Title:ECiB 60-3_05 Page:35 Date: 2022/07/04 Mon 17:19:05

Short Communication Environ. Control Biol., 60 (3), 187-190, 2022

DOI: 10.2525/ecb.60.187

Production of Doubled Haploid through Anther Culture of the Male-Fertile Lines

in the CMS System of Eggplant with the Cytoplasm of Solanum grandifolium

Md Mizanur Rahim KHAN1, Takumi MATSUYAMA1, Ichiro NAKAMURA1, Kenji URESHINO1,

Takashi ARITA2, Masaki IWAYOSHI2, Yuki OGURA-TSUJITA2 and Shiro ISSHIKI2
1 Laboratory of Horticulture, Faculty of Agriculture, University of the Ryukyus, Okinawa 903―0213, Japan
2 Vegetable and Flower Horticulture Laboratory, Faculty of Agriculture, Saga University, Saga 840―8502, Japan

(Received October 25, 2021; Accepted February 18, 2022)

To produce doubled haploid homozygous male-sterile line of eggplant with the cytoplasm of Solanum grandifolium,
anther culture of the male-fertile (MF) BC5 in the S. grandifolium induced CMS system was done. Anthers were cultured
in an embryo induction medium and were heated to 35℃ for 3 d in the dark and then transferred to 25℃ under a 16 h
photoperiod. After 20 d the anthers were transferred to a germination medium. The presence of pollen in the anther, number
of chromosomes in the root tip cells, size of the stomatal guard cells and pollen fertility of the regenerated plants were
investigated. The frequency of embryo formation in the MF line was 1.02%. From the embryos, one regenerated plant
showed pollen non-formation type male sterility. Root tip chromosome number and stomatal guard cell size confirmed that
the regenerated male-sterile plant is a microspore originated doubled haploid through spontaneous chromosome doubling.
The present study demonstrates the first successful production of a doubled haploid homozygous male-sterile line in a CMS
system of eggplant with the cytoplasm of S. grandifolium. Therefore, this material could be a useful breeding material for
eggplant and improve the speed of conventional breeding.

Keywords : androgenesis, cytoplasmic male sterility, homozygous, pure line, male-sterile

A pollen non-formation type promising cytoplasmic

INTRODUCTION
Eggplant (Solanum melongena L.) is one of the most
extensively cultivated vegetable crops worldwide belong-
ing to the family Solanaceae. Most of the intervarietal
hybrids of eggplant are reported to have exhibited consider-
able vigor in economic characters, particularly the yield
(Sambandam, 1962). For this reason, the main breeding
method in eggplant has been F1 hybrid seed breeding. In F1
hybrid breeding, first of all, homozygous pure parent lines
are needed. Pure lines have traditionally been obtained
through 6–10 self-crossing generations (Prigge et al.,
2012). Instead, completely homozygous, doubled haploid
(DH) individuals can be induced from haploid cells of the
germ line in a single generation. This decreases the number
of generations needed to produce a pure line, thus reducing
the costs of breeding programs (Germana, 2011). In addi-
tion, the fully homozygous condition of DHs makes them a
valuable tool to facilitate the establishment of chromosome
maps, mapping of genetic markers, marker-trait associa-
tion and identification of recessive mutations, etc. (Forster
et al., 2007). Currently, the DH technique is used in egg-
plant for androgenesis. Generally, anther culture, isolated
microspore culture, and shed-microspore cultures were
used for the induction of androgenesis. Anther culture is
the most widely applied method for DH production
because of its practical applicability.
male sterility (CMS) system of eggplant was reported to be
developed by substituting the cytoplasm of eggplant by
that of ‘Taibyo VF,’ which is indicated to contain the cyto-
plasm of S. grandifolium C.V. Morton (Saito et al., 2009;
Hasnunnahar et al., 2012). ‘Taibyo VF’ is a commercial
rootstock hybrid cultivar between S. grandifolium and S.
melongena released by Takii & Co., Ltd. (Kyoto, Japan),
resistant to Verticillium wilt. Backcross (BC) progeny of
S. grandifolium induced CMS line of eggplant were segre-
gated into male-sterile (MS) and male-fertile (MF) types
(Fig. 1). Anther of the male-sterile line is completely
devoid of pollen. Two independent dominant fertility
restorer genes were reported to control the pollen forma-
tion in this CMS system. Pollen can form if plants contain
the Rf gene at either or both of these loci, while recessive-
ness for both results in failure of pollen formation.
Several researchers reported on the production of hap-
loid and DH plants from anthers of eggplant (Isouard et al.,
1979; De Vaulx and Chambonnet, 1982; Salas et al., 2012).
In contrast to the extensive application of anther culture to
common eggplant, very little information is available
regarding anther culture of eggplant containing the cyto-
plasm of wild Solanum species. A DH male-fertile plant
was reported from anther culture of the male-fertile lines
in S. grandifolium induced CMS system of eggplant (Khan
et al., 2013) but to the best of our knowledge, there is no
report of DH homozygous male-sterile line production of

Corresponding author : Shiro Isshiki,

e-mail : isshiki@cc.saga-u.ac.jp

Vol. 60, No. 3 (2022) （ 35 ）187

Title:ECiB 60-3_05 Page:36 Date: 2022/07/04 Mon 17:19:05
M. M. R. KHAN ET AL.
Fig. 1 Development of the cytoplasmic substitution line of eggplant containing the cytoplasm of Solanum grandifolium by continuous
backcrossing. Progenies were selected in the two directions of male-fertile and sterile types during backcrossing.
this CMS system through anther culture. Therefore, in the
present study, our main goal is to produce DH homozy-
gous male-sterile line, through anther culture of the MF
line in the CMS system of eggplant containing the cyto-
plasm of S. grandifolium.
MATERIALS AND METHODS
S. melongena ‘Uttara’ and the MF line of BC5 prog-
eny containing the cytoplasm of S. grandifolium were used
as plant materials. Cytoplasmic substitution line of egg-
plant was previously developed (Hasnunnahar et al., 2012)
by continuous backcrossing of an interspecific hybrid
‘Taibyo VF’ (S. grandifolium⫻S. melongena) with recur-
rent pollen parent S. melongena ‘Uttara’ (Fig. 1).
Anthers of the MF line containing microspores at the
uninucleate stage were selected for culture. The unopened
flower buds were sterilized with 70% ethanol for 10 s and
then with 1% sodium hypochlorite solution for 10 min.
They were then rinsed 3 times in sterile water. After sterili-
zation of the unopened flower buds, anthers were cultured
in an embryo induction medium and were heated to 35℃
for 3 d in a dark and then transferred to 25℃ under a
16 h photoperiod. Embryo induction medium consists of
Murashige and Skoog (MS) medium supplemented with
3% sucrose, 0.3% gellan gum, 1% charcoal, 10 mg/L
AgNO3, 0.1 mg/L both of 2,4-D and kinetin. The medium
was adjusted to pH 5.8. After 20 d the anthers were trans-
ferred to a germination medium and cultured in the same
temperature and photoperiod condition. Germination
medium consists of MS medium supplemented with 3%
sucrose, 0.3% gellangum supplemented with 0.1 mg/L
kinetin. The embryos derived from anthers were trans-
planted to a hormone-free regeneration medium until roots
appeared. Regeneration medium consists of MS medium
supplemented with 3% sucrose and 0.8% agar. The regen-
erated plants were transplanted in a plastic pot filled with
vermiculite. For ploidy determination, the number of chro-
mosomes in the root tip cells and the size of the stomatal
guard cells were investigated in the regenerated plants. In
188（ 36 ）
addition, pollen formation ability and pollen stainability of
the regenerated plants were investigated.
To investigate the number of chromosomes in the root
tip cells, roots were cut approximately 1 cm from the tips
and pretreated with 0.05% colchicine for 2.5 h at 20℃ and
kept in the mixture of acetic acid and ethyl alcohol
(1:3 v/v) before hydrolyzing at 60℃ for 10 min in 1N
HCL. The root tips were then stained with leucobasic
fuchsine for 20 min and placed separately on a glass slide
with the addition of a drop of 45% acetic acid on them. A
coverslip was placed over the root tip and gentle pressure
was applied to the coverslip to smear the root tip. The num-
ber of chromosomes in the root tip cells was observed and
counted under a microscope. The size of stomatal guard
cells was measured from 5 well-expanded leaves of each
plant. Three samples of epidermal cells were collected
from the lower surface (abaxial side) by using the nail var-
nish technique. A small area of the abaxial side of the
leaves was covered with a thin layer of clear nail polish
and left to dry. A piece of scotch tape was firmly attached
to one end of the nail polish and the polish was carefully
pulled off the leaf by the tape. Each peel with tape was
placed on microscope slides, then covered with a cover
glass and observed under a microscope. Guard cells were
randomly selected, and their length and width were meas-
ured in units of an ocular micrometer that were calibrated
with a calibration slide.
To investigate pollen formation ability, anthers from
freshly opened flowers were squashed in acetocarmine and
examined for the presence of pollen. Anthers from 5 flow-
ers were examined for each plant material. The plant which
did not produce any pollen was defined as MS and the
plant which produce pollen was defined as MF. To investi-
gate pollen stainability, pollen grains from freshly opened
flowers were extracted from the anthers by dissection and
then smeared in 1% acetocarmine to assess their staining
ability following Singh (2002). At least 500 pollen grains
per flower were observed in five flowers per plant for
assessing pollen fertility.
Environ. Control Biol.
Title:ECiB 60-3_05 Page:37 Date: 2022/07/04 Mon 17:19:06

DH BY ANTHER CULTURE OF CMS EGGPLANT

Table 1 Response of anther culture of three male-fertile (MF) lines of eggplant with the cytoplasm
of Solanum grandifolium.
Plant materials No. of Frequency of Frequency of Ploidy
anthers embryo regenerated
cultured formation (%) plants (%) Haploid Diploid

Male-fertile BC5 394 1.02 0.51 0 2

S. melongena ‘Uttara’ 560 0.36 0 ― ―

A B C

Fig. 2 Embryoid and regenerated plant formation of a doubled haploid homozygous male-sterile (MS) plant from anther culture of male-
fertile (MF) line of eggplant with the cytoplasm of Solanum grandifolium. Thirty-seven days after anther culture (A), 80 d after anther
culture (B) 140 d after anther culture (C).

MF line of eggplant was 2n ⫽ 24 (Table 2). Root tip cells

RESULTS AND DISCUSSION
The MF BC5 plant showed lower pollen stainability
with acetocarmine and pollen germination ability than S.
melongena ‘Uttara.’ The frequency of embryo formation in
the MF line was 1.02% and in the S. melongena ‘Uttara’
was 0.36% (Table 1, Fig. 2). The frequency of embryo for-
mation from anther culture in the cytoplasmic substitution
lines containing the cytoplasms of S. anguivi and S. grandi-
folium was reported 0.39% and 0.26%, respectively (Khan
et al., 2013). Higher embryo formation in our study might
be due to supplemented AgNO3 in embryo induction
medium as reported in the past (AboShama and Atwa,
2019; Buyukalaca et al., 2004). Previous studies on egg-
plant anther culture reported maximal responses of 14.2
embryos (Başay et al., 2011) and 3.67 embryos/100
anthers with the most responsive of the genotypes used
(Alpsoy and Seniz, 2007). Compared to common eggplant
low frequency of embryo formation obtained in the present
study might be due to lower pollen stainability of the
anther donor MF line of BC5 progeny.
From the embryos, two plants were regenerated
(Table 1). Chromosome numbers of the original diploid
of both the regenerated plants have 24 chromosomes
(Table 2). The length and width of the stomatal guard cells
of these doubled haploids were almost similar to its mother
plant and S. melongena ‘Uttara.’ Thus, from the root tip
chromosome number and stomatal guard cell size, it is con-
firmed that both of the regenerated plants are diploid.
Lower frequency of embryo formation in S. melongena
‘Uttara’ might be due to genotypic variation to anther cul-
ture response. Plant donor genotype is one of the most
important factors affecting androgenic capacity. There is
variation in the androgenic response among genotypes in
eggplant exposed to the same set of inductive and cultural
conditions (Rotino et al., 1987; Başay et al., 2011). In gen-
eral, genotype, developmental stage of the microspore and
different culture conditions are the three main factors that
prevent many species from being deviated to androgenesis
(Salas et al., 2012).
Investigation of pollen formation ability revealed that
among the two regenerated plants, anthers of the regener-
ated plant No.1 was smaller in size and did not contain any
pollen. However, anthers of the regenerated plant No.2
were normal in size and contain the substantial amount of
pollen. As we obtained male-sterile plant from anther cul-

Table 2 Characteristics of the regenerated plants obtained from anther culture of the male-fertile BC5 of eggplant with
the cytoplasm of Solanum grandifolium.
Male- Pollen Stomatal guard cell size (mm) Chromosome
Plant materials fertile/male- stainability number in the
sterile (%) Length Width root tip cells
S. melongena ‘Uttara’ MF 95.5⫾1.01 a 18.68⫾0.45 a 13.8⫾0.30 a 2n ⫽ 24
Male-fertile BC5 MF 55.18⫾3.71 b 19.33⫾0.35 a 13.44⫾0.24 a 2n ⫽ 24
Regenerated plant No.1 MS ― 19.25⫾0.31 a 13.17⫾0.25 a 2n ⫽ 24
Regenerated plant No.2 MF 64.14⫾1.40 b 20.03⫾0.36 a 14.72⫾0.30 a 2n ⫽ 24
a
Values represent the mean⫾SE, and those with the same letters within a column are not significantly different accord-
ing to Tukey’s HSD tests (P ⬍ 0.05).

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Title:ECiB 60-3_05 Page:38 Date: 2022/07/04 Mon 17:19:08
M. M. R. KHAN ET AL.
ture of MF line, it is confirmed that the male-sterile plant
is of haploid origin and chromosome doubling of the regen-
erated plant might have occurred spontaneously. There-
fore, regenerated plant No.1 obtained in the present study
is a doubled haploid homozygous male-sterile line of egg-
plant containing the cytoplasm of S. grandifolium.
Through spontaneous chromosome doubling, duplication
of the haploid genome of pollen-derived individuals has
been thought to occur. Mainly, cellular processes such as
nuclear fusion are the cause of spontaneous genome dou-
bling (Kasha, 2005), and their occurrence is highly depend-
ent on plant species. Spontaneous chromosome doubling
has been reported in barley 70–90%, bread wheat 25–70%,
rice 50–60% and maize 20% (Maluszynski et al., 2003;
Martin and Widholm, 1996).
Pollen stainability of S. melongena ‘Uttara’ was very
high (Table 2). Pollen stainability of the regenerated MF
plant was not different from its mother plant but differed
with S. melongena ‘Uttara.’ This might be due to dishar-
mony between the cytoplasm of S. grandifolium and the
nucleus of S. melongena in the regenerated MF plant. In
this study, we could not confirm that the regenerated MF
plant is of microspore origin. A DH male-fertile plant was
reported in S. grandifolium induced CMS system of egg-
plant from anther culture of the MF lines (Khan et al.,
2013) but they could not able to develop DH male-sterile
plant. In this study, a DH male-sterile plant is successfully
produced through anther culture.
The present study demonstrates successful production
of a DH homozygous male-sterile line in a CMS system of
eggplant with the cytoplasm of S. grandifolium. To the
best of our knowledge, this is the first report of DH male-
sterile line in the S. grandifolium induced CMS system of
eggplant. Therefore, this material would improve the effi-
ciency and the speed of the usually cumbersome, time-con-
suming, laborious conventional breeding.
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