Plant Cell Reports

Plant Cell Reports (1988) 7:123-126

© Springer-Verlag1988

Production, maintenance and plant regeneration from cell suspension cultures of Stevia rebaudiana (Bert.) Bertoni C. M. Ferreira and Walter Handro Plant Tissue Culture Laboratory, Department of Botany, Institute of Biosciences, University of Sao Paulo, C.P. 11461, 05499 Sao Paulo, Brazil Received June 12, 1987 / Revised version received December 3, 1987 - Communicated by I. K.Vasil

ABSTRACT A method i s d e s c r i b e d f o r producing and m a i n t a i n i n g S t e v i a r e b a u d i a n a suspens i o n s and regenerat-i-6-n- o7 p l a n t s from c a l l i d e r i v e d from c e l l s u s p e n s i o n s . S u s p e n s i o n c u l t u r e s composed o f i s o l a t e d c e l l s (ca. 10%) and c e l l u l a r aggregates (5-100 cells) were o b t a i n e d i n 20-30 days by u s i n g f r i a b l e cal l u s as the i n i t i a l i n o c u l u m i n l i q u i d medi~ w i t h BA ( 0 . 5 m g / l ) + 2 , 4 - D ( I . 0 m g / l ) , and periodic filtering ( 1 0 0 - 5 0 0 ~m s i e v e s ) w i t h 6-7 days i n t e r v a l between s u b c u l t u r e s . Cult u r e s derived from a c t i v e l y growing calli are m a i n l y d i p l o i d (2n = 22) whereas those d e r i v e d from s e n e s c e n t c a l l i showed a wide variation i n chromosome number ( 5 5 - 2 0 0 ) Stock c e l l s u s p e n s i o n s w h i c h had been m a i n t a i n e d f o r 3 y e a r s were p l a t e d on basal LS agar medium w i t h BA ( 0 . 5 m g / l ) + 2,4D ( 0 . 5 m g / l ) to form c a l l u s . Calli originati n g from p r e d o m i n a n t l y 2n c e l l suspensions when t r a n s f e r r e d to medium w i t h K ( 2 . 0 mg/ I ) + NAA ( 0 . 0 2 m g / l ) were a b l e to form buds. Shoot e l o n g a t i o n and f u r t h e r r o o t i n g of i s o l a t e d s h o o t s was b e t t e r on LS medium devoid of growth regulators. Variation in rooting capacity, plant vigour, morphological c h a r a c t e r s and chromosome number was f o u n d amongst r e g e n e r a t e d p l a n t s . Abbreviations: BA - B e n z y l a m i n o p u r i n e ; 2 , 4 ~---- 2~4 Dichlorophenoxyacetic a c i d ; GA3Gibberellic a c i d ; IAA - I n d o l e a c e t i c acid; IBA - I n d o l e b u t y r i c acid; K - Kinetin; LS L i n s m a i e r & Skoog. INTRODUCTION Stevia rebaudiana (Compositae) a perennia-]--FF6-Fb~guay and S o u t h e r n Bra z i l has become e c o n o m i c a l l y v a l u a b l e as a s o u r c e o f n a t u r a l s w e e t e n e r s . The s w e e t e n i n g power o f i t s most i m p o r t a n t p r i n c i p l e , the s t e v i o s i d e , ranges from lO0 to 300 t i mes h i g h e r than s u c r o s e ( I s h i m a and Kataya ma 1976, Tanaka 1 9 8 2 ) . T h u s , commercialcultivation has become a t t r a c t i v e , and t h e r e i s a need f o r the p r o d u c t i o n o f homogeneous p o p u l a t i o n s composed o f improved genotypes. Unfortunately, the p r e s e n c e of a h i g h degree o f s e l f i n c o m p a t i b i l i t y in

Offprint requests to: W. Handro

S t e v i a r e s u l t s i n wide v a r i a t i o n in import a n t c h a r a c t e r s such as the g l y c o s i d e cont e n t i n l e a v e s (Sumida 1980, Nakamura and Tamura 1985) and i m p a i r s the e f f i c i e n c y of c o n v e n t i o n a l b r e e d i n g methods. R e s u l t s from t i s s u e c u l t u r e s t u d i e s of t h i s s p e c i e s i n d i c a t e t h a t t h i s technique may be a v i a b l e a l t e r n a t i v e to c o n v e n t i o n a l p r o p a g a t i o n and b r e e d i n g methods (Handro e t a l . 1977, Yang and Chang 1979, Yang e t a-T.-l-981, Czechowiak e t a l . 1984, T a m u r a - e-t a l . 1984a, b, Handro and F e r r e i r a 1986, Efya-gawa e t alo I ~ 6 , F e r r e i r a and Handro 1987a, b, as w e l l as f o r d i r e c t p r o d u c t i o n of s w e e t e n e r s i n v i t r o (Komatsu et a l . 1976, K o t a n i I~--80, Lee e t a l . 1982, Hsin9 et a l . 1 9 8 3 ) . However, the use o f t i s s u e and c e l l culture in Stevia for studying variability produced i n v--#Tt-Fo remains u n e x p l o r e d . Recent rep-6rts-s'-h-a-#e shown t h a t p l a n t p o p u l a t i o n s produced by d i r e c t o r g a n o g e n e s i s from s h o o t t i p s and l e a f e x p l a n t s are a p p a r e n t l y homogeneous (Tamura e t a l . 1984a, Miyagawa e t a l . 1986, F e r r e i r a and Handro 1987b) . TT~us~, the use of c a l l u s and c e l l s u s p e n s i o n s may be a d v a n t a g e o u s as an a l t e r n a t i v e to c o n v e n t i o n a l b r e e d i n g methods to e x p l o r e the n a t u r a l a n d / o r i n d u c e d v a r i a t i o n . These c u l t u r e s may a l s o be used to s e l e c t u s e f u l traits a t the c e l l u l a r l e v e l and to o b t a i n improved p l a n t s or s t a b l e c e l l l i n e s cap a b l e o f i n v i t r o p r o d u c t i o n o f the s w e e t e n i n g prin-c-ip--les. S u c c e s s f u l u t i l i z a t i o n o f t h i s system depends o r i m a r i l y on the rapid production of well dispersed cell suspensions with high growth rate which a l s o a l l o w the m a i n t e n a n c e o f g e n e t i c a l l y stable cells. In t h i s paper we d e s c r i b e procedures f o r the p r o d u c t i o n and m a i n t e n a n c e of suitable stocks of Stevia rebaudiana c e l l s u s p e n s i o n s as weT-T as s o m e basic i n f o r m a t i o n on t h e i r p h y s i o l o g i c a l and genetic features. The r e g e n e r a t i o n of p l a n t s from c e l l s u s p e n s i o n d e r i v e d c a l l i is also described. MATERIAL AND METHODS Cell suspension culture - Plants of Stevia r e b a u d i a n a o b t a i n e d by v e g e t a t i v e p r o p a g a -

124 t i o n from m a t e r i a l b r o u g h t from Paraguay in the e a r l y s i x t i e s and c u l t i v a t e d in the D e p a r t m e n t of Botany± I n s t i t u t e of B i o s c i e n ces, U n i v e r s i t y o f Sao P a u l o , were used in t h i s s t u d y . The b a s i c c u l t u r e medium was composed o f LS macro - and m i c r o - n u t r i e n t s ( L i n s m a i e r and Skoog 1 9 6 5 ) , v i t a m i n s ( N i t s c h 1 9 6 9 ) , 3% s u c r o s e , and pH a d j u s t e d to 5.5 before autoclaving. Two t y p e s of c a l l i were employed as i n i t i a l i n o c u l u m f o r c e l l suspen sion: a) f r i a b l e actively growing callus o b t a i n e d from f o l i a r d i s c s , c u l t u r e d on basi c LS medium w i t h BA ( 0 . 5 m g / l ) + 2 , 4 - D ( 0 . 5 m g / l ) + GA3 ( I . 0 m g / l ) ( F e r r e i r a and Handro 1 9 8 7 a ) ; and b) a w h i t e f r i a b l e call u s , o b t a i n e d from the above c a l l i i n the s e n e s c e n t s t a g e . The c a l l i were d i s p e r s e d i n l i q u i d medium and kept as 50 ml b a t c h suspen s i o n i n 125 ml e r l e n m e y e r f l a s k s on an o r b i t a l s h a k e r ( I 0 4 rpm) a t 26±2°C i n the l i g h t (5W.m - 2 , 1 6 h / d a y ) . Stock s u s p e n s i o n s were s u b c u l t u r e d e v e r y 6-7 days by t r a n s f e r r i n g I / 8 to I / 4 of the o r i g i n a l volume to empty f l a s k s and a d d i n g new medium to 50 ml. At each s u b c u l t u r e the s i z e o f c e l l u l a r aggregates was c o n t r o l l e d by f i l t r a t i o n in sieves o f known mesh (100-500 pm, s u p p l i e d by Bro~ z i n o x , Sao P a u l o ) , i n f i l t e r s specially d e s i g n e d f o r t h i s p u r p o s e . The assessment o f g r o w t h r a t e was made by c u l t u r i n g c e l l sus p e n s i o n s i n e r l e n m e y e r f l a s k s adapted for t h i s p u r p o s e , the t o t a l volume o f c e l l mass b e i n g r e c o r d e d e v e r y day. C a l l u s c u l t u r e and p l a n t r e g e n e r a t i o n - For plating, stock cell suspensions 3 years old, 2-3 days a f t e r s u b c u l t u r e , were f i l t e r e d in a I00 ~m s i e v e and mixed w i t h an equal v o l u me (ca. I0 ml) o f g e l l e d medium (0.8% a g a r ) a t 40°C and poured i n t o P e t r i d i s h e s (15 x I00 mm). Small c a l l i (ca. 2 mm i n d i a m e t e r ) , d e v e l o p e d a f t e r 2 weeks, were t r a n s f e r r e d to the same medium f o r g r o w t h . To i n d u c e buds fragments of these calli were s u b c u l t u r e d on media c o n t a i n i n g K ( 2 . 0 m g / l ) + NAA ( 0 . 0 2 m g / l ) and kept under l i g h t (14 W.m - L , 16h/ day, 2 6 ° C ) . C a l l i w i t h buds were t h e n t r a n s f e r r e d to media c o n t a i n i n g o n l y BA ( 0 . 0 2 mg/~ where bud f o r m a t i o n i s enhanced, and f u r t h e r to media w i t h o u t g r o w t h r e g u l a t o r s f o r s h o o t elongation. A f t e r I - 2 weeks the e l o n g a t e d s h o o t s (2-3 cm l e n g h t ) were i s o l a t e d and c u l t u r e d i n the same medium f o r rooting. When r o o t e d (ca. 2 weeks l a t e r ) plantlets are t r a n s f e r r e d to pots and kept f o r 2 weeks under humid c o n d i t i o n s a t a b o u t 25°C, 16 h light/day, before being transferred to the g r e e n h o u s e or to beds i n open a i r . Cytological o b s e r v a t i o n s - For chromosome c o u n t s c e l l s were p r e - t r e a t e d w i t h 0.1% aqueous c o l c h i c i n e f o r 6 h, f i x e d and s t o r e d i n 50% a c e t i c a c i d . C e l l s were h y d r o l i z e d i n 5N HCI a t 20-25°C f o r 20-30 m i n , squashed with a coverslip , and s t a i n e d w i t h 2% Giemsa ( p h o s p h a t e b u f f e r pH 6 . 8 ) . RESULTS AND DISCUSSION The f o l l o w i n g p r o c e d u r e s were f o u n d to be i m p o r t a n t f o r o b t a i n i n g s u i t a b l e c e l l s u s p e n s i o n s : a) use o f f r i a b l e callus as initial i n o c u l u m ; b) s e q u e n t i a l f i l t r a t i o n t h r o u g h 500-100 ~m s i e v e s p e r f o r m e d e v e r y 2-3 s u b c u l t u r e s ; c) 6-7 days i n t e r v a l between subcultures; d) and 2 , 4 - D ( I . 0 m g / l ) . The

d e v i c e used f o r f i l t e r i n g proved to be q u i te s u i t a b l e f o r work under s t e r i l e condi t i o n s and a l l o w s the s e l e c t i o n o f c e l l or aggregate populations differing by s i z e , by u s i n g e i t h e r the f i l t e r e d or the r e t a i n e d material, and c o m b i n i n g d i f f e r e n t sieves. By t h i s method s u s p e n s i o n c u l t u r e s composed o f i s o l a t e d c e l l s (10-20%) and small c e l l u l a r a g g r e g a t e s ( 5 - 5 0 c e l l s ) were o b t a i n e d i n 20-30 d a y s , w i t h d o u b l i n g time o f a p p r o x i m a t e l y 2-3 d a y s . C e l l s u s p e n s i o n s d e r i v e d from a c t i v e l y growing calli (Fig. I ) have d i s t i n c t f e a t u r e s as compared w i t h t h o s e d e r i v e d from s e n e s c e n t c a l l i ( F i g . 5). Cytological o b s e r v a t i o n s of the f o r m e r show ed the p r e d o m i n a n c e of c e l l s w i t h normal chromosome number (2n = 22) a f t e r 6 months i n c u l t u r e ( F i g . 2, 7 ) ; some m i t o t i c abnormalities such as l a g g i n g chromosomes and anaphase b r i d g e s were o b s e r v e d ( F i g . 3, 4 ) . In s u s p e n s i o n s d e r i v e d from s e n e s c e n t c a l l i , h o w e v e r , a wide v a r i a t i o n i n p l o i d y was o b s e r v e d and no d i p l o i d c e l l s were f o u n d ( F i g . 6, 7 ) . After plating, Calli developed in a p p r o x i m a t e l y 20 d a y s , and by t r a n s f e r to the same medium y e l l o w f r i a b l e calli were o b t a i n e d ( F i g . 8 ) . Fragments o f t h e s e c a l l i became green when s u b c u l t u r e d on medium c o n t a i n i n g K ( 2 . 0 m g / l ) + NAA ( 0 . 0 2 m g / ! ) . In c u l t u r e s d e r i v e d from p r e d o m i n a n t l y d i p l o i d c e l l s u s p e n s i o n s , compact dark green portio~were also present. Selection of t h e s e by s u c c e s s i v e i s o l a t i o n and s u b c u l t u r e (3-5 t i m e s on the same medium) r e s u l t ed i n bud n e o f o r m a t i o n ( F i g . 9 ) . I f this organogenetic callus is transferred to media c o n t a i n i n g BA ( 0 . 0 2 m g / l ) as the s o l e growth regulator, the number o f neoformed buds i n c r e a s e s . A f u r t h e r s u b c u l t u r e to basal medium d e v o i d o f g r o w t h r e g u l a t o r s was n e c e s s a r y to a c c e l e r a t e s h o o t d e v e l o p m e n t ( F i g . I 0 ) . I s o l a t e d s h o o t s showed a t e n d ency to form c a l l u s a t t h e base and f o r t h i s r e a s o n the r o o t i n g medium was d e v o i d of growth regulators. In t h i s r e s p e c t t h i s material differs from s h o o t s o r i g i n a t i n g by d i r e c t o r g a n o g e n e s i s from l e a f e x p l a n t s , w h i c h were f o u n d to r o o t b e t t e r i n media c o n t a i n i n g IBA ( F e r r e i r a and Handro 1 9 8 7 b ) . A preliminary s c r e e n i n g o f the r e g e n e r a t e d p l a n t s showed a wide variation in several characteristics such as l e a f and stem shape, c o l o r , pubescence, v i g o u r and development patterns of the stem apex (Fig. 11-14). This variation has n o t been seen to o c c u r i n p l a n t s o b t a i n e d e i t h e r by d i r e c t o r g a n o g e n e s i s or v e g e t a t i v e p r o p a g a t i o n (Tamura e t a l . 1984a, b, F e r r e i r a and Handro 1 9 8 7 b ) . Amongst the few r e g e n e r a t e d p l a n t s w i t h abnormal c h a r a c t e r i s t i c s in w h i c h chromosome number was r e c o r d e d , it was v e r i f i e d that tetraploids and a n e u p l o i d s ( F i g . 15) are f r e q u e n t . E f f o r t s are now underway to char acterize in detail the p l a n t s recovered from c e l l c u l t u r e s for morphological aspects, stevioside content, ploidy level , and m a i n t e n a n c e of their characteristics, a f t e r v e g e t a t i v e or s e x u a l r e p r o d u c t i o n . Acknowledgements - The a u t h o r s t h a n k FINEP ~Brasil) f o r a g r a n t ; C.M.F. t h a n k s CNPq (Brasil) for a scholarship.

125

Figure 1 Cell suspension derived from actively growing callus. F i g u r e 2 = C h r o m o s o m e s ~rum a diploid cell (2n=22). F i g u r e 3 , 4 - L a g g a r d s and a n a p h a s e b r i d g e s , observed in cell suspensions derived from actively growing callus. Figure 5 - Cell suspension derived from a senescent callus. Figure 6 - Chromosomes from a polyploid cell.

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F i g u r e 8 - F r i a b l e c a l l u s d e r i v e d from p l a t e d c e l l s . F i g u r e 9 - C a l l u s showing n e o - f o r m e d buds. F i g u r e I0 - E l o n g a t e d s h o o t s . F i g u r e 11-14 - C h a r a c t e r i s t i c s o f some r e g e n e r a t e d p l a n t s . F i g u re I I Normal m o r p h o l o g y . F i g u r e 12 - Abnormal m o r p h o l o g y w h i t h a p i c a l g r o w t h a r r e s t e d . F i g u re 13 - Normal f l o w e r i n g apex. F i g u r e 14 - Abnormal apex d e v e l o p m e n t . F i g u r e 15 - Chromosomes ( 4 n - I = 4 3 ) i n r o o t t i p o f the p l a n t shown i n F i g u r e 13. REFERENCES Czechowiak C, Dubois J, Vasseur J (1984) C. R. Seances Acad. S c i . Ser. I I I S c i . Vie 298: 173-176.

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