Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake
Descripción
PlantCeU Reports
Plant Cell Reports (1991) 10:375-379
9 Springer-Verlag 1991
Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake Arundhati Mukhopadhyay 1, Reinhard TSpfer z, Akshay K. Pradhan i, Yaspal S. Sodhi 1,
Hans-Henning Steinbifl 2, Jeff ScheU 2, and Deepak Pental 1 1 Tara Energy Research Institute, 90 Jot Bagh, New Delhi 110 003, India 2 Max-Planck-Institut ffir Zfichtungsforschung, Carl-von-Linn6-Weg I0, W-5000 KSln, F R G Received June 11, 1991/Revised version received July 17, 1991 - Communicated by H. LSrz
Summary. E f f i c i e n t r e g e n e r a t i o n (80%) and high f r e q u e n c y g e n e t i c t r a n s f o r m a t i o n (10-33%) were a c h i e v e d by culturing protoplasts isolated from hypocotyl t i s s u e s of six day old B r a s s i c a o l e r a c e a s e e d l i n g s and by s u b j e c t i n g t h e s e p r o t o p l a s t s to PEG m e d i a t e d d i r e c t plasmid uptake. Three different plasmid vectors c a r r y i n g marker g e n e s for r e s i s t a n c e to m e t h o t r e x a t e (dhfr), hygromycin ( h p t ) and p h o s p h i n o t r i c i n (bar) were c o n s t r u c t e d and u s e d for t r a n s f o r m a t i o n . Large number of normal, f e r t i l e t r a n s f o r m a n t s were o b t a i n e d with v e c t o r s c a r r y i n g h p t and b a r genes. No t r a n s formants could be regenerated for resistance to methotrexate as it severely suppressed shoot differentiation.
bialaphos resistance gene/ phosphinotricin acetyltransferase; 2,4-D 2,4-dic h l o r o p h e n o x y a c e t l c acid; dhfr/DHFR dihydrofolate reductase gene/enzyme; gus/GUS p-glucuronidase gene/enzyme, hpt/HPT - hygromycin phosphotransferase g e n e / e n z y m e ; Kn - k i n e t i n ; PEG - p o l y e t h y l e n e glycol; RH - r e l a t i v e humidity. Abbreviations.
bar~PAT
-
The s u c c e s s of g e n e t i c t r a n s f o r m a t i o n by d i r e c t plasmid u p t a k e d e p e n d s on t h e a v a i l a b i l i t y of a s u i t a b l e protocol for p l a n t r e g e n e r a t i o n from p r o t o plasts. Several papers describe plant regeneration from p r o t o p l a s t s of B. o l e r a c e a with v a r y i n g degree of s u c c e s s (Xu e t al. 1982, Glimelius 1984, R o b e r t s o n and Earle 1986, Lillo and Olsen 1989, Yamashita and Shimamoto 1989, J o u r d a n e t al. 1990, Kao e t al. 1990). As d i f f e r e n t i a t i o n i n v i t r o is g e n e r a l l y known to be genotype-specific and B. oleracea types are no e x c e p t i o n ( J o u r d a n and Earle 1989, J o u r d a n e t al. 1990), it is d e s i r a b l e to d e t e r m i n e an e f f i c i e n t r e g e n e r a t i o n protocol for t h e s p e c i e s to be worked upon. In t h i s p a p e r we r e p o r t high f r e q u e n c y r e g e n e r a t i o n and g e n e t i c t r a n s f o r m a t i o n t h r o u g h d i r e c t DNA u p t a k e by h y p o c o t y l p r o t o p l a s t s of B r a s s i c a oleracea var. Early Kunwari, an e a r l y t y p e h e a t t o l e r a n t c a u l i flower, s u i t a b l e for s u b t r o p i c a l climate. We h a v e u s e d t h r e e d i f f e r e n t plasmid v e c t o r s c a r r y i n g g e n e s t h a t c o n f e r r e s i s t a n c e to m e t h o t r e x a t e (dhfr), hygromycin (hpt) and p h o s p h i n o t r i c i n (bar) and compared t h e r e l a t i v e e f f i c i e n c y of t h e s e m a r k e r s for t h e s e l e c t i o n of t r a n s f o r m e d cell colonies and p l a n t s .
Key words: B r a s s i c a o l e r a c e a - Direct gene t r a n s f e r Protoplast regeneration - Transformation M a t e r i a l s and m e t h o d s Introduction
The g e n u s Brassica includes m a n y economically important vegetable and oilseed crops amongst which B. oleracea constitutes one of the largest group of vegetables, having a worldwide distribution. For the improvement of yield and quality of different Brassics species, the classical methods of plant breeding are being supplemented with in v i t r o techniques and genetic manipulation. Genetic manipulation can be achieved either t h r o u g h transformation using Agrobacterium based vectors or by direct uptake of plasmid DNA. Direct gene transfer has been reported to yield normal, fertile transformed plants in many species, e . g . tobacco (Hain et al. 1985, Potrykus et al. 1985), maize (Rhodes et al. 1988), Arabidopsis (Datum et al. 1989), rice (Shimamoto et al. 1989, Datta et al. 1990). However, amongst Brassica species normal, f e r t i l e t r a n s f o r m e d p l a n t s h a v e so f a r b e e n r e p o r t e d only in B. n a p u s (Golz e t al. 1990, Guerche e t al. 1987, 1990), w h e r e t r a n s f o r m e d p l a n t s were r e c o v e r e d a t low f r e q u e n c i e s .
O f f p r i n t r e q u e s t s to: A. Mukhopadhyay
Plasmid constructions. Plasmid vectors were constructed following the cloning techniques given by Sambrook et al. (1989). The dhfr gene was taken as a XbaI-NsiI fragment of pFR400 (Simonsen and Levinson 1988), treated w i t h Sl nuclease and cloned into the expression vector pRTI00 (Tbpfer et al. 1987) which was digested with NcoI-Xbal and made blunt-end by SI nuclease treatment. The recombinant plasmid was named pRTl00dhfr. For the hpt gene a 1055 bp BamHI fragment from pHYMI8 (B. Nelsen-Salz, unpublished) was treated w i t h SI nuclease and cloned into pRTI00 restricted with NcoI-Xbal and treated with S1 nuclease resulting in plasmid pRTl00hpt. The gene encoding phosphinotricin acetyltransferase (PAT) was subcloned in pRTI04 (Tbpfer et al. 1987) as a NcoI-BamHI fragment, subsequently restricted with BamHI-XbaI (the 5' p r o t r u d i n g e n d s were c o n v e r t e d to b l u n t e n d s by S1 n u c l e a s e t r e a t m e n t ) p r o v i d i n g t h e plasmid pRT103pat. F i n a l l y , to p r o d u c e t h e v e c t o r s pRT58, pRT66 and pRT77 respectively, the selectable marker genes together w i t h t h e i r p o l y - A s i g n a l s were t a k e n as X h o I - P s t I f r a g m e n t s from pRT100dhfr, pRT100hpt and pRT103pat (pRT100hpt was p a r t i a l l y c u t w i t h PstI) and cloned
376 pRTSSgusR (6.6kb)
p,~)'66gusR ( "]I kb)
pRT??QusR (6 Skb)
Table 1. Media regeneration. medium
used
basalcomposition
for
protoplast
hormones
culture
and
reference
pm~0plastcuitu~
Ps~
FOl
ho + 04 } glurose
2,4-D(1O rag/l) + 1(~(I_~mg/ll
Glime~us(1984)
EL'2
ho + OJ [ ~ucrose
2,4-D([.0rag/l) + Kn(LO m~)
Glime~us(!984)
PO~
K3 + 0,25~ agarose + 0,! M sucrose
2,4-D(I0 rag/l) § l(n(l0 rag/l)
Nagy and MaHga (IH6)
F04
~S + 0.8 ~ ~g~t
2,4-I)((.0mdt) + Kr,(l.Omgl~)
+ 3 ~ s~crose
Fig. 1. S c h e m a t i c diagram of pRT55gusR, pRT66gusR and pRT77gusR vectors used for transformation. The plasmids carry the reporter genes coding for ~ - g l u c u r o n i d a s e (GUS) combined with r e s i s t a n c e g e n e s e n c o d i n g d i h y d r o f o l a t e r e d u c t a s e (DHFR), h y g r o m y c i n p h o s p h o t r a n s f e r a s e (HPT) or p h o s p h i n o t r y c i n a c e t y l transferase (PAT), r e s p e c t i v e l y . Both t h e reporter g e n e and t h e r e s i s t a n c e gene are u n d e r t h e c o n t r o l of t h e 35 S RNA p r o m o t e r of c a u l i f l o w e r mosaic v i r u s (CaMV) ( d o t t e d a r e a ) and i t s c o r r e s p o n d i n g p o l y - A s i g n a l s e q u e n c e ( h a t c h e d area). Bar r e p r e s e n t s 100bp.
into v e c t o r pRT99 (Tbpfer et al. 1988a). Based on these three vectors, constructs for direct gene t r a n s f e r (pRT55gusR, pRT66gusR, pRT77gusR; F i g . l ) were made by cloning t h e ~ - g l u c u r o n i d a s e (gus) g e n e from pRT103gus (TOpfer et el. 1988b) as a PstI f r a g m e n t i n t o t h e Nsi] s i t e of t h e v e c t o r s .
P r o t o p l a s t i s o l a t i o n , c u l t u r e a n d p l a n t r e g e n e r a tlon. Seeds of BrasMca oler~ce~ var. E a r l y Kunwari were g e r m i n a t e d a s e p t i c a l l y on MS (Murashige and Skoog t962) b a s a l medium in d a r k for two d a y s , and t h e n t r a n s f e r r e d to l i g h t (200 ~Em-2s-L 16h p h o t o p e r i o d ) and m a i n t a i n e d at 25"C. P r o t o p l a s t s were i s o l a t e d from h y p o c o t y l e x p l a n t s of 4 - 7 day old s e e d l i n g s . Media u s e d for p r o t o p l a s t c u l t u r e and r e g e n e r a t i o n are l i s t e d in Table 1. Hypocotyl e x p l a n t s were f l o a t e d in CPW ( F r e a r s e n e t al. 1973) c o n t a i n i n g 9% m a n n i t o l (cPWeM), cut i n t o 0 . 5 - 1 . 0 mm s e g m e n t s , p l a s m o l y s e d for l h and t h e n p u t for o v e r n i g h t enzyme i n c u b a t i o n a t 25~ w i t h slow s h a k i n g (50 rpm). The enzyme s o l u t i o n c o n t a i n i n g 1% Cellulase R - 1 0 and 1% Macerozyme R - 1 0 (Yakult H o n s h a Co. Ltd.), 1 mg/i 2 , 4 - D and 1 mg/i k i n e t i n (Sigma) was p r e p a r e d in cPWeM (40 rag/1 t e t r a cyclin, 20 mg/1 ampicillin, 20 mg/1 g e n t a m y c i n were i n c l u d e d p r o p h y l a c t i c a l l y to p r e v e n t b a c t e r i a l c o n t a m i n a t i o n s ) . I s o l a t e d p r o t o p l a s t s were s i e v e d t h r o u g h 0.16 mm mesh, w a s h e d twice in CPW9M, c e n t r i f u g e d (580 x g, 10 rain.) and r e s u s p e n d e d in 2 ml of cPWeM. P r o t o p l a s t s u s p e n s i o n was g e n t l y o v e r l a y e r e d on CPW 18S (18% s u c r o s e ) and c e n t r i f u g e d (400 x g, 10 rain.). Purified protoplasts, gathered as a b a n d at the i n t e r f a c e , were h a r v e s t e d , w a s h e d once and c u l t u r e d in PCI medium a t a d e n s i t y of 3-5x104/ml in 55 mm petridishes (Costar) in dark. Division frequencies ( t h e r a t i o of t h e n u m b e r of p r o t o p l a s t s d i v i d i n g to t h e t o t a l p r o t o p l a s t s p l a t e d ) were r e c o r d e d on t h e 5 th day of culture. After I0 days of culture initiation, PCI medium was progressively diluted w i t h 0.7 m] of PC2 medium every third day over a period of 9 days, The microcolonies w e r e then plated on PC3 medium. Cultures w e r e maintained at low light intensity (60 ~Em-2s-x) for 4 weeks and then transferred t o PC4 medium and kept at a light intensity of 200 ~Em-2s-I.
shootregene~tio~ !
PC@ [
{
MS+O.SWagar
Kn(lO m~/l)
Murmshige~,Skoog(1962)
+ 3 ~ sucrose
rootingof shoots i
PC6
[
{
Mg+0.8~agar
Wumhige &Sk00g(!962)
+ ! ~{ Sllcfose
A f t e r a n o t h e r 4 - 5 weeks t h e growing c o l o n i e s t r a n s f e r r e d to PC5 medium for s h o o t r e g e n e r a t i o n .
were
R e g e n e r a t e d s h o o t s were r o o t e d on PC6 medium. Shoots regenerated from p u t a t i v e transformed calli were r o o t e d u n d e r s e l e c t i o n p r e s s u r e in t h e p r e s e n c e of appropriate antibiotics (see below). P l a n t l e t s (3-5 cm) were t r a n s f e r r e d to a p o t t i n g mix ( a u t o c l a v e d soil : p e a t m o s s in 1:1 ratio) and were m a i n t a i n e d in a growth c h a m b e r a t 10h/15"C and 14h/8~ cycle with 90% r e l a t i v e h u m i d i t y (RH), A f t e r one m o n t h t h e growth c o n d i t i o n s were c h a n g e d to 14h/25r l i g h t and 10h/15~ dark cycle with 60% RH. S e e d s were o b t a i n e d by bud pollination.
Uptake of plasmid DNA and
transformation frequency.
For plasmid uptake, freshly harvested protoplasts were washed once and suspended in W5 medium (Menczel et al. 1981). Vector pRT66gusR was linearized w i t h HindIII, whereas Xbal was used for pRT55gusR and pRT77gusR. The PEG m e d i a t e d DNA d e l i v e r y was p e r f o r m e d a c c o r d i n g to Saul e t el. (1988) u s i n g 40 ~l of DNA s o l u t i o n containing 12 ~g o f I i n e a r i z e d t r a n s f o r m i n g DNA. C o n t r o l s were s e t w i t h u n t r e a t e d p r o t o p l a s t s and t h o s e t r e a t e d with only PEG in t h e a b s e n c e of plasmid DNA. The D N A - t r e a t e d p r o t o p l a s t s were c u l t u r e d as t h e u n t r e a t e d p r o t o p l a s t s . S e l e c t i o n was a p p l i e d 6 - 7 weeks after t h e i n i t i a t i o n of c u l t u r e . According to t h e plasmid used for t r a n s f o r m a t i o n , calti of 1 to 2 mm in size were t r a n s f e r r e d to PC4 medium c o n t a i n i n g 0.01 ~g/ml m e t h o t r e x a t e , 10 ~g/ml hygromycin, and 5 ~g/ml p h o s p h i n o t r i c i n ( k i n d l y p r o v i d e d by H o e c h s t AG), r e s p e c t i v e l y . S u r v i v i n g c o l o n i e s were s u b s e q u e n t l y m a i n t a i n e d on r e s p e c t i v e s e l e c t i o n media d u r i n g f u r t h e r growth and regeneration. To c a l c u l a t e t r a n s f o r m a t i o n f r e q u e n c i e s , calli (< 1 mm d i a m e t e r ) d e r i v e d from D N A - t r e a t e d p r o t o p l a s t s growing on n o n - s e l e c t i v e PC3 medium were picked i n d i v i d u a l l y a n d t r a n s f e r r e d to s e l e c t i o n p l a t e s and s c o r e d . The n u m b e r of growing c o l o n i e s were c o u n t e d a g a i n 4 weeks a f t e r t h e t r a n s f e r . The r e l a t i v e f r e q u e n c y of t r a n s f o r m a t i o n (RTF) was e x p r e s s e d as:
377
number of c o l o n i e s s u r v i v i n g s e l e c t i o n x 100 RTF = number of c o l o n i e s t r a n s f e r r e d to s e l e c t i o n
Analysis
of transformants.
DNAs
were
extracted
Table 2. Division f r e q u e n c y of u n t r e a t e d , P E G - t r e a t e d and PEG+ v e c t o r DNA (pRT66gusR) t r e a t e d p r o t o p l a s t s as c o u n t e d a f t e r 5 d a y s of p l a t i n g . 5x104 p r o t o p l a s t s were u s e d for e a c h t r e a t m e n t .
from
l e a v e s of p u t a t i v e t r a n s f o r m a n t s following D e l l a p o r t a e t al. (1984), p u r i f i e d on CsC1 d e n s i t y g r a d i e n t , and d i g e s t e d with r e s t r i c t i o n e n d o n u c l e a s e s a c c o r d i n g to the m a n u f a c t u r e r ' s i n s t r u c t i o n . Spermidine (0.01 M) was a d d e d to e n s u r e p r o p e r r e s t r i c t i o n of t h e DNAs. R e s t r i c t e d DNAs were e l e c t r o p h o r e s e d on 0.8% or 1.0% a g a r o s e gels and b l o t t e d onto n y l o n membrane ( H y b o n d N, Amersham). Probes were l a b e l l e d with [32PadCTP] u s i n g multiprime l a b e l l i n g kit (Amersham). H y b r i d i z a t i o n was done o v e r n i g h t a t 420C in t h e p r e s e n c e of 50% formamide (Perbal 1988). Hybridized filters were w a s h e d u n d e r s t r i n g e n t c o n d i t i o n s (2 x 15 rain. a t room t e m p e r a t u r e and 1 x 15 rain. a t 65"C in 2 x SSC, 0 . 1 % SDS; l x 30 rain. and 1 x 10 min. a t 65 oC in O.2 x SSC, 0 . 1 % SDS) and e x p o s e d to Kodak X-OMAT AR films. Pieces of calli growing on s e l e c t i o n media were s t a i n e d for {3-glucuronidase a c t i v i t y following J e f f e r son (1987) and s c o r e d for t h e p r e s e n c e of blue colour. Leaf disc a s s a y s were c a r r i e d out on PC4 medium c o n t a i n i n g 0, 10, 20, 50, and 100 {Jg/ml h y g r o m y c i n or 0, 5, 10, 20, and 50 {Jg/ml p h o s p h i n o t r i c i n to t e s t t h e r a n g e of t o l e r a n c e of t h e t r a n s f o r m e d p l a n t s to t h e selection agents.
untreated exp no.
no. of dividL~g pr0topiasts ~
I 2 3 4 6 6 ?
1.85xI04 !.66x!0~ 1,66xI04 !.50x104 1.76xI04 1.56xI0~ 1,66xi0'
PE + DNA treated
PEG treated
d[v~inn frequency (~)
no, of division dividing frec!ueney protoplasts, (~) 1.40x10~ L20xl0* h06xl0* 1.20x10' !.16x10' !.10xl0* 1,10xl0~
07 33 ~3 30 36 31
~2
no. of dividing protopiasts *
28 24 21 24 23 22 22
division frequency (~)
0.60x]04 0,60x10~ 0.45xI0' 0,6~x10W 0.70x104 0,60xi0' 0.65xi0'
12 I0 9 II !4 12 II
B.
oleraeea
a averageof ten readings
T a b l e 3. T r a n s f o r m a t i o n f r e q u e n c i e s of p r o t o p l a s t s u s i n g d i f f e r e n t plasmid v e c t o r s . piasmid
selectionfor
vector
pRT55gesR methotrexate Results
exp. no,
no. of colonies transferred to selectionmedia
no. of colonies after fourleeks of selection
1107
330 468 842
! 2 0
1260 !086
I 2 3
1314 1!70 !224
!06
i 2 3
!080 1125 1224
!26 279 262
transformation frequency(~)
Protoplast culture and plant regeneration pRT66gusR hygromycin
H e a l t h y , v i a b l e p r o t o p l a s t s could be i s o l a t e d only from h y p o c o t y l t i s s u e s of six day old s e e d l i n g s . Any variation in t h e age of t h e s e e d l i n g s d r a s t i c a l l y r e d u c e d t h e yield. A d d i t i o n of h o r m o n e s of PC1 medium in t h e enzyme s o l u t i o n a d v a n c e d t h e o n s e t of d i v i s i o n of p r o t o p l a s t s from 7 2 - 9 6 h to 2 4 - 4 8 h . Optimum d i v i s i o n f r e q u e n c y of p r o t o p l a s t s was 33% when p l a t e d a t a density of 3-5x104/ml. Periodic dilutions of t h e c u l t u r e medium with PC2 medium h e l p e d in b e t t e r growth and s u r v i v a l of t h e microcolonies. In t h e a b s e n c e of s u c h d i l u t i o n s , a c c u m u l a t i o n of p h e n o l i c compounds was o b s e r v e d in t h e medium l e a d i n g to d r a s t i c r e t a r d a t i o n of t h e growth of t h e microcolonies. The s e q u e n t i a l t r a n s f e r of p r o t o c a l l i to PC3 and PC4 media and t h e g r a d u a l e x p o s u r e to s t r o n g e r l i g h t also h e l p e d in b e t t e r growth of t h e calli. Colonies ( 2 - 3 ram), when t r a n s f e r r e d to t h e r e g e n e r a t i o n media, d i f f e r e n t i a t e d m u l t i p l e s h o o t s in a b o u t 80% of t h e c u l t u r e s in 3 - 4 weeks. T h e s e s h o o t s were r o o t e d with 100% e f f i c i e n c y on PC6 medium w i t h i n 7 d a y s of t r a n s f e r . P l a n t s t r a n s f e r r e d to g r o w t h c h a m b e r s showed normal v e g e t a t i v e growth, flowering and s e e d s e t . M a i n t a i n i n g high RH (90%) a t i n i t i a l v e g e t a t i v e g r o w t h s t a g e was found to be n e c e s s a r y , as otherwise the plants remained significantly retarded.
Genetic transformation T r e a t m e n t of p r o t o p l a s t s with PEG r e d u c e d t h e d i v i s i o n f r e q u e n c y by an a v e r a g e of 10% compared to t h e u n t r e a t e d c o n t r o l s . A d d i t i o n of DNA along w i t h PEG further lowered t h e d i v i s i o n f r e q u e n c y (Table 2). A d d i t i o n of DNA s o l u t i o n in e x c e s s of 50 {Jl was h i g h l y deleterious. Sensitivity
assay
of
the
microcolonies
derived
pRT77gusR phosph[n0tricin
!!7 144
n o t r i c i n . Hence, for t h e s e l e c t i o n and m a i n t e n a n c e of the transformed calli 0.01 {Jg/ml methotrexate, 10 tJg/ml hygromycin and 5 ~g/ml p h o s p h i n o t r i c i n were used. Colonies d e r i v e d from u n t r e a t e d or only PEGtreated p r o t o p l a s t s did n o t grow on a n y of t h e s e l e c t i o n p l a t e s . The t h r o u g h p u t of r e s i s t a n t c o l o n i e s t h a t s u r v i v e d on s e l e c t i o n media was f o u n d to be 33%, 10% and 22% for m e t h o t r e x a t e , hygromycin and p h o s p h i n o t r i c i n , r e s p e c t i v e l y (Table 3). Colonies t r a n s f o r m e d for all t h e t h r e e m a r k e r g e n e s grew a t r a t e s similar to t h a t of t h e u n t r a n s f o r m e d colonies. When t r a n s f e r r e d to PC5 medium c o n t a i n i n g respective selection agents, colonies resistant to h y g r o m y c i n and p h o s p h i n o t r i c i n r e g e n e r a t e d a t f r e q u e n cies c o m p a r a b l e to t h e r e g e n e r a t i o n from n o n - t r e a t e d calll. D i f f e r e n t i a t i o n , h o w e v e r , was d e l a y e d by 7 - 1 0 days. T h e s e s h o o t s grew n o r m a l l y and were r o o t e d with 100% e f f i c i e n c y in the presence of appropriate s e l e c t i o n a g e n t s . P r e s e n c e of m e t h o t r e x a t e , h o w e v e r , s e v e r e l y i n h i b i t e d d i f f e r e n t i a t i o n and d e v e l o p m e n t of s h o o t s . A l t h o u g h a b o u t 35% of t h e c u l t u r e s s h o w e d d i f f e r e n t i a t i o n of small s h o o t - l i k e p r o j e c t i o n s , n o n e of these developed further. Even withdrawal of m e t h o t r e x a t e a t t h i s s t a g e f a i l e d to i n d u c e f u r t h e r d e v e l o p m e n t of s h o o t s .
from
non-treated protoplasts showed that the growth of the colonies was completely inhibited by 0.005 ~g/ml methotrexate, 8 ~g/ml hygromycin and 3 tJg/ml phosphi-
In t o t a l , 98 p l a n t s c a r r y i n g hpt a n d 61 p l a n t s c a r r y i n g bar m a r k e r g e n e s were t r a n s f e r r e d to p o t s . Of t h e s e , 70% and 93% of t h e p l a n t s w i t h hpt and b a r
378
Fig. 2. S o u t h e r n a n a l y s i s of p h o s p h i notricin resistant transgenic plants. Genomic DNAs were c u t w i t h X h o I - P s t I r e l e a s i n g a 790 bp f r a g m e n t ( b a r g e n e and t e r m i n a t o r ) a n d p r o b e d w i t h 3~p l a b e l l e d bar coding region. Lane 1, DNA of a u n t r a n s f o r m e d p l a n t + 50 pg of probe DNA; l a n e 2, DNA of a u n t r a n s f o r m e d p l a n t and l a n e 3 - 7 , DNA from f i v e t r a n s g e n i c p l a n t s .
Fig. 3a,b. S o u t h e r n blot a n a l y s i s of t o t a l DNA i s o l a t e d from h y g r o m y c i n r e s i s t a n t t r a n s g e n i c p l a n t s . DNAs were d i g e s t e d w i t h (a) XhoI-PstI ( r e l e a s i n g a 400 bp and a 900 bp f r a g m e n t , r e s p e c t i v e l y ) and (b) HindlII ( c u t t i n g in t h e p l a n t DNA and once in t h e c o n s t r u c t ) . The blot was probed w i t h 32p l a b e l l e d s t r u c t u r a l hpt gene. Lane 1, DNA of a u n t r a n s f o r m e d p l a n t + 50 pg of probe DNA; l a n e 2, DNA of a u n t r a n s f o r m e d p l a n t and lane 3 - 8 , DNA from six transgenic plants.
m a r k e r s , r e s p e c t i v e l y , were f o u n d to be f e r t i l e and formed s e e d upon s e l l i n g . However, pollen p r o d u c t i o n in some of t h e s e p l a n t s was lower t h a n t h a t in n o n t r a n s f o r m e d f e r t i l e p l a n t s . Seeds o b t a i n e d from some of t h e t r a n s f o r m e d p l a n t s a f t e r c r o s s p o l l i n a t o n with pollen from another flower of the same plant g e r m i n a t e d and grew on media c o n t a i n i n g t h e s e l e c t i o n a g e n t s . D e t a i l e d a n a l y s i s on s e g r e g a t i o n p a t t e r n is b e i n g c a r r i e d out.
t e s t e d . P l a n t s t r a n s f o r m e d w i t h bar gene s h o w e d normal c a l l u s g r o w t h on 5 ~g/.ml p h o s p h i n o t r i c i n c o n c e n t r a t i o n at which colonies were selected initially, but displayed variability in the extent of callus formation at higher concentrations. GUS a s s a y was c a r r i e d out on calli o b t a i n e d from l e a f t i s s u e s of 51 p l a n t s t r a n s f o r m e d with pRT66gusR and grown on s e l e c t i o n media. C o e x p r e s s i o n f r e q u e n c y was o b s e r v e d to be 80~.
Confirmation of transformation Total genomic DNAs were isolated from f i v e of phosphinotricin and six of hygromycin resistant primary regenerants and digested with XhoI-PstI. Hybridization of restricted DNAs to radiolabelled BamHI f r a g m e n t s of s t r u c t u r a l b a r g e n e (580 bp) from pRT101pat (T6pfer, u n p u b l i s h e d ) a n d hpt gene (1055 bp) from pHYM13 r e s p e c t i v e l y , s h o w e d t h e p r e s e n c e of m a r k e r g e n e s in t h e t r a n s f o r m e d p l a n t s (Fig. 2, 3a) a t t h e e x p e c t e d p o s i t i o n s (790 bp f r a g m e n t s for b a r gene a n d 920 bp and 360 bp for hpt gene, F i g . l ) . In addition, two out of t h e f i v e pat t r a n s f o r m a n t s ( n u m b e r s 4 (weak s i g n a l a t 2.3 kb) and 5 in Fig. 3a) and all t h e hpt t r a n s f o r m a n t s showed t h e p r e s e n c e of 1 - 7 a d d i t i o n a l f r a g m e n t s of h i g h e r m o l e c u l a r weight, i n d i c a t i n g p l a s m i d r e a r r a n g e m e n t - p r i o r to or during i n t e g r a t i o n , When DNAs from t h e same p l a n t s were d i g e s t e d with HindIII (which c u t s t h e v e c t o r once o u t s i d e t h e s t r u c t u r a l g e n e s ) and h y b r i d i z e d to t h e same p r o b e s , t h e hpt t r a n s f o r m a n t s s h o w e d one to m u l t i p l e copy (Fig. 3b) and b a r t r a n s f o r m a n t s s h o w e d one or two copy i n t e g r a t i o n ( s ) per p l a n t genome. DNA from n o n - t r a n s f o r m e d p l a n t did n o t h y b r i d i z e to t h e l a b e l l e d probe. Leaf disc a s s a y s were c a r r i e d o u t on 51 p l a n t s t r a n s f o r m e d with hpt and 13 p l a n t s w i t h b a r g e n e s . All the plants carrying hpt gene, produced callus u n i f o r m l y on all t h e c o n c e n t r a t i o n s of h y g r o m y c i n
Discussion P r o t o p l a s t s h a v e b e e n i s o l a t e d and r e g e n e r a t e d from d i f f e r e n t t i s s u e s in B. oleracea. Of t h e s e , h y p o c o t y l s h a v e b e e n d e s c r i b e d as t h e most s u i t a b l e e x p l a n t (Glimelius 1984, Y a m a s h i t a and Shimamoto 1989). We h a v e d e v e l o p e d a simple p r o t o c o l for high f r e q u e n c y (80%) r e g e n e r a t i o n of h y p o c o t y l p r o t o p l a s t s by m o d i f y i n g t h e hormone s u p p l e m e n t s u s e d by Glimelius (1984) to optimize c u l t u r e c o n d i t i o n s for t h e v a r i e t y E a r l y Kunwari. Age of t h e e x p l a n t was f o u n d to be c r u c i a l for o b t a i n i n g maximum y i e l d s of p r o t o p l a s t s . Compared to t h e e x i s t i n g r e p o r t s on r e g e n e r a t i o n of h y p o c o t y l p r o t o p l a s t s in B. oleracea (Glimelius 1984, Lillo and Olsen 1989, Y a m a s h i t a and Shimamoto 1989, Kao e t al. 1990) our protocol shows a considerably higher f r e q u e n c y of r e g e n e r a t i o n . T r a n s f o r m a t i o n f r e q u e n c y is c a l c u l a t e d e i t h e r on t h e b a s i s of t h e n u m b e r of p r o t o p l a s t s i n i t i a l l y c u l t u r e d ( a b s o l u t e t r a n s f o r m a t i o n f r e q u e n c y ) or as a r a t i o of t h e n u m b e r of c o l o n i e s growing on s e l e c t i o n media to t h o s e on n o n - s e l e c t i o n media ( r e l a t i v e t r a n s f o r m a t i o n f r e q u e n c y , RTF). As t h e P E G - m e d i a t e d d i r e c t DNA u p t a k e s e v e r e l y a f f e c t s t h e v i a b i l i t y of p r o t o p l a s t s ( p r e s e n t s t u d y , Datum e t al. 1989, D a t t a e t al. 1990), i t is d e s i r a b l e to employ RTF c a l c u l a t i o n to o b t a i n more accurate d a t a u n d e r s u c h c o n d i t i o n s . In t h e p r e s e n t
379 s t u d y we d e f e r r e d t h e a p p l i c a t i o n of s e l e c t i o n to facilitate transfer of individual colonies on s e l e c t i o n media. More t h a n 1000 colonies were s c r e e n e d in t h i s f a s h i o n d u r i n g e a c h e x p e r i m e n t . This method s h o u l d give more a c c u r a t e a s s e s s m e n t of t r a n s f o r m a t i o n f r e q u e n c y , as it a v o i d s t h e p o s s i b i l i t y of m i s c a l c u l a t i o n due to o v e r c r o w d i n g of colonies on t h e n o n selection plates. Our r e s u l t s show t h a t B. oleracea h y p o c o t y l p r o t o p l a s t s can be t r a n s f o r m e d a t high f r e q u e n c i e s (10-33%) u s i n g P E G - m e d i a t e d u p t a k e of plasmid v e c t o r s . This v a l u e is high compared to r e p o r t s on P E G - m e d i a t e d t r a n s f o r m a t i o n f r e q u e n c i e s ( b a s e d on RTF c a l c u l a t i o n ) in other plant species, e. g. Nicotana tabacum (Negrutiu et al. 1987) Arabidopsis thallana (Damm et al. 1989), Brassica n a p u s (Golz e t al. 1990), Zea mays (Rhodes e t al. 1988), Oryza s a t i y a (Yang e t al. 1988) where t h e v a l u e s r a n g e from 0.03% to 5% in dicots and from 0.02% to 2.5% in monocots. The h i g h e s t t r a n s f o r m a t i o n f r e q u e n c i e s (26-28%) for d i r e c t u p t a k e of DNA r e p o r t e d so f a r are in rice (Zhang et al. 1988, u e t al. 1988) o b t a i n e d t h r o u g h e l e c t r o p o r a t i o n of protoplasts. Among t h e t h r e e v e c t o r s u s e d in t h e p r e s e n t s t u d y , we found pRT66gusR and pRT77gusR to be more s u i t a b l e . pRT55gusR could not be u s e d to t r a n s f o r m B. oleracea as it s e v e r e l y s u p p r e s s e d d i f f e r e n t i a t i o n and d e v e l o p m e n t of s h o o t s in v i t r o . However, m e t h o t r e x a t e h a s b e e n r e p o r t e d to be an e f f e c t i v e s e l e c t i o n a g e n t for B. napus (Pua e t al. 1987). Similarly, in c e r t a i n Brassica species kanamycin was found to either i n t e r f e r e or i n h i b i t r e g e n e r a t i o n p r o c e s s (Thomzik and Hain 1990), whereas in others this was used e f f e c t i v e l y for s e l e c t i n g t r a n s f o r m a n t s (De Block et al. 1989, Boulter e t al. 1990). The s e n s i t i v i t y to d i f f e r e n t a n t i b i o t i c s t h u s may v a r y a m o n g s t s p e c i e s or even varieties. S o u t h e r n blot a n a l y s i s and l e a f disc a s s a y s in t h e p r e s e n t s t u d y , r e v e a l e d t h a t t h e r e is no c o r r e l a t i o n b e t w e e n t h e copy n u m b e r and t h e r e s i s t a n c e l e v e l s of t h e t r a n s f o r m a n t s to h y g r o m y c i n or p h o s p h i n o t r i c i n . Similar o b s e r v a t i o n s were made by De Block e t al. (1989) in t h e i r s t u d i e s with B. oleracea and B. n a p e s using Agrobacterlum based vectors. However, in c o n t r a s t to our o b s e r v a t i o n s De Block e t al. (1989) r e p o r t e d s u b n o r m a l c a l l u s f o r m a t i o n in 25% of t r a n s f o r m a n t s on p h o s p h i n o t r i c i n or k a n a m y c i n c o n c e n t r a t i o n s a t which t h e y were o r i g i n a l l y s e l e c t e d . In t h e p r e s e n t s t u d y , all t h e t r a n s f o r m a n t s d i s p l a y e d normal c a l l u s f o r m a t i o n on c o n c e n t r a t i o n s of a n t i b i o t i c s a t which t h e y were s e l e c t e d . Moreover, in all t h e h y g r o mycin transformants, normal callus formation was o b s e r v e d a t t e n t i m e s h i g h e r c o n c e n t r a t i o n (100 ~g/ml) t h a n t h a t u s e d for i n i t i a l s e l e c t i o n . The r a t h e r high t r a n s f o r m a t i o n f r e q u e n c y (>10%) coupled w i t h t h e r e g e n e r a t i o n c a p a c i t y of t h e t r a n s formed calli l e a d i n g to normal f e r t i l e p l a n t s in B. oleracea var. Early Kunwari, could be u s e f u l for the i n t r o d u c t i o n of d e s i r a b l e g e n e s of commercial i n t e r e s t in t h i s crop s p e c i e s .
A c k n o w l e d g e m e n t s . T e c h n i c a l a s s i s t a n c e was p r o v i d e d by Mr. B. S. Yadav. The r e s e a r c h was s u p p o r t e d by a CEC g r a n t n u m b e r ECII-0193-IND(BA) a n d a Rockefeller B i o t e c h n o l o g y F e l l o w s h i p to D.P..
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