Improved Androgenesis of Broccoli (Brassica oleracea var italica)
Anthers Using Sucrose and Growth Regulators
Magdi A.A. Mousa,
Ashraf G. Haridy,
Hassan S. Abbas
Mohammed F. Mohammed
The presented study was conducted to study the effects of BAP and its combinations
with 2, 4-D and sucrose concentrations on androgenesis of broccoli cv. Arcadia.
The MS Basel media supplemented with AC (0.5 g L-1) and 3% sucrose
was used. The experiment was laid out in Completely Randomized Design (CRD)
using 3 replicates. The effects of BAP in combination with 2, 4-D and sucrose
concentrations on androgenesis of broccoli (cv Arcadia) anther cultures were
investigated. The MS basal media supplemented with activated charcoal (AC) (0.5
g L-1) was used. The medium complemented with BAP (1 mg L-1)
plus 2, 4-D (0.5 mg L-1) significantly increased the percentage of
anthers developing embryos (90%). Increasing 2, 4-D concentration (1 mg L-1)
significantly decreased the percentage of anthers formed embryos (10%). Regarding
the regeneration of the formed embryos, the culture medium containing BAP (1
mg L-1)+2, 4-D (0.5 mg L-1) or BAP (1 mg L-1)+2,
4-D (1 mg L-1) produced the high percentages of embryos induced callus.
The maximum percentage of embryos developed into plantlets were obtained with
culture medium with BAP (1 mg L-1)+2, 4-D (0.5 mg L-1)
followed by BAP (1 mg L-1) and 2, 4-D (1 mg L-1). Concerning
sucrose concentration, the culture medium with low sucrose concentration (20
g L-1) enhanced the percentage of anthers developed embryos. The
higher sucrose concentrations (50 and 60 g L-1) increased percentages
of dead anthers and anthers formed callus and decreased the percentage of embryos
developing plantlets. BAP in combinations with 2, 4-D and low concentrations
of sucrose can be successfully used to perform embryos and/or callus from the
callus induction and embryos development either to callus or plants.
Received: August 14, 2013;
Accepted: January 16, 2014;
Published: March 08, 2014
Another culture is a useful plant tissue culture technique that can be successfully
be used for production of homozygous lines. The production of double haploid
lines using anther culture includes three steps: (a) Initiating androgenetic
embryos, (b) Regenerating haploid plantlet from androgenetic embryos and (c)
Doubling the chromosome number of the haploid plants (Lichter,
1982; Zhao et al., 1996; Palmer
et al., 1996; Gorecka and Krzyzanowska, 2007).
Anther and microspore cultures were used to produce double haploids in broccoli
(Duijs et al., 1992; Lee
and Nam 1995; Yuan et al., 2011), Brussels
sprouts (Biddington et al., 1993; Ockendon
and McClenaghan, 1993) and cabbage (Higdon et al.,
2007; Zhao et al., 2007; Yuan
et al., 2011). Broccoli (Brassica oleracea var italica)
(2n = 2x = 18) is widely consumed and grown vegetable crops worldwide. During
the last decade, broccoli received a great attention of seed companies due to
the increase production and its association with lower incidence of certain
cancers including lung, colon, breast, ovarian and bladder cancers (Higdon
et al., 2007; Zhao et al., 2007; Yuan
et al., 2011). Broccoli is a cross-pollination crop and the production
of high yield and quality broccoli homozygote/pure lines required intensive
work and time. High genetically uniform lines/cultivars (pure line and homozygote
line) with desired traits are urgently required before start plant breeding
program. The response of broccoli anther culture varied based on crop genotypes,
growth conditions of donor plants, microsporogensis developmental stage, growth
medium and pretreatments of the flower buds (Higdon et
al., 2007; Zhao et al., 2007; Yuan
et al., 2011). Embryo production of broccoli anther culture could
be improved via combined cold and heat shock pretreatments of the flower (Yuan
et al., 2011) and addition of activated charcoal to the culture medium
(Da Silva Dias, 1999). Heat shock treatments and NLN-13
medium was successful to enhance emberyogenesis of 6 subspecies of Brassica
oleracea (Duijs et al., 1992). Da
Silva Dias 1999 reported that the percentage of embryogenesis increased
in broccoli by the addition of activated charcoal to the culture medium. The
present study aimed to investigate the effects of BAP in combination with 2,
4-D and the media sucrose content on androgenesis of broccoli.
MATERIALS AND METHODS
Plant materials: The first formed flower buds of 2-3 mm were collected
from the donor plants for anther culture. The donor plants belong to the broccoli
cv. Arcadia which is grown in autumn and winter seasons in Saudi
Arabia. The stage of microsporogenesis was determined by microscopic observation
of crushed specimens in hematoxyline (Fig. 2a-b).
Sterilization protocol for flower buds: The flower buds were left under
running tap water for 30 min, then buds were transferred to the laminar flow
hood and rinsed in 70% ethanol for 1 min. The flower buds were rinsed in 10%
Clorox for 10 min and finally buds were washed four times in double distilled
Culture media and growing conditions: The Murashige and Skoog Basel
medium (MS) supplemented with 0.5 g L-1 Activated Charcoal (AC) was
used. The media pH was adjusted to 5.8±0.1 prior to adding agar and the
media were autoclaved at 121°C and 1.05 kg cm-2 for 15 min. Thermo-labile
vitamins and growth regulators were add to the autoclaved media through membrane
filters (Millex-GS 0.20 μm filter unit) under controlled conditions. Media
were handed out in 10-ml into sterilized petri dishes and ten anthers were cultured
in each petri dish. The petri dishes subsequently were plugged and bundled with
one layer of parafilm (Pechiney Plastic Packaging, Inc, Chicago, IL.60631).
The petri dishes with anthers, callus, embryos and plantlets were incubated
under 16 h lights (white fluorescent light with intensity of 55 μmol m-2
sec-1) and 8 h dark at 24±1°C.
Experiment of plant growth regulators (PGRs): The effects of BAP in
combinations with 2, 4-D on the production of androgenric broccoli plants using
anther culture. The experiment was laid out in Completely Randomized Design
(CRD) using 3 replicates with 5 petri dishes of each treatment. The applied
plant growth regulators treatments for anther induction and embryos development
were as follow: (1) 1 mg L-1 BAP+0.5 mg L-1 2, 4-D, (2)
1 mg L-1 BAP+1 mg L-1 2, 4-D, (3) 3 mg L-1
BAP+0.5 mg L-1 2, 4-D, (4) 3 mg L-1 BAP+1 mg L-1
2, 4-D, (5) 5 mg L-1 BAP+0.5 mg L-1 2, 4-D and (6) 5 mg
L-1 BAP+1 mg L-1 2, 4-D.
Experiment of sucrose media concentrations: Regarding sucrose effects
on androgensis of cabbage anthers, four sucrose treatments 20, 30, 40, 50 and
60 g L-1 were tested using the culture medium supplemented with 1
mg L-1 BAP in combinations with 0.5 mg L-1 and 1 mg L-1
2, 4-D. The experiment was laid out in Completely Randomized Design (CRD) using
3 replicates with 5 petri dishes of each treatment. The applied sucrose treatments
were as follow: (1) 20 g L-1 sucrose+1 mg L-1 BAP+0.5
mg L-1 2, 4-D, (2) 20 g L-1 sucrose+1 mg L-1
BAP+1 mg L-1 2, 4-D, (3) 30 g L-1 sucrose+1mg L-1
BAP+0.5 mg L-1 2, 4-D, (4) 30 g L-1 sucrose+1 mg L-1
BAP+1 mg L-1 2, 4-D, (5) 40 g L-1 sucrose+1 mg L-1
BAP+0.5 mg L-1 2, 4-D, (6) 40 g L-1 sucrose+1 mg L-1
BAP+1mg L-1 2, 4-D, 7) 50 g L-1 sucrose+1 mg L-1
BAP+0.5 mg L-1 2, 4-D and (8) 50 g L-1 sucrose+1 mg L-1
BAP+1 mg L-1 2, 4-D.
Assessed parameters: The following parameters were assessed from clean
cultures: No. of anthers developed embryos, No. of undeveloped anthers, No.
of embryos formed callus, No. of embryos formed plantlets and No. of undeveloped
Data analysis: Analysis of variance relevant to Completely Randomized
Design (CRD) experiments as described by Gomez and Gomez (1984)
were conducted. The treatment means were compared by the Least Significant Differences
test (LSD) at 5% probability level.
Effects of plant growth regulators (PGRs): The development of broccoli
anther was significantly influenced by the applied PGRs combinations. AS presented
in Table 1 there are significant differences due to the effects
of plant growth regulators combinations at all assessed traits. The applied
growth regulators combinations significantly affected the anther induction and
plantlets development. The culture medium with low PGRs concentrations (1 mg
L-1 BAP+0.5 mg L-1 2,4-D and 1 mg L-1 BAP+1
mg L-1 2, 4-D) significantly increased number of anthers induced
embryos comparing other applied PGRs treatments (Fig. 1a and
2c, d). The least number of developed anthers
were produced when broccoli anthers were cultured on the medium supplemented
with high concentrations of BAP (5 mg L-1 BAP+0.5 mg L-1
2, 4-D ). On the other hand, the culture medium with lower concentration of
BAP reduced significantly the number of both undeveloped anthers and dead anthers
(Fig. 1b, c). Medium containing 3 and 5
mg L-1 BAP reduced anthers development and subsequently increased
number of undeveloped and dead anthers. As presented in Fig. 1d
and 2f, the highest values of embryos formed callus were obtained
when the embryos were transferred to culture medium supplemented with low BAP
and 2, 4-D concentrations (46.47 and 44.67 for 1 mg L-1 BAP+1 mg
L-1 2, 4-D and 1 mg L-1 BAP+0.5 mg L-1 2, 4-D,
respectively). Moreover, medium with lower concentration of BAP and 2, 4-D enhanced
direct differentiation of the formed embryos to plantlet (Fig.
1e and 2e-i). The highest numbers of
embryos formed plantlets were 23.2 and 17.74 for culture medium 1 mg L-1
BAP+0.5 mg L-1 2, 4-D and 1 mg L-1 BAP+1 mg L-1
2, 4-D, respectively.
Effects of plant growth regulators on
androgenesis of broccoli anthers. The MS basal medium (Murashige and Skoog)
supplemented with 0.5 g activated charcoal (AC) was used. Different plant
growth regulators (PGRs) combinations were applied: (a) No. of anthers
developed embryos (LSD0.05 = 1.04), (b) No. of not developed
anthers (LSD0.05 = 0.261), (c) No. of dead anthers (LSD0.05
= 0.667), (d) No. of embryos formed callus (LSD0.05 = 0.728),
(e) Embryos formed plantlets (LSD0.05 = 0.302) and (f) No.
of dead embryos (LSD0.05 = 0.266)
Contrary, increasing BAP in the culture medium form (regardless from 2, 4-D
concentrations) reduced the numbers of embryos formed plantlets and increased
numbers of dead embryos (Fig. 1e, f). The
highest number of dead embryos were 40.66 and 39.12 for culture medium supplemented
with 3 mg L-1 BAP+1 mg L-1 2, 4-D and 5 mg L-1
BAP+1 mg L-1 2, 4-D (Fig. 1f and 2g).
||Mean squares for the effects of growth regulators combinations
on induction of broccoli anther
|*,***Significant at p≤0.05 and 0.001, 1PGR =
Plant growth regulators, 2dead anthers after the development
||Mean squares for the effects of sucrose concentrations in
the culture medium on induction of broccoli anther
|*,***Significant at p≤0.05 and 0.001, 1Suc =
sucrose concentrations in the culture medium, 2dead anthers after
the development to embryos
Effects of sucrose concentrations: The results revealed that anther
induction and development were significantly affected by sucrose concentrations
in the culture medium. There are significant differences due to the effects
of sucrose concentrations at all assessed traits (Table 2
and Fig. 3a-e). As presented in Table
2 the applied sucrose concentrations (20, 30, 40, 50 and 60 g L-1)
significantly differed in their effects on anthers induction and plantlets development.
The low sucrose concentrations inspired anthers induction and embryos formation
comparing to high sucrose concentrations. The highest numbers of developed anthers
were 90, 90 and 89 and produced on culture medium supplemented with 20, 30 and
40 g L-1 of sucrose, respectively (Fig. 3a). Contrary
high sucrose concentration inhibited development of broccoli anthers since the
lowest number of developed anthers was observed with 60 g L-1 sucrose
(35 anthers). High sucrose concentrations significantly increased the numbers
of dead anthers after induction (Fig. 3c). Regarding embryos
development, low sucrose concentration significantly increased number of embryos
developed plantlets with 26.94 plantlets (from 90 embryos), 24.00 plantlets
(from 89 embryos) and 17.74 plantlets (from 90 embryos) and produced by 20,
40 and 30 g L-1, respectively) (Fig. 3e). Thus,
low sucrose concentration increased percentages of dead embryos which were 24.78%
(22.13 from 90), 23.70% (21.33 from 90) and 23.42% (20.84 from 89) for 20, 40
and 30 g L-1, respectively (Fig. 3f). Contrary,
the numbers of embryos performed callus were significantly increased with increase
sucrose concentrations. The culture medium supplemented with 60 g L-1
with 83.00% produced the highest percentage of embryos formed callus (29.06
from 35 developed embryos) followed by 50 g L-1 with 71.70% (39.25
from 54.67 developed anthers) (Fig. 3d). However, high sucrose
concentrations significantly decreased percentages of dead embryos comparing
to low concentration of sucrose (Fig. 3f).
High percentages of broccoli anthers were induced and developed embryos when
cultured on induction medium complemented with low BAP (1 mg L-1)
and 2,4-D (0.5 and 1 mg L-1). In cabbage anther culture, (Gorecka
and Krzyzanowska, 2007) found that MS medium with low BA concentration (1
mg L-1) produced the maximum percentages of embryos formed shoots
(8.3%), embryos formed callus (25%) and embryos developed into plants (25%).
Androgenesis of broccoli anthers cultured
on Murashige and Skoog basal medium (MS) supplemented with 0.5 g L-1
activated charcoal (AC) and 3% sucrose, BAP in combinations with 2, 4-D
and different sucrose concentrations: (a) Mature pollen grain, (b) The
stage of microsporogenesis, (c and d) Developed anthers, (e) Anthers developed
embryos, (f) Embryos formed callus, (g) Dead embryos and (h and i) Embryos
Hu et al. (1993) cultured anthers of carrot
cv. Senkou 5 Sun on solid MS medium containing various combinations
of growth regulators. The highest rate of embryoid formation (15%) was obtained
on medium containing 2, 4-D (1.0 mg L-1). The low concentration of
BAP and 2,4-D increased percentage of embryos formed callus and plantlet, while
high BAP concentrations increased percentage of dead embryos. The results of
the presented study were partially compatible to the results of Dore
and Boulidard (1988) who found that embryos on a medium without growth substances
transformed into single complete plants, whereas from embryos placed on a medium
with 0.1 mg L-1 BA emerged shoots. George et
al. (2008) found that BAP was most effective in enhancing shoot multiplication
and elongation. Moreover, they found that BAP enhance first the differentiation
of cell into shoot and subsequently the formation of shoots.
||Effects of sucrose concentrations (20, 30, 40, 50 and 60 g
L-1) on androgenesis of broccoli anthers. The MS basal medium
(Murashige and Skoog) supplemented with 0.5 g activated charcoal (AC) was
used. Different plant growth regulators (PGRs) combinations were applied:
(a) No. of anthers developed embryos (LSD0.05 = 1.053), (b) No.
of not developed anthers (LSD0.05 = 0.593), (c) No. of dead anthers
(LSD0.05 = 0.850), (d) No. of embryos formed callus (LSD0.05
= 0.686), (e) Embryos formed plantlets (LSD0.05 = 0.509) and
(f) No. dead embryos (LSD0.05 = 0.489)
Ravanfar et al. (2009) reported that high concentration
of BAP (5 mg L-1) decreased the mean number of shoots formed per
explant due to the toxicity to the shoot growth. The results of the five sucrose
concentrations showed that high percentages of anthers induction and embryos
development were observed when broccoli anthers were cultured on medium supplemented
with low sucrose concentrations. Low sucrose concentration significantly increased
the No. of embryos produced plantlets, while increased the No. of dead embryos.
The results were partially agreed with that reported by Roulund
et al. (1990). The authors studied the effects of sugar concentrations
and types on another culture of head cabbage. They reported that a higher average
response was on the sucrose containing media (3.4 embryos/100 anthers) compared
to the media with maltose (1.6 embryos/100 anthers). Also, Zhang
et al. (2006) reported that the B5 media supplemented with 2 mg L-1
2, 4-D, 2 mg L-1 kinetin and 60 g L-1 sucrose was the
optimal medium for embryoid induction of cabbage anthers. The induction and
stimulation of broccoli anthers were significantly restricted by
increasing the sucrose concentrations. High sucrose concentrations increased
percentages of embryos performed callus, while decreased percentages of dead
embryos comparing to low concentration of sucrose. However, Gorecka
and Krzyzanowska (2007) found that B5 medium without amino-acids and hormones
and containing 20 g L-1 sucrose revealed the lowest percentages of
embryos formed shoots (0%), embryos formed callus (0%) and embryos developed
into plants (0%). Krzyzanowska et al. (2006)
reported that MS with 20 g L-1 sucrose, 1 mg L-1 BA and
0.001 mg L-1 NAA showed the highest percentages of embryos producing
As conclusion, BAP in combinations with 2, 4-D can be successfully used to
perform embryos and/or callus from the callus induction and embryos development
either to callus or plants. The low concentrations of sucrose revealed the superlative
results of percentages of induced anthers and embryos developed into callus
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