PlantCell Reports

Plant Cell Reports (1985) 4:348-350

© Springer-Verlag 1985

Isolation of protoplasts and regeneration of callus from suspension cultures of cultivated beets S. R. Bhat, B. V. Ford-Lloyd, and J. A. Callow Department of Plant Biology, University of Birmingham, P.O. Box 363, Birmingham B15 2T1~, UK Received August 19, 1985 / Revised version received November 8, 1985 - Communicated by B. L. Miflin

ABSTRACT C o n d i t i o n s necessary f o r the isolation and c u l t u r e o f p r o t o p l a s t s from suspension cultures of sugar, f o d d e r and garden b e e t s were i n v e s t i g a t e d . Good y i e l d s o f p r o t o p l a s t s were o b t a i n e d by t r e a t i n g c e l l s w i t h a m i x t u r e o~ c e l l u l a s e , Macerozyme and D r i s e l a s e enzymes. Nutritional requirements of beet p r o t o p l a s t s were found t o be q u i t e simple: protoplasts c o u l d be c u l t u r e d i n MS, B5 o r PGo based media w i t h 0 . 4 M g l u c o s e w i t h the optimum r e s u l t b e i n g produced on KM8p medium. P l a t i n g e f f i c i e n c y (P.E) was g e n o t y p e - d e p e n dent w i t h t h e sugar b e e t g i v i n g b e t t e r P.E. than the f o d d e r o r garden b e e t s used, and h i g h e r v a l u e s b e i n g a c h i e v e d w i t h t h e use o f d e s a l t e d D r i s e l a s e f o r i s o l a t i o n f o l l o w e d by c u l t u r e on KM8p medium. Abbreviations : 2,4-D - 2 , 4 - d i c h l o r o p h e n o × y acetic acid ; BAP - N~ b e n z y l a m i n o p u r i n e ; NAA - n a p h t h a l e n e a c e t i c a c i d ; P.E. - p l a t i n g efficiency ; ~ - University of Birmingham b e e t germplasm a c c e s s i o n number.

INTRODUCTION (Beta vulgaris L.) is the most Beet i m p o r t a n t economic p l a n t o f t h e f a m i l y Chenop o d i a c e a e . Both d i p l o i d and p o l y p l o i d h y b r i d v a r i e t i e s a r e bred and c u l t i v a t e d . Beet b r e e ding can g r e a t l y benefit from protoplast technology especially in the p r o d u c t i o n of n o v e l c y b r i d s and t r a n s f e r o f genes from w i l d species f o r d i s e a s e and p e s t resistance, Considering the a g r i c u l t u r a l importance of beet, literature on t i s s u e c u l t u r e i s very limited, even t o t h e e x t e n t t h a t very few o t h e r members o f t h i s f a m i l y have been c u l t i v a t e d in v i t r o , P r e v i o u s r e p o r t s (Smolenskaya and R a l d u gina, 1981~ Szabados and Gaggero, 1985) have d e m o n s t r a t e d t h e c u l t u r e o f suspension c e l l derived protoplasts of sugar b e e t t o the c a l l u s s t a g e o f s e l e c t e d g e n o t y p e s . However, a comparative s t u d y of the usefulness of different enzymes and media f o r m u l a t i o n s on the i s o l a t i o n and c u l t u r e o f p r o t o p l a s t s in

Offprintreques~to:B.V. Ford-Lloyd

d i v e r s e beet genotype sources i s not yet available. Here we d e s c r i b e t h e conditions suitable for t h e i s o l a t i o n and c u l t u r e of protoplasts from suspension c u l t u r e s o÷ a range o f sugar b e e t , garden b e e t and f o d d e r beet genotypes.

M A T E R I A L S AND METHODS

1. E s t a b l i s h m e n t o f

suspension c u l t u r e

Suspension c u l t u r e o f sugar beet cv. Bush M o n o (B382)* was e s t a b l i s h e d from friable callus d e r i v e d from h y p o c o t y l e×plants c u l t u r e d on PGo medium (De Greef and aacobs, 1979) supplemented w i t h 0 . 2 mg 1 -~ each o f 2 , 4 - D and BAP. Garden b e e t c v . P i a t t a d'Egitto (B799) * and f o d d e r b e e t cv. White Knight (B1064) ~ suspension c u l t u r e s were initiated from f r i a b l e c a l l u s o b t a i n e d from shoot cultures growing on MS medium (Nuras h i g e and Skoog, 1 9 6 2 ) c o n t a i n i n g 0.25 mg 1- I BAP. Suspension c u l t u r e s were grown i n 250 ml Erlenmeyer f l a s k s c o n t a i n i n g 50 ml medium and were k e p t on a o r b i t a l shaker a t 70 rpm i n the d a r k a t 26° C. A l l t h e suspension c u l tures were m a i n t a i n e d i n PGo medium w i t h 0.1 mg l - j each o f 2 , 4 - D and BAP w i t h s u b - c u l t u r e e v e r y 7 days. 2.

Protoplast isolation

Cells from 4 o r 5 day o l d suspension cultures were c o l l e c t e d and p r e - p l a s m o l y s e d in CPW 13M medium (Frearson e t al, 1973). After 1 h t h i s medium was r e p l a c e d by an enzyme m i x t u r e c o n t a i n i n g c e l l u l a s e Onozuka RIO, Macerozyme RIO ( Y a k u l t Pharmaceutical I n d u s t r y Co. Ltd., Japan) and D r i s e l a s e o r pectinase (Sigma Chemical Company) p r e p a r e d in CPW 13M ( t a b l e 1). Both commercial and d e s a l t e d D r i s e l a s e were employed. Desalting was a c h i e v e d by p a s s i n g t h e enzyme s o l u t i o n t h r o u g h a B i D - g e l P6 column (Evans and B r a v o , 1984). Cells were i n c u b a t e d i n t h e r e l e v a n t enzyme m i x t u r e f o r 12-14 h on a shaker (40-50 rpm) a t 18~'C a t t h e end o f which t h e c e l l and protoplast m i x t u r e was passed t h r o u g h two l a y e r s of m u s l i n and then t h r o u g h a nylon mesh (40 Hm). P r o t o p l a s t s and c e l l s were

pelleted

by

centrifugation

at

100 g

for

5

349 min. P r o t o p l a s t s were washed t h r e e t i m e s in CPW 13M t o remove t h e enzymes and f l o a t e d on CPW 21S. F i n a l l y p r o t o p l a s t s were washed once i n c u l t u r e medium and resuspended i n I ml of f r e s h c u l t u r e medium. P r o t o p l a s t d e n s i t y was d e t e r m i n e d u s i n g a haemocytometer. V i a b i l i t y and presence o f c e l l w a l l were checked by staining w i t h f l u o r e s c e i n d i a c e t a t e and C a l c o f l u o r White, r e s p e c t i v e l y .

3.

Protoplast culture

P r o t o p l a s t s were c u l t u r e d i n 100-200 ul d r o p s a t d i f f e r e n t d e n s i t i e s t Le. 10=, 104 , 5x104, I 0 ~, 2 . 5 x I 0 ~ and 10a p r o t o p l a s t s ml --~. Culture media c o n t a i n e d B~ (Gamborg e t al, or MS f o r m u l a t i o n s a t half 1968), PGo strength, with glucose (0.3, 0 . 4 , o r 0 . 5 M) and s u c r o s e a t 15 g l -~ Casein h y d r o l y s a t e (250 mg I - * ) and y e a s t e x t r a c t (200 mg I - ' ) were a l s o added t o MS and PGo based media. Growth r e g u l a t o r s 2 , 4 - D and BAP were added i n different combinations. Additionally, KM8p medium was used (Kao and M i c h a y l u k , 1975) without casamino a c i d s b u t c o n t a i n i n g 1.0 mg 1 -j NAA, 2,4-D and BAP at 0.2+0.25, 0 . 4 5 + 0 . 2 5 , 0 . 7 0 + 0 . 5 and 1 . 2 + 0 . 5 mg 1-~. P r o t o p l a s t s were c u l t u r e d i n t h e d a r k a t 2b°C. In a l l c a s e s , gradual d i l u t i o n of the osmoticum was a c h i e v e d by t h e addition of f r e s h medium (25-50 HI) w i t h l o w e r osmoticum, o v e r a p e r i o d o f 21 days. Plating efficiency was e x p r e s s e d as t h e p e r c e n t a g e o f protoplasts which d e v e l o p e d t o form c e l l c o l o n i e s a f t e r 21 days o f c u l t u r e .

ed, glucose at 0.3 M r e s u l t e d in extensive budding, giving rise to enlarged, highly i r r e g u l a r l y shaped c e l l s . At 0 . 4 M and 0 . 5 M g l u c o s e l e v e l s good r e g e n e r a t i o n o f c e l l w a l l was o b t a i n e d w i t h resultant regular cell shape. However, a few c e l l s s t i l l showed budding. P r o t o p l a s t s c u l t u r e d i n B5, PGo o r MS based media a t densities below 105 m l - * failed t o show s u s t a i n e d d i v i s i o n s w h i l e in KM8p medium c o l o n y f o r m a t i o n was o b t a i n e d a t d e n s i t i e s o f 104 o r 5.10 ~ ml - j . Among t h e v a r i o u s combinations of the hormones t e s t e d , t h e optimum 2 , 4 - D and BAP c o n c e n t r a t i o n s were found t o be 1.0 and 0 . 5 mg I -'I r e s p e c t i v e l y f o r P8o, MS and 85 based media. At t h i s c o n c e n t r a t i o n about 11% o f t h e c e l l s showed m i t o t i c a c t i v i t y . Densely c y t o plasmlc c e l l s underwent r a p i d mitotic divi s i o n t o g i v e r i s e t o c e l l c o l o n i e s w i t h i n 10 days w h i l e h i g h l y v a c u o l a t e d c e l l s remained quiescent or entered i n t o d i v i s i o n very slowly. With KM8p medium optimum hormone concentrations were (mg 1- I ) 1.0 NAA p l u s 0 . 2 to 0 . 5 2,4-D and 0.25 BAP. The p l a t i n g e f f i c i e n c y o f p r o t o p l a s t s o f d i f f e r e n t g e n o t y p e s as i n f l u e n c e d by c u l t u r e media and i s o l a t i o n c o n d i t i o n s a r e p r e s e n t e d i n t a b l e 2. P.E. was found t o be dependent upon g e n o t y p e , media and t h e enzymes used i n protoplast isolation. The h i g h e s t P.E. was o b t a i n e d i n g e n o t y p e s o u r c e B382, w h i l e KMSp medium was s u p e r i o r t o o t h e r media t e s t e d . B5 and PGo based media were n e a r l y e q u a l i n P.E. f o r B382. Use o f d e s a l t e d D r i s e l a s e f o r p r o toplast i s o l a t i o n r e s u l t e d i n improved P.E. in sources B382 and B1064 w h i l e i n 8799 it was s l i g h t l y l o w e r .

RESULTS Four different enzyme m i x t u r e s were assessed f o r p r o t o p l a s t i s o l a t i o n from the suspension-cultured cells. Release o f p r o t o plasts was q u i t e s a t i s f a c t o r y i n a l l enzyme mixtures tested (table I). A p p r o x i m a t e l y lOfold loss o f p r o t o p l a s t s was q u i t e common d u r i n g washing. Viability of i s o l a t e d p r o t o plasts was a p p r o x i m a t e l y 80% i n all enzyme mixtures. The enzyme m i x t u r e c o n t a i n i n g 2% cellulase, 1% Macerozyme and 0.5% commercial D r i s e l a s e or 0.05% d e s a l t e d D r i s e l a s e was used i n subsequent i n v e s t i g a t i o n s . Protoplasts cultured in l i q u i d medium lost t h e i r s p h e r i c a l shape w i t h i n 24 h and c o m p l e t e r e g e n e r a t i o n o f c e l l w a l l s was o b s e r v e d i n 80% o f t h e v i a b l e p r o t o p l a s t s after 96 h. Of t h e t h r e e l e v e l s o f osmoticum t e s t -

Table 1. E f f e c t o f enzyme c o n c e n t r a t i o n (per cent w/v) on y i e l d of protoplasts from suspension c u l t u r e d c e l l s . E1

E2

E3

E4

C e l l u l a s e Onozuka RIO

2.0

2.0

2.0

2.0

Macerozyme RIO

1.0

1.0

1.0

1.0

D r i s e l a s e (Sigma)

0.1

0.5

1.0

Enzyme

P e c t i n a s e (Sigma) Y i e l d of p r o t o p l a s t s per g of c e l l s ( a f t e r (xlO 5) ~ashing)

-

1.0

-

-

b.O

5.4

-

1.0

3.4

T a b l e 2. P l a t i n g e f f i c i e n c y o f p r o t o p l a s t s o f b e e t i n d i f f e r e n t c u l t u r e media. 8enotype Source

B5

Medium PGO MS

8382

1.8

1.7b

8799

-

B1064

-

KM8p (a)

(b)

2.38

-

7.30

-

1.96

-

1.12

-

0.40

0.78

3.50

(a) - Commercial D r i s e l a s e (b) - D e s a l t e d D r i s e l a s e

DISCUSSION In t h e c u l t u r e and m a n i p u l a t i o n o f p l a n t protoplasts, c o n s i s t e n t h i g h y i e l d s a r e important. These were o b t a i n e d i n t h e present s t u d y by t r e a t i n g r a p i d l y g r o w i n g suspension c u l t u r e d c e l l s w i t h a m i x t u r e of c o m m e r c i a l l y available c e l l w a l l d i g e s t i n g enzymes. However, commercial enzymes, e s p e c i a l l y D r i s e l ase a r e known t o c o n t a i n impurities which adversely affect the protoplasts during iso1981; Santos e t a l , lation (Patnaik et al, 1980). T h i s was found t o be t h e case i n the present investigation where i n c r e a s e d P.E. was o b t a i n e d when d e s a l t e d D r i s e l a s e was used for protoplast isolation. P r o t o p l a s t membranes a r e l e a k y r e s u l t i n g i n l e a c h i n g of c y t o p l a s m i c c o n t e n t s i n t o the medium (Evans and C o c k i n g , 1977). Therefore nutritional and hormonal r e q u i r e m e n t s of p r o t o p l a s t s o f t e n d i f f e r from t h o s e o f their

350 previous c e l l s t a t e . N u t r i t i o n a l requirements of b e e t p r o t o p l a s t s of different genotype sources used were found t o be q u i t e s i m p l e . Popular media f o r m u l a t i o n s such as Bs, PGo and MS w i t h 0 . 4 M g l u c o s e as t h e osmoticum and (mg I - * ) 1.0 2,4-D and 0 . 5 BAP as h o r mones were s u f f i c i e n t t o o b t a i n s o m e c e l l c o l o n i e s . However, w i t h t h e s e media a minimum of 102 p r o t o p l a s t s m l - " were necessary t o obtain sustained d i v i s i o n s . Previous r e p o r t s have a l s o found t h e same o p t i m a l d e n s i t y f o r t h e c u l t u r e o f sugar b e e t suspension p r o t o p l a s t s (Smolenskaya and R a l d u g i n a 1981; Szabados and Gaggero 1983,1985). In c o n t r a s t t o t h e Szabados and Gaggero o b s e r v a t i o n (1985), we found no p r o m o t i v e e f f e c t of c a s e i n h y d r o lysate, yeast e x t r a c t or c o n d i t i o n e d medium on p l a t i n g e f f i c i e n c y . The hormone-autonomous c e l l suspension o f sugar b e e t used by Szabados and Gaggero (1985) i s p r o b a b l y p h y s i o l ogically d i f f e r e n t from suspension c u l t u r e s used i n t h e p r e s e n t s t u d y which may e x p l a i n the d i f f e r e n c e s in r e s u l t s . G e n e r a l l y however, we found t h a t when n u t r i t i o n a l l y r i c h KM8p m e d i u m was used, minimum p r o t o p l a s t d e n s i t y f o r o b t a i n i n g c e l l c o l o n i e s was r e duced t o below 105 ml - ~ , and a l s o a h i g h e r P.E. was a c h i e v e d . As w e l l as b e i n g a f f e c t e d by medium, t h e P.E. of b e e t p r o t o p l a s t s was found t o be genotype dependent. P r e v i o u s r e p o r t s have each o n l y d e a l t w i t h one g e n o t y p e . Szabados and 8aggero (1985) r e p o r t e d up t o 35% P.E. The P.E. in the present study i s the actual number o f p r o t o p l a s t s t h a t developed t o form c o l o n i e s , whereas Szabados and 8aggero (1985) counted any c e l l which showed a t least one mitotic d i v i s i o n by t h e e i g t h day f o r e s t i m a t i n g P.E. In t h e p r e s e n t s t u d y , although initially about 10% of c e l l s showed m i t o t i c activity in a l l genotypes, the a c t u a l P.E. was d i f f e r e n t f o r each g e n o t y p e . From t h e present study, we can c o n c l u d e t h a t despite g e n o t y p i c a l l y dependent v a r i a t i o n i n p l a t i n g efficiency, t h e t e c h n i q u e of p r o t o p l a s t c u l t u r e from suspension c u l t u r e d c e l l s o f beet can be a p p l i e d o v e r a range o f genotypes. A l t h o u g h c a l l u s d e r i v e d from suspension c u l t u r e p r o t o p l a s t s can now be produced q u i t e freely, as y e t i t has n o t proved p o s s i b l e t o induce o r g a n o g e n e s i s using such c e l l systems in t h e s a m e way t h a t c a l l u s d e r i v e d from o t h e r sources can be used (Saunders and Daub,

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