the stevioside content (5-15~ of dry weight), as well as, in such morphological characters as the shape and color of leaves. Since Sievia is a highly self-incompatible plant, most of the variation probably is due to gene segregation. To avoid segregation and %o improve the yield of stevioside, it is necessary to propagate a genetically homogeneous population from a selected plant with desirable characters. Clonal propagation of Sievia in vitro may serve this purpose. There have been reports of the growth of Stevia in vitro. Callus tissue has been induced from Stevia leaf or stem explants (Handro et al. 1977), the detection of stigmasierol (Nabeta et al. 1976) and rutin (Suzuki et al. 1976) in callus cultures, and the formation of shoots (Yang and Chang 1979) and roots (Wada et al. 2981) from callus tissue has also been reported. However, the propagation of plants from callus tissue is not only slow, but may bring about frequent variability in the regenerated plants.

Yang e~ a l . lary shoots but only a propagated

(1981) have s u c c e e d e d i n o b t a i n i n g a x i l from t h e e x c i s e d stem nodes of S i e v i a , l i m i t e d number of c l o n a l p l a n t s c o u l d be from each node.

The aim of this study was to establish an effifor propagating Stevia plants with genetcient method ically uniform characteristics.

MATERIALS AND METHODS

Plant Material : A perennial clone of S. rebaudiana which had been cultivated for 6 years in the field at the Medicinal Plant Garden of Kyo%o University was used for culture experiments. Culture Method : The distal parts (I-5 cm long) of stems were surface-sterilized with 70~ ethanol for I0 see, followed by immersion in 1.5~ sodium hypochlorite solution for 15 min, and then were rinsed with sterile water three times. The stem-tips with 2 to 8 leaf primordia (0.2-2.0 mm long) were dissected out with a piece of razor blade under a binocular microscope in a elean bench, and each tip was placed on culture medium (15 ml) in a test tube (18 x 180 ~) capped with a Morton culture tube closure. Excised stem-tips were cultured on LinsmaierSkoog basal agar (i~) medium (LS) (Linsmaier and Skoog 1967) supplemented with 3% sucrose and various concentrations of plant growth regulators, i.e. lnaphthaleneacetic acid (~) and kinetin (Ky;r Nbenzyladenine (BA). The pH of the medium was adjusted to 6.0 with 1N Na0H before autoclaving. The cultures were incubated at 25°C and irradiated with light (5.0 klx) from cool white fluorescent lamps for 14 h per day. The multiple shoots that developed after ca 50 days of culture were transferred to the same medium (40 ml) in i00 ml Erlenmeyer flasks. About 30 days later the multiple shoots in the flasks were separated into 2-4 pieces at the base and subeultured on the same medium in i00 ml flasks to form numerous new shoots. To obtain normal growth, the multiple shoots were dissected and each shoot was transferred separately to LS medium containing a lower concentration (i mg/l) of K in I00 ml flasks. For root formation, these shoots were cultured on 1/4 strength LS medium (1% sucrose) supplemented with NAA (0.i mg/l) in I00 ml flasks. Rooted plantlets, which were taken out from the culture flasks and carefully washed with

184 Table l.

Relationship between stem-tip size and shoot formation in stem-tip cultures of Stevia a)

Size of stem-tip

(mm) 0.2

- 0.3

0.3

-

No. of

No. of

leaf

stem-tip

primordia

cultured

0

1.5

2 -

1.5 - 2.0

Frequency of shoot

f o r m a t i o n (%)b)

I0 4

6 - 8

0

30

80

(multiple

I0

90 (single shoots)

shoots)

C u l t u r e medium was LS medium + 10 mg/1 k i n e t i n + 3% s u c r o s e + 1% a g a r . C u l t u r e p e r i o d , 50 days ; Temperature, 25°C ; I l l u m i n a t i o n , 5.0 k l x , 14 h p e r day. P e r c e n t o f c u l t u r e d s t e m - t i p s t h a t formed s h o o t s .

a) b)

Table 2.

Effect of plant growth regulators on shoot formation in stem-tip cultures of Stevia a)

P l a n t growth r e g u l a t o r s added to t h e mediumb) No. of shoots formed per stem-tip

(mg/1) None

0

Kinetin

1

1 -

2

i-3

I0 Benzyladenine

3

2

0 - 4

lO

1

Kinetin

2

+

NAA

0.02

2

+

NiA

0.2

b)

~0 (multiple shoots)

1

Kinetin a)

2

- 5

0 - ~ (callus formation) 1 (callus formation)

Culture period, 50 days ; Temperature, 25oc ; Illumination, 5.0 klx, 14 h per day ; Number of replications, 10. Basal medium was LS medium + 3% sucrose + 1% agar.

water to remove agar, were transplanted to a mixture of vermiculite and sand (I:I) in pots and grown in the greenhouse.

RESULTS AND DISCUSSION

Relationship between stem-tip size and shoot formation: Table 1 shows the relationship between the initial size of the stem-tip explan%s and the frequency of shoot formation on LS medium supplemented with I0 mg/l K. The stem-tips without leaf primordium (less than 0.3 n~n in length) failed to grow and turned brown. However, 80% of the stem-tips with 2 to % leaf primordia (0.3-1.5 mm in length) developed multiple shoots. Ninety percent of the explants with 6 to 8 leaf primordia (1.5-2.0 mm in length) formed single shoots instead of multiple shoots. It seems as if the larger explants contain an unknown substance that inhibits the formation of multiple shoots as in the expression of apical dominance. There was no callus formed in these experiments. These results suggest that the ability to form multiple shoots is dependent on the size of excised stem-tip and/or the number of leaf primordia. Effect of cytokinin of shoot formation : To examine the effect of plant growth regulators on shoot format%on, stem-tips were cultured for 50 days on LS

medium supplemented with K or BA singly or in combination with NAA. As shoran in Table 2~ with the limited range of BA and K concentrations used the high level of 10 mg/l K ~as most stimulatory to shoot format%on while the low level of 1 mg/l BA was most stimulatory. The shoots that developed in the presence of BA were generally thicker and shorter than those produced in the presence of K as observed in apple by Lundergan and Janick (1980). Neither roots nor callus developed when stem-tips were grown on a medium supplemented with cytokinin only. The addition of NAA to K containing media did not increase shoot formation, but did induce callus formation. The culture of stem-tips having a few leaf primordia on a medium containing I0 mg/l K proved s u i t able for efficient vegetative propagation of Stevia, yielding 50-100 shoots from a single stem-tip in 80 days (Fig. i). Each one of these multiple shoots, when transplanted to a fresh medium containing i0 mg/] K, formed multiple shoots again. Since this asexual cycle of shoot multiplication can be repeated every 30 days~ it is estimated that more than one million shoots could be obtained from an original stem-tip within 12 months.

Development o f p l a n t l e t s : Shoots (1-10 mm l o n g ) which had b e e n m u l t i p l i e d on LS medium w i t h l0 mg/l K i n f l a s k s were s e p a r a t e l y t r a n s f e r r e d t o LS medium w i t h 1 m g / 1 K to promote growth f o r a p e r i o d o f 30 days. They were t h e n t r a n s f e r r e d t o LS medium w i t h 0.1 mg/1NAA where r o o t s d e v e l o p e d i n 30 days. A l l s h o o t s r o o t e d on t h e NAA-medium ( F i g . 2).

185 T w e n t y - f o u r p l a n t l e i s (3-5 cm h i g h ) were t a k e n from t h e f l a s k s , washed w i t h w a t e r to remove a g a r , and t h e n t r a n s p l a n t e d t o a m i x t u r e o f v e r m i c u l i t e and sand (1:1). The r a t e o f s u c c e s s f u l t r a n s p l a n t a t i o n t o s o i l was 85~. The p l a n t s grown i n t h e g r e e n h o u s e a p p e a r e d t o be normal and u n i f o r m i n m o r p h o l o g i c a l c h a r a c t e r s . C y t o l o g i c a l o b s e r v a t i o n o n %he r o o t - t i p c e l l s o f i0 r e g e n e r a t e d p l a n t s showed t h a i t h e y had t h e normal chromosome number (2n = 22). F u r t h e r s t u d i e s on t h e uniformity of various traits including the constitut i o n o f d i t e r p e n e g l u c o s i d e s a r e u n d e r way, u s i n g t h e c ] o n a l p l a n t s t r a n s p l a n t e d to t h e f i e l d . Processes of multiple-shoot formation : In conec%ion with multiple shoot formation in the stem-tip cultures, i% was found t h a t a number o f s h o o t s d e v e l o p e d from t h e margin o f a l e a f l e t (ca 5 mm long) t h a t had been c u t from t h e m u l t i p l e - s h o o t and s u b c u l t u r e d on LS medium with I0 mg/l K for ~0 days (Fig. 3). This observation s u g g e s t e d thai multiple-shoot formation was initiated in the leaflet in contact with a medium containing a high amount of K. These shoots are apparently adventitious, since no shoot formation was observed on the axil of leaf but along the margin of the leaf. Anatomy of the cultured leaflets showed that groups of small meris%ema%ic cells occur at various

sites of the epidermal or suhepidermal region to organize buds (Fig. ~), which will develop into shoot apices accompanied hy leaf primordia (Fig. 5). It is likely~ %herefore~ that the multiple shoots formed by the stem-tip cultures also originate from these buds.

Fig.

I

Multiple shoots developing from a single stemtip on LS medium supplemented wiih ]0 mg/l kinetin after 80 days of culture.

RE~NCES Handro W, Hell KG, Kerbauy GB (1979) Planta Medica

52:115-117. Linsmaier EM, Skoog F (1967) Physiol.

Yig. 2

Pig. 3

Fig.

Stevia planilets regenerated from a stem-tip culture on i/~ strength LS medium (l~ sucrose) supplemented with N~ (0.1 mg/1). Shoot formation from the margin of a leaf which was cut from the multiple-shoot and cultured on LS medium supplemented with I0 mg/l kine%in for ~0 days.

$ and 5

Section of a cultured leaflet on LS medium supplemented with i0 mg/l kinetin. Arrows indicate merisiematie regions.

Plant. 51:100-

127. Lundergan CA, J a n i c k J (1980) Hor%. Res. 20:19-2!÷. Nabeta K, Kasai T, Sugisawa H (1976) A g r . - - B i o l . Chem. 40:2105-2105. Suzuki H, I k e d a T, Ma±sumoto T, Noguchi M (1976) Agr. Biol. Chem. ~0:819-825. Wada Y, Tamura T, Kodama T, Yamaki T, Uehida Y (1981) Yukagaln~ 50:115-119. Yang YW, Chang WC (1979) Z. Pflanzenphysiol. 95:337-

5~5. Yang 7flg, Hsing liT, Chang WC (1981) Bot. Bull. Academia Sini ca 22:57-62.