Euphytica 30 (198 1) 239-246

CHROMOSOME DOUBLING THROUGH

ADVENTITIOUS SHOOT FORMATION ON IN
VITRO CULTIVATED LEAF EXPLANTS FROM
DIPLOID INTERSPECIFIC POTATO HYBRIDS

J. G. Th. HERMSEN’, M. S. RAMANNA’, S. ROEST’ and G. S. BOKELMANN’

‘Institute of Plant Breeding (IvP). Agricultural University, Wageningen, the Netherlands

‘Foundation ITAL, Wageningen, the Netherlands.

INDEX WORDS

Solunum, potato, explant culture, interspecific hybrid, adventitious shoots, in vitro, chromosome doubling,
solid mutants.

SUMMARY

Nearly 450 plantlets were produced from 51 diploid Solunum etuherosum x S. pinnutisectum F, hybrids
through adventitious shoot formation on in vitro cultivated rachis and petiole explants.
On the basis of phenotypical assessment of the ploidy level of 425 plants, 84.7 ‘>(; of the plants were scored
as doubled or doubled twice. A cytological analysis of ploidy in the three layers L,, L, and L, of 1 I2 plants
revealed 83.9 ‘A complete doubling: periclinal ploidy chimeras were not found and only two sectorial ploidy
chimeras were detected. Doubled plants were obtained from all 51 clones.
Various flower colours and epinastic leaves (in I clone) may be indications of mutagenesis through the
treatment.

INTRODUCTION

When somatic chromosome numbers are to be doubled in potato for experimental as

well as breeding purposes, there are a few conventional methods of colchicine treat-
ment. These methods involve the treatment of either the seeds (FRANDSEN, 1967;
HERMSEN & DE BOER, 1971) or the axillary buds (Ross et al., 1967) with an aqueous
solution of colchicine.. The seed treatment is not practical when clones with specific
genotypes are to be doubled, because the seeds of clones are not true to the genotype of
the parent. Therefore, the treatment of axillary buds is the only suitable method for
doubling clones of specific genotypes.
But this method is rather laborious and time-consuming for the following reasons:
(a) After treatment, owing to the effect of the drug, the axillary shoots do not develop in
large numbers. (b) The developed shoots must be rooted and grown to maturity for
tuber production. A considerable proportion of these may not tuberize. (c) The plants
that originate from such a procedure often are ploidy chimeras.
The use of an in vitro technique may be more efficient and less laborious than such in
vivo methods. Doubling of dihaploid potato clones via leaf tissue culture has already
been described by JACOBSEN (1977).
In our experiment we tested the explant culture technique developed by ROEST &
BOKELMANN( 1976, 1980).

239
 J.G.TH.HERMSEN,M.S.RAMANNA,S.ROEST AND G.S.BOKELMANN

According to this method a vegetative propagation of the potato can be achieved

through adventitious shoot formation on rachis and petiole explants and subsequent
root formation of subcultured shoots. After irradiation this so-called ‘in vitro adven-
titious bud technique’ leads to the production of normal (non-mutated) and almost
exclusively solid (non-chimeral) mutants (VAN HARTEN et al., 198 l), which indicates
that a great majority of adventitious shoots can be traced back to single cells. When cells
are cultured in vitro, depending upon cultural conditions a wide range of instabilities
may occur during their division and development. One of the most commonly observed
instabilities is the occurrence of endomitosis and consequently polyploidy (see SUN-
DERLAND, 1973, for references). Hence, regeneration of adventitious shoots and plants
may be a good source for obtaining completely polyploid plants.
Such in vitro regenerated plants were tested for their ploidy level, chimerism and the
occurrence of mutants, in order to evaluate the practicability of this method for a
routine chromosome doubling in potato. The results are reported in this article.

MATERIALSANDMETHODS

A total of 51 diploid, sterile F, clones from the cross Solanum etuberosum x S.

pinnatisectum (HERMSEN&TAYLOR, 1979; RAMANNA~HERMSEN, 1979) weregrownin
4 liter pails in the greenhouse at 12 h daylength. The clones were obtained from cuttings
of the original seedlings. One compound leaf was collected from each clone.
With respect to the preparation of plant materials, the composition of culture media
and the climatic conditions may be referred to ROEST& BOKELMANN(1980). Only, BA
was replaced by zeatin at 1 mg/l in this experiment.
Plantlets, produced through adventitious shoot formation on in vitro cultivated
rachis and petiole explants (Fig. 1) and root formation of subcultured shoots, 3 months
after explant incubation, were transplanted in small 180 ml pots filled with fertile soil of
53 % organic matter and a pH of 5.6. After transplantation the plantlets were covered
with plastic (high humidity), which was lifted daily. The plastic was removed after one
week and the survived plantlets were tested for chromosome doubling.
Phenotypic assessment of ploidy level was made on the basis of width of leaflets and
spontaneous berry and seed set and, moreover, cytological studies were carried out.
L, was studied by counting chloroplasts in guard-cells of 10 stomata of young leaves
after staining with potassium iodide. Ploidy in L, was determined indirectly by estimat-
ing pollen stainability with lactophenol acid fuchsine. Stainability is a measure for
pollen fertility and this is brought about by regular meiotic behaviour as a consequence
of chromosome doubling.
Direct assessment of L, was done by counting chromosomes in pollen mother cells at
meiosis. To this end young flower buds were fixed in a 3: 1 solution of ethanol
propionic acid saturated with ferric acetate for at least 48 h.
Fixed anthers were squashed in 1% acetocarmine. The somatic chromosome num-
bers in L, were counted in root tip cells. Root tips were collected from young potted
plantlets. They were then treated with &hydroxyquinoline for about 24 h at room
temperature and fixed in a 3 : 1 solution of ethanol acetic acid.
After 24-72 h of fixation the root tips were hydrolysed in 1 N HCl at 60°C for 8 min
and squashed in propionic acid haematoxylin according to HENDERSON& Lu (1968).
240 Euphyrica 30 (1981)
 INTERSPECIFIC POTATO HYBRIDS

Table I, Summary of experiment and results obtained from culturipg explants of 5 I F,-clones of Solununt
etuberosum x S. pinnatisectum.

Parts of experiment Number Remarks and results

Total explants taken 510 14 explants contaminated

Cultures obtained 496 I8 cultures without shoots
Explants with shoots 478
Explants used for subculturing 236 including explants from all S I clones
242 explants not used
Excised shoots subcultured 492 7 shoots contaminated
Rooted shoots after 3 weeks 341
Not-rooted after 3 weeks I44 44 unrooted shoots not planted
Plants potted 441 2 plants died
Viable plants 439 I4 viable plants not assesed
Phenotypically assessed 425 36 plants probably 8x (doubled twice)’
324 plants 4x (doubled once)
65 plants 2x (undoubled)
Cytologically assessed
all three layers I I2 plants
L, and L, 52 plants 164 94 plants all three layers 4x
I8 plants all three layers 2x
SO plants L, and L, 4x*
2 plants L, and L, 8x2

r Plants with a phenotype similar to that of two cytologically checked octaploids.

* In these cases L, probably had the same ploidy level as L, and L, because among I I2 fully assessed plants
not a single case of periclinal chimerism was observed.

RESULTS

The results from culturing 510 explants of 51 F,-clones of Sofanum etuberosum x S.

pinnatisectum are summarized in Table 1 together with detailed data on the experiment.
The percentage of the 496 uncontaminated rachis and petiole explants with adven-
titious shoots were scored each week until 8 weeks after incubation and finally after an
incubation for 19 weeks (Fig. 2).
It can be observed that the first explants initiated adventitious shoots after 2 weeks,
while 478 explants, representing all F, clones, had initiated adventitious shoots 19
weeks after incubation.
In order to restrict the number of plantlets to be handled 492 shoots (> 1 cm),
including all 5 1 F, clones, were excised from a limited number (236) of explants and
subcultured for rooting, with an average of 9.6 shoots per F, clone and 2.1 shoots per
explant, 10 weeks after incubation. After a subculture for 3 weeks, 341 of these shoots
were rooted and transplanted to soil; of the 144 unrooted shoots 100 were transplanted
to soil; 7 subcultured shoots were contaminated. So, 441 subcultured shoots in all were
transplanted to soil for the ultimate production of plantlets.
Of the 441 transplanted shoots 439 in all survived and 425 could be assessed
phenotypically. Cytological assessment of all three layers (L,-L,-L,) was carried out
in 112 plants, whereas only L, and L, could be assessed in 52 plants. The results are
included in Table 1 and allow the following conclusions.
Euphytica30 (1981) 241
 Fig. 1. Adventitious shoot formation on an in vitro cultivated
potato leaf explant of a Solarium etuberosum x S. pinnatisec-
turn hybrid (clone EP5), about 7 weeks after incubation.

percentage shmt fwmatiw,

100-
.
90 - .
.
so - .
70

60 -
.
50
40 -

30- ’

20 -
lo- l

.
I Fig. 2. Time course of adventitious shoot formation on rachis
0 2 4 6 8 10 12 14 16 18 20 and petiole explants of Solarium etuberosum x S. pinnatisec-
weeks of incubation turn hybrids.

242 Euphytica 30 (1981)

 INTERSPECIFIC POTATO HYBRIDS

Fig. 3. Representative corolla from Solarium etuberosum (E) and S. pinnatisectum (P) and from six character-
istic doubled (4x) F, plants (E x P).

1. On the basis of phenotypical assessment 360 out of 425 plants (= 84.7%) were
doubled once or twice.
2. Based on cytological observations 94 out of the 112 completely studied plants
(= 83.9 %) were doubled and no periclinal chimeras were detected. Including the
incompletely assessed plants, and assuming that L, in these plants is also doubled, 146
out of 164 plants (89.0 %) would have been doubled.
3. Each of the 51 treated F,-clones yielded doubled plants.
The frequency of ploidy chimeras is extremely low when compared with the method
ofcolchicine treatment. As stated before, no periclinal chimeras were detected, whereas
only two sectorial ploidy chimeras were found, one of which showed three different
flower colours. Flower colour among the doubled clones displayed more. variation
(Fig. 3) than that among the non-treated diploid counterparts (HERMSEN & TAYLOR,
1979). This suggests the presence of mutagenic effects of the treatment. Another
indication of a mutagenic effect was the occurrence of two tetraploid plants of one
clone showing epinastic leaves (Fig. 4). It is being investigated whether this epinasty is
an after-effect of the treatment or whether it is genetically determined.
The octaploid plants were far less vigorous than the tetraploids and diploids and
generally had malformed leaves (Fig. 5), like is often found after treatment of potato
plants with colchicine.

Euphytica 30 (1981) 243

 J. G. TH. HERMSEN, M. S. RAMANNA,?,. ROEST AND G. S. BOKELMANN

Fig. 4. Leaves of tetraploid Solanut~ ctuhu~~sun~ x S. pinnatisrcmn hybrids. Left: normal leaf; right:
epinastic leaves of clone EP 13 (lower leaf upside down).

Fig. 5. Representative leaves from Solarium efuberosum (E), S. pinnatisectum (P) and from a diploid (2x),
tetraploid (4x) and octaploid (8x) EP clone.

244 Euphytica 30 (1981)

 INTERSPECIFIC POTATO HYBRIDS

DISCUSSION

Since adventitious shoots and plantlets have been produced from every 2x-EP clone
and also from monohaploid S. tuberosum (unpubl. data) it can be concluded that this
method can be applied for diverse genotypes. Moreover, this propagation method
makes a fast multiplication possible since plantlets were produced 3 months after in
vitro incubation of rachis and petiole explant*.
Both the rate of plant regeneration and the rate of doubling are at a very high level. It
should be emphasized that the above mentioned results were obtained from highly
vigorous F, clones. When applied to weak material like potato monohaploids (2n =
12) or other inbred clones (JACOBSEN,1977) the regeneration and survival rate of
plantlets is clearly lower. But even with such material the in vitro methods are much
more efficient than colchicine treatment and yield a higher frequency and a larger
number of doubled (and even twice doubled) plants than the colchicine method.
The leaf tissue culture method (JACOBSEN,1977) is much more complicated and time-
consuming than the explant culture technique (ROEST& BOKELMANN, 1980). Accor-
ding to JACOBSEN(1977) plantlets are produced through 4 stages: callus induction
on leaf explants, separation of callus from the explants for further growth, transfer
of callus for shoot regeneration and root formation of subcultured shoots, which
procedure takes 5-6 months.
Using the explant culture technique of ROEST& BOKELMANN (1980) plantlets are
produced after 3 months through 2 stages: shoot formation on rachis and petiole
explants and root formation of subcultured shoots. A clear additional advantage of the
explant culture technique is the low frequency of chimeras (cf. VAN HARTEN et al.,
1981).
Last but not least, for the in vitro culture method only one leaf is needed, which can
be taken from young plants. Once the explants are in culture, there is hardly any danger
of infection by viruses or other potato diseases during the treatment, whereas with the
colchicine method according to Ross et al. (1967) the plants first have to be grafted
onto tomato and the grafts have to develop to a certain height; after the treatment of
the plant axils, shoots have to develop, cuttings of the shoots have to be made and after
selection of the doubled cuttings, these have to grow onto maturity. And during all
these manipulations the plants are exposed to diseases and pests in the greenhouse.
The regenerated plantlets from test tubes can be planted in sufficient numbers in one
run, whereas the shoot cuttings have to be made over an extended period.
In view of the single cell origin of a great majority of the adventitious shoots the
ploidy level in all three layers of a plant is basically equal. Therefore, assessment
of the ploidy level of a plant may in principle be restricted to one layer only.
Considering all these aspects, the explant culture technique is a valuable tool for
propagating potato plants and simultaneously doubling their number of chromo-
somes, which is especially important for workers in interspecific hybridization and in
haploids.

* This propagation however, is not fully true to genotype (cf. VAN HARTEN et al., 1981).

Euphytica 30 (1981) 245

 J. G. TH. HERMSEN,M. S. RAMANNA,S. ROEST AND G. S. BOKELMANN

REFERENCES

FRANDSEN,N. O., 1967. Chromosomenverdopplung und Chimarenbildung nach Colchicinbehandlung

haploider Kartoffelsamen. Eur. Potato J. 10: I-15.
HARTEN,A. M. VAN, H. BOUTER& C. BROERTJES, 1981. In vitro adventitious bud techniques for vegetative
propagation and mutation breeding of potato. (Solarium tuberosum L.) II. Significance for mutation
breeding. Euphytica 30: 1-8.
HENDERSON.S. A. & B. C. Lu, 1968. The use of haematoxylin for squash preparations ofchromosomes. Stain
Techn. 43: 233-236.
HERMSEN,J. G. TH. & A. J. E. DE BOER, 1971. The effect of colchicine treatment of Solunum acauk and S.
bulbocasranum; a complete analysis of ploidy chimeras in S. bulbocastanum. Euphytica 20: 171-180.
HERMSEN,J. G. TH. & L. M. TAYLOR, 1979. Successful hybridization of non-tuberous Solarium etuberosum
LIND. and tuber-bearing S. pinnatisectum DUN. Euphytica 28: 1-7.
JACOBSEN,E., 1977. Doubling dihaploid potato clones via leaf tissue culture. 2. Pflanzenziichtg 80: 8c-82.
RAMANNA,M. S. &J. G. TH. HERMSEN,1979. Unique meiotic behaviour in F, plants from a cross between a
non-tuberous and a tuberous Solunutn species in section Petota. Euphytica 28: 9-15.
ROEST,S. & G. S. BOKELMANN,1976. Vegetative propagation of Solarium ruberosum L. in vitro. Potato Res.
19: 1733178.
ROEST, S. & G. S. BOKELMANN, 1980. In vitro adventitious bud techniques for vegetative propagation and
mutation breeding of potato, Solarium tuberosum L. I. Vegetative propagation in vitro through adven-
titious shoot formation. Potato Res. 23: 167-181.
Ross, R. W., L. A. DIONNE& R. W. HOUGAS,1967. Doubling the chromosome number of selected Solarium
genotypes. Eur. Potato J. 10: 37-52.
SUNDERLAND,N., 1973. Nuclear cytology. In: H. E. STREET(Ed.), Plant tissue and cell culture, Chapter 7.
Blackwell Sci. Publ., Oxford, 1973, 503 pp.

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