Download Double Haploid Technique: In Soybean and Other Species

International Journal of Applied Agricultural Research
ISSN 0973-2683 Volume 5 Number 5 (2010) pp. 649–655
© Research India Publications
http://www.ripublication.com/ijaar.htm
Double Haploid Technique: In Soybean and
Other Species
Saleem Khan*, Abdul Latif, Sahibzada Qayyum Ahmad,
Farhad Ahmad and Mehnaz Ghaffar
Agricultural Research Station, Baffa, Mansehra, KPK, Pakistan
*Corresponding Author E-mail: [email protected]
Abstract
Recent work however, has been directed towards development of new plant
varieties by using double haploid technology. Research has resulted in
successful development of haploid and doubled haploid plants from several
crop species. Double haploid technology refers to the use of the microspore or
anther culture (ovary culture in few species) to obtain haploid embryos from
microspores. The ploidy level could be doubled autonomously or by chemical
treatments to obtain plants with 100% homozygosity. This technology is
available in some crops including soybean to speed up the breeding procedure.
The anther culture technique used in Glycine max, (2n = 40) results in haploid
plants. The chromosome number could be doubled by chemical treatment i.e.
Colchicine treatment results in double haploid plants with 100%
homozygosity.
Keywords: Doubled haploid, soybean, anther culture.
Introduction
Oil seeds are important source of edible oil production and are cultivated on a large
scale in the world. It plays important role in the country’s economy. However, in
Pakistan its production is very low and hence Pakistan is the third largest importer of
edible oil in the world.
Soybean (Glycine max. L.) is the most important crop among the oil seed crops.
Soybean originated in north eastern china and was domesticated about 1100 B.C.
(Hymowitz, 1970). The major producing countries of soybean are United States of
America, China, Brazil, and Argentina. Soybean seed contain about 40% protein and
20% oil on dry weight basis. Although soybean is a highly self pollinated crop,
however, insects may carry the pollen but the frequency of cross pollination is less
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than 1%.
Soybean is a member of the genus Glycine and family leguminosea and has a
chromosome number of 2n=40 (Hadley and Hymowitz., 1973). The cultivated
soybean is erect, bushy and leafy, varies in height (from 30 cm to 1 m). It is an annual
crop having different varieties. Every locality has its own indigenous germplasm.
Different breeding methods are used by the soybean breeders to develop soybean
varieties. Introduction, selection and hybridization are the principal breeding methods.
The conventional breeding of soybean takes several years for variety development.
For this purpose, the soybean breeders have turned to the non conventional methods
to develop 100% homozygous varieties in shortest possible time.
Double haploid technology is one of the non conventional methods which refer to
the use of microspore or anther culture to obtain haploid embryo. Guha and
Maheshwari (1964) first reported the direct development of haploid embryo from
microspores of Datura innoxia by anther culture in vitro.
Anther culture technique has been refined and extended to 200 species belonging
to 60 genera and 26 families of Angiosperm. The first pollen derived plants obtained
from cultured anthers in China were rice and wheat. The anthers of a floret contain
microspores, the male sexual cells. In contrast to vegetative cells, in microspores the
number of chromosomes is reduced by half. On specific media microspores within the
anther, regenerate into so called haploid plants having single set of unpaired
chromosome which are sterile, using colchicines for chromosome doubling i.e.
produce fertile homozygous doubled haploids.
Advantages of double haploid plants in plant breeding are:
1. Saving time in producing homozygous plants
2. Easy selection of characteristics i.e. genotype or phenotype
3. Reduced time to develop new variety
4. Use of double haploid as basic germ plasm for breeding new variety.
The objective of this review paper is to collect the relevant literature, on the title
of double haploid technique in soybean, and to get the latest information in the
discipline of plant breeding and genetics, which will be helpful for understanding of
the researcher and onward access of the researcher working in this particular area of
the discipline of plant breeding.
Fundamental Informations
Haploids may occur spontaneously in nature, or they may be induced artificially.
Induced haploids can generally be obtained by the stimulation of the egg, synergids,
or sperm by a number of methods, including ionizing irradiation and radioisotopes,
thermal shocks, distant hybridization, delayed pollination, application of abortive
pollen, spraying with various chemicals.
Protocol for anther culture
The immature anthers containing uninucleate pollen at the time of first mitosis are the
most suitable material for the induction of androgenesis. The flower buds are
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sterilized with 1% (w/v) solution of sodium hypochlorite, or 5% (v/v) solution of a
commercially available disinfectant such as Clorox. They are washed a couple of
times with sterile distilled water, and the anthers are dissected out and cultured on
agar solidified medium. The flower buds obtained from plants grown in greenhouses.
An excision is made on one side of the flower bud, the stamens are gently squeezed
out and collected in a sterile Petri dish.
Basal medium of MS with various additives have been employed. The usual level
of sucrose is 2-4%; however, higher concentrations (8-12%) favor androgenesis in
cereals. The cultures are incubated at 24-28 oC in a 14-hr daylight regime at about
2000 lux.
The anthers normally start to undergo pollen embryogenesis within 2 weeks, and
either directly develop into haploid plants in about 6 weeks (in tobacco) and are
capable of transferring to the soil, or undergo proliferation to form callus which can
be induced to differentiate plants.
Induction of androgenesis
Haploid plantlets are formed in two distinct ways, by direct androgenesis or by
organogenesis from haploid callus tissue.
The anthers containing uninucleate pollen cultured on basal medium turn
brownish within 2 weeks without any visible signs of growth. After 3-4 weeks in
culture, small white protuberances, the embryos, appear which eventually develop
into plantlets.
Microscopic observations reveal that within the excised anthers the microspores
exhibit various modes of development. The microspore nucleus either undergoes a
normal mitosis and forms a vegetative and a generative nucleus or divides to form two
“similar looking” nuclei. In some pollen the vegetative nucleus, while in others the two
“similar looking” nuclei, undergo further repeated divisions to form multinucleate pollen.
The generative nucleus remains quiescent or divides a couple of times and aborts. As a
result of repeated division pollen with up to 30 nuclei may be formed with no walls
separating the nuclei. Such pollen do not take part in androgenesis and generally abort.
At the early stages of development it is not possible to differentiate between the
multinucleate type of development and that which leads to the formation of callus.
However, after about 2 weeks, pollen are observed to be larger than the multinucleate
and the embryonal type and contain only a few cells. These cells increase in size,
exerting pressure on the exine which bursts open and the contents are released in the
form of callus. At the same time, in some cases, shoots differentiate from the callus.
The plantlets originating from the callus are generally undesirable, as they exhibit
various levels of ploidy.
Culture of Isolated Pollen
A hanging drop method were used to culture isolated pollen of Brassica oleracea and
Brassica oleracea x Brassica alboglabra. The method involved placing a drop of
medium containing 50-80 grains on the cover glass, which is then inverted over a
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cavity slide and sealed with paraffin. Before inverting the cover glass, a column of
paraffin is raised in the center, so that when inverted, it touches the bottom of the
cavity slide. This facilitates the aeration as well as the movement of the pollen when
the slide is rotted. Cell clusters were formed from isolated pollen after 4 weeks in a
medium containing coconut water. Making use of the observations that thermal
shocks given to plants can change the mode of division of the pollen nuclei. It was
reported that trauma given to pollen at the time of the first mitosis considerably
enhanced the number of pollen cells undergoing androgenesis.
Partial success along the same lines was also reported in tomato. Later a fully
synthetic medium was substituted for the anther extract and obtained plantlets from
Nicotiana tabacum cv. Red Flowered and Coulo. This work was then extended to
another commercially important tobacco cultivar.
The isolated pollen can be cultured by the methods which follow:
Nurse culture technique
The isolated pollen of Lycopersicon esculentum was induced to form haploid callus.
In this method the anthers are placed horizontally on top of the basal medium within a
French square container. A filter paper disk is placed over the intact anther, and about
10 pollen grains (in suspension) are then placed on the filter paper disk.
The controls are prepared in exactly the same way, except that the pollen are
placed on a filter paper disk kept directly on the agar. With this method the control
did not grow at all, while the pollen kept on the nurse cultures had a planting
efficiency of up to 60%, and clusters of green parenchymatous cells were formed on
the filter paper disk in 2 weeks. These clones were observed to be haploid.
Androgenesis was successfully induced in Nicotiana tabacum microspores using
petunia callus as a nurse tissue.
Preculture Method
The technique for the culture of isolated pollen involves the following steps. The
anthers are aseptically removed and cultured after 4 days of incubation at 27 0C, the
pollen are squeezed out of the anther into the liquid medium. One liter of the medium
contains KNO3 (8.9 mM), NH4 NO3 (8.9 mM), MgSO4 7H2O (0.75 mM), CaCI2 (1.42
mM), KH2PO4 (0.50 mM), FeEDTA (100 mM), glutamine (5.5 mM), serine (0.95
mM), Myoinositol (0.03 M), Zeatin (0.046 mM), indoleacetic acid (0.57 uM), and
sucrose (0.06-0.23 M). After filtration the pollen are centrifuged to form a pellet. The
pollen are washed twice, centrifuged again, and suspended in fresh medium with a
density of about 5 x 104 pollen per ml. Aliquots of 2 ml are then dispensed in thin
layers in small Petri dishes or 25 ml Erlenmeyer flasks. However, if the material is
available in microquantities, it can be grown in drop cultures. To prepare such
cultures, a drop of silicon is placed in the center of a small (5 cm) sterile plastic Petri
dish, and a cover slip (22 x 22 mm) is gently lowered onto the drop. Then a drop of
250-500 μl of the pollen suspension is pipetted onto the cover slip. To prevent
dessication, the Petri dishes are sealed with “Parafilm M” and the cultures incubated
for the first 4-6 days under low light (500 lux), and then maintained in 14-hr daylight
regime of 2000 lux at 28 0C. The induced pollen undergo normal androgenesis and
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eventually produce haploid plants. Recently these techniques have been considerably
modified and refined to obtain high percentages of pollen embryogenesis. For
instance, rye pollen suspension may be purified to separate the viable microspores.
Likewise, androgenesis can be induced in pollen cultures of tobacco. The serial
culture of anthers in the liquid medium enables the embryogenic pollen to discharge
in the medium in good quantities. Such pollen can then be filtered and cultured.
Following are some of the important points which have emerged from the work on
the culture of isolated pollen of Nicotiana and Datura.
Growth and morphogenesis are adversely affected if the isolated pollen are not
washed before culturing. Unwashed pollen either fail to grow or have a tendency
toward callus formation.
Cold treatment has been used to increase the frequency of androgensis. Close
examination reveals that even in cold-treated pollen, normal mitosis usually takes
place to form a vegetative and a generative nucleus. The cold treatment does not
induce androgenesis, but it enhances the viability of cultured pollen, and causes
repression of the gametophytic differentiation which results in higher frequency of
androgenesis.
Androgenesis in isolated tobacco pollen can be induced at various stages of
development (ranging from uninucleate microspore, during and after first mitosis to
late binucleate), but frequency of success is most frequently achieved using anthers
taken at late uninucleate to binucleate pollen stage.
The frequency of androgenesis can be enhanced considerably by raising the
sucrose and myo-inositol concentrations in the medium.
Homozygous plants
Homozygous plants can be obtained in a relatively short time by the production of
haploids and by doubling their chromosomes. This technique has been successfully
used in china where several varieties of wheat and rice have been developed. The
duplication of the chromosomes can be achieved by a number of methods.
Endomitosis
Haploid cells are, in general unstable in culture and have a tendency to undergo
endomitosis to form diploid cells. This property of cell cultures has been exploited for
obtaining homozygous plants. A small segment of stem from a haploid plant is grown
on an auxin cytokinin medium to induce callus formation. During callus growth and
differentiation there is a doubling of the chromosomes by endomitosis to form diploid
homozygous cells and ultimately plants.
Colchicine Treatment
Colchicine has been used extensively as a spindle inhibitor to induce chromosome
duplication and to produce polyploid plants. This has been employed for obtaining
homozygous diploid plants from haploid cultures. While still enclosed by the anther,
the young plantlets are treated with 0.5% colchicine solution for 24-48h washed
thoroughly, and replanted. The fertile homozygous plants thus obtained can be used
for producing inbred lines used to produce hybrids.
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Acknowledgements
I am thankful to Prof. Dr. Farhatullah, Department of Plant Breeding & Genetics,
NWFP, Agricultural University Peshawar, for their special support and critical
suggestions in writing this review
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