Resistant or tolerant cultivars are mainly uti- lized for disease management since chemicals are
not effective and sanitation measures difficult to apply. Therefore, the worldwide control strategy
has consisted of plant breeding for resistance to bacterial wilt [6]. Using resistant cultivars to con-
trol bacterial wilt has been successful for tobacco and peanut. Since immunity was not identified in
potato, only tolerant cultivars were selected [7], such as cultivar Prisca mainly cropped in Mada-
gascar, cultivar Ndinamagara in Burundi, Rwanda and Zaire and cultivar Achat in Brazil [4,8]. To
better control bacterial wilt, continuous develop- ment of resistant or tolerant varieties is needed.
Some wild or related cultivated species are known to be resistant or highly tolerant to bacte-
rial wilt and thus are potential sources for resis- tance. Unfortunately, hybrids of potato with
resistant genotypes
of Solanum
chacoense, Solanum sparsipillum and Solanum multidissectum
revealed some traits of wildness such as a high glycoalkaloid content besides a moderate level of
resistance to bacterial wilt [4]. Unlike these wild species which have been classified as wild Tuberosa
[9], Solanum phureja cultivated Tuberosa
is phyl- logenetically close to Solanum tuberosum and dis-
plays resistant
traits, dominant
and readily
transmitted to progeny. Some clones of S. phureja with high degree of resistance to bacterial wilt
have been used for crossing with commercial culti- vars of S. tuberosum [4,10]. Resistance to bacterial
wilt derived from S. phureja was first described as dominant and controlled by three unlinked genes
[6,11,12]. More recently, at least four major genes have been reported to be involved in potato resis-
tance to bacterial wilt [13].
The introgression of multiple resistance genes from wild Solanum species into S. tuberosum by
classical breeding methods is time-consuming, la- borious and may encounter difficulties because of
sexual incompatibilities, particularly due to differ- ences in ploidy level or in endosperm balance
numbers. Somatic fusion is expected to provide a new possibility for increasing genetic variability,
and also a means of transferring desirable agro- nomic traits into potato. The potential use of
somatic hybridisation has been demonstrated by the successful introduction of traits such as resis-
tance to viruses [14,15] and frost [16] from Solanum bre6idens, resistance to Phytophtora infes-
tans and Globodera pallida from Solanum circaei- folium
[17], insect
resistance from
Solanum berthaultii [18], and resistance to bacterial wilt
from Solanum commersonii [19], into the cultivated potatoes. In other studies of fusion between
potato and S. phureja, partial elimination of chro- mosomes was reported in the resulting somatic
hybrids. The nuclear chromosomes of S. phureja were preferentially eliminated [20,21]. So far, no
information has been available about evaluation of the somatic hybrid clones for the introgression
of resistance against bacterial wilt from S. phureja into potato.
In this study, somatic hybridisation between the dihaploid S. tuberosum cv. BF15 and the diploid
S. phureja was performed. The somatic hybrids were identified and characterised as well as evalu-
ated for resistance to race 1 and race 3 strains of R. solanacearum.
2. Materials and methods
2
.
1
. Plant material A clone of dihaploid S. tuberosum L. 2n = 2 ×
= 24 chromosomes, cv. BF15, obtained from the
Institut National de a Recherche Agronomique INRA, Landerneau, France, and a diploid clone
of the wild tuber-bearing S. phureja, SVP5PH77- 1445-2242, provided by the Centre for Genetic
Resources, Wageningen, the Netherlands, were used. Plants were maintained in vitro from axillary
shoot tips on MS basal medium [22] as described by Chaput et al. [23]. The clones were subcultured
at 4-week intervals. Environmental conditions were 14 h day
− 1
illumination at 55 mmol m
− 2
s
− 1
, 20°C and 60 relative humidity.
2
.
2
. Protoplast isolation and electrofusion The protocol for protoplast isolation was
derived from Chaput et al. [23]. Leaves from 4- week-old in vitro plants were scarified and placed
in an enzyme solution containing CPW salts [24], 1 wv cellulase R-10 Yakult, Tokyo, Japan,
0.05 wv pectolyase Y-23 Sheishin, Tokyo, Japan, 0.5 M mannitol and 0.05 wv 2-N-
morpholino ethanesulfonic acid MES buffer, pH 5.5. At the end of the digestion period, proto-
plasts were separated from undigested material through metallic sieves 100-mm mesh, and the
resulting suspension was purified and rinsed by centrifugation in successive 0.6 M sucrose, and 0.5
M mannitol + 0.5 mM CaCl
2
solutions. Prior to electrical fusion, the protoplasts were suspended in
the last washing solution, and the density for both species adjusted to 4.0 × 10
5
protoplasts ml
− 1
. Electrical apparatus and fusion procedure were
described previously [25]. The movable multi-elec- trodes were placed in a 15 × 50-mm Petri dish
containing 500 – 700 ml of a mixture 1:1 of proto- plasts from both parents. The protoplasts were
aligned for 15 s by the application of an a.c. field at 230 V cm
− 1
and 1 MHz. Subsequently, one square pulse developing 1.2 kV cm
− 1
for 40 ms was applied to achieve protoplast fusion. After
application of the square pulse, the a.c. field was reduced routinely to 20 V cm
− 1
to keep alignment of protoplast chains and to estimate fusion fre-
quency by observing fusion events through an inverted microscope.
2
.
3
. Protoplast culture and plant regeneration The culture medium was VKM medium [26]
supplemented with 250 mg l
− 1
PEG, 0.2 mg l
− 1
2,4-D, 0.5 mg l
− 1
zeatin, 1 mg l
− 1
NAA, 0.2 M mannitol and 0.2 M glucose as osmotic agents and
0.05 wv MES. The pH was adjusted to 5.8 prior to sterilizing by filtration 0.22-mm filter,
Millipore. After electrical treatment, 6 ml of cul- ture medium were added progressively to the Petri
dish containing the fused protoplast mixture. Cul- tures were kept in the dark for 7 days prior to
putting them into the light. On day 15, cultures were diluted eight times with fresh VKM medium
supplemented with 2 mg l
− 1
BAP and 0.1 mg l
− 1
2,4-D, pH 5.8. Calli 3 – 4 mm diameter were then transferred onto the regeneration medium: MS
basal medium, plus vitamins [27], to which was added 2 wv sucrose, 2 mg l
− 1
zeatin and 0.1 mg l
− 1
IAA and solidified with 7 g l
− 1
agar Difco. Emerging shoots were excised from callus
and plantlets were multiplied by subculturing leafy node cuttings on hormone-free MS medium. Both
parental and selected hybrid plants were trans- ferred to the greenhouse. Environmental condi-
tions were 14 h day
− 1
illumination at 55 mmol m
− 2
s
− 1
, 20°C and 60 relative humidity for in vitro cultures and 16 h day
− 1
illumination at 180 m
mol m
− 2
s
− 1
, 25 – 30°C and 70 – 85 relative humidity in the greenhouse.
The selection and identification of somatic hy- brids was based on the analysis of plant morphol-
ogy and ploidy level. Evidence for the hybrid nature of selected plants was provided by examin-
ing isoenzyme patterns and DNA analysis.
2
.
4
. Determination of ploidy le6el and pollen 6
iability Flow cytometry was used to quantify DNA for
the determination of ploidy level. Using a razor blade 1 cm
2
of leaf material from in vitro plants was chopped in 1 ml buffer containing CPW salts
[24], 0.5 M mannitol, 0.25 wv PEG, 0.5 wv Triton X-100, 0.25 vv mercaptoethanol
at pH 6.5 – 7. Crude samples were filtered through a 40-mm mesh nylon and stained with 4,6 di-
amidino-2-phenylindole DAPI, 5 mg ml
− 1
. Nu- clei were analysed on a PARTEC CA II flow
cytometer Chemunex, Maisons-Alfort, France equipped with a 100-W mercury lamp type HBO.
Fluorescence at 455 nm was recorded as a func- tion of the DNA content. The DNA distribution
was analysed by using DPAC software on his- tograms generated from at least 10
4
nuclei. The dihaploid parental plants were used as external
references to calibrate fluorescence scale. Pollen viability was evaluated by staining pollen
with fluorescein diacetate FDA, 5 mg ml
− 1
.
2
.
5
. Isoenzyme analysis Crude extracts were prepared using leaves from
in vitro-grown plants [23]. A total of 20 ml of each sample was loaded on polyacrylamide gels run-
ning gel: 7.5 acrylamide + 0.2 bisacrylamide; stacking gel: 4.5 acrylamide + 0.12 bisacry-
lamide, and electrophoresis was performed for 3 h at 4°C and 20 mA. Staining for esterases E.C.
3.1.1.2 and peroxidases E.C. 1.11.1.7 was done as described previously [28].
2
.
6
. DNA analysis Total DNA was obtained from frozen leaf tissue
extracted with the DNeasy plant mini kit Qiagen following the manufacturer’s instructions. PCR
reactions leading to RAPD markers contained 30 ng DNA 0.5 ml of DNA sample, 10 × Taq poly-
merase buffer including 1.5 mM MgCl
2
, 0.3 mM each dNTP, 0.2 mM of 10-mer primer Fisher and
1 U of Taq polymerase Appligene in a total volume of 25 ml. A total of 20 decamer oligonu-
cleotide primers from the kit AB-0320-1 Fisher and 14 primers previously described for potato
by Xu et al. [29] A02, A05, A10, A12, A16 and Baird et al. [30] SC10-01, SC10-02, SC10-03,
SC10-04, SC10-12, SC10-20, PBI-R005, ST1, ST2 were used. The thermal cycling profile,
derived from Baird et al. [30], included 3-min denaturing at 92°C followed by 45 cycles of 92°C
for 1 min, 37°C annealing for 1 min, and 72°C extension for 1 min. A final extension for 8 min
at 72°C was the last step of the amplification program which was performed with a Touch
Gene thermal cycler. Amplification products were electrophoresed onto 1.4 agarose gels. After
staining with ethidium bromide, gels were pho- tographed on an UV box with Polaroid 665
films.
Polymorphic simple sequence repeat SSR markers in chloroplast genomes of Solanum
plants were recently described [31]. Several pairs of primers given by these authors NTCP 6-1
and – 1 bis; NTCP 9-1 and – 1 bis; NTCP 12-1 and – 1 bis; Genaxis have been used to distin-
guish the chloroplast genomes of the parents and to characterise the ct genome type of the corre-
sponding hybrids.
PCR amplification of chloroplast microsatel- lites was performed with the reaction as de-
scribed above, primers excepted. The thermal cycling profile was that of Bryan et al. [31], in-
cluding: 4 min denaturing at 94°C, followed by 30 cycles of 94°C for 1 min, annealing tempera-
ture for 1 min, 72°C extension for 1 min and a final extension for 5 min at 72°C. Amplification
products were electrophoresed onto 1.8 agarose gels which were stained and photographed as de-
scribed above.
2
.
7
. Determination of in 6itro resistance to bacterial wilt
Two strains of R. solanacearum, G14 race 1, biovar 3; isolated from Pelargonium asperum in
Reunion and PDT-5 race 3, biovar 2; isolated from potato in Reunion, provided by CIRAD
Centre de
Coope´ration Internationale
en Recherche Agronomique pour le De´veloppement,
Saint-Pierre, Reunion Island were used to inoc- ulate both parental lines and the somatic hy-
brids. Cultures of R. solanacearum were routinely grown 24 h, 28°C on basal medium, i.e. YPGA
medium yeast extract, 7 g l
− 1
; peptone, 7 g l
− 1
; glucose, 7 g l
− 1
; agar, 15 g l
− 1
; pH 7.2. Tests to evaluate sensitivity to bacterial wilt were per-
formed on in vitro plants, using roots inoculated with suspension containing 10
7
colony forming units cfu per ml.
After cutting a part of the roots, the parental lines and somatic hybrids were soaked in the
bacterial suspension for 30 min. Control plants were inoculated using sterile water. After inocu-
lation, plants were placed in MS medium liquid, and exposed to 14 h day
− 1
illumination at 55 m
mol m
− 2
s
− 1
, 20°C and 60 relative humidity in a controlled environment chamber. The tests
for bacterial resistance were done by using 36 plants per clone, distributed over three replicates.
Each replicate of 12 plants was distributed at random in the chamber. Plants were observed
weekly and symptoms recorded using a disease index ranging from 0 to 4: 0 = no wilted leaves,
1 = up to 25 wilted, 2 = up to 50 wilted, 3 = up to 75 wilted and 4 = plants entirely wilted.
Disease indices were calculated, according to Winstead and Kelman [32], as the ratio between
the sum of the products of each disease number, divided by 4, and the corresponding number of
plants and the total number of inoculated plants.
Disease incidence was evaluated at d15 and d30 15 days and 30 days after inoculation, re-
spectively by estimating the percentage of wilted plants that is with a disease index = 4. Values of
disease indices and disease incidence recorded at d15 and d30 were compared using a GStat test
[33]. At d15, three inoculated but healthy looking plants belonging to the parental lines BF15 and
S. phureja and to the hybrid BP9 were sampled and washed three times in sterile Tris buffer 10
mM, pH 7.2 to remove superficial bacterial pop- ulations. Roots and stems of each plant were
separately weighed and then crushed with a pestle in 5 ml of Tris buffer. To determine cfu
mg
− 1
fresh weight, an aliquot of the resulting suspension and of its suitable dilutions were
spread onto YPGA medium plates two repli- cates and incubated at 28°C for 3 days. Means
of bacterial populations were compared by using Duncan’s multiple range method after having
performed an analysis of variance.
3. Results
