3. Results
3
.
1
. Protoplast culture and plant regeneration At 3 weeks after dilution with fresh VKM
medium, dividing cells gave rise to hundreds of microcolonies, which developed rapidly into calli
after the cultures were transferred onto the soli- dified growth medium. Early selection of the puta-
tive somatic hybrids was based on the apparent difference in the cultural behavior of the parental
and hybrid calli, particularly the ability of the latter to grow faster and regenerate early [34].
Therefore, 2 weeks later, only calli 2 – 3 mm in size were selected to be transferred onto the regenera-
tion medium. Their growth was at least twice as rapid as that of the parental lines in the control
cultures. After 5 weeks on the regeneration medium, some selected calli produced shoots with
a frequency of one to five shoots per callus. Only one shoot was excised from the regenerating callus
and multiplied by subculture on hormone-free MS medium. Finally, 55 of 322 selected calli produced
shoots, representing a percentage of 17.0.
3
.
2
. Ploidy le6el of the selected putati6e hybrids The ploidy level was determined by comparing
the position of dominant peaks corresponding to nuclei at G0-G1 phase of the cell cycle, between
putative hybrid and parental plants Fig. 1. Clear differences in peak position are shown in Fig. 1
between diploid parents and their somatic hybrids. The analysis of plants recovered from the fusion
experiments showed that more than 80 were diploids. In total, ten plants were found to have a
higher ploidy level. These plants were retained, as they were putative somatic hybrids. Among the
selected plants, six BP3, BP4, BP6, BP14, BP15, BP16 were tetraploids, two BP1, BP8 mixo-
ploids, one BP9 amphiploid \ 4 × ; 48 and one BP13 octoploid 8 × ; 96. Chromosome counts
made on root tips of a sample of the selected plants confirmed the ploidy level determined by
flow cytometry.
3
.
3
. Morphological analysis Both parental and putative somatic hybrid
plants were grown up to maturity in the green- house. Most putative hybrid plants grew vigor-
ously and were larger than the parental plants. Their morphology was relatively homogeneous
and intermediate between the parental lines, in- cluding leaves, flowers and tubers, all of which
were larger than those of the parents Fig. 2, except for the mixoploid clones having small
leaves with irregular forms.
Pollen viability of putative hybrid flowers varied widely, ranging from 20 to 60 compared to
50 – 70 viable pollen in the parental plants.
3
.
4
. Isoenzyme analysis The hybrid nature of the selected putative hy-
brids was confirmed by examining the elec- trophoretic patterns for esterases and peroxidases
Fig. 3. Each isoenzyme system revealed differ- ences between potato BF15 and S. phureja. They
also distinguished somatic hybrids from the par- ents. The somatic hybrid pattern for esterases
contained bands which were identical to those found in the mixed extracts of the parents Fig.
3A. For peroxidase system in addition to the sum of the parental bands, the hybrid pattern showed
additional bands, which were specifically relevant to the hybrid nature and not found in the mixed
extracts of the parents Fig. 3B.
Fig. 1. Histograms of fluorescence intensities associated with 10 000 nuclei isolated from leaves of in vitro dihaploid
potato, cv. BF15, S. phureja, and their somatic hybrids. After DAPI staining, fluorescence is proportional to nuclear DNA
quantity and the position of the dominant G0 and G1 peak reflects the ploidy level.
Fig. 2. Plants: S. tuberosum St, S. phureja Sp and four somatic hybrids SH; the mixoploid is indicated by the arrow A; leaves and flowers: S. tuberosum cv. BF15 B, E, S. phureja D, G and their somatic hybrids C, F.
3
.
5
. DNA analysis Out of 34 ten-mer primers tested, eight gave
reproducible RAPD patterns showing polymor- phism between S. tuberosum cv. BF15 and S.
phureja. Three of them led to clear identification of the somatic hybrids. RAPD patterns obtained
with the primer AB1-12 5-CCTTGACGCA-3 are given in Fig. 4A. Parental patterns can be
distinguished by DNA bands 500 – 1000 bp in size. The ten putative somatic hybrids showed similar
patterns with specific parental bands and with an additional band 1.5 kb in size which was absent
from the parents.
The NTCP6
primers 5-GGTTCGAATC-
CTTCCGTC-3 and 5-GATTCTTTCGCATCTC- GATTC-3 led to distinction between the two
parents of the fusion experiments. Unique and
specific ctDNA bands of 180 and 130 bp were amplified with the cultivated as expected
from Ref. [31] and the wild species, respectively. All the somatic hybrids examined showed the
ctDNA pattern of either one or the other parent. The ctDNA of potato was found in two somatic
hybrids and the remaining eight hybrids possessed the S. phureja ct type Fig. 4B.
Taking into account the intermediate morphol- ogy, the ploidy level and the analysis of nuclear
and chloroplast genomes of the selected plants by examining the isoenzymes and DNA markers, it
was concluded that the ten selected plants were somatic hybrids between S. tuberosum and S.
phureja.
3
.
6
. E6aluation of resistance to bacterial wilt S. tuberosum cv. BF15 was susceptible to both
races, as 71.8 and 100 of plants were wilted 30 days d30 after inoculation with races 1 and 3
strains, respectively, and disease indices reached 0.87 and 1.00 Tables 1 and 2. Moreover, charac-
teristic symptoms of bacterial wilt necrosis and wilting occurred earlier and stronger for the di-
Fig. 4. Electrophoresis profiles of PCR amplification prod- ucts. A RAPD patterns. DNA from S. tuberosum St, S.
phureja Sp and their somatic hybrids BP3, BP4, BP6, BP15 and BP9 lanes 1 – 5, respectively was amplified using the
primer AB1-12. Fractionation was on 1.4 agarose gels. The other five hybrids not shown led to patterns identical to the
above hybrids. B Chloroplast microsatellite patterns. DNA from the same seven genotypes as above was amplified using
NTCP6 primers. Fractionation was on 1.8 agarose gels. The other five hybrids not shown led to patterns identical to S.
phureja L.: 100-bp DNA ladder Biolabs.
haploid potato line than for the S. phureja line. S. phureja appeared to be tolerant to race 1 strain,
showing a low disease indices 0.36 and no wilted plants at d30. S. phureja was moderately suscepti-
ble to race 3 strain with 50 of wilted plants at d30 but with a rather high disease indices 0.85
Tables 1 and 2.
A total of six somatic hybrid plants, including five tetraploids and one amphiploid, were checked
for resistance to bacterial wilt using race 1 and race 3 strains. Race 3 strain induced plant wilting
earlier and with a higher frequency than did race 1 strain Tables 1 and 2. Three tetraploid hybrids
BP3, BP6 and BP16 appeared moderately sus- ceptible to race 1 strain with 30.6 – 48.6 of wilted
plants but their disease indices were not signifi- cantly different from that of the susceptible parent
0.78 – 0.80 versus 0.87. Two hybrids BP4 and BP15 were less susceptible with 7.9 and 20.5 of
wilted plants at d30, respectively, and their disease indices were not significantly different from that of
the wild parent Table 1. Only the amphiploid hybrid BP9 was found at least as tolerant to race
1 strain as S. phureja since no wilted plants were recorded for both. However, its disease indices
0.08 was significantly lower than that of S.
Fig. 3. Electrophoresis banding patterns of A esterases EST E.C. 3.1.1.2. and B peroxidases PRX E.C.
1.11.1.7. for S. tuberosum cv. BF15 St, S. phureja Sp, a mixture of parental extracts M and their somatic hybrids
BP3, BP4, BP6, BP15 and BP9 lanes 1 – 5, respectively. The other five hybrids not shown led to patterns identical to the
above hybrids.
phureja 0.36. All tetraploid hybrids were either more or at least similarly susceptible to race 3
strain compared to BF15 Table 2. They were more susceptible than S. phureja, except BP4
which showed a similar wilting rate but a higher disease indices than the wild parent. Interestingly,
the wilting rate of the amphiploid hybrid was low 2.8 and not significantly different from zero.
However, the disease indices 0.52 though signifi- cantly lower from that of S. phureja 0.85, would
indicate that the hybrid was tolerant rather than resistant Table 2.
Within roots of apparently healthy looking plants belonging either to both parental lines or to
the amphiploid hybrid high populations expressed as logarithms of cfu g
− 1
fresh weight were recov- ered, ranging from 7.27 to 7.93 whatever the clone
or the strain. However, significant differences were recorded within stems of plants inoculated with
race 1 strain between BF15 on the one hand 7.13 and S. phureja 3.74 and BP9 3.20 on the other
hand. For plants inoculated with race 3 strain, the bacterial
populations within
stems of
both parental lines were significantly different although
the difference was lower than one logarithmic unit. In contrast, the population within stem of the
Table 2 Disease indices and disease incidence recorded 15 days and 30
days after root
inoculation by race 3 strain of
R. solanacearum
a
Disease indices Disease incidence
d15 d30
d15 d30
0.97
b
1.00
b
BP3 2n = 4× 88.9
b
100.0
b
0.88
bc
68.4
c
0.75
cd
BP4 2n = 4× 26.3
c
86.1
b
100.0
b
BP6 2n = 4× 0.96
bc
1.00
b
0.48
d
0.52
d
BP9 2n\4× 0.0
d
2.8
d
BP15 2n = 4× 0.96
bc
1.00
b
86.1
b
100.0
b
1.00
b
100.0
b
1.00
b
100.0
b
BP16 2n = 4× 100.0
b
0.96
bc
BF15 1.00
b
86.1
b
2n = 2× = 24 S. phureja
50.0
c
0.64
d
0.85
c
11.1
c
2n = 2× = 24 0.00
e
0.0
d
0.0
d
Non-infected 0.00
e
controls
a
Disease indices is the weighted average of the disease index. Disease index ranges 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 incidence is the
percentage of inoculated plants displaying a disease index of 4. Values followed by the same letter are not significantly
different at P = 0.05.
amphiploid hybrid was significantly reduced com- pared to both parents Table 3.
4. Discussion
Kategori