ACTA
PHYSIOLOGIAE
Plant regeneration from the protoplasts of Solanum tuberosum, S. nigrum
and S. bulbocastanum
Anna 52czerbakowa. M a r i a Borkowska*, B e r n a r d Wielgat
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Pawifiskiego 5A, Poland
* We regret to report the death of M. Sc. Maria Borkowska after the completion of this work.
Key words: protoplast regeneration, Solanum bul-
bocastam#n, Solanum nigrum, Solanum tuberosum
Abstract
The lnesophyll protoplasts were isolated from the Solanum
tuberosum (S. tbr) clones of different ploidy level (4x Bzura
cv., 2x H-8105, and 2x ZEL- 1.136) as well as from the wild spe-
cies: S. bulbocastamlm (S. blb, 2x) and two accessions of S.
~ligrum (S. ngr, 6x). Additionally, the protoplasts were isolated
from the cell suspensions of Bzura cv. and H-8105 clone. The
conditions of protoplast isolation as well as the media for their
culturing and regeneration, were selected and optimized for the
studied gcnotypes. For mesophyll protoplasts, the shooting
calli were produced by all the cultured protoclones except that
of S. bulbocastamm~. The shoots excised from the protoplast-
derived calli developed into whole plants in all the studied po-
tato clones but only in one accession of S. nigrum, i.e.S, ngr
var. gigar~tea. As for suspension-cell-derived protoplasts, only
H-8 i05 clone produced the regenerative type of calli, though
normal shoots could not be obtained. The regenerative capacity
of the protoplasts isolated from leaves and cell suspensions is
compared and discussed.
Introduction
It is well k n o w n that the regenerative capacity of
diffcrent genotypes varies significantly even for the
closely related species or individual lines within the
PLANTARUM
Vol. 22. No. 1. 2000:3-10
same species (e.g. M a s s o n et aL 1987). T h o u g h the
literature on methods of plant regcncration from the
protoplasts of potato and wild reprcsentatives of
Solanaceae family is abundant, the selection and
modification of the p r o p o s e d techniques is needed
in ordcr to apply t h e m to any particular species or
line.
W i l d Solanum species, S. bulbocastanum (diploid,
2x) and S. nigrum (hexaploid, 6x), both resistant to
different pathogens, could be used as donors of the
resistance by m e a n s o f somatic l~lsion with potato
protoplasts (Austin et al. 1993, H o r s m a n et al.
1997). The regeneration systems for mesophyll
protoplasts o f those species have been described
(Nehls 1978, Sihachakr & Ducreux 1987) though
the protoplasts of S. bulbocastanum usually failed
to regenerate (Austin et al. 1993). Since our objec-
tive was to i m p r o v e the late blight resistance of do-
mestic potato cultivars by the somatic hybridization
technique, it was first inevitable to determine the re-
sponses to culturing and the regenerative capacity
of their protoplasts. For that purpose we have se-
lected the potato lines with different ploidy level:
the tetraploid cv. Bzura, relatively resistant to late
blight, and two diploid clones susceptible to that
A. SZCZERBAKOWA, M. BORKOWSKA & B. W1ELGAT
disease: H-8105 - a dihaploid, and ZEL-1136 - an
interspecific hybrid between S. tuberosum and S.
chacoense. The protoplasts of those potato lines
have not been previously used in fusion experi-
ments and their culture requirements were not
known. For Bzura cv. and H-8105 clone, the regen-
eration of both leaf- and suspension-cell-derived
protoplasts were examined. In the present work we
describe the culturing of protoplasts isolated from
leaves and cell suspensions of S. tuberosum, from
leaves of S. nigrum and S. bulbocastanum, and
compare the ability of the cultured protoplasts to re-
generate plants in selected culture media.
Materials and methods
Genotypes
Wild
S o l a n u m species completely resistant to Phy-
tophthora infestans:
- S. nigrum L. (hexaploid, 2n=6x=72, non-tube-
rizing)
- S. nigrum var. gigantea (hexaploid, 2n=6x=72,
non-tuberizing)
- S. bulbocastanum (PI243512, clone 112/23, dip-
loid, 2n=2x=24, tuber-bearing)
Cultivated potato
- Bzura cv. (tetraploid, 2n=4x=48), a late-maturing
cv. belonging to the family PG-232 and expressing
field resistance to Ph. infestans scored 8 in 1-9
grade scale (9=resistant in leaflet test); origin of the
family: [(MPI 55.957.24 x USDA 96-56) x Ora] x
Prosna
- H-8105 (dihaploid, 2n=2x=24), obtained from
crosses of the secondary dihaploids of the parental
forms MH-73-17-12-77 x SH-77-45-35 by Het-
tema, Holland; mid-early maturing, susceptible to
Ph. infestans, field resistance scored 2 in 1-9 grade
scale
- ZEL-1136 (diploid, 2n=2x=24), originated from
crosses between dihaploids of S. tbr and wild spe-
cies S. chacoense; ZEL-1136=HS106 x 15/39-
40;63 (H8106=S. tbr dihaploid from Holland);
4
clone 15/39-40 is the clone No.52 from self-
fertilizing of OPS 5; origin of self-compatible pa-
rental clone OPS5:F1LI356 x (B 16 x G609)P127;
LI356=chcCPC-3785 x yunGLK-s67.107/3R ;
B16, G609 - dihaploids of S. tbr of Dutch origin;
ZEL-1136 is susceptible to Ph. infestans, scored 2
in 1-9 grade scale; yun=S, yungasense
Plant material
The plants were propagated in vitro on hormone-
free MS medium (Murashige & Skoog 1962) with
2 % sucrose and 0.6 % agar. The fully expanded
leaves of 3-4 week-old plants were excised and pre-
conditioned according to Haberlach et al. (1985).
Cell suspensions of Bzura cv. and H-8105 clone,
were cultured for 5-7 days in MS medium enriched
with Gamborg vitamins lmg/ml, glycine 2 mg/ml,
casein hydrolysate 0.5 mg/ml, NAA 5 mg/ml, BAP
0,1 mg/ml and 3 % sucrose, at26 °C and 100 rpm in
the dark.
Protoplast isolation
Suspension-cultured cells and the preconditioned
leaves were digested overnight in the dark, in K4
medium (Nagy & Maliga 1976) with 1.6 % cdlu-
lase Onozuka R-10 and 0.6 % macerozyme R-10
(suspension cells) or 0.4 % cellulase and 0.2 %
macerozyme (leaves), at 28 °C (suspension cells) or
23 °C (leaves). Following the cell wall degradation,
the protoplasts were poured through steel filters of
74 gm or 100 ~m pore size, and centrifuged at 80 g
for 15 min in MPW-340 centrifuge with 1 ml W5
(Menczel et al. 1981) applied to the top of suspen-
sion. Protoplasts were collected from K4/W5 inter-
phase and suspended in W5 mcdium.
Protoplast culture
The composition of the media used is given in the
table. Liquid media were sterilized by filtration
through 0.2 tam filter. Agar media were autoclaved
at 121 °C for 15 min. Filter-sterilized vitamins and
hormones were added to agar media after autoclav-
ing. The SKM medium (Hunt & Helgeson 1989)
was found to be the most effective when used ac-
cording to the protocol of Austin et al. (1993[) .
Briefly, the protopIasts were cultured for 3-5 days
in liquid SKM medium without BSA, with two
50 % dilutions with fresh SKM medium on the 1st
 REGENERATION OF SOLANUM PROTOPLASTS

Table. Composition of the media used

Media components K4 MS SKM Cul MSR1 .__ MSR2 _..
__Mac(0 elements (m_~/1final concentration) ......... _.
KNO~ 2500 1900 1900 1900 1900 1900
NHqNOa 250 1650 825 1650
CaCI2x2H20 900 440 600 440 44{) 44(}
MgSOax7H20 250 370 350 370 370 37(.I
(NIG}2SO4 250
K HaPO4 170 680 170 170 170
NaH2POax]-]20 1~(
KCI 3{}0
NH4C1 107 . . . . . . .
_Micro eleme.nt_s (mg/l final concentration}
Na2EDTA 37.3 37,3 37,3 18,64 37,3 37,3
FeSOax7H20 27,8 27,8 27,8 13,94 27,8 27,8
HaBOa 3 6,2 3 3,1 6,2 6,2
KJ 0,75 0.83 0,75 0,42 0.83 0,83
MnSO~xH20 10 I6.9 10 8.45 16,9 16.9
ZnSO4x7H20 2 8,6 2 4,3 8,6 8,6
CuSO4x5H20 0,025 (},{)25 (/,025 (},013 0.{t25 I),(}25
NazMoO4x2HzO 0.25 {/,25 0,25 0,13 0.25 0,25
C.oCtzx6tt,O . . . . . . . . 0,(}25 0,025 0.025 ~ {},013 0,(.}25 _._ (~,025 . . . . .
Qarboh~drates£~/1 t~inal concentration}
Sucrosc 136,92 30 10 2,5 2,5 10
Glucose 10
IVlannitol 40 54,7 35
Sorbitol 0,25
Celh}biosc 0,25
Fructose 025
Mannose (},25
Rhamnose 0,25
Ribose 0,25
Xylose 0.25 0.25
~Wo-Im}sitol . . . . . . . . . . {}_.1. . . . 0,1 . . . . 0.05 0,1 0, I (},1
y i t a m i n s ~ / 1 linal concentration)
Pyridoxinc HC1 1 {),5 0,5 1),5 0,5 0,5
Thiamine HCI 10 0,1 2 {},5 0,5 0,5
Nicotinic acid 1 0,5 5 5 5 5
Folio acid {).5 0.5 (}.5
D-Ca-Pantothenate (},5
p-Aminobenzoic acid 0,01
Choline chloride 0,5
P.iboflavin (), 1
Ascorhic acid t 0,5 1
Biotilk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0,005 {),{)5 0,05 {},{}5
HpLmp_w.s~/1 final concentration)
NAA (oc maphthaleneacetic acid} 1 5 1 0. I 0~025
gAP (6-benzylaminopurine) 0.2 0,25 (}.4 0.5 0,5
GA~ (gibberellic acid) 0,5 2
IAA (indole-3-acetic acid} 0,1
Kinetin (6 furfurylaminopurine) 0.5
*rons-Zeatin 1 2 2
_Other 9rgan!c~..(~pg~ fijml c_'5?m~entratkm)........................................................................................
Glycine 2 2
Casein hydrolysate 50{) 250 100 100 100
L-Glutamine /46,2 146,2
Adenine sulfate 40 4(} 40 40
Coconut water, ml/l 22
pH 5,8 5,8 5,6 ~,8 ........................ _5,8 5,8

5
A. SZCZERBAKOWA, M. BORKOWSKA & B. WIELGAT
and 4th day after isolation. O n the 4th or 5th day, the
cultures were solidified with 0.5 % SeaPlaque aga-
rose and further cultured on SKM+ 1% B S A+0.8 %
agar layer for at least 2 weeks at 26 °C (lst week in
the dark, 2nd week in weak light). Calli of about
lmm in diameter were transferred to the culture
(Cul) medium (Haberlach et al. 1985) for growth
and greening for 2-3 weeks.
Plant regeneration
The green calli of 3-4 mm in diameter were trans-
ferred to MSR1 medium (SA4 medium of Austin et
al. 1993) for shoot initiation. After 2 weeks, the
calli were transferred to MSR2 medium with 2 rag/1
t-zeatin and 2 mg/1 GA 3, for shoot elongation. The
differentiating calli were incubated at 22 °/19 °C
and a 16h/8h day/night cycle with illumination of
150 btE.m-2.s -l .
Results
Protoplast culturing
O n average, the protoplast yield ranged from 0.5 x
106 to 1.5 x 106 per g fresh weight of leaves or
suspension-cultured cells. After plating at an initial
density of about 105 protoplasts.m1-1 and two feed-
ing 50 % dilutions with SKM(-BSA) medium (on
the following day and on the 4th day after isola-
tion), the protoplasts started to divide in 3-4 days
and in 8-10 days the multicellular colonies were
formed. The colonies were formed both in liquid
and semi-solid medium, though in the latter the
cells were more uniform and white. In fast growing
cultures overcrowding and successive browning of
colonies occurred. A positive effect of additional
dilution at ratios 1:3 and 1:6 immediately before so-
lidification, was found. The diluted and solidified
cell clusters grew faster and remained white with
the exception of S. blb microcalli which turned tan-
nish on solidified SKM medium.
Callus growth and shoot production
W h e n the leaf protoplast-derived microcalli were
transfen'ed to solid medium for successive growth,
the three Solarium species reacted differently. Most
of the S. bib calli on Cul medium became brown and
ceased growth. Calli that were not completely
6
brown were successively transferred to shoot initia-
tion and elongation media (MSR1 and MSR2, re-
spectively) but no greening and shoot initiation was
observed in more than 300 calli tested (Fig, 1A). By
contrast, the calli of potato clones, as well as of S.
ngr, continued to enlarge in semi-solid SKM me-
dium, greened on Cul medium, produced shoot
meristems on shoot initiation medium MSR1 and
distinct shoots in 3-4 weeks after transfer to shoot
elongation medium MSR2 (Fig. 1B-E). Shoot elon-
gation was hampered only in S. ngr L. calli (Fig.
1F), while a significant part of shoots in all the po-
tato clones and S. ngr vat. gigantea grew long
enough to be detached for rooting. An increase in
shoot regeneration was obtained when the sucrose
concentration in MSR2 was reduced from 2 % to
1%. According to Masson et al. (1987), a sucrose
concentration greater than 10 g-I q enhanced the
callus growth but was detrimental to regeneration
and shoot formation.
Callus growth dynamics and shooting potential
varied for different Solanum genotypes. The
number of shoots in individual leaf protoplast-
derived callus ranged from 1 to more than 10. The
wide range of shoot morphology was also ob-
Fig. 1 (A-F). Mesophyll protoplast-derived calli on shoot elon-
gation medium (MSR2). First shoot initials were noticed in 10
weeks (E), 12 weeks (C, D, F) and 16 weeks (B, without solidi-
fication) after protoplast isolation, while no shoot morpho-
genesis occurred in (A). Bars = 1 cm.
Fig. 2. Suspension cell protoplast-derived calli on shoot elon-
gation medium (MSR2) after 7 weeks (Bzura) and 11 weeks
(H-8105) of culturing with (+sd) or without (-sd) a solidifica-
tion step. The positive effect of solidification on regeneration
of Bzura calli is evident. Bars = I cm.
Fig. 3. Plants regenerated Fig. 1A [-F~g.2
from mesophyll proto-
plasts of Bzura, H-8105, Fig. IB
ZEL-1136 and S. nigrum
vat. gigantea.
Fig. 1C Fig. 3
Fig. ID
Fig. 1E
Fig. IF
 REGENERATION OF SOLANUM PROTOPLASTS

...B
.. zura ~

d : ,,~ A ¸

S.ngr L. : i !i;ii i!~; i!~:~iilII~¸ F

~~ !i!IIi~i~i
A. SZCZERBAKOWA, M. BORKOWSKA & B. WIELGAT
served. Generally, the shoots from dihaploid
protoplast-derived calli were less vigorous than
those from tetraploid or hexaploid protoplast-
derived calli. Namely, the shoots on H-8105 calli
were multiple but mostly abnormal (thin and leaf-
less). The calli of ZEL- 1136, bright green in color
with further bleaching, were the only ones that con-
tinued their enlargement on MSR2 medium, turned
extremely soft and friable, and produced shoots at
rather low frequency. In other words, ZEL-1136
calli had a tendency to grow without redifferentia-
tion. The largest calli of ZEL-1136 achieved ca.
3 cm in diameter and did not regenerate. On the
contrary, practically all the Bzura calli produced
abundant shoots with heavy purple pigmentation,
long internodes, and poor leaf development, as can
be seen in Fig. lB. The most vigorous calli ofS. ngr
vat. gigantea produced many shoots with thickened
stems, short internodes, and wrinkled, dark green,
broad leaves (Fig. 1E). Typical abnormalities of S.
ngr shoots included the lack of clear main stem, dis-
torted leaves, and, more rarely, albinism.
It was found to be untrue that larger calli would
yield more shoots, and, moreover, in the case of
ZEL-1136 the opposite effect was noticed. On the
other hand, there surely was a critical size of the cal-
lus (not less than 2 mm in diameter after two weeks
of culturing on Cul medium) that seemed to be
obligatory for further shoot organogenesis. Too
small, unsufficiently developed calli died inevita-
bly after transfer from Cul to MSR1 medium.
A s for suspension cell-derived protoplasts of
Bzura, the spreading translucent calli were mostly
formed that failed to green on Cul medium and
turned brown on MSR1 and MSR2 media (Fig. 2A,
right part). Those were our first experiments on pro-
toplast regeneration when the protoplast cultures
were not yet solidified with SeaPlaque agarose.
When a solidification step was included on the
4th-5th day after protoplast isolation, a few green,
granular calli were produced on MSR1/MSR2 me-
dia, though no shoots were developed (Fig. 1A, left
part). The suspension cell-derived protoplasts of
H-8105 formed the regenerative type of calli more
frequently but the last ones were also without
shoots (Fig. 2B).
8
Rooting
The tbrmation of roots was routinely induced by
plating shoot cuttings on hormone-free medium
with 0.6 % agar (Nehls 1978). For better rooting,
the percentage of sucrose was again increased up to
2 %. The temperature of 25 °C was found to be opti-
mal for rooting, while at 22 °/18 °C cycle the root
formation was inhibited. Morphologically normal
shoots ~brmed roots in 2-3 weeks (Fig. 3), but some
shoots formed calli instead of roots, or formed both.
In such cases, the shoots were once more detached
and rerooted, often with success. Shoots with
grossly abnormal appearance usually failed to root
(see also Austin et al. 1986). Those aberrant forms
of regenerated plants that succeded in rooting, often
recovered a normal habitat after further passages
(similarly to observations of Sihachakr & Ducreux
1987).
Discussion
The media for protoplast culturing and regenera-
tion are usually based on compositions proposed by
Kao and Michayluk (1975) though many various
modifications were later introduced by different rc-
search groups. Our own experience confirmed the
necessity of the following steps in the protocol for
successful protoplast regeneration of the studied
genotypes. (1) The preconditioning of in vitro
grown leaves according to Haberlach et aI. (1985)
was essential for viability of isolated protoplasts.
(2) The conditions of protoplast isolation (enzyme
concentration, temperature, and duration of diges-
tion) should be selected individually for each used
line or species. (3) The initial density of 105 proto-
plasts per ml promoted the onset of cell divisions,
while the feeding dilutions up to the final density of
104 protoplasts per ml helped to avoid the further
browning of cell colonies caused by their over-
crowding. Such a dilution before plating could,
however, be detrimental in the case of protoplasts
with low plating efficiency (i.e. low division capac-
ity). (4) The growing of cell aggregates in solidified
(soft) medium was crucial for microcalli formation.
As in a study of Bokelmann & Roest (1983), the cell
aggregates did not survive if they were transferred
directly from the liquid culture medium (either
KM-8p or SKM) to solid MS medium. Semi-solid

medium probably fixes the location of cell aggre-
gates thus affecting their future differentiation. In
our experiments only S. ngr var. gigantea calli re-
generated after direct transfer from liquid K3 me-
dium (Nagy & Maliga 1976) to solid regeneration
medium. (5) The microcalli transferred to Cul me-
dium could grow and green at continuous weak
light and temperature 25 °C, but full transformation
into regenerative type of callus and shoot initiation
on MSR1 medium occurred only after lowering the
temperature to 22 '718 °C day/night cycle, Both vi-
tality and size of the calli were important factors for
successful regeneration (see also Boehmer et al.
1995,/'or protoplast-derived pea callus). The calli
smaller than lmm (as most of S. blb calli) failed to
develop further on Cul medium, turned brown, and
died.
The wild protoclones used in fusion combinations
by other authors usually did not regenerate them-
selves undcr culture conditions. Austin et al. (19861)
observed only three shooting calli ofS. blb per sev-
cral thousands cultured. The protoplasts of S. pin-
natisectum (S. pnt) failed even to divide sustainably
(Sidorov et al. 1987). Also Hunt & Helgeson
(1989) as well as Menke et al. (1996) found S. pnt to
be a recalcitrant species in respect to protoplast cul-
ture and plant regeneration. The same was true for
S. brevidens Phil. (Rokka et al. 1994) and for a di-
haploid clone of S. acaule, a Peruvian specics (Ya-
mada et al. 1997). The protoclones of S. p n t and S.
tbr (dihaploids) did not regenerate even after sclffu-
sion (Ward et al. 1994). The S. tbr parents selected
for hybridization also did not regenerate in the
study of Rokka et al. (1994). On the other hand, the
inability of the parental protoplasts to produce calli
and to regenerate, usually favoured the initial selec-
tion of their somatic hybrids.
As for protoplasts isolated from the cell suspen-
sions, their plating efficiency (i.e. division fre-
quency) could be higher than the plating efficiency
of mesophyll protoplasts, due to the previous adap-
tation to culture conditions, as shown by Anjum
(1998) for two potato cultivars. Nevertheless, the
calli derived fi'om mesophyll protoplasts differenti-
ated two weeks earlier than those from suspension
cell protoplasts, as shown by the same author. In our
case, the suspension cell-derived protoplasts of
both dihaploid H-8105 clone and tetraploid Bzura
REGENERATION OF SOLANUM PROTOPLASTS
cv. also seemed to start division earlier and with
higher frequency than the mesophyll protoplasts.
But only the H-8105 protoctone produced signifi-
cant number of calli of regenerative type: green,
tight, with shoot initials, which, however, did not
develop into normal shoots. The capacity for or-
ganogenesis was certainly reduced during pro-
longed culturing of the cells in liquid medium ,
though it was still expressed at least in H-8105 pro-
toclone to a considerable extent.The elongatcd
shoots could probably be observed if larger popula-
tion of H-8105 calli was screened. As for Bzura cv.,
the dominating spreading, amorphous, translucent
calli derived from its suspension-cell protoplasts,
restricted the possibility of usage of this protoclone
as a recipient of wild partner's genome in fusion ex-
periments. Concurrently, the advanced regenera-
tion of H-8105 suspension cell protoplasts indi-
cated their possible usefulness lbr somatic hybridi-
zation, since they would enable the early selection
of interspecific fusants with mesophyll protoplasts
of wild species by their intermediate morphology.
The evident diffcrence in vigour was observed be-
tween the regenerants from the mesophyll proto-
plasts of dihaploid potato lines and their counter-
parts from in vitro cultures: the protoplast-derived
plants were weaker and their rooting lasted longer
(about two weeks instead of one for normally
propagated plants). Those diffcrences were not so
noticeable in Bzura protoplast-derived plants,
while the protoplast-derived regcnerants of S. nig-
rum were as vigorous as in vitro cultured plants and
rooted rapidly (in a week) on condition that the ex-
cised shoots had normal morphology. Shoots with
grossly abnormal appearance usually failed to root
(see also Austin et al. 1986). The aberrant forms of
regenerated plants that succeeded in rooting, often
recovered a normal habitat (Sihachakr & Ducreux,
1987}. We also noticed the stabilization of the nor-
mal shoot phenotypes after several propagations.
Thus, the tetraploid potato line and hexaploid wild
species did not lose the capacity for organogenesis
during protoplast isolation and culturing, while this
capacity was reduced and even completely lost in
case of the studied dihaploid potato lines and wild
S. blb species, respectively.
The demonstrated differences in regeneration of
protoplasts isolated both from leaf mesophyll and
9
A. SZCZERBAKOWA. M. BORKOWSKA & B. WIELGAT
•: : :: ~:.~.
suspension cells o f three potato lines and two re-
lated wild S o l a n u m species, S. bib and S. ngr, could
be critical for success in interspecific hybridization
between them. We consider the presented data as a
baseline for further study o f the value of those lines
as partners in protoplast-fusion experiments,
Acknowledgements
The authors are grateful to Prof. J. Jakubiec and M. Sc. Anna
Pawetczak (Warsaw Agricultural University) for the axenic
shoot cultures of S. bulbocastaman, H-8105 and ZEL-1136
clones of S. tuberosum, as well as the seeds of S. nigrum L., to
Bonin and Mtoch6w Research Centers (IHAR, Poland) for
Bzura cv. and the seeds of S. nigrum var. gigantea. We also
thank M. Sc. L. Laskowski for the photographic plates and
Prof. L.D. Wasilewska for the critical reading of the manu-
script. This work was supported in part by a grant from the
State Committee for Scientific Research (KBN) No. 6 PO4B
021 12.
References
Anjum M.A. 1998. Effect of protoplast source and me-
dia on growth and regenerability of protoplast-derived
calluses of Solanum tuberosum L. Acta Physiol. Plant.,
20: 129-133.
Austin S., Ehlenfeldt M. K., Baer M. A., Helgeson
J.P. 1986. Somatic hybrids produced by protoplast fu-
sion between S. tuberosum and S. brevidens: phenotypic
variation under field conditions. Theor. Appl. Genet.,
71: 682-690.
Austin S., Pohlman J.D., Brown C.R., Mojtahedi H.,
Santo G.S., Douches D.S., Helgeson J.P. 1993. Inter-
specific somatic hybridization between Solanum tubero-
sum L.. and S. bulbocastanum Dun. as a means of trans-
ferring nematode resistance. Am. Potato J., 70: 485-495.
Boehmer P., Meyer B., Jacobsen H.-J. 1995.
Thidiazuron-induced high frequency of shoot induction
and plant regeneration in protoplast derived pea callus.
Plant Cell Rep., 15: 26-29.
Bokelmann G.S., Roest S. 1983. Plant regeneration
from protoplasts of potato ( S o l a n u m t u b e r o s u m
cv.Bintje). Z. Pflanzenphysiol., 109: 259-265.
Haberlach G.T.,Cohen B.A., Baer M.A.,Towill L.E.
Helgeson J.P. 1985. Isolation, culture and regeneration
of protoplasts from potato and several related Solanum
species. Plant Sci., 39: 67-74.
Horsman K., Bergervoet J.E.M., Jacobsen E. 1997.
Somatic hybridization between Solanum tuberosum and
species of the S. nigrum complex: Selection of vigor-
ously growing and flowering plants. Euphytica, 96:
345 -352.
10
:: : =======================================================. •,
Hunt G.J., Helgeson J.P. 1989. A medium and silnpli-
fled procedure for growing single cells from Solarium
species. Plant Sci., 60:251-257.
Kao K.N., Michayluk M.R. 1975. Nutritional require-
ments for growth of Vicia hajastana cells and proto-
plasts at a very low population density in liquid media.
Planta, 126: 105-110.
Masson J., Lecerf M., Rousselle P., Perennec P., Pel-
letier G, 1987. Plant regeneration from protoplasts of
diploid potato derived from crosses of Solanum tubero-
sum with wild Solanum species. Plant Sci., 53: 167-176.
Menczel L., Nagy F., Kiss Z.R., Maliga P. 1981. Strep-
tolnycin resistant and sensitive somatic hybrids of Nico-
tiana tabacum + Nicotiana knightiana: Correlation of re-
sistance to N. tabacum plastids. Theor. Appl. Genet., 59:
191-195.
Menke U., Schilde-Rentschler L., Ruoss B., Zanke
C., Hemleben V., Ninnemann H. 1996. Somatic hy-
brids between the cultivated potato Solanum tuberosum
L. and the 1EBN wild species Solanum pinnatisectum
Dun.: morphological and molecular characterization.
Theor. Appl. Genet., 92: 617-626.
Murashige T., Skoog F. 1962. A revised medium for
rapid growth and bioassays with tobacco tissue culture.
Physiol. Plant., 15: 473-497.
Nagy J.I., Maliga P. 1976. Callus induction and plant
regeneration from mesophyll protoplasts of Nicotiana
sylvestris. Z. Pflanzenphysiol., 78: 453-455.
Nehls R. 1978. Isolation and regeneration of protoplasts
from Solanum nigrum L. Plant Sci. Lett., 12: 183-187.
Rokka V.M., Xu Y.S., Kankila J., Kuusela A., Pulli
S., Pehu E. 1994. Identification of somatic hybrids od
dihaploid Solanum tuberosum lines and S. brevidens by
species specific RAPD patterns and assessment of de-
sease resistance of the hybrids. Euphytica, 80:207-217.
Sidorov V.A., Zubko M.K., Kuchko A.A., Komarnit-
sky I.K., Gleba Y.Y. 1987. Somatic hybridization in po-
tato: use of y-irradiated protoplasts of Solanum pinnati-
sectum in genetic reconstruction. Theor. Appl. Genet.
74: 364-368.
Sihachakr D., Ducreux G. 1987 Variations ol"morpho-
genetic behavior and plant regeneration in cultured pro-
toplasts of Solanum nigrum. Plant Sci., 52:117-126.
Ward A.C., Phelpstead J..St-J., Gleadle A.E., Black-
hall N.W., Cooper-Bland S., Kumar A., Powell W.,
Power J.B., Davey M.R. 1994. Interspecific somatic
hybrids between dihaploid Solanum tuberosum L. and
the wild species, S. pinnatisectum Dun. J. Exp. Bot., 45:
1433-1440.
Yamada T., Misoo S., Ishii T., Ito Y., Takaoka K.,
Kamijima O. 1997. Characterization of somatic hybrids
between tetraploid Solanum tuberosum L. and dihaploid
S. acaule. Breeding Sci., 47: 229-236.
Received July 06, 1999; accepted October 26, 1999