Plant Cell Reports
Plant Cell Reports (1987) 6:67-69
© Springer-Verlag 1987
Protoplast culture and plant regeneration from Brassica carinata Braun Phan V. Chuong, K. P. Pauls, and W. D. Beversdorf Department of Crop Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received August 14, 1986/Revised version received October 29, 1986 - Communicated by F. Constabel
ABSTRACT Protoplasts isolated from hvDocotvls of three-day-old seedlinqs of Brassica carinBta (Rraun) cv R-212~ were cultured in a modified Nitsch and Nitsch l i n u i d medium containinQ 13% sucrose, 0.4% F i c o l l , 0.25 mq/l BA, 0.5 mq/l NAA and Q.5 mg/l 2,4-D. The densit~ of medium caused the DrotoDlasts and the developinn m i c r o c a l l i to f l o a t on the surface of the l i n u i d medium whereas a l l debris and lysed c e l l s sank to the bottom of the culture plate. After 4-6 weeks developing m i c r o c a l l i were approximately O. 5 mm in diameter and were transferred onto MS medium containinq 3% sucrose, ~.4% agarose, 200 m~/l casein h~drolysate~ 5 mq/l ~A and ~.5 mq/l NAA, pH 5.7. Approximately 20% of the c a l l i transferred to this medium produced plantlets. Abbreviations: RA, 6-~enzylaminoDurine; NAA, 1-naDhthaleneacetic acid; IAA, indole-3-acetic acid; 2,4-~, 2,4-dichloronhenoxvacetic acid; ~S, ~urashiqe-Skoo~ INTR~ntICTIF)~ The development of plant regeneration procedures for Rrassica DrotoDlasts is an active area of researc--~-~--~-cause this Qenus includes several economically important vegetable and oilseed crop species and because the research to date indicates that, in general, Brassica species resoond well to a wide v a r i e t y of tissue culture manipulations. Success with anther culture ( K e l l e r and Armstrong, 1978, 1983; Litcher, 1981), microsDore culture (Litcher, 1 9 8 2 ; Chuong and Beversdorf, 1985), protoplast fusion (Rarsby et a l . , 1986; P e l l e t i e r et al., 1983 and Ti ~ d ~ t e d transformation - ~ o l b ~ o k , personal communication) has been reported. The culture procedures for reQenerating mature plants from u n d i f f e r e n t i a t e d callus tissue and single c e l l s are central to the a p p l i c a t i o n of biotechnologv to plant improvement. In the qenus Brassica olant regeneration has been reported from p r o s of B. napus (Kartha et a l . , 1974; Thomas et a l . , 1976; ~u e-6~'-'~T., ig82; ~ d Kohlenbach, 198~,']~hlenbach et a-~F.~--l~82; Lu et a l . , 1982; Glimelius, Ig84 an--~ C--~uong et a l , , 19-~.5T~,B. oleracea (Xu et a l . , 1982; Lu et a-~T.~--Ig82; G l i ~ - e l ~ u s , ~ 4 ; R~-cF6e~'-et a l . , Ig83TF~--et a l . , 19R5), B. ~uncea ( C h a t t e r ~ e e ~ t a-~F., 1985) a n ~ . - - ~ a m D e s t r i s T G l ~ s , 1984). In m o s t cases the frefluencv of plant regeneration from orotoDlast-derived c a l l i is low.
Offprint requests to." K.P. Pauls
Xu et a l . , (ig82) found that 20% of the c a l l i developinn from root ~rotoDlasts of R. napus regenerated shoots. Glimelius (1984) was- a b ~ to increase the regeneration frequency to approximately 70% by using h~Docotyl DrotoDlasts from the Swedish rapeseed l i n e , O I Q a . Robertson and Earle (1986) reported that 77% of c a l l i derived from l e a f protoplasts of R. oleracea var. i t a l i c a regenerated shoots. Consi~erab-~Tv more d e v i s a l work is requ~red to obtain con,sistent plant regeneration from protoplasts of a l l economically important Brassica species. This is the f i r s t report to d e s c r i ~ - ~ reqeneration from DrotoDlasts of 8. carinata, an amDhidiDloid species. ~ATERIALS AND ~ETHODS Seeds of B. carinata Braun cv. R-2128 were s t e r i l i z e d by w ~ - t t i ~ 0 % ethanol for 30 seconds followed by immersion in f u l l strenqth commercial bleach (5.25% sodium hvpochlorite) for I0 min. The seeds were rinsed several times with sterile, deionized water and germinated on 0 . 8 % aQar containing ~.2% sucrose in the dark for 2 days at 25°C. Approximately 200 hvpocotyl hooks were excised and Dlasmolysed for I h in 5 ml of CPW 13 M solution containing 27.2 mq/l KH2Pn4, 101 mg/l KN03, I~RO ma/l CaCI2.2H2~, 24Q mg/l MQS04, 0.16 mq/l KI, 0.~?~ mn/l CuS~4.~H2~, in 13% mannitol, DH 5.7. SubseQuently they were digested for 16 h in an enzyme mixture containing 2% (w/v) Rhozyme HPI50 (Genencor, New York, USA), 4% Meicelase (Meiji Seika Co., Tokyo, Japan), 0.3% Macerozyme RIO (Yakult Pharmaceutical Industry, Nishinomiya, Japan), 400 mg/l amDicillin, i0 mg/l qentamycin, i0 mg/l tetracyclin in CPW 13 M (DH=5.7) to release protoplasts as reported previously (Chuong et a l . , 1985). The enzyme mixture containing Drotoplasts was f i l t e r e d through 2 layers of nylon screen (a 63 um pore screen on top of a 44 um pore screen) and the protoplasts were collected by c e n t r i f u g a t i o n at 100 x g for 3 min. The orotoDlasts were washed 3 times with CPW 13 M solution and cultured in 2.5 ml modified Nitsch and Nitsch (1967) medium, supplemented with 13% sucrose, 0.4% F i c o l l 400 (Pharmacia Fine Chemicals, UDosala, Sweden), 0.25 mq/l BA, 0.5 mg/l NAA and 0.5 mQ/l 2,4-D in 15 x 60 mm Falcon p l a s t i c ~etri dishes at a density of 2 x 105 cells/ml and incubated at 25"C in the dark. Four weeks a f t e r p l a t i n g , f l o a t i n g m i c r o c a l l i were transferred onto regeneration medium based on MS medium (~urashige and Skooq, 1962) supplemented with
68 3% sucrose, 0.4% agarose (Electrophoresis grade RRL, Gaithersburg, ~D, USA), 200 mg/l casein hydrolysate, 5 mg/l BA and~O.5 mg/l: NAA,-oDH 5.7. The fr~quency:of plant regeneration was scored 4 weeks after the f i r s t subculture onto regeneration medium. In addition, after all floating microcalli were transferred onto regeneration medium, the microcolonies which had sunk were collected with a pipette and placed onto rescue medium Z6, which is based on MS medium supplemented with 3% sucrose, 0.5% agarose, 0.8 mg/l zeatin, 0.1 mq/l IAA, DH 5.7. To obtain Dlantlets frm the abnormal structures, which sometimes arose from the calli on the regeneration medium, t h e y were transferred onto RSM medium which contaiDs MS basal ingredients olus 200 m g / l myoinositol, 2 mq/l nicotinic acid, 2 mn/l Dvridoxine HCI, I0.5 mq/l thiamine HCI, 1.10 mq/l BA, 0.~2 mQ/l IAA, 30 nil sucrose and 7 ~/l aqar, pH 5.7 RESULTS AND DISCUSSION
Protoplast Division and Callus Development Immediately. after plating, most of the hypocot~l ~rotoDlasts rose to the surface of the medium (Fiq.1). A small fraction of small Drotoplasts with dens.~e cytoplasm as well as broken cells and cell fragments, sank to the bottom of the culture dishes. Cytoplasmic streaming was readily apparent in most of the viable Drotoplasts. During the f i r s t few days, protoplasts qradually increased in volume and sometimes became irregularly
shaped. F i r s t d i v i s i o n s were observed a f t e r 2-3 days in c u l t u r e ( F i g . 2} and second d i v i s i o n s u s u a l l y occured a f t e r 4~5 days~ ( F i g . . 3 } . Cell ~ d i v i s i o n frequencies in the ~ r o t o D l a s t c u l t u r e s ranged from 10-15% at day f i v e . Only the s w o l l e n , small or medium sized p r o t o p l a s t s underwent d i v i s i o n . Cells which became extremely large did not divide. Non-vacuolated, densely cytoplasmic D r o t o p l a s t s t h a t sank to the bottom of the c u l t u r e dishses only occasionally divided (approx. 2%}. The s i n k i n g p r o t o p l a s t s t h a t d i v i d e d g e n e r a l l y stopoed a f t e r the second or the t h i r d d i v i s i o n . Some n o n - d i v i d i n g protoplasts, e s p e c i a l l y those t h a t sank to the bottom, turned brown and lysed to release t h e i r g r a n u l a r contents i n t o the medium. This ohenomenon of browning also occured in f l o a t i n q proto~lasts which underwent 2-3 d i v i s i o n s and then stoDned. C l u s t e r s of 8-20 c e l l s were often observed a f t e r 7-10 days ( F i g . 4 } . Four weeks a f t e r i n i t i a t i n g the cultures, microcalli 400-800 um in size were o b t a i n e d . These were l a r g e enough to be t r a n s f e r r e d onto r e g e n e r a t i o n medium. Microcalli t h a t were c l o s e l y c l u s t e r e d due to the uneven d i s t r i b u t i o n of p r o t o p l a s t s on the surface of the medium stayed s m a l l e r than those derived from D r o t o p l a s t s well separated in the c u l t u r e p l a t e . T y p i c a l l y 1000-3000 m i c r o c a l l i developed on the surface of the medium in each o l a t e a f t e r 4 weeks ( F i g . 5 } . This corresponds to an o v e r a l l p l a t i n g e f f i c i e n c y between 0.2-0.6% in these c u l t u r e s . The f l o a t i n g c u l t u r e method has been w i d e l y used f o r anther c u l t u r e IKao, 1981; L i t c h e r , 1981; K e l l e r LEGENDS
~
-I~-~;~,I~ ~i!~:
Fig. i: Freshly i s o l a t e d p r o t o p l a s t s from seedlings of Rrassica c a r i n a t a cv. 2128~50" urn. F i n . 2: F i r s t d i v i s i o n a f t e r 3 days in c u l t u r e . Rat : 50 Umo Fi~. 3: Second d i v i s i o n a f t e r 5 days in c u l t u r e . Rat = 50 um. F i g . 4: C l u s t e r of c e l l s a f t e r 10 days in c u l t u r e . Rar = 50 um. F i g . 5: Protopl a s t - d e r i v e d microcalli 4 weeks a f t e r culture. I~ar : 1000 urn. F i g . 6: P l a n t l e t reqenerated from protopl ast-derived callus. Bar : 5 ram. F i g . 7: Abnormal s t r u c t u r e regenerated from cal I us. Bar : 5 ram. F i g . 8: Rescue of abnormal s t r u c t u r e regenerated from callus after subculturinq on RSM medium showing the i n d u c t i o n of many shoots. Rat = I0 mm. F i g . 9: Protopl a s t - d e r i v e d mature p l a n t of ~. c a r i n a t a .
69 and ArmstronQ, 19R3; Dunwell et a l . , lq@5; Cardv, (19R6). Kao (IQRI) added Fico-l-I -To harley anther c u l t u r e media to suDoort the anthers plated onto the surface of the l i n u i d and reported a s i g n i f i c a n t enhancement of c a l l i production from pollen which was a t t r i b u t e d to enhanced qas exchanae. Recently a modified B. napus microspore c u l t u r e medium ( L i t c h e r , 19R2} w a s - - u s ~ cul%ure Rrassica protoplasts at the a i r / l i n u i d interface ( C h U g et a l . , 1985). The present report indicates a ~mi~iar procedure is e f f e c t i v e in inducing sustained d i v i s i o n and callus development in B. carinata protoplasts.
P l a n t l e t Reqeneration. M i c r o c a l l i transferred onto regeneration medium developed into c a l l i , 2-3 mm in size, and plant regeneration occurred w i t h i n 4 weeks. Twenty one percent of the c a l l i oroduced plants. Plant reqeneration was observed only from c a l l i that developed from f l o a t i n g Protoplasts. None of the calli derived from rescued submeraed m i c r o c a l l i reqenerated into plantlets after transfer to reqeneration medium. Plant reaeneration could be categorized as complete reoeneration of a perfect shoot (Fla. 6) or abnormal reqeneration of structures that did not resemble any normal Dlant orqan (Fiq. 7). Transfer of complete shoots renenerated from callus onto hormone-free MS medium induced rootinq p r i o r to transolantina to neat pellets. Abnormal structures could be induced to form normal shoots by subculturina them onto RS~¢ medium (Fig. 8). Shoots resultinq from these l a t t e r treatments also formed roots after transfer to hormone-free MS medium. Plantlets were acclimatized to growth room conditions and were transplanted into Dots to Qrow to maturity (Fie. 9).
The results of the present study increase the possibilities for in v i t r o manipulation of the Brassicaceae by demo~trat-~g t o t i p o t e ~ t c y in another species of the genus.
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