Plant Cell Reports (1981) 1 : 77-79

Plant Cell Reports © Springer-Verlag 1981

Hoechst 33258 as a Vital Stain for Plant Cell Protoplasts M. G. Meadows and I. Potrykus Friedrich Miescher-Institut, Postfach 273, CH-4002 Basel, Switzerland Received September 15, 1981; November 30, 1981

~bstract: A m e t h o d for staining nuclei in u n f i x e d p r o t o p l a s t s using H o e c h s t 33258 is described. It was found that the dye was not taken up by all p r o t o p l a s t s e x c e p t in the p r e s e n c e of a d e t e r g e n t and that the s t a i n i n g of the nuclei was pH- and c o n c e n t r a t i o n - d e p e n d e n t . The use of vital f l u o r e s c e n t p r o b e s w i t h p l a n t protoplasts m a y he h e l p f u l for the i d e n t i f i c a t i o n , s e l e c t i o n and s u b s e q u e n t c u l t u r e of h y b r i d fusion p r o d u c t s in a u t o m a t i c cell sorting systems. Introduction: It w o u l d be of g r e a t i n t e r e s t for p l a n t s c i e n t i s t s to be able to use flow c y t o m e t r i c t e c h n i q u e s w i t h p l a n t cells and p r o t o p l a s t s . These t e c h n i q u e s w o u l d be useful to a n a l y z e cell cycle events in c u l t u r e d cells and also to s o r t c y c l i n g from n o n - c y c l i n g cells. More importantly, f l o w c y t o m e t r y m a y provide~a~ g e n e r a l m e t h o d for s e p a r a t i n g h y b r i d fusion p r o d u c t s from p a r e n t a l fusion p r o d u c t s and non-fused protoplasts. F l o w c y t o m e t r y req u i r e s the use of f l u o r e s c e n t dyes w h i c h bind to the cells to be analyzed. A prerequisite for sorting and f u r t h e r c u l t u r i n g of the cells is that the f l u o r e s c e n t dye be a vital and n o n - t o x i c stain. The b i s - b e n z i m i d a z o l e dyes, H o e c h s t 33258 and H o e c h s t 33342, are vital, n o n - t o x i c in c u l t u r e d m a m m a l i a n cells and are considered to be q u a n t i t a t i v e l y s p e c i f i c for DNA (Arndt-Jovin and J o v i n 1977; C e s a r o n e et al. 1979). P r e v i o u s uses of H o e c h s t 33258 in p l a n t cells for n u c l e a r and c h r o m o s o m e studies (Filion et al. 1976; L a l o u e et al. 1980; G a l b r a i t h et al. 1981) have used m a t e rial fixed prior to staining. We d e s c r i b e a p r o c e d u r e for the vital s t a i n i n g w i t h the dye, H o e c h s t 33258, of n u c l e i of p r o t o p l a s t s derived from c u l t u r e d p l a n t cells. M a t e r i a l s and Methods: P r o t o p l a s t Isolation: The p r o t o p l a s t s were d e r i v e d from the m a i z e cell line, inbred B73, e s t a b l i s h e d by P o t r y k u s et al. (1977). The cells w e r e r o u t i n e l y m a i n t a i n e d in susp e n s i o n c u l t u r e as d e s c r i b e d by P o t r y k u s et al. (1979). To i s o l a t e p r o t o p l a s t s from the

cells, a s t a t i o n a r y phase c u l t u r e was d i l u t e d 1:4 w i t h fresh m e d i u m and c u l t u r e d overnight. The cells were then h a r v e s t e d by g e n t l e c e n t r i f u g a t i o n and r e s u s p e n d e d in an isoo s m o t i c s o l u t i o n c o n t a i n i n g 1% C e l l u l a s e O n o z u k a R-10 (Kinki Yakult, N i s h i n o m i y a , Japan), 0.5% P e c t i n o l F e s t (R~hm, D a r m s t a d t , FRG) and 0.5% H e m i c e l l u l a s e (Rhizopus, Sigma H2125, St. Louis, USA) in 0.3M m a n n i t o l and 0.04M CaCI_ at pH 5 8. After incubating with o g e n t l e sha~in@ for i to 3 h at 24 C in the dark, p r o t o p l a s t s w e r e s e p a r a t e d from r e m a i n i n g cell clumps and large debris by s u c c e s s i v e p a s s a g e s t h r o u g h 250~m, 100zm and 50~m s t a i n l e s s steel sieves. T h e y w e r e then harvested, w a s h e d three times in 0.16M CaCl 2 + 0.5% MES ( 2 - N - m o r p h o l i n o e t h a n e s u l f o n i c acid), pH 5.8, and c u l t u r e d in NN medium (Nitsch and N i t s c h 1967) + 0 . 1 7 M 6 ~ a n n i t o l , 2% W / V s u c r o s e and 2 mg/£ 2,4-D (2,4-dichlor o p h e n o x y a c e t i c acid). DNA Staining: The H o e c h s t 33258 (H33258) was a g i f t from Dr. H. Loewe, H o e c h s t AG, Frankfurt, FRG. It was stored as a 1 m g / m l stock s o l u t i o n in w a t e r and stored o at 4 C in the dark. H33258 was found to p r e c i p i t a t e from s o l u t i o n at 4°C when dissolved in p h o s p h a t e - c o n t a i n i n g buffers. To e s t a b l i s h the r e l a t i o n s h i p b e t w e e n pH, c o n c e n t r a t i o n of the dye, time in stain and other c o n s t a n t a s p e c t s of the s t a i n i n g procedure, the m a n i p u l a t i o n s w e r e c a r r i e d out w i t h the p r o t o p l a s t s r e s u s p e n d e d in 0.16M CaCl 2. The pH was a d j u s t e d w i t h KOH. Microscop~: The m i c r o s c o p i c e x a m i n a t i o n of the p r o t o p l a s t s was done on an O r t h o p l a n m i c r o s c o p e e q u i p p e d w i t h an O r t h o m a t autom a t e d c a m e r a (Leitz). The light source was a 200 W HBO high p r e s s u r e m e r c u r y lamp used w i t h e x c i t a t i o n b a r r i e r filter U V - U G I and d i c h r o i c filter a s s e m b l y P l o e m p a k A. T o x i c i t y of H 3 3 2 5 8 to P r o t o p l a s t s : H o e c h s t 33258 is known to be n o n - t o x i c to m a m m a l i a n cells b u t has not been tested on p l a n t cells. Petunia hybrida protoplasts w e r e s t a i n e d w i t h H33258 a c c o r d i n g to the p r o c e d u r e e s t a b l i s h e d b e l o w for 1 to 20 min. The s u r v i v a l and p l a t i n g e f f i c i e n c y of s t a i n e d cultures w e r e i d e n t i c a l to those of control cultures.

0721-7714/81/0001/0077/$ 01.00

78 R e s u l t s and D i s c u s s i o n : E f f e c t of pH: The relationship between the p H of the m e d i u m a n d H 3 3 2 5 8 u p t a k e is s h o w n in Fig. i. T h e d y e is o p t i m a l l y taken u p a t p H 7.5; h o w e v e r , even a t this pH, no m o r e than 80% of the p r o t o p l a s t s s t a i n e d . A t y p i c a l p r e p a r a t i o n of p r o t o p l a s t s is s h o w n in Fig. 2 w h e r e some p r o t o p l a s t s h a v e s t a i n e d and o t h e r s h a v e not.

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F i g u r e 3: T h e e f f e c t of 2 n o n - i o n i c d e t e r g e n t s on H 3 3 2 5 8 u p t a k e i n t o the p r o t o plasts. T w e e n 80 p r e v e n t s u p t a k e a t all concentrations whereas Triton X-100 enhances uptake.

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i___ 80 F i g u r e l: T h e e f f e c t of p H on the u p t a k e of H 3 3 2 5 8 b y the p l a n t p r o t o p l a s t s . Even a t the o p t i m u m pH, o n l y 80% of p r o t o p l a s t n u c l e i stain.

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~5 U s e of S u r f a c t a n t s : To increase prot o p l a s t u p t a k e of H 3 3 2 5 8 , two n o n - i o n i c d e t e r g e n t s w e r e a d d e d to the p r o t o p l a s t s o l u t i o n s at v a r y i n g c o n c e n t r a t i o n s in the p r e s e n c e of the d y e a t p H 7.5. The results are in Fig. 3. T w e e n 80 i n h i b i t e d the u p t a k e of the d y e a t all c o n c e n t r a t i o n s whereas Triton X-100 increased nuclear s t a i n i n g to 100% a t c o n c e n t r a t i o n s f r o m 0 . 0 0 6 % and h i g h e r . T a y l o r a n d M i l t h o r p e (1980) h a v e r e c e n t l y r e p o r t e d s i m i l a r r e s u l t s for T r i t o n X - 1 0 0 w i t h m a m m a l i a n c e l l s s t a i n e d w i t h H o e c h s t 33342. H33258 Concentration: T h e e f f e c t of H 3 3 2 5 8 c o n c e n t r a t i o n on the p e r c e n t a g e of p r o t o p l a s t n u c l e i s t a i n i n g w i t h and w i t h o u t T r i t o n X - 1 0 0 t r e a t m e n t is s h o w n in Fig. 4. The m e a s u r e m e n t s w e r e m a d e a f t e r the p r o t o p l a s t s w e r e in the s t a i n for 1 h. In sepa r a t e e x p e r i m e n t s , it w a s f o u n d that g o o d s t a i n i n g w a s o b t a i n e d in 15 m i n w i t h an H 3 3 2 5 8 c o n c e n t r a t i o n of 2 0 ~ g / m l , and a T r i t o n X - 1 0 0 c o n c e n t r a t i o n of 0.006%.

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H 33258 concentration (#g/ml) F i g u r e 4: T h e e f f e c t of H 3 3 2 5 8 c o n c e n t r a t i o n o n the p e r c e n t a g e of n u c l e i s t a i n i n g a t p H 7.5. T h e l o w e s t c o n c e n t r a t i o n to g i v e m a x i m u m s t a i n i n g is 20 ~ g / m l H33258. Solid circles ( 0--0 ) with 0.006% Triton X-100; Open circles ( O O ) without added surfactant.

Background Staining: W e f o u n d t h a t the m e m b r a n e s of the p r o t o p l a s t s f l u o r e s c e d slightly after treatment with H33258. This b a c k g r o u n d s t a i n i n g w a s e l i m i n a t e d if the p r o t o p l a s t s w e r e t r e a t e d w i t h D N A s e (50 ~g/ml f i n a l c o n c e n t r a t i o n ) for 20 m i n a t 37°C b e f o r e e x p o s u r e to the stain.

79

F i g u r e 2: P l a n t p r o t o p l a s t s do n o t a l l s t a i n w i t h H 3 3 2 5 8 in the a b s e n c e of d e t e r g e n t . a: Transmitted-light photomicrographs of B73 p r o t o p l a s t s , b: Fluorescence photomicrographs of the same p r o t o p l a s t s s h o w n in p a n e l a. The protoplasts l a b e l l e d "i" have n o t t a k e n up the stain. Arrows indicate stained nuclei. Conclusions: In a n t i c i p a t i o n of u s i n g f l o w c y t o m e t r i c techniques with plant protoplasts, we have d e f i n e d c o n d i t i o n s for the s t a i n i n g of p l a n t protoplast nuclei with H33258, a vital f l u o r e s c e n t s t a i n for DNA. The following protocol summarizes these conditions: (i) P r o t o p l a s t s in m e d i u m a r e t r e a t e d w i t h D N A s e (50 ~g/ml f i n a l conc e n t r a t i o n ) for 20 m i n a t 37°C. (2) A f t e r w a s h i n g , the p r o t o p l a s t s are r e s u s p e n d e d in 0 . 1 6 M C a C I _ , p H 7.5, in the p r e s e n c e of 0 . 0 0 6 % T r i t o n X-100. (3) H 3 3 2 5 8 is a d d e d to a f i n a l c o n c e n t r a t i o n of 20 ~g/ml. (4) A f t e r 15 min, the p r o t o p l a s t s a r e w a s h e d and r e s u s p e n d e d in m e d i u m for s o r t i n g or for m i c r o scopic examination. Acknowledgements: MGM gratefully acknowledges receipt of the M a r g a r e t M c W i l l i a m s T r a v e l l i n g

F e l l o w s h i p g i v e n b y the C a n a d i a n A s s o c i a t i o n of U n i v e r s i t y Women. We thank Pam S i m p s o n for e x p e r t s e c r e t a r i a l a s s i s t a n c e . References:

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