Plant Cell Reports (1994) 13:657-660

PlantCell
Reports
9 Springer-Verlag1994

Intergeneric protoplast fusion between Brassica carinata

and Camelina sativa
S.B. Narasimhulu 1, p.B. Kirti 1, S.R. Bhatt 2, Shyam Prakash 1, and V.L. Chopra 1
1 Biotechnology Centre, Indian Agricultural Research Institute, New Delhi-ll0012, India
2 National Bureau of Plant Genetic Resources, New Delhi, India

Received 7 December 1993/Revised version received 11 April 1994- Communicated by G. C. Phillips

Abstract Hybridization attempts between

C a m e l i n a sativa is a wild crucifer that is cultivated Brassicas and Camelina sativa
reported to be resistant to Alternaria have been u n s u c c e s s f u l due to the o c c u r r e n c e
blight. Polyethylene glycol m e d i a t e d fusion of strong reproductive barriers. Because
was attempted between protoplasts from introgression of genes conferring resistance
etiolated h y p o c o t y l s of Brassica carinata to Alternaria blight ranks high among
and mesophyll protoplasts of Camelina breeding objectives for crop Brassicas, we
sativa. The mean frequency of h e t e r o k a r y o n s have attempted somatic hybridization to
was 6.8%. Three hybrid shoots were ~ombine the genomes of B.carinata with
regenerated, each from a single fusion- C.sativa. This paper r e p o r t s results of
these experiments.
derived callus. These shoots failed to
produce roots capable of withstanding
Materials and Methods
transplantation. C o n f i r m a t i o n of h y b r i d i t y
was obtained from the m o r p h o l o g y of in vitro Plant material: The two g e n o t y p e s used in
produced leaves, somatic chromosome---number the protoplast fusion experiment are
in leaf tips, and restriction fragment Brassica carinata (A. Br.) BCR 171 (2n=34)
length polymorphism for a nuclear rDNA and C a m e l i n a sativa (L.) Crantz. (2n=40).
probe. A n a l y s i s for organelle constitution Seed material of C.sativa was kindly
using RFLPs indicated that the hybrid provided by Dr S.S.Banga.
contained chrloroplasts derived from the
wild species and mitochondria from the P r o t o c o l s for the p r e p a r a t i o n of leaf
cultivated Brassica species. protoplasts and protoplast fusion were
described by Kirti et al (1992a).
Abbreviations: 2,4-D, 2 , 4 - d i c h l o r o p h e n o x y - Protoplast fusion product---s we--~e cultured
-acetic acid; IAA, Indole'3-acetic acid; according to the r e g e n e r a t i o n m e t h o d o l o g y
NAA, - N a p h t h a l e n e a c e t i c acid: IBA, Indole-3- described for B.carinata protoplasts
butyric acid; GA 3, g i b b e r e l l i c acid: BAP, 6- (Narasimhulu et al. 1992).
B e n z y l a m i n o p u r i n e : MS, Murashige and Skoog
(1962) basal medium. For the induction of roots on the hybrid
shoots a variety of factors were tested.
Introduction These included: i) six different basal
media, viz., MS, B.(Gamborg et al. 1968), SH
Alternaria blight, caused by Alternaria (Schenk and Hildebrandt 1972), NN (Nitsch
brassicae, is among the most important and Nitsch 1969), Blaydes (Blaydes 1966),
biotic factors reducing yields of crop and White's (White 1954); ii) growth
Brassicas. No source of resistance against regulators IAA, IBA, NAA and G A ~ in
this disease is so far available among the c o n c e n t r a t i o n s ranging from 0.i to i0 ~ g / l ;
Crop Brassicas. iii) modification in concentrations of
Camelina sativa is a wild crucifer that inorganic nitrogen and sucrose: iv)
occurs as an annual weed throughout Europe. modification of osmotic potential using
It has been reported to possess a high mannitol or increased agar concentration; v)
degree of resistance to A l t e r n a r i a blight culture in liquid m e d i u m on filter paper
(Tewari 1991). The a n t i m i c r o b i a l component bridges; vi) MS basal medium of varying
responsible for its resistance has been strengths and vii) in vitro grafting onto
traced to two new phytoalexins, camelexin B.carinata.
and methoxycamelexin. The molecular
structure of these p h y t o a l e x i n s is similar Cytology: Leaf apices from old shoot
to thiabendazole, a commercial fungicide cultures were treated for 1.5 h in a
(Browne et al 1991). saturated solution of p - d i c h l o r o b e n z e n e and
fixed for 24 h in Carnoy's solution.
Mitotic squashes were prepared from leaf
Correspondence to." S. Prakash
658
tips h y d r o l y z e d in IN HCI at 60~ for 7 min combining a h e r b a c e o u s species with a tree
and stained with 1% aceto-orcein. species (Schieder 1980). Grafts in this
case showed several a b n o r m a l i t i e s i n c l u d i n g
DNA a n a l a y s i s was carried out following tumorous outgrowths, While no such
the protocols outlined in Kirti et al. abnormalities were evident in the non-
(1992a). The probes used were:i) a full- grafted hybrid shoots of C . s a t i v a + B.
length rDNA sequence (18S-25S) of wheat carinaca, failure to survive t r a n s p l a n t a t i o n
n u c l e a r genome (Gerlach and B e d b r o o k 1979); suggests incomplete graft union or
ii) a m i t o c h o n d r i a l maize gone for 58-18S prevalence of strong genetic
rRNA (Chao et al. 1983); and iii) incompatibility.
chloroplast gene for large subunit of
ribulose b i s p h o s p h a t e carboxylase-oxygenase Hybridity of fusion products was
(rbcL) (Gatenby et al, 1981). Because of e s t a b l i s h e d On the basis of m o r p h o l o g y and
contamination problems, only one of the karyology of r e g e n e r a t e d shoots, and DNA
three hybrids could be m a i n t a i n e d in culture analysis.
and was used in these analysis.
M o r p h o l o g y : Because wh0~le plants could not
Results and D i s c u s s i o n be raised, our o b s e r v a t i o n s were r e s t r i c t e d
to morphological characteristics of in
R e c o v e r y of hybrids: The mean f r e q u e n c y of v i t r o p r o d u c e d leaves. The hybrid leaves
h e t e r o k a r y o n s among the cultured p r o t o p l a s t s combine features of both p a r e n t a l species.
of the two species was 6.8%. M o r p h o l o g i c a l Thev were l a n c e o l a t e w i t h s e r r a t e d margin~,
distinction of fusion products and cell

colonies derived from h y p o c o t y l p r o t o p l a s t s thick and brittle with an acute tip.

could not be d e t e r m i n e d beyond the third Cultu 9 seedling leaves of C . s a t i v a
cell division. Mesophyll protoplasts not are l a n c e o l a t e having entire margins. They
involved in fusion were unstable and are thin when compared to the h y b r i d and
c o l l a p s e d within 24 h. Out of a total of the 9 other parent B.carinata. B. carinata
227 calli o b t a i n e d from three independent grown in culture produced broad, oval-
fusion experiments, three individual calli shaped, thick and brittle leaves with
regenerated shoots over a period of 6 s e r r a t e d m a r g i n s (Fig. i).
months. Each callus piece p r o d u c e d a single
shoot. Shoots t r a n s f e r r e d to h o r m o n e - f r e e Cytology: Leaf tip m i t o s i s of two hybri d
MS basal m e d i u m failed to g r o w further. A shoots was carried out to d e t e r m i n e the
low concentration of BAP (0.i mg/l) was chromosome number. The e x p e c t e d mitotic
found essential for shoot p r o l i f e r a t i o n and c h r o m o s o m e n u m b e r of the h y b r i d is 74, the
maintenance. sum of its parental chromosomal
constitution; viz., B . c a r i n a t a (2n= 34) and
Efforts in inducing roots from the C . s a t i v a (2n = 40). The m i t o t i c m e t a p h a s e
somatic hybrid shoots initially in MS m e d i u m showed about 70 c h r o m o s o m e s in both cases
s u p p l e m e n t e d with 1 mg/l IBA failed. This (Figs 2, ~3). Analysis revealed size
is in contrast with the ease with which d i f f e r e n c e b e t w e e n the c h r o m o s o m e s of the
other somatic hybrids (Trachystoma ballii + wild and crop species. Chromosomes of
B. juncea, M o r i c a n d i a a r v e n s i s + B. juncea C . s a t i v a were d i s t i n c t l y longer c o m p a r e d to
and B . s p i n e s c e n s + B.3uncea) could be rooted B.carinata. While the 40 long c h r o m o s o m e s
and t r a n s p l a n t e d to soil (Kirti et al. 1991; of C.sativa were clearly seen, it was
1992a, b). Growth r e g u l a t o r s IAA, IBA, NAA difficult to account for all 34 s m a l l - s i z e d
and . GA 3 were tested in concentrations B . c a r i n a t a c h r o m o s o m e s in the h y b r i d cells.
ranglng from 0.i to i0 mg/l, either
i n d e p e n d e n t l y or in combination, on clonal Molecular analysis: Restriction fragment
derivatives of the hybrid shoots. length polymorphism was used in the
Occasionally, 1 or 2 slender roots were m o l e c u l a r c o n f i r m a t i o n of the h y b r i d n a t u r e
obse r v e d in m e d i u m with 2 mg/l IBA/IAA and of r e g e n e r a t e d shoots as well as the origin
in 0.5 mg/l NAA. Concentrations exceeding 4 of their organelles. Species-specific
mg/l IAA or IBA, or 1 mg/l in the case of Southern hybridization patterns were
NAA, led to callus formation at the base of e s t a b l i s h e d by c o m p a r i n g DNA samples of the
the shoot with s i m u l t a n e o u s root formation parental species and of the hybrid
in 8-15% of the cases. But these did not following Eco RI and Hpa II enzyme
survive when t r a n s p l a n t e d in soil. Five d i g e s t i o n and h y b r i d i z i n g with a p p r o p r i a t e
other basal media as listed e a r l i e r with IBA probes. A heterologous n u c l e a r probe of
at 2 or 4 mg/l also failed to induce rooting f u l l , l e n g t h n u c l e a r rDNA of wheat was used
in the h y b r i d shoots. for c o n f i r m i n g the n u c l e a r h y b r i d i t y using
Eco RI d i g e s t e d DNAs . It h y b r i d i z e d to a
Experiments attempting rhizogenesis by 3.7 kb fragment specific to C.sativa, and
supplementation of mannitol, use of h i g h e r to 1.8 and 1.0 kb f r a g m e n t s s p e c i f i c to
agar c o n c e n t r a t i o n , culture in liquid m e d i u m B.carinata. The h y b r i d had the 3.7 kb
on filter paper b r i d g e s in MS basal m e d i u m C . s a t i v a s p e c i f i c fragment, and the 1.8 and
of varying strengths, and 9 onto 1.0 kb B.carinata specific fragments,
B . c a r i n a t a stocks in culture did not y i e l d t h e r e b y c o n f i r m i n g its h y b r i d n a t u r e (Fig.
plants capable of growth in soil after 4)
transplantation. To our knowledge, the only
instance where somatic hybrids failed to The chloroplast composition of the
produce transplantable grafts is a somatic hybrid shoots was determined by
protoplast fusion derivative of Datura probing the Hpa II d i g e s t e d total DNAs with
 659

Fig.l In vitro grown shoots of : a) B. carinata, b) somatic hybrid B. ca]~inata + C. sativa

(SHI), and c) C. sativa. Fig.2 Chromosomal analysis of leaf tip of hybrid SH]. Flg.3
Chr6mosomal ana~si--~o-~--leaf tip of hybrid SH 2. Fig.4-6 Southern hybridization o f ~ N A . The
lanes are : a) B. carinata, b) somatic hybrld B. carinata + C. sativa ( S H ~ , and c) C.
sativa. 4. Eco - ~ digested DNA samples hybrid~ze-d ~ i--8~-25S rDN-A p--~be (p~A71). 5. Hp-a
II digested DNA samples hybridized with chloroplast-encoded gene for the large subunit o f
ribulose biphosphate c a r b o x y l a s e - m o n o x y g e n a s e (pZmBIB). 6. Hpa II digested DNA samples
hybridized with m i t o c h o n d r i a l - e n c o d e d 5S-18S rRNA gene probe.
660
a c h l o r o D l a s t - e n c o d e d gene, rbcL. The probe Kirti PB, N a r a s i m h u l u SB, Prakash S, ChQpra
h y b r i d i z e d to two fragments, i.5 and 0.96 VL (1992a) Plant Cell Rep ii: 90-92
kb, s p e c i f i c to B . c a r z n a t a [Fig. 5). In C.
sat~iva the probe h y b r i d i z e d t o 1.4 and 0.~-2 Kirti PB, N a r a s i m h u l u SB, Prakash S,Chopra
kb" s p e c i f i c fragments. The somatic hybrid VL (1992b) P l a n t Cell Rep ii: 3 1 8 - 3 2 1
shoots h a v e the two Camelina specific
fragments, s u g g e s t i n g that c h l o r o p l a s t s are Kumar A, Cocking EC (1987) Amer J Bot 74:
of the wild species origin. 1289-1303

Origin of m i t o c h o n d r i a in the somatic Murashige T, Skoog F (1962) Physiol Plant

hybrid was d e t e r m i n e d by probing the total 15:473-479
DNk with the m i t o c h o n d r i a l - e n c o d e d gene for
5S-18S rRNA. Hpa II enzym e digest shows the N a r a s i m h u l u SB, Kirti PB, Prakash S, Chopra
probe b i n d i n g to t w o fragments, 1.8 and 1.0 VL (1992) Plant Cell Rep ii: 159-162
kb, s p e c i f i c to B.carinata. There is no
fragment specific for C.sativa in this Nitsch JP, Nitsch C (1969) Science 163:85-87
restriction (Fig.6). The hybrid had the
B.carinata specific fragments, indicating Schenk RU, Hildebrandt AC (1972) Can J Bot
the p r e s e n c e of m i t o c h o n d r i a of B . c a r i n a t a 50:199-204
origin.
Schieder O (1980) Z Pflanzenphysiol 98: 119-
Somatic hybridization brings together 127
three g e n e t i c systems, namely, the nuclear,
montochondrial and chloroplast g e n o m e s of Tewari JP (1991) Current u n d e r s t a n d i n g of
two genetically different parents in a resistance to Alternaria brassica in
h e t e r o k a r y o n (Eberhard 1981). This leads to crucifers. In: Rapeseed in a changing
complex somatic incompatibility reactions world, McGrego--r D I (Ed). Proc Eighth
due to nuclear, i n t e r - o r g a n e l l a r and n u c l e o - Internat Rapeseed Cong, Saskatoon,
organellar genomic interactions in Saskatchewan, Canada, vol 2, pp 471-476
subsequent cell d i v i s i o n s (Kumar and Cocking
1987). In the absence of selection White PR (1954) The cultivation of animal
pressure, random sorting of organelles and plant cells. The Ronald Press Co, New
e v e n t u a l l y leads to new, stable o r g a n e l l e York
a s s o r t m e n t s as seen in the present study.
The outcome of the present fusion event has
led to a c o m b i n a t i o n of parental nuclei,
with m i t o c h o n d r i a of the c u l t i v a t e d B r a s s i c a
species and chloroplasts from the alien
species. Efforts are being made to produce
plants that can be used in further genetic
studies.

Acknowledgements: Financial assistance of

the Department o f B i o t e c h n 0 1 o g y , Govt. of
India is g r a t e f u l l y a c k n o w l e d g e d . We are
grateful to the technical assistance
p r o v i d e d by Mrs Seema Dargan and Mr D a y a n a n d
Verma.

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