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1 FigureIn Vitro Modification of Sex Expression in Mulberry (Morus Alba) by Ethrel and Silver Nitrate
Abstract
Mulberry (Morus alba L.) is a dioecious plant. In vitro sex modification in mulberry by using two chemicals, viz., ethrel and silver nitrate, is described. 2-Chloroethylphosphonic acid (ethrel) and silver nitrate were filter sterilized and added to Murashige and Skoog (1962) medium supplemented with 5 M 6-benzyl amino purine (BA). Ethrel applied at different concentrations produced female, male and mixed inflorescences in male mulberry nodal cuttings. Maximum 13.6% female inflorescences were observed on media containing 2000 g l–1 ethrel. The induction of male, female and mixed inflorescences was observed in female plants after applying silver nitrate to the culture medium. The number of male inflorescences increased with the silver nitrate concentration to a maximum of 22.4% induction on 2500 g l–1. Bisexual flowers were also observed along with male and female flowers in silver nitrate treated plants.
Figures
Plant Cell, Tissue and Organ Culture 77: 277–281, 2004.
© 2004 Kluwer Academic Publishers. Printed in the Netherlands. 277
Research note
In vitro modification of sex expression in mulberry (Morus alba)
by ethrel and silver nitrate
T. Dennis Thomas
Post Graduate and Research Department of Botany, St. Thomas College, Pala, Arunapuram (P.O.) 686574,
Kottayam, Kerala, India (Fax +91-95482-2216313; E-mail: den_thuruthiyil@yahoo.com)
Received 24 April 2003; accepted in revised form 5 October 2003
Key words: ethrel (ethephon), Morus alba, mulberry, sex expression, silver nitrate
Abstract
Mulberry (Morus alba L.) is a dioecious plant. In vitro sex modification in mulberry by using two chemicals,
viz., ethrel and silver nitrate, is described. 2-Chloroethylphosphonic acid (ethrel) and silver nitrate were filter
sterilized and added to Murashige and Skoog (1962) medium supplemented with 5µM 6-benzyl amino purine
(BA). Ethrel applied at different concentrations produced female, male and mixed inflorescences in male mulberry
nodal cuttings. Maximum 13.6% female inflorescences were observed on media containing 2000 µgl
−1ethrel.
The induction of male, female and mixed inflorescences was observed in female plants after applying silver nitrate
to the culture medium. The number of male inflorescences increased with the silver nitrate concentration to a
maximum of 22.4% induction on 2500 µgl
−1. Bisexual flowers were also observed along with male and female
flowers in silver nitrate treated plants.
Abbreviations: BA – 6-benzylaminopurine; Ethrel – 2-chloroethylphosphonic acid (2-CEPA); MS – Murashige and
Skoog (1962) medium
Mulberry (Morus alba L.) is an important tree in seri-
culture industry since its foliage is used for rearing
the silkworm larva (Bombyx mori). The genus Morus
belongs to the family Moraceae which is comprised of
nearly 35 species (Anonymous, 1975). Several species
of this genus are cultivated on a commercial scale in a
number of countries including China, Japan, Korea,
former USSR and India for the sericulture industry
(Oka and Ohyama, 1986).
Camerarius (1964) demonstrated sex in plants for
the first time in mulberry. Mulberry is a highly het-
erozygous plant and shows diverse sex behavior. Sex
expression is a prerequisite component to determine
an effective breeding programme for the improvement
of the mulberry. Mulberry is a dioecious plant with
male and female plants occurring separately so that
the breeding work in mulberry is affected heavily. The
breeding work in this plant is hampered due to the ex-
tremely asynchronous flowering especially in tropical
varieties (Narayan et al., 1993). Also the production
of homozygous lines of mulberry by inbreeding is
not possible because of its dioecious nature and male
and female lines being genetically diverse. There are
several reportssuggesting the role of ethylene in modi-
fying the sex expression in plants (Mohan Ram and
Jaiswal, 1970; Takahashi and Jaffe, 1984; Saito
and Takahashi, 1987; Trebitsh et al., 1987; Stankovic
and Prodanovic, 2002). Therefore, the present study
was performed with the objective to find out the
chances of modifying the sex expression in mulberry
in vitro by ethrel and silver nitrate.
Two cultivars of mulberry (M. alba L.) were used
for the present study. Of this, cv. S-13 produces
male and cv. K-2 (M-5) female flowers only. Ethrel
was used to change the sex expression of the male
cultivar (i.e., S-13) and silver nitrate for the female
cultivar (i.e., K-2). The plants were established in the
Botanical garden of the Institute and were pruned peri-
278
odically to allow the fresh shoots to emerge so that
they can be collected for in vitro experiments. Fresh
shoot tips containing axillary buds were collected for
the experiments.
The explants (i.e., single node cuttings) were col-
lected from cultivar S-13 and K-2 of M. alba.The
young healthy twigs (30–40 cm length) cut from the
top portions of the branches were trimmed and shoots
were cut into 5 cm pieces having a single axillary
bud. The explants were dipped in 1% Savlon germi-
cide (Johnson and Johnson, Mumbai) for 15min and
washed thoroughly under sterile distilled water for
three times. They were then surface sterilized with
0.1% aqueous mercuric chloride (HgCl2) solution for
8 min, rinsed three times with autoclaved distilled wa-
ter and were allowed to surface dry inside a laminar
flow cabinet. Such surface sterilized nodal cuttings
were slightly trimmed at the cut ends to expose fresh
tissue and planted on MS (Murashige and Skoog,
1962) medium fortified with different concentrations
of BA. For inflorescence induction, 5 µMBAwas
found optimum.
Both ethrel and silver nitrate used to modify sex
expression was filter sterilized before adding. Six con-
centrations of each chemical ranging from 500 to
3000 µgl
−1were tried along with 5 µM BA. After 45
days the nodal cuttings containing the inflorescences
were taken out of the test tube and observed using
a dissection microscope. Each flower from the inflo-
rescence was separated with the help of a needle and
forceps and observed under the microscope for sex
determination.
The media were solidified with 8 g l−1agar and
the pH was adjusted to 5.8 before autoclaving at
1.5kgcm
−2and 121 ◦C for 15 min. All cultures were
maintained at 25 ±2◦C under a 16-h photoperiod sup-
plied by two Philips TL 40 W fluorescent tubes. At
least 24 cultures were raised for each treatment and
all experiments were repeated three times. Analysis of
variance and Duncan’s multiple range test was used
for comparison among treatment means.
For sectioning, the flowers were fixed in FAA
(Formalin: glacial acetic acid: 70% ethanol in a 1:1:18
ratio by volume) for at least 24h. Following dehy-
dration in tertiary butanol, the material was infiltrated
and embedded in paraffin wax (mp 60 ◦C) (Johansen,
1940). Sections (8 µm thick) were cut on a rotary
microtome and stained with safranin-astrablau for
photography.
The single node cuttings measuring about 3–4cm
in length were cultured on MS medium supplemented
with BA alone or in combination with either ethrel
(500–3000 µgl
−1) or silver nitrate (500–3000g l−1)
and showed bud break after 1 week. The axillary bud
elongated and gave rise to a well-developed shoot. The
inflorescences emerged from the axil of this newly
formed shoot. One or more inflorescences were ob-
served in a single nodal cutting culture. The mean
number of inflorescences observed in the control treat-
ment (BAP 5 µM)was4.5incv.K-2,and4.8in
cv. S-13. The number of florets in each inflorescence
varied from 25 to 30 in female and 31 to 36 in male
cultivars. The female inflorescences turned light red 8
weeks after culture. The male inflorescences showed
anther dehiscence in the test tube. As in the case of
in vivo formed inflorescences, the male inflorescences
are comparatively longer than the female ones. All the
cultured nodal cuttings performed equally well irre-
spective of their position in the twig and there was no
significant difference in the number of inflorescences
formed per cutting.
The inflorescences at different stages of develop-
ment were observed 45 days after initiation of culture.
In both female and male cultivars, the axillary bud
growth was fast. The addition of BA did not affect the
sex of the male and female plants studied. In cv. S-13
100% male inflorescences were observed on medium
containing 5 µM BA. Similarly 100% female inflores-
cences were observed in cv. K-2 on the same medium
(Figure 1A). However the addition of ethrel and silver
nitrate changed the sex expression of both cultivars
significantly.
In cv. S-13 the maximum number (13.6%) female
inflorescences was observed on medium containing
2000 µgl
−1ethrel (Table 1). The induction of female
flowers was suppressed at 500–1000 µgl
−1ethrel con-
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 1. (A) Single node cutting culture of cultivar K-2 on MS
medium supplemented with 5 µM BA 4 weeks after culture. Sev-
eral female flowers have developed from the axil of the leaves.
Bar =1 cm. (B) A mixed inflorescence of cv. S-13 showing both the
male and female flowers on MS medium supplemented with BAP
(5 µM) and ethrel (2000 µgl
−1). Note the position of the flowers.
The male flowers (top arrow) are seen towards the tip of the in-
florescence and female flowers (bottom arrow) towards the base.
Bar =0.5 cm. (C) Two bisexual flowers of cv. K-2 on MS medium
fortified with BAP (5 µM) and silver nitrate (2500 µgl
−1) showing
the ovary and the anther (arrows). Bar =0.5 cm. (D) An LS of a
bisexual flower showing the ovule (top arrow) and anther (bottom
arrow). The flower was taken from MS medium supplemented with
BAP (5 µM) and silver nitrate (2500 µgl
−1). Bar =715 µm. (E)
Two abnormal flowers of cv. S-13 on MS medium supplemented
with BAP (5 µM) and ethrel (1500 µgl
−1) showing the fusion of
the ovaries (left) and elongation of the ovary (right). Bar =0.5 cm.
279
280
Table 1. Effect of various concentrations of ethrel on percent distribution of inflorescences in male plants of
mulberry (M. alba cv. S-13) in vitroa
Concentration Male Female Mixed Abnormal
(µgl
−1) inflorescences (%) inflorescences (%) inflorescences (%)binflorescences (%)
Control 100 – – –
500 88.0 ±5.6 c 4.7 ±0.7 a 4.1 ±0.08 a 3.2 ±0.9 a
1000 80.6 ±4.8 c 8.1 ±1.2 b 6.8 ±1.3 b 4.5±0.3 b
1500 74.8 ±4.4 b 12.3 ±1.3 c 7.4 ±1.1 c 5.5 ±0.6 c
2000 68.3 ±5.1 a 13.6 ±1.2 c 10.5 ±1.4d 7.6 ±1.4 d
2500 67.6 ±3.9 a 12.8 ±0.8 c 11.7 ±1.3 e 7.9 ±1.0 e
3000 67.5 ±4.3 a 12.0 ±1.4 c 11.4 ±1.2 e 9.1 ±1.7 f
aMedium: MS; control MS +BA (5 µM); culture period: 45 days. Means within a column followed by the
same letter are not significantly different by Duncan’s multiple range test (p > 0.05).
bInflorescences having female and male flowers.
Table 2. Effect of various concentration of silver nitrate on percent distribution of inflorescences in female plants of
mulberry (M. alba cv. K-2) in vitroa
Concentration Female Male Mixed inflorescences (%)
(µgl
−1) inflorescences (%) inflorescences (%)
FandB
bMandB
bFandM
bF, M an d B b
Control 100 f – – – – –
500 86 ±4.5 d 4.1 ±0.6 b 2.3 ±0.5 a 1.0 ±0. 2 a 4.4 ±0.5 b 2.2 ±0.4 b
1000 72 ±3.3 c 12.8 ±1.1 c 3.1 ±0.3 b 2.1 ±0.1 b 6.9 ±0.5 c 3.1 ±0.5 c
1500 66 ±4.8 b 20.8 ±1.8 d 2.2 ±0.7 a 1.6 ±0.3 b 6.1 ±0.4 c 3.3 ±0.3 c
2000 58 ±4.4 a 21.6 ±2.1 e 3.8 ±0.2b 3.2 ±0.3 c 7.1 ±0.6 d 6.3 ±0.2 d
2500 54 ±4.3 a 22.4 ±2.2 e 4.7 ±0.5 c 3.5 ±0.2 c 8.7 ±0.4 e 6.7 ±0.3 d
3000 54 ±4.4 a 22.3 ±1.9 e 4.8 ±0.4 c 3.3 ±0.4 c 8.9 ±0.5 e 6.7 ±0.4 d
aMedium: MS; control MS +BA (5 µM); culture period: 45 days. Means within a column followed by the same letter are
not significantly different by Duncan’s multiple range test (p > 0.05).
bF: female flower, B: bisexual flower, M: male flower.
centrations (Table 1). Mixed inflorescences having
both male and female flowers were also observed on
all the ethrel concentrations tried and their percent also
varied with the concentration of the ethrel. A max-
imum of 11.7% mixed inflorescences was observed on
2500 µgl
−1ethrel containing media. On higher con-
centrations (above 3000 µgl
−1) of ethrel the axillary
buds showed poor growth and hence it affected the
inflorescence development. On this medium, the tip
of the developing shoot became brown and eventually
died.
The arrangement of the female and male flowers
in the mixed inflorescences of ethrel treated cultures
showed variation. In 48% of the cultures the male
flowers were seen towards the base of the inflores-
cence and female flowers towards the tip whereas the
rest of the 52% inflorescences gave female flowers to-
wards the base of the inflorescences and male flowers
towards the tip (Figure 1B).
Six concentrations of silver nitrate were tried for
the present experiment (Table 2). The concentration
of silver nitrate considerably affected the development
of the female inflorescences in cv. K-2. The num-
ber of male inflorescences increased with the silver
nitrate concentration with a maximum of 22.4% in-
duction on 2500 µgl
−1(Table 2). In male flowers,
pollen viability was analyzed by tetrazolium test us-
ing 2,3,5-triphenyltetrazolium chloride (TTC) and an
average 91% of the pollen grains were found to be
viable. The effect of silver nitrate on male flower in-
duction is well documented in cucumber, Cucumis
sativus (Lower et al., 1978). Besides this, silver ni-
trate reduces female flowers in cucumber (Stankovic
and Prodanovic, 2002). Silver nitrate is known to be a
potential inhibitor of ethylene action. (Ockendon and
Clenaghan, 1993; Ghaemi et al., 1994). The inhib-
itors of ethylene action suppress the development of
female flowers and induce the male flowers (Beyer,
281
1976; Atsmon and Tabbak, 1979; Takahashi and Jaffe,
1984).
Another important observation during the investi-
gation was the origin of bisexual flowers (Figure 1C)
in silver nitrate treated cultures. Mulberry is dioecious
and the male and female flowers are normally seen
in separate plants. The bisexual flowers never occur
under natural conditions. By treating the nodal cut-
tings with silver nitrate it was found that bisexual
flowers could be induced in female plants. The histo-
logical analysis of such bisexual flowers showed both
ovule and anther in the same flower (Figure 1D). The
presence of such induced bisexual or hermaphrodite
flowers was observed in cucumber as a result of silver
nitrate treatment (Stankovic and Prodanovic, 2002).
The presence of ethrel in medium promoted the
development of abnormal female flowers in some in-
florescences. A maximum of 9.1% of flowers was
abnormal on 3000 µgl
−1ethrel. On this medium,
67.5% of male inflorescences was formed (Table 1).
Among the abnormalities, fusion of ovaries and ab-
normal elongation of the ovary were very common
(Figure 1E).
The present study, using ethrel as an ethylene
source, confirmed the possible role of ethylene in
in vitro sex expression of dioecious trees of M. alba.
Ethylene has been shown to induce female flowers
on dioecious male plants of Cannabis sativa (Mohan
Ram and Jaiswal, 1970). In flowering plants the sex
expression is controlled by the balance between endo-
genous auxin and gibberellin and/or between gibber-
ellin and ethylene. Ethylene and auxin promote the
formation of female flowers, whereas gibberellins pro-
mote the formation of male flowers (Mohan Ram
and Jaiswal, 1970; Saito and Takahashi, 1987). The
enhancement of femaleness by auxin possibly oc-
curs through the induction of ethylene biosynthesis
(Takahashi and Jaffe, 1984; Trebitsh et al., 1987). It is
believed that ethylene act more directly than gibberel-
lins on sex expression in some plants (Yin and Quinn,
1995). Ethylene evolution is highly correlated with
sex expression in plants and dioecious plants produce
more ethylene than monoecious ones (Rudich et al.,
1972; Trebitsh et al., 1987).
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- ... Controlled starvation of the plants in culture is often considered as a useful method for root induction in several systems. Our result is in agreement with previous observations by several workers242526 . In the present study ABA in combination with BA and NAA were responsible for in vitro flowering. ...
- ... Controlled starvation of the plants in culture is often considered as a useful method for root induction in several systems. Our result is in agreement with previous observations by several workers [24][25][26]. In the present study ABA in combination with BA and NAA were responsible for in vitro flowering. ...
- ... Controlled starvation of the plants in culture is often considered as a useful method for root induction in several systems. Our result is in agreement with previous observations by several workers242526 . In the present study ABA in combination with BA and NAA were responsible for in vitro flowering. ...
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