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Haploid Induction in Spring Onion (Allium fistulosum L.) via Gynogenesis

Article  in  Biotechnology(Faisalabad) · January 2016

DOI: 10.3923/biotech.2016

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Universiti Malaysia Kelantan, Jeli Campus, Kelantan, Malaysia Universiti Pendidikan Sultan Idris (UPSI)
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 OPEN ACCESS Biotechnology
ISSN 1682-296X
DOI: 10.3923/biotech.2016.

Research Article
Haploid Induction in Spring Onion (Allium fistulosum L.) via
Gynogenesis
1 1 1 2
Ahmed Mahmood Ibrahim, Fatimah Binti Kayat, Dwi Susanto, Pedram Kashiani and
1
Mohammed Arifullah

1
Faculty of Agro Based Industry, University Malaysia Kelantan, Jeli Campus, Locked Bag 100, 17600, Jeli, Kelantan, Malaysia
2
Department of Crop Science, Faculty of Agriculture, University Putra Malaysia, UPM Serdang, 43400, Selangor, Malaysia

Abstract
Regeneration into haploid plantlets had been obtained in spring onion using flower and ovary culture. Flowers and ovaries were cultured
into media using two protocols (A and B) and the ability to produce callus or somatic embryogenesis were invistigated. Flowers around
3.0-5.0 mm long were collected, whole flower or ovary which were excised from flowers using dissecting microscope were cultured into
BDS medium supplemented 2.0 mg LG1 2, 4-D and 2.0 mg LG1 BAP fortified with 100 g LG1 sucrose, 200 mg LG1 proline, 500 mg LG1
myo-inositol (protocol A) or into BDS media supplemented 2.0 mg LG1 2,4-0 and 2.0 mg LG1 BAP for 14 days, then sub-cultured by
regeneration medium (BDS) supplemented with 1.0 mg LG1 NAA and 2.0 mg LG1 2iP (protocol B). Embryos were emerged from ovary wall
after around 4-5 months, high frequency of embryogenesis induction was produced from ovaries that were using protocol A. While the
less percentage observed from flower culture using protocol B.

Key words: Ovule, ovary, embryogenesis, BDS media, shoot regeneration

Received: Accepted: Published:

Citation: Ahmed Mahmood Ibrahim, Fatimah Binti Kayat, Dwi Susanto, Pedram Kashiani and Mohammed Arifullah, 2016. Haploid Induction in Spring
Onion (Allium fistulosum L.) via Gynogenesis. Biotechnology, CC: CC-CC.

Corresponding Author: Fatimah Binti Kayat, Faculty of Agro Based Industry, University Malaysia Kelantan, Jeli Campus, Locked Bag 100, 17600, Jeli,
Kelantan, Malaysia

Copyright: © 2016 Ahmed Mahmood Ibrahim et al. This is an open access article distributed under the terms of the creative commons attribution License,
which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

Competing Interest: The authors have declared that no competing interest exists.

Data Availability: All relevant data are within the paper and its supporting information files.
 Biotechnology 2016

INTRODUCTION gametophyte (gynogenesis) is an alternative method in onion

for haploid creation. Several authors has been reported
Spring onion (Allium fistulosum L.) is a biennial successfully by (Bohanec et al., 1995; Geoffriau et al., 1997;
monocotyledonous plant is one of the most important Martinez et al., 2000; Michalik et al., 2000; Fayos et al., 2015).
vegetable crops around the world in terms of crop value. Geoffriau et al. (1997) reported that the main limitation of
Spring onion is grown widely in Cameron Highlands gynogenesis in onion is the low percentage of embryogenesis
particularly in the Tringkap and Kea Farm areas (Ramasamy, formation, chromosome doubling and plant survival from
1991). The demand for specific varieties accommodate to local most of the materials.
environment is too high since onion is a photoperiodically The first research on onion gynogenesis were applied
sensible plant and forms bulbs only after special with ovule and ovary culture, most of cases, protocol from two
environmental conditions among genotypes. According to step was applied, a preculture of the flower buds prior ovule
the inbreeding method, two types of onion varieties are or ovary excised. In these studies MS (Murashige and Skoog,
cultivated: hybrid varieties and open pollinated (Jakse and 1962), B5 media (Gamborg et al., 1968) and BDS (Dunstan and
Bohanec, 2003). Approximately 170 countries grow onions for Short, 1977) were used (Muren, 1989; Keller, 1990;
their own domestic use and many are also involved in Campion and Alloni, 1990; Campion et al., 1992). later, new
international trade. Onion ranking coming second after protocols were improvment dependce on the first protocol
tomato in the list of vegetables cultured worldwide, It is on those used for ovule and ovary culture with some
estimated that over 90 million tons on 4.7 million ha (FAO., modifications on culture medium, including PGR
2013). The production of F1 hybrids is considered one of the (Bohanec et al., 1995; Martinez et al., 2000; Michalik et al.,
main goals in onion breeding program. The main restriction of 2000), afterward on a simplified by one step protocol,
this field is the length of the time needed to produce inbred. containing of culturing the completed flower bud in an
The most time-consumption and work-intensive aspect of induction media until the embryo induction (Bohanec and
improvment these hybrids is the conventional breeding Jakse, 1999; Jakse and Bohanec, 2003).
process that requires manual self-pollination essential to The objective of this study was to evaluate gynogenesis
generate pure lines of homozygous parent. This way requires ability of spring onion gynotype using flower bud and ovary
eight or more off-springs of inbreeding to establish sufficient culture and to optimize the frequency of embrayo induction
regular lines that can be applied in hybrid production. In and acclimated haploid plants. two protocols, (A) described by
many varieties in vitro methods have provided to speed up Jakse and Bohanes (2003) and (B) described by Michalik et al.
the development of homozygous lines, as an alternative to the (2000) were examined in this study.
slower conventional inbreeding process. Haploid plants may
be accomplished by using anther, unfertilized ovule, ovaries MATERIALS AND METHODS
or entire flowers buds culture. The number of haploid
chromosome means that meiotic recombination's and This study was carried out in the tissue culture laboratory
recessive gene effects are manifested at plant level. And of Faculty of Agro Based Industry, University Malaysia
spontaneous or induced chromosome doubling consent the Kelantan.
regeneration of doubled haploids (DHs) homozygous
materials, with restored fertility that can be used in different Plant material and sterilization: Whole umbel of spring
breeding strategies because all the alleles of DHs lines are onion (Allium fistulosum L.) was taken from donor plants
fixed and aid efficient selection for quantitative traits in when the flowers were at 3-4 days before anthesis (Fig. 1),
breeding. Induction of superior onion hybrids rely on the individual flower 3-5 mm with pedicel were removed and
advancement of high-quality, fertile inbred lines to use as sterilized with 70% alcohol for 45 sec then surface-sterilized
parents. These lines should be sufficiently inbred, because with 5% sodium hypochlorite (NaClO) with a few drops of
commercially suitable hybrids should be uniform for tween 20 for 15 min and rinsed four times with sterile distilled
horticultural characterizes such as bulbs shape, bulbs size and water.
time to maturity. By rising homozygosity of the inbred parent
lines this uniformity can be achieved through cycles of Culture media: Two protocols were used in this study, first
self-pollination. However, such as many cross-pollinated protocol (A) described by Jakse and Bohanes (2003) was
crops, onions suffer from hard inbreeding depression when followed in this study, whole flower buds or ovaries after
self-pollinated for several generations (Bohanec, 2002). Female excised from flower were cultured in Petri dish 100×15 mm

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were transferred to 7 cm plastic pots containing peat moss

and covered with plastic cap having small hole in top for
gaseous exchange for one weeks and we started remove the
cap for one hour per days and increase uncovered time daily
two, three and four hours for two weeks, finally the cap was
removed.

Flow cytometry: Flow cytometer BD Accuri™ C6 was used for

ploidy test and the following procedure were used to isolate
the nuclei from haploid and diploid onion leaf (a) 150 mg of
onion leaves were weighted, (b) Covered with 1 mL ice-cold
tris Mgcl2 buffer (200 mM tris, 4 mM MgCl 2.6 H2O, 0.5% (v/v)
triton X-100, pH 7.5), (c) Chopped with scalpel edge blade,
worked quickly, (d) Added 1 mL PI/RNase staining and mix
promptly and kept it in cool and dark place for 2 h before run
and (e) Filtered the suspension through a 40 µm mesh nylon
filter in ice-cool cytometer sample tube and run the sample.
Fig. 1: Plant material of spring onion, umbel 3-4 days before Leaves from young seedlings were used as control
anthesis (Galbraith et al., 1983).

contained BDS medium (Dunstan and Short, 1977) Data analysis: All experiments were designed according to
supplemented 2.0 mg LG1, 2, 4-d and 2.0 mg LG1 BAP fortified Completely Randomized Design (CRD). The data was analyzed
with 100 g LG1 sucrose, 200 mg LG1 proline, 500 mg LG1 using one-way analysis of variance (ANOVA) using the General
myo-inositol, 10 mg LG1 adenine sulfate and vitamins, pH was Linear Model (GLM) and post hoc multiple comparisons for
adjusted to 5.8 prior to addition 6 g LG1 agar, then autoclaved observed mean, significant differentiations were compared by
for 15 min at 121EC. Ten flowers or ovaries were inoculated in Duncan Multiple Range Test (DMRT).
each Petri dish and placed in growth room with light The following Eq. 1-3 were used to calculated percentage
illumination (16/8 h) under 25EC until embryo production. The of callus, shoot and root induction:
percentage of gynogenic embryos, entire plants and haploid
plantlets were scored and percentage of acclimated. Second No. of callus induction
 100
Callus induction = (1)
protocol (B) described by Michalik et al. (2000), flowers or No. of explants
ovaries were inoculated into BDS media supplemented
2.0 mg LG1 2,4-0 and 2.0 mg LG1 BAP for 14 days, then No. of shoot induction
Shoot induction =  100 (2)
sub-cultured by regeneration medium (BDS) supplemented No. of calli
with 1.0 mg LG1 NAA and 2.0 mg LG1 2iP. Embryo was broken
through the ovary wall was sub cultured into to medium No. of root induction
Root induction =  100 (3)
having MS media without growth regulators and No. of calli
supplemented with 30 g LG1 sucrose until plant development.
RESULTS
In vitro rooting and acclimatization: Embryo formation is
awaited about four to six months in culture. Regenerated Haploid production: The flowers, ovaries, anthers and ovules
plants were recorded around 130 days. At that time of embryo excised from the chosen developmental stage were subjected
emergence mostly of ovaries will change color from green to to two different protocols (A and B) before being cultured
yellow. New embryos were transferred to MS media half onto BDS media. Flowers began to bloom after 4-5 days
strength, supplemented 1.0 mg LG1 IBA+1.0 mg LG1 KIN with cultured in BDS medium and swelled up to two times of their
0.5% charcoal for root induction and shoot development for original size. Since there was no anther opening being
3-4 weeks, then were transferred to tubes having tap water observed, it was assumed that self pollination do not occur.
and covered by piece of cotton and put in growth room for Callus or shoot formation appeared after four to six months of
1-2 weeks to help for autotrophic. Next step new plantlets inoculation. Calli were recorded around 90 days after flower

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(a) (b)

Fig. 2(a-b): (a) Calli produce from the ovary cultures after 90 days in BDS media and (b) Shoots regeneration observed from the
ovary cultures after 60 days in BDS media

(a) (b)

5 mm 5 mm

Fig. 3(a-b): (a) Callus induction from ovule after 90 days and (b) Shoot induction after 60 days of callus induction

Table 1: Effect of two types of media on callus induction of flower, ovary and anther culture from spring onion
Calli induction Shoot induction Acclimated plants
----------------------------------------- --------------------------------------------- ---------------------------------------
Culture media Expnats No. of explants No. of calli Calli/flower (%) Embryos Embryos/flowers (%) Plants Plants/embryo (%)
Protocol A Flower 650 5 0.77b 3 0.51b 1 33.3
Ovary 300 7 2.33a 4 1.52a 2 50.0
Protocol B Flower 550 5 0.91b 3 0.69b 0 0.0
Ovary 250 6 2.40a 4 1.33a 2 50.0
Values followed by the same letters within a column are not significantly different (#0.05), Duncan s Multiple Range Test (DMRT)

and ovary inoculation and shoot induction was observed after protocols. Higher percentage of callus induction had been
60 days of callus induction in BDS media (Fig. 2). The cultures recorded for ovary culture by using protocol B with 2.4%
were green in color during the first three months and compared to protocol A with 2.33%. The flower culture also
gradually turned to yellowish as the culture progressed showed higher percentage of callus induction in protocol B
(Fig. 3). Calli from the ovule were easily recognized with burst compared to protocol A with 0.91 and 0.77%, respectively
of ovary after 4-5 months of inoculation. (Table 1). However, no callus being produced for the anther
Results obtained showed that there was a significant cultures in both protocol A and B. The shoot regeneration had
difference in the percentage of callus produced from the two been observed from the callus produced (Fig. 4). A significant

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(a) (b)

5 mm
10 mm

Fig. 4(a-b): Shoots regeneration observed from callus of the ovule culture (b) Shoot development observed after 150 days of
culture

(a)

Anther

Callus from
Ovary nectary

5 mm

(b)

Fig. 5: Callus and shoot regeneration from the septal nectaries

region of the flower culture (discard)

response towards shoot regeneration had also been observed

with 1.52% for protocol A and 1.33% for protocol B while, the
percentage of shoot produced from the flower culture was
higher in protocol B compared to protocol A with 0.51% and
0.69%, respectively (Table 1). No shoot being produced from
the anther cultures for both protocols. Beside calli production
and shoot regeneration from the flower culture, direct shoot
regeneration had also been observed from the septal
nectaries region of the flower (Fig. 5).
Fig. 6(a-b): (a) In vitro rooting and (b) Acclimatization of
In vitro rooting and acclimatization: Root and shoot plantlets produce from both protocols
development had been obtained by using half strength MS
media supplemented with 1.0 mg LG1 IBA+1.0 mg LG1 KIN and added with tap water to decrease plant hyper hydricity
added with 0.5% activated charcoal. Rooted plantlets (Fig. 6). Then, the acclimatized plantlets were let to grow for
produced form both protocols were acclimatized in tubes ploidy determination (Fig. 7).

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(a) (b)

Fig. 7(a-b): Acclimatized haploid and diploid plants, (a) Diploid and (b) Haploid

100 (a) 100 (b)

P1
P2
Total number of nucleate

Count

Count

50 50

0 0
0 1,000,000 2,000,000 3,000,000 4,000,000 0 1,000,000 2,000,000 3,000,000 4,000,000
PI-A PI-A
Relative nuclear DNA-content

Fig. 8(a-b): Flow cytometry profiles showing the nuclear DNA content of the spring onion plantlets, (a) Haploid (n) and (b) Control
(2nd) diploid

Ploidy test: Leaves of spring onion were collected and their DISCUSSION
ploidy was examined by flow cytometry. The flow cytometry
profiles of the plantlets generated from ovule culture showed Gynogenesis capacity from spring onion has been
a single peak at around 1.000 PI-A (P1) while, the plantlets evaluated and two protocols have been assayed in order to
generated from diploid (control) showed a single peak at produce haploid plant. Genetic factors, including varieties,
around 2.000 PI-A (P2) (Fig. 8). Therefore, it is confirmed that geographic origin, plant genotype and genetic structure are
plantlets generated from the ovule culture contained half of believed to be very important for the achievement of
DNA content compared to plantlets generated from diploid. gynogenesis formation (Campion and Alloni, 1990;
Based on the amount of DNA content, it is appeared that Geoffriau et al., 1997; Bohanec and Jakse, 1999; Michalik et al.,
plantlets generated from the ovule culture were haploid, by 2000; Chen et al., 2011). Bohanec and Jakse (1999) found
comparing to the control diploid control plantlets. indicating of gynogenesis is strongly affected by the

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genotype. In the present study, the addition of proline with Campion, B., M.T. Azzimonti, E. Vicini, M. Schiavi and A. Falavigna,
BAP+2, 4-D to the medium induced the onset of 1992. Advances in haploid plant induction in onion
embryogenesis and raise the amount of gynogenic embryos (Allium cepa L.) through in vitro gynogenesis. Plant Sci.,
gained. The embryo initiation phase is crucial to develop the 86: 97-104.
Chen, J.F., L. Cui, A.A. Malik and K.G. Mbira, 2011. In vitro haploid
yield of regenerated plantlets as the higher gynogenic embryo
and dihaploid production via unfertilized ovule culture.
number and the great probability of getting haploid plants.
Plant Cell Tissue Organ Cult., 104: 311-319.
The mean percentage of embryos induction, which developed
Dunstan, D.I. and K.C. Short, 1977. Improved growth of tissue
into entire haploid plantlets was 0.46%. This confirm the
cultures of the onion, Allium cepa. Physiol. Plant., 41: 70-72.
results of Bohanec et al. (1995), Phillips and Luteyn (1983) and FAO., 2013. FAOSTAT. Food and Agricultural Organization of the
Geoffriau et al. (1997), who demonstrate that the haploid United Nations, Rome, Italy.
plants production in onion was quite affected by the Fayos, O., M.P. Valles, A. Garces-Claver, C. Mallor and A.M. Castillo,
population. Genetic factors, including variety, geographic 2015. Doubled haploid production from Spanish onion
origin, genotype of donor plant and genetic structure are (Allium cepa L.) germplasm: Embryogenesis induction, plant
thinking to be the high importance for the success of regeneration and chromosome doubling. Front. Plant Sci.,
gynogenesis production (Campion and Alloni, 1990; Vol. 6. 10.3389/fpls.2015.00384
Geoffriau et al., 1997; Bohanec and Jakse, 1999; Michalik et al., Galbraith, D.W., K.R. Harkins, J.M. Maddox, N.M. Ayres, D.P. Sharma
and E. Firoozabady, 1983. Rapid flow cytometric analysis of
2000; Chen et al., 2011). In Spanish germplasm, variety and
the cell cycle in intact plant tissues. Science, 220: 1049-1051.
plant genotype as well has a strong effect on gynogenesis
Gamborg, O.L., R.A. Miller and K. Ojima, 1968. Nutrient
formation, two Valencia-type varietes Rita and Recas exhibted
requirements of suspension cultures of soybean root cells.
the highest embryogenesis frequency (0.87-2.09%) then,
Exp. Cell Res., 50: 151-158.
pungent landrace BGHZ1354 (0.40-1.28%) and lastly the sweet Geoffriau, E., R. Kahane and M. Rancillac, 1997. Variation of
cultivar Fuentes de Ebro (0.27-0.75%) (Fayos et al., 2015). gynogenesis ability in onion (Allium cepa L.). Euphytica,
94: 37-44.
CONCLUSION Jakse, M. and B. Bohanec, 2003. Haploid Induction in Onion via
Gynogenesis. In: Doubled Haploid Production in Crop Plants,
Our successful haploid induction plant from spring onion Maluszynski, M., K.J. Kasha, B.P. Forster and I. Szarejko (Eds.).
using whole flower buds and ovaries allow the use this Kluwer Academic Publishers, Dordrecht, Netherlands,
pp: 281-285.
method in programs of onion breeding, further studies need
Keller, J., 1990. Culture of unpollinated ovules, ovaries and flower
to produce double habloid plants by dublicate number of
buds in some species of the genus Allium and haploid
chromosome in haploid plants to fast homozygous ilnes
induction via gynogenesis in onion (Allium cepa L.).
production for new varieties production.
Euphytica, 47: 241-247.
Martinez, L.E., C.B. Aguero, M.E. Lopez and C.R. Galmarini, 2000.
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