Plant Cell Reports

Plant Cell Reports (1985) 4:33-35

© Springer-Verlag 1985

A method for the microinjection and culture of protoplasts at very low densities W . A. L a w r e n c e and D . R. D a v i e s John Innes Institute, Colney Lane, Norwich NR4 7UH, UK Received December 6, 1984 / Revised version received December 20, 1984 - Communicated by M. H. Zenk

ABSTRACT

MATERIALS AND METHODS

A m e t h o d has b e e n d e v e l o p e d which allows the r e c o v e r y of c a l l i f r o m a h i g h p r o p o r t i o n of individual, injected, m e s o p h y l l p r o t o p l a s t s of N i c o t i a n a t a b a c u m c.v. Xanthi. A small drop of l o w m e l t i n g p o i n t a g a r o s e is u s e d b o t h to h o l d p r o t o p l a s t s d u r i n g m i c r o i n j e c t i o n a n d for their s u b s e q u e n t c u l t u r e in feeder dishes. The f e e d e r dishes c o n s i s t of "beads" of p r o t o p l a s t s at a h i g h d e n s i t y set in a g a r o s e to "feed" the i n f e c t e d p r o t o p l a s t s across a liquid medium. The m e t h o d has b e e n u s e d s u c c e s s f u l l y b o t h w i t h normal p r o t o p l a s t s and p r o t o p l a s t s f r o m which the vacuole has b e e n removed.

I) P r e p a r a t i o n of P r o t o p l a s t s Plants of N i c o t i a n a t a b a c u m cv. X a n t h i were grown f r o m seed as d e s c r i b e d b y Watts et al (1980). Mesop h y l l p r o t o p l a s t s were p r e p a r e d from leaves, a p p r o x imately 75% expanded, which h a d b e e n s t e r i l i z e d in 10% D o m e s t o s (Lever B r o t h e r s Ltd.) for i0 minutes. Leaf sections stripped of the lower e p i d e r m i s were f l o a t e d on 0 . 5 M m a n n i t o l for 30 minutes, a n d then on a 0.3 strength NT m e d i u m in 0 . 5 M m a n n i t o l a n d containing 0.05% M a c e r o z y m e Rt0 and 0.25% O n o z u k a RI0 c e l l u l a s e (Yakult M a n u f a c t u r i n g Co., Japan) o v e r n i g h t at 25oc in the dark. P r o t o p l a s t s were f i l t e r e d through a 100U mesh, c o l l e c t e d b y c e n t r i f u g a t i o n at 600 r/min for 4 minutes and r e s u s p e n d e d in a 0 . e M sucrose ~olution. T h e y were th~n spun for a further 4 minutes, the p r o t o p l a s t s r e m o v e d and w a s h e d twice in 0 . 5 M m a n n i t o l and once in NT medium. They were then left in the NT m e d i u m c o n t a i n i n g 3mgl -I N A A and imgl -I B A P for at least i h o u r b e f o r e b e i n g t r a n s f e r red to N T m e d i u m c o n t a i n i n g 0.1mgl -I NAA. This m e d i u m w i t h a low level of N A A was u s e d for all s u b s e q u e n t culture u n l e s s o t h e r w i s e stated. M e s o p h y l l p r o t o p l a s t s contain a large vacuole, a n d since it is d e s i r a b l e to avoid injecting D N A into this volume, e v a c u o l a t e d p r o t o p l a s t s h a v e been u s e d for some of our work. T h e y were p r e p a r e d by the m e t h o d of G r i e s b a c h and Sink (1983), except that c e n t r i f u g a t i o n through the P e r e o l l g r a d i e n t was for i h o u r at 30,000 r/min u s i n g a SW50.1 rotor (Beckman). W a s h e d e v a c u o l a t e d p r o t o p l a s t s w e r e then t r e a t e d as normal v a c u o l a t e d ones.

ABBREVIATIONS N T m e d i u m = N a g a t a - T a k e b e m e d i u m (Nagata and Takebe, 1971), MS m e d i u m = M u r a s h i g e - S k o o g m e d i u m (Murashige and Skoog, 1962), N A A : l - N a p h t h a l e n e a c e t i c acid, BAP = 6 - B e n z y l a m i n o p u r i n e , LMP a g a r o s e = l o w m e l t i n g p o i n t agarose. INTRODUCTION Methods of m i c r o i n j e c t i n g p l a n t p r o t o p l a s t s a k i n to those r e a d i l y a v a i l a b l e for animal cells h a v e not b e e n d e v e l o p e d to a stage where t h e y can be r o u t i n e l y u s e d for the i n t r o d u c t i o n of D N A or o t h e r m a c r o molecules. T h e r e are m a n y t e c h n i c a l r e a s o n s for this b u t one of the m o s t i m p o r t a n t d i f f e r e n c e s b e t w e e n c u l t u r e d animal cells and p l a n t p r o t o p l a s t s is the ease with w h i c h m a n y of the former attach to solid s u r f a c e s when grown in liquid media. This i m m o b i l ises t h e m and a l l o w s a m i c r o n e e d l e to be introduced, whereas p f o t o p l a s t s h a v e to b e a r t i f i c i a l l y i m m o b i l ised or p h y s i c a l l y held. Steinbiss a n d Stabel (1983) h a v e p r o v i d e d the o n l y d e s c r i p t i o n of a m e t h o d of h o l d i n g p r o t o p l a s t s for injection, b y a t t a c h i n g t h e m to p o l y - L - l y s i n e t r e a t e d glass. We h a v e d e v e l o p e d an a l t e r n a t i v e t e c h n i q u e w h i c h in our e x p e r i e n c e has advantages over m e t h o d s w h i c h use c o m p o u n d s such as p o l y - L - l y s i n e for attachment; it involves the use of a g a r o s e m i c r o d r o p s on to which the p r o t o p l a s t s are plated. This m e t h o d a l l o w s m i c r o i n j e c t i o n to be u n d e r t a k e n as a r o u t i n e procedure, h i g h rates of survival are obtained, p r o t o p l a s t s can be c u l t u r e d at v e r y low densities, the fate of i n d i v i d u a l p r o t o p l a s t s can be f o l l o w e d a n d plants can b e r e g e n e r a t e d f r o m them.

2)

P r e p a r a t i o n of m i c r o d r o p s The u n i t d e v e l o p e d for p r o t o p l a s t c u l t u r e and i n j e c t i o n c o n s i s t s of a sterile c o v e r s l i p (22 x 22mm) on which is p l a c e d a sterile nylon ring of 9mm inner d i a m e t e r and lo5mm deep (Fig. la). The ring is filled almost to the top with N T m e d i u m c o n t a i n i n g 0.9% LMP a g a r o s e (Bethesda R e s e a r c h L a b o r a t o r i e s ) , the a g a r o s e b e i n g a l l o w e d to a l m o s t set b e f o r e use. Equal v o l u m e s of p r o t o p l a s t s at an a p p r o x i m a t e c o n c e n t r a t i o n of 2 x 104ml -I in NT m e d i u m h e l d at 20°C, and N T m e d i u m c o n t a i n i n g 0.6% LMP agarose as well as a small a m o u n t of sterile c h a r c o a l h e l d b e l o w 40oc are m i x e d a n d I~i p l a c e d in the centre of the a g a r o s e f i l l e d ring. The n u m b e r s of p r o t o p l a s t s p l a t e d in o u r e x p e r i m e n t s v a r i e d f r o m 5 to 15, a n d they are h e l d just u n d e r the surface of the 0.3% agarose. A b o u t 12 of these units, r e f e r r e d to h e n c e forth as microdrops, are p r e p a r e d at any given time and h e l d for up to 2 h o u r s in 9cm p e t r i dishes lined

34 with m o i s t filter paper. 3)

Mieroinjection M i c r o i n j e c t i o n is carried out u s i n g a Leitz m i c r o m a n i p u l a t o r with a fixed stage m i c r o s c o p e h o u s e d in a laminar flow cabinet and is c o n t r o l l e d by a h e a v y liquid p a r a f f i n filled system a t t a c h e d to a Beaudcu/n syringe (Microinstruments Oxford). N e e d l e s are p r e p a r e d f r o m 2mm outer d i a m e t e r "electrode tubing", inner diameter I to 1.25mm (Jencons, Hemel Hempstead), siliconized using "Sigmacote" (Sigma) and dried at 60°C. A "needle puller", made by Mr. T. Parker of this Institute, a l l o w e d needles of I~ tip d i a m e t e r or less to be produced. The needles are a p p r o x i m a t e l y 4 . 5 c m long and are b a c k filled with h e a v y p a r a f f i n oil; the injection solution is introduced to the tip simply by lowering the oil filled needle into the solution on a slide. The approximate volume i n j e c t e d into each p r o t o p l a s t is b e t w e e n 10 -8 and 10-9ml. 4)

C u l t u r e of m i c r o i n j e c t e d p r o t o p l a s t s Microdrops are r e p l a c e d in the moist p e t r i dishes i m m e d i a t e l y after injection; injected protoplasts are covered with N T m e d i u m containing 0.9% LMP agarose at <40°C within 20 to 30 minutes, sufficient agarose b e i n g added to form a meniscus above the ring. Once the agarose has set the m i c r o d r o p is p l a c e d in a feeder dish system (Fig. ib) we have d e v e l o p e d for low d B n s i t y culture and which exploits the p g i n c i p l e of a "bead" culture system (Shillito et al; 1983). To prepare beads, 5ml of p r o t o p l a s t s at 5x104 ml -I in NT m e d i u m with 0.7% LMP agarose are set in 5cm p e t r i dishes, the agarose cut into 8 sections and 2 of these p l a c e d in a 5cm p e t r i dish containing 3.5mi N T medium. These b e a d s "feed" the few p r o t o p l a s t s i n

the microdrop; p r o t o p l a s t s u s e d to p r e p a r e b e a d s may be up to 3 days old. Feeder dishes are shaken slowly (40 rpm) at 25°C for I week, then the m i c r o drop is t r a n s f e r r e d to a freshly p r e p a r e d feeder dish. A f t e r a further 2 weeks, when the calli in the m i c r o drop are u s u a l l y visible to the n a k e d eye, microdrops are removed, the ring lifted off and the entire m i c r o d r o p p l a c e d on MS m e d i u m containing 0 . 2 5 M mannitol and 0.7% LMP agarose (Fig. Ic). The surface of the m i c r o d r o p is cut at this stage to a l l o w better aeration of die calli. If the calli have not grown sufficiently a t h i r d feeder dish is u s e d for a further i week before transfer to the MS medium. Once calli are large enough to be p i c k e d up individually, they are t r a n s f e r r e d onto MS m e d i u m again with 0.7% LMP agarose b u t with no m a n n i t o l (Fig. id). RESULTS AND D I S C U S S I O N The m i c r o d r o p m e t h o d is easy to o p e r a t e and is now a routine p r o c e d u r e in our laboratory; its a d v a n t a g e s over other methods that we have tried for h o l d i n g cells for m i c r o i n j e c t i o n are the following. A large number of m i c r o d r o p s can be quickly and easily p r e p a r e d in one b a t c h and h e l d with p r o t o p l a s t s on t h e m for up to two hours in a h u m i d atmosphere in a sterile petri dish. This separates m i c r o i n j e c t i o n and p r o t o p l a s t p r e p a r a t i o n into two d i s t i n c t o p e r a t i o n s and allows a c o n t i n u o u s p e r i o d o{ injection without the n e e d to p r e p a r e successive b a t c h e s of protoplasts. The surface of the drop is almost flat and allows the needle to be kept in one plane~ which eases the m a n i p u l a t i v e o p e r a tions. By incorporating a small amount of charcoal

Figure la. A g a r o s e m i c r o d r o p s c o n t a i n i n a protoplasts, enclosed by nylon rings on glass coverslips in a h u m i d i fied p e t r i dish. Figure lb. Feeder dish with two feeder beads containing a high density of p r o t o p l a s t s and an agarose microdrop with injected protoplasts. Figure ic. A g a r o s e m i c r o d r o p s containing small calli d e r i v e d f r o m injected protoplasts, p l a c e d on MS medium. Figure id. Calli from individual m i c r o i n j e c t e d p r o t o p l a s t s removed from the agarose m i c r o d r o p s and p l a t e d on MS medium.

35 with the p r o t o p l a s t suspension, the area of the microdrop c o n t a i n i n g p r o t o p l a s t s is q u i c k l y identified. The needle can be m o v e d through the 0.3% agarose easily, yet the p r o t o p l a s t s are h e l d firmly enough to a l l o w needle penetration. Optical q u a l i t y of, and light t r a n s m i s s i o n through, the agarose p o s e no problems. The m i c r o d r o p s can be e x p o s e d in the m i c r o m a n i p u l a t o r in a laminar flow c a b i n e t for at least i0 min w i t h o u t h a r m i n g the protoplasts. A f t e r injection the latter can once m o r e be h e l d in the h u m i d i f i e d petri dishes; our routine p r o c e d u r e is to w a i t until up to eight m i c r o d r o p s are ready, and then cover t h e m with agarose. Yet a n o t h e r advantage is the ease with w h i c h m i c r o d r o p s can b e subsequently handled; the w h o l e u n i t i.e. the g l a s s coverslip, m i c r o d r o p and ring are p l a c e d in a 50mm p e t r i dish c o n t a i n i n g the two feeder b e a d s and l i q u i d medium. When the p e t r i dishes are p l a c e d on a shaker, the m i c r o d r o p floats off the coverslip but the ring u s u a l l y remains a r o u n d it m a k i n g transfers to fresh dishes very easy. M i c r o c a l l i v i s i b l e to the n a k e d eye develop within 3 to 4 weeks. Since v e r y low numbers of p r o t o p l a s t s are p l a t e d (from 5 to 15 per microdrop) they c o u l d be r e a d i l y p h o t o g r a p h e d to establish their r e l a t i v e positions, and the fate of individuals followed. It is worth noting that 5 to 15 per m i c r o d r o p represents 50 to 150 p e r ml, an e x t r e m e l y l o w d e n s i t y but one which allows g o o d rates of survival. O t h e r methods of a t t a c h i n g p r o t o p l a s t s for m i c r o i n j e c t i o n were tried b e f o r e the m i c r o d r o p m e t h o d was developed. P o l y - L - l y s i n e was found to be toxic to p r o t o p l a s t s at the c o n c e n t r a t i o n s n e c e s s a r y to attach t h e m to glass. Steinbiss and Stabel (1983) report that the t o x i c i t y is o v e r c o m e if a p e r i o d of 24 h p r e c u l t u r e is used, but we find that cell wall r e f o r m a t i o n a f t e r 24 h u n d e r s t a n d a r d culture c o n d i t i o n s limits the n u m b e r of successful injections which can b e made. P r o t a m i n e sulphate was also u s e d f o r a t t a c h m e n t but was less e f f i c i e n t and also appeared to be d e l e t e r i o u s to p r o t o p l a s t recovery. T h r e e o t h e r c h e m i c a l means of a t t a c h m e n t were tested, the lectin c o n c o n a v a l i n A, L y s o z y m e and P o l y e t h y l e n e Glycol but none was satisfactory. It was p o s s i b l e to h o l d the p r o t o p l a s t s in an electric field, but the technique was too c u m b e r s o m e and slow to be adopted. P r o t o p l a s t s can be i n j e c t e d for up to 24 h after p r e p a r a t i o n if h e l d at 20°C although wall f o r m a t i o n b e g i n s within a few h o u r s of p l a t i n g in NT medium. To extend the p e r i o d available for injection, the p r o t o p l a s t s are k e p t r o u t i n e l y at 10°C; this delays wall formation and allows up to 48 h o u r s for injection.

Needles must b e p r e p a r e d on the day of use, o t h e r w i s e they become d i f f i c u l t to fill. Sterilisation is u n n e c e s s a r y in our experience, p r o v i d e d the needles are kept in a clean closed container. One of the m a j o r p r o b l e m s we have e n c o u n t e r e d in the whole m i c r o i n j e c t i c n p r o c e d u r e is that of needles blocking; b e c a u s e of this, they can rarely be u s e d for m o r e than 20 injections. With the p r o c e d u r e s o u t l i n e d above, 50 p r o t o p l a s t s can b e injected routinely and consistently, per hour. Initially dyes were i n j e c t e d into the p r o t o p l a s t s to establish the n e c e s s a r y protocol; after t h a t we t e s t e d the integrity and v i a b i l i t y of the cells by injecting a virus, B r o m e mosaic virus, and a s s a y e d for infection 48 h o u r s later with a f l u o r e s c e n t l a b e l l e d antibody. I n j e c t e d p r o t o p l a s t s were infected, w h e r e a s controls g r o w n in the p r e s e n c e of, but not injected with virus were not infected. To further test the m i c r o d r o p system, 144 p r o t o p l a s t s were injected with 2mM citrate buffer, pH 5.5; from these, 84 calli were r e c o v e r e d (58% survival) and plants have b e e n r e g e n e r a t e d f r o m them. Survival rates vary with p r o t o p l a s t batch, with the b e s t values b e i n g up to 71% of those injected surviving to give calli, although the mean value is nearer to 40%. Evacuolated p r o t o p l a s t s have a lower survival rate. Having e s t a b l i s h e d a standard procedure, we are n o w engaged in a p r o g r a m m e of m i c r o i n j e c t i o n with a g e n e t i c a l l y m a r k e d plasmid.

ACKNOWLEDGEMENTS W.L. a c k n o w l d g e s the receipt of a post g r a d u a t e s t u d e n t s h i p from the John Innes Foundation; we are grateful to Dr. J. Watts for much helpful advice, and for p r o v i d i n g a sample of Brome mosaic virus and antibody to the virus.

REFERENCES G r i e s b a c h RJ Sink KC (1983) P l a n t Sci Lett 30: 297301 M u r a s h i g e T Skoog F (1962) Physiol Plant 1 5 : 4 7 3 - 4 9 7 N a g a t a T Takebe I (1971) Planta 99: 12-20 Shillito RD P a s z k o w s k i J Potrykus I (1983) Plant Cell Reps 2 : 2 4 4 - 2 4 7 Steinbiss HH Stabel P (1983) P r e t o p l a s m a 116: 223227 Watts JW Dawson JRO King J M (1980) In: Ingram DS H e l g e s o n JP (eds) T i s s u e C u l t u r e M e t h o d s for Plant Pathologists, Oxford, Blackwell, pp 97-101