Isolation, culture and plant regeneration of colt cherry (Prunus avium × pseudocerasus) protoplasts

Plant Sctence, 50 (1987) 139~143 Elsewer Scmntffic Pubhshers Ireland Ltd

139

ISOLATION, CULTURE AND PLANT REGENERATION A VIUM × PSEUDOCERASUS) PROTOPLASTS

OF COLT CHERRY (PRUNUS

S J OCHATT, E C COCKING and J B POWER Plant Genetic Man~pulatmn Group, Department of Botany, Unwers~ty of Nottingham, Nottingham NG7 2RD (U K ) (Received November 28th, 1986) (Revision received January 27th, 1987) (Accepted January 27th, 1987) Large numbers of viable protoplasts were isolated from leaf mesophyll tissues and cell suspension cultures of Colt cherry using 1% (w/v) Onozuka R-10, 0 2% (w/v) Macerozyme R-10, 0 1% (w/v) Drlselase, 1% (w/v) polyvmylplrrohdone (av MW 10 000) (PVP-10) and 2% (w/v) Melcelase, 2% (w/v) Rhozyme HP-150 and 0 03% (w/v) Macerozyme R-10 Culture media, based on Murashlge and Skoog's (MS) salts, supplemented with 9% (w/v) manmtol and various combmatmns of ~-naphthalene acetic acid (NAA), 6-benzylamlnopurme (BAP) and zeatm (Z) promoted cell wall regeneration followed by cell colony and callus formatmn Protoplasts of both sources were compared in relatmn to their cultural requirements Protoplast-derlved callus underwent organogenems Key words woody fruit trees, cherry rootstock, tissue culture, protoplasts, callus and plant regeneration

Introduction Plant breeders working with sweet cherry ( P r u n u s av~um L ) a r e c o n t i n u a l l y s e e k i n g m e a n s of c o n t r o l h n g t r e e size a n d b r i n g i n g orc h a r d s i n t o e a r l y p r o d u c t m n I n t h i s respect, t h e i m p r o v e m e n t of e x i s t i n g r o o t s t o c k s , as well as t h e p r o d u c t i o n of n e w ones, p l a y s a n import a n t r o l e [1] Tissue culture and somatic hybndlzatmn p r o v i d e a l t e r n a t i v e a p p r o a c h e s to the generat i o n of g e n e t i c n o v e l t y v i a s o m a c l o n a l v a r l a t m n or s o m a t i c h y b r i d p r o d u c t m n [2,3] H o w e v e r , for w o o d y p e r e n n m l s p r o t o p l a s t t e c h n o l o g y is n o t m u c h b e y o n d the p r e p a r a t i v e stage, w i t h p l a n t r e g e n e r a t m n f r o m p r o t o p l a s t s b e i n g r e s t r i c t e d to a few s p e c m s [4-9] A m o n g t h e r o s a c e o u s f r u i t crops, p r o t o p l a s t c u l t u r e h a s b e e n a t t e m p t e d for a p p l e [10-12]

Abbreviations BAP, 6-benzylamlnopurme, FDA, fluroesceln dlacetate, fresh wt, fresh weight, MES, 2-N-morphohno ethane sulfomc acid, MS, Murashlge and Skoog, NAA, ~-naphthalene acetic acid, PVP-10, polyvmylpyrrohdone (av MW 10 000), Z, zeatm

wild p e a r [5], a l m o n d [13] a n d p e a c h [14]. However, p l a n t r e g e n e r a t i o n was r e p o r t e d o n l y for wild p e a r [5] T h e c h e r r y r o o t s t o c k Colt ( P r u n u s a v i u m × pseudocerasus) is of c o m m e r c i a l i m p o r t a n c e since it h a s a n u m b e r of d e s i r a b l e t r a i t s I t is also a trlploid a l l o p o l y p l o l d (2n = 3 × = 2 4 ) and is t h e r e f o r e sterile a n d c a n n o t be s u b j e c t e d to c o n v e n t i o n a l b r e e d i n g a p p r o a c h e s T h e p r e s e n t w o r k was s t i m u l a t e d b y r e p o r t s on p l a n t r e g e n e r a t i o n f r o m Colt c h e r r y callus [15,16] a n d t h e a b i h t y to i s o l a t e p r o t o p l a s t s f r o m this h y b r i d [10] T h i s p a p e r describes t h e i s o l a t i o n a n d c u l t u r e of Colt c h e r r y p r o t o p l a s t s a n d t h e i r d e v e l o p m e n t to the callus stage. This callus u n d e r w e n t o r g a n o g e n e s i s . T h e estabh s h m e n t of Colt c h e r r y as a dividing p r o t o p l a s t s y s t e m will p r o v i d e a basis for ~ts s u b s e q u e n t genetic mampulatlon.

Materials and m e t h o d s P l a n t mater~al Callus w a s p r o d u c e d f r o m m v i t r o g r o w n r o o t s of Colt c h e r r y on a s e m i s o h d P1 m e d m m

0618-9452/87/$03 50 © 1987 Elsevmr Scmntffic Pubhshers Ireland Ltd Pubhshed and Printed m Ireland

140

(MS salts and organics [17] with 2.0 rag/1 NAA, and 0 5 mg/1 BAP and 0.8% agar, Sigma) at 25°C and under constant illumination (1000 lux daylight fluorescent tubes). Cell suspensmn cultures were lnitmted in the same hquid medium and under the same cultural conditions Cultures were shaken at 80 cycles/mln and subcultured at 3-weekly intervals. Shoot cultures of Colt cherry were maintained by monthly subculturmg on MS salts with 0 I mg/1 4-(indole-3yl)-butync acid, 1 0 mg/1 BAP and 0 1 rag/1 gibberelhc acid, and could be rooted m half-strength MS medium with 0.5mg/1 NAA as prewously reported [18]. Shoot cultures were maintained under the same conditions as for root callus Cell suspension cultures (14 or 21 days after subculture) or fully expanded leaves (10mm x 5ram) of m vitro shoot cultures (rooted or unrooted) of the Colt cherry rootstock (P av~um × pseudocerasus) were used as sources of protoplasts Protoplast ~solatwn Both leaves and cell suspensions were sometimes plasmolized for 1 h, in CPW 13M

Table I. cherry

solution [19] prior to enzymatic digestion Peeled or chopped leaves (1 g fresh wt.) or 1.0 ml (packed cell volume) of the cell suspension were incubated in 10-ml ahquots of several enzyme mixtures (Table I). All tissues were incubated, overnight, in the appropriate enzyme solution at 25°C, In the dark or with a continuous illumination (500 lux). The influence of agitation on protoplast release was also assessed (40 or 80 cycles/rain) After a minimum of 17 h incubation period the digested cells (leaf or cell suspension) were passed through two nylon reeves (100 pm followed by 64 #m pore size) Any undigested tissues were rinsed repeatedly with CPW 13M solution and the filtrate, containing protoplasts, was then centrifuged (10 mIn, 100 × g) The supernatant was removed and protoplasts resuspended m CPW 21S solution (CPW salts [19] with 21% (w/v) sucrose) Following centrifugatlon (200 × g, 10 min) floating protoplasts were resuspended m 10 ml of CPW 9M (CPW salts with 9% (w/v) mannitol) solution whereupon the yield was determined and their viability assessed using fluroescem diacetate (FDA) [19]

Composltmn of enzyme solutions, yields and vlablhtms of cell suspensmn and leaf mesophyll protoplasts of Colt

Enzyme code

Compomtlon a (%-w/v)

Protoplast ymld (Y) and viability (V) Leaf

A B C D E F G H I J

0 5 0 N O , 0 1 MAC 1 0 0 N O , 1 0 MAC 1 0 ONO, 0 01 PEC 1 0 ONO, 1 0 HEM, 1 0 MAC 1 0 0 N O , 1 0 HEM, 0 1 PEC 1 0 0 N O , 0 2 MAC, 0 1 DRI 2 0 0 N O , 1 0 HEM, 0 05 PEC 2 0 0 N O , 0 1 PEC, 05 DRI 2 0 MEI, 2 0 RHO, 0 03 MAC 3 0 MEI, 0 5 MAC

Cell suspension

Y ( × 106/g fresh wt )

V (%)

Y ( × 107/g fresh wt )

V (%)

241 2 09 2 19 287 0 98 5 22 2 23 2 31 0 76 0 93

35 9 36 1 38 6 39 5 37 0 44 2 34 8 39 7 33 3 34 1

1 14 4 60 2 63 5 90 2 11 3 87 1 96 2 14 15 61 8 53

89 91 77 90 76 85 81 79 98 94

DRI, Drmelase, HEM, Hemlcellulase, MAC, Macerozyme R-10, MEI, Melcelase, ONO, Onozuka R-10. PEC, Pectolyase Y-23 RHO, Rhozyme HP-150 Enzymes were dissolved m CPW 13M solution [19J supplemented with 5 mM MES (pH 5 6) For leaf protoplast ~solatlons, all enzyme solutions were also tested wlth the addition of 1% (w/v) PVP-10

142

[15,16]. Root r e g e n e r a t m n was observed m 10% of the cell suspension protoplast derived callus and on MS medmm with 0 05 rag/1 NAA, 5 rag/1 BAP and 100 mg/1 caseme hydrolysate.

Leaf mesophyll protoplasts Protoplasts generally failed to float in the CPW 21S solutmn Thin was rectified by the addltmn, to the enzyme solutmn, of 1% (w/v) PVP-10 The best results were obtained when preplasmohzed leaf tmsues were incubated with slow agitation and m the h g h t m enzyme solution F (Table I), where both peeled or chopped leaves gave comparable results m terms of yield and wabfllty (5.1 × 106 protoplasts/g fresh wt - 45% w a b i h t y and 5 2 x 106 protoplasts/g fresh wt. - 44 2% viability, respectlvely) Likewise leaves from m w t r o cultured plants, whmh had been rooted, gave a similar response As for cell suspensmn protoplasts, KmP8 medium d~d not support dlwsmn of leaf protoplasts F o u r MS-based medm were capable of supporting initial protoplast dlwsmn" 2.0 mg/1 NAA, 1 0 mg/1 Z, 2 0 mg/1 NAA, 0 5 mg/1 BAP, 0 5 rag/1 Z, 1 0 rag/1 NAA, 0.25 rag/1 BAP, 0 5 rag/1 Z; and 0.5 rag/1 NAA, 0 1 rag/1 BAP, 1 0 rag/ 1 Z In all four medm cell walls were regenerated within 7-9 days, with first mitosis commencing 24 h later. The optimum plating denslty for leaf protoplasts was 5 × 105 protoplasts/ ml and m the h g h t No differences were detected for h q m d or agar os e- c ont a m m g media. Within 14 days of plating the media were diluted w~th the approprmte manmtol-free medium m a 1 3 r a t m as described e a r h e r At th~s t~me, plating efficmncy (percentage of the m~tlal protoplasts which had dlwded at least once) was 16.2% with colomes of 4 ~ cells. The m a n m t o l c o n c e n t r a t m n was f ur t her reduced at 21 and 28 days by adding mannitol-free medmm m the r atms 1 : 2 and 1 1, respectively. F~ve weeks after molatmn colonies were of a urnform sine (20 cells), whereupon they were transferred to P1 medmm with 0 3% (w/v) agar It was only after a f u r t h e r 2 weeks t hat differences between the four medm became apparent Cell growth ceased in those colonies that had grown m the medmm c o n t m m n g 2.0 mg/1

Fig. 1. Shootbud regeneration on a leaf mesophyllprotoplast-derlved callus of Colt cherry (2 × ) NAA and 1 0 mg/1 Z. Colomes from the three remaining media were successfully transferred, at 2-weekly intervals, to P1 medmm with 0.6% (w/v) agar. U l h m a t e l y those colomes originally grown on MS medmm with 2.0mg/1 NAA, 0 5mg/1 BAP and 0.5rag/1 Z proved best, with an overall plating efficmncy of 0 01% (week 9) as based on the original number of plated protoplasts (zero time) These protoplast-denved calh have been subsequently maintained separately, for an assessment of regeneration potential. Shoot buds ( X + S . E from n = 20 calluses = 5 9 + 0.8 shoots per callus) were obtained from 40% of these calh after transfer to MS medmm with 0 1 rag/1 NAA, 0.75 rag/1 BAP, 0 1 mg/l Z and 50 rag/1 caseme hydrolysate (Fig. 1). These regenerated shoots were subsequently handled and maintained as for the shoot cultures t hat provided the protoplasts

Discussion In previous reports on the isolation of protoplasts from Prunus spp, Pectolyase Y-23 was mentioned as optimal [10] and indeed essential [14] for the efficmnt molahon of protoplasts This enzyme offered no improvement m terms of y~eld or viablhty for Colt protoplast isolation Thin same principle seems to apply, m relation to the source for leaf tmsues, since, m spite of a previous report [10] to the contrary: ymld and/or viability were not influenced either by source or whether explants were mdeed rooted.

143

Agarose gave good results for the culture of Colt cherry protoplasts from both sources, this was consistent with the wild pear protoplast system [5] and those of other deciduous woody species [21]. In addition, the fact that KmP8 medium and the complex organic mixture of Gamborg et al. [3] were detrimental to protoplast growth, suggests that Colt cherry protoplasts have a low reqmrement for organic components in the culture medium Moreover, the absence of whole plant regeneration from Colt protoplast derived calli, on media which normally supported plant production directly from explant-callus of this hybrid [15,16], emphasizes the fact that the callus source can influence regeneration capacity and presumably nutritional and hormonal requirements. This is also ewdenced by the fact that different cultural responses were exhibited by the leaf mesophyll and cell suspension protoplasts. Colt cherry protoplasts had a relatively high initial plating density requirement which often made the assessment of plating efficiency difficult. Even though plating efficiency was numerically low this still represented the productlon of very large numbers of calli from protoplasts. Similar plating efficmncms were reported for shoot tip protoplasts of the related woody rosaceous species, S o r b u s a u c u p a n a [22]. These results for Colt cherry, coupled with recent developments m protoplast culture for several other fruit tree specms [4~,8] suggest that woody fruit crops will, in the near future, be amenable to techniques of genetic manipulation based on protoplasts Provided that efficient selection strategies can be developed, natural barriers to hybridization are likely to be broken down, through cell fusion, thus facilitating gene transfer in relation to rooting capability and disease and stress tolerance.

Acknowledgements S.J.O. was supported by a fellowship of the Consejo Naclonal de Investlgaclones Cmntlfi-

cas y Tecnlcas de la Republica Argentina (CONICET) under its joint exchange agreement with The Royal Society

References 1 R E C Layne and W B Sherman, InterspeclfiC hydrldlzatlon of Prunus, in Proc Workshop Overcoming barriers to mterspecffic hybridization of perenmal fruit crops, Hortm Scl, 21 (1986) 48 2 E M Frearson, J B Power and E C Cocking, Dev Blol, 33 (1973) 130 3 O L Gamborg, J P Shyluk and E A Shahln, Isolation, fusion and culture of plant protoplasts, in T A Thorpe (Ed), P l a n t Tissue Culture Methods and Applications in Agrmulture, Academic Press, New York, 1981, p 115 4 S Kobayashl, H Uchlmlya and I Ikeda, J p n J Breed, 33 (1983) 119 5 S J Ochatt and O H Caso, J P l a n t Physlol, 122 (1986) 243 6 S Oka and K Ohyama, J Plant Physlol, 119 (1985) 455 7 P S Rao and P Ozlas-Akms, Protoplasma, 124 (1985) 80 8 A Vardl, Protoplast derived plants from different Citrus species and cultlvars, in Proc Int Soc Cltrlculture, 1981, p 149 9 J A Russell and B H McCown, P l a n t Scl, 46 (1986) 133 10 D J James, A J Passey and S B Malhotra, Isolation and fusion of protoplasts, in Rep E M a l h n g Res Stn for 1983, p 63 11 M Komder, R Hauptmann, J M Wldholm, R M Sklrvln and S S Korban, P l a n t Cell Rep, 3 (1984) 142 12 M Nllzekl, Y Hldano and K Salto, J p n J Breed, 33 (1983) 369 13 S C Wu and A H Kumyukl, Plant Scl, 41 (1985) 55 14 N Matsuta, T Hlrabayashl, T Aklhama and I Kozakl, Scl Hortlc, 28 (1986) 59 15 D J James, A J Passey and S B Malhotra, Plant Cell Tissue Organ Culture, 3 (1984) 333 16 O P Jones, J A Gayner and R Watkms, J Hortlc Scl, 59 (1984) 463 17 T Murashlge and F Skoog, Physml P l a n t , 15 (1962) 473 18 S J Ochatt and O H Caso, Rev Inv Agrop I N T A , XIX (1984) 24 19 J B Power, J V Chapman and D Wilson, Laboratory M a n u a l P l a n t Tissue Culture P l a n t Genetic Manipulation Group, Dep Botany, U m v Nottingham 1984 20 K N Kao and M R Mlchayluk, Planta, 125 (1975) 105 21 F M Tremblay, J B Power and M Lalonde, Plant Scl, 41 (1985) 211 22 H Binding, J Jorgensen, G Krumblegel-Schroeren, J Finger, G Mordhorst and G Suchowlat, Culture of apical protoplasts from shoot cultures in the orders Fabales, Rosales and Caryophyllales, m 6th Protoplast Syrup, Basel, 1983, p 38