Journal of Alumni Association of Biotechnology (2023) 5(1):1-4
Review on double haploid in rice plant (Oryza sativa L.)
P. B. Ingle1, S. S. Rokade2*, R.V.Raut3
Innani Mahavidyalaya, Karanja (Lad), Dist. Washim (M.S.) India.
of Botany Late Pundalikrao Gawali Arts and Science Mahavidyalaya, Shirpur
(Jain) Dist. Washim (M.S.) India.
Seeds Pvt.Ltd. Pachora Dist. Jalgaon (M.S.) India.
*Corresponding Author Email- firstname.lastname@example.org
14 April 2023
Received in revised form
27 May 2023
Rice (Oryza sativa L.) is an important and indigenous crop in India.
Rice is a plant belonging to the family of grasses, Gramineae (Poaceae). It
is a major food crop of the world and forms the staple diet of about half of
the world's population. Rice is repeatedly
mentioned in ancient Indian texts, including the Yajur Veda. The area
under cultivation of rice in India is 45416 thousand hectare, Total
production of Rice during 2020-21 is estimated at record 122.27 million
tonnes. Two major Asian cultivated varieties of rice have been historically
recognized as japonica and indica subspecies. Japonica rice is
sticky and indica rice is non sticky. Obtaining high quality homozygous pure
lines by classical breeding methods is very time consuming and labour
intensive. Due to uncontrolled pollination, the results are hard to obtain
through this method. In-vitro haploid production offers an excellent
solution for development of high quality homozygous pure lines in lesser
time. Indica rice is mainly
cultivated in tropical and subtropical environments at lower latitudes or
altitudes, whereas japonica rice is
grown mainly in more temperate environments at higher latitudes or altitudes.
Anther culture is easy in japonica rice, but
its application in Indica rice is
limited as it is difficult to breed through anther culture. Indica cultivars of rice showed low
anther culturability (1.2% callus induction) whereas Japonica cultivars had 20-fold higher (28.1%) anther
Rice (Oryza sativa L.) is an
important and indigenous crop in India. Rice is a plant belonging to the family
of grasses, Gramineae (Poaceae). It is a major food crop of the world and forms
the staple diet of about half of the world's population. Archaeological
evidence suggests that this grain was the basis of India’s ancient
civilizations. The rice is supposed to have been cultivated in the valley of
the Ganges River as far back as 6500 BC. India is the second-largest producer
of rice in the world, and it also is among the largest consumers of this grain.
Around over 50% of India’s people depend on rice for sustenance. Rice is
repeatedly mentioned in ancient Indian texts, including the Yajur Veda. It is
closely associated with fertility and health across India’s many cultural
traditions. (Asia Society 2023). The area under cultivation of rice in India is
45416 thousand hectare, Total production of Rice during 2020-21 is estimated at
record 122.27 million tonnes. It is higher by 9.83 million tonnes than the last
five years' average production of 112.44 million tonnes (Annual Report
2021-22). Rice is a highly important cereal in the world, with a total of 490.9
million tonnes produced in 2015-16 of which more than 80% was destined to
direct human consumption (FAO Trade and Market Division 2017). Rice is supposed
to have undergone single domestication event around ∼8,200–13,500 year ago,
estimated on the basis on the molecular clock with the help of resequencing 630
gene fragments on chromosomes 8, 10, and 12 that is supported through
Demographic modeling based on SNP data and a diffusion-based approach (Molina,
J et al., 2011). Higher plants show two
alternations of generations i.e. sporophytic and gametophytic generation. The
sporophytic form is somatic (soma=body) having a complete set of chromosomes
usually diploid (2n) and the gametophytic form is haploid (n) in genomic
constitution. Haploid plants have gametophytic (n) chromosomes and
doubled-haploid (DH) plants have sporophytic (2n) chromosome numbers. The
haploid plants derived from a diploid are known as monoploid, while a haploid
plant derived from a polyploid is a polyhaploid. Haploid plants occur
spontaneously or haploid can be induced by in-vivo
or in-vitro methods (Dwivedi et al., 2015). Natural sporophytic
haploids in the higher plants were first spotted in Jimson weed (Datura stramonium L.) (Blakeslee et al., 1922). Guha and Masheswari made
the first breakthrough in the production of pollen embryogenesis by
successfully producing anther culture of Datura
innoxiaat the Department of Botany in the University of Delhi, India (Guha
and Masheswari 1964). The rice (Oryza
sativa) genome is composed of 12 chromosomes (2n = 24) which has a total length of 430 Mb. (Kurata et al., 1994). 95% of the genome of
rice is mapped and it can be studied as a model plant for the grasses.
(International Rice Genome Sequencing Project, 2005). The anther culture
technique was first developed by Niizeki and Oono in rice (Niizeki and Oono
Homozygous pure lines
high quality homozygous pure lines by classical breeding methods is very time
consuming and labour intensive. Due to uncontrolled pollination, the results
are hard to obtain through this method. In-vitro
haploid production offers an excellent solution for development of high quality
homozygous pure lines in lesser time. In-vitro
methods for haploid plant production shorten time up to a year by using
androgenesis to obtain haploid plants through tissue culture. Microspore
embryogenesis is an important and useful culture technique to obtain full
homozygous lines from only the male gametes. Plant regenerated with a single
chromosome set, originating from microspores of in-vitro cultivated anthers, are ideal for genetic analysis due to
the variety of possible expressions of the genetic makeup. The doubling of the
haploid genome results in fully homozygotic lines. It shortens the period which
is important for creation of genetic diversity. It also forms a base of new
varieties with higher quality for crop development.
The use of
haploid technique among modern breeding techniques provides tremendous
advantages for breeders, especially since it allows the production of 100%
homozygous pure lines only in one generation (Kurtar et al. 2010). After a haploid plant is produced, the plant can
undergo diplodizaiton (2n), where the chromosome number is doubled upon
stimulation with colchicines (Mohammadi et
al. 2007). This method is advantageous when compared to the alternative
method and is used by plant breeders or institutions around the world. Doubled
haploid (DH) technology allows for the production of pure lines, useful for
plant breeding through a one-generation procedure that considerably reduces the
time and resources needed to produce them. The doubled haploid technique aims
to generate pure inbred lines for basic research and as commercial plant
varieties. The doubled haploid technique first generates haploid plants which
is followed by chromosome doubling. DH Technology is of core importance in
breeding programs, since it allows the production of true breeding (pure)
lines, homozygous for all traits, in a way faster and cheaper than classical
breeding procedure. This reduces considerably the number of generations needed
to produce a pure line, thus decreasing the cost of breeding programmes with
increase in variability in germplasm.
breeding activities cannot respond quickly to market mobility but with the
integration of dihaploidization methods into the breeding cycles, breeding
programs have gained significant momentum. Present efforts are an attempt to
standardize development of an efficient in-vitro
development of double haploid in Rice crop using anther culture. Two major Asian cultivated verities of rice
have been historically recognized as
Japonica and Indica subspecies. Japonica rice is sticky and Indica rice is non sticky (Yanget al., 2014). Indica rice is mainly cultivated in tropical and subtropical
environments at lower latitudes or altitudes, whereas Japonica rice is grown mainly in more temperate environments at
higher latitudes or altitudes. Anther culture is easy in Japonica rice, but its application in Indica rice is limited as it is difficult to breed through anther
culture. Indica cultivars of rice
showed low anther culturability (1.2% callus induction) whereas Japonica cultivars had 20-fold higher
(28.1%) anther culturability (Grewal et
al., 2011) because of early anther necrosis, poor callus proliferation and
regeneration of albino plantlets (Chen et
For a long
time, conventional breeding methods have been used to obtain pure, 100%
homozygous lines for hybrid seed production in crops of agronomic interest.
However, by double haploid technology, it is possible to produce 100%
homozygous plants derived from precursors of male gametophytes (androgenesis),
to accelerate the production of pure lines, which implies important time and
cost savings. Producing homozygous breeding lines via in-vitro haploid techniques are much more efficient in terms of
time and cost as compared to other methods (Bajaj et al., 1990). Anther culture protocol in eggplant is being already
reported till regeneration and acclimation of doubled haploid plants (Calabuig et al., 2020).Adopting the method of
haploid identification, from undesired heterozygote plants will substantially
affect the success of androgenesis in breeding programs (Ahmadi & Ebrahimzadeh,
2020).The future for double haploid breeding is promising as vigorous DH
protocols are available for a growing number of crops and future applications
will witness a closer integration with molecular-marker and gene-splicing
technologies (Humphreys and Knox, 2015).
analysis is done by flow cytometry and SSR based PCR markers. Flow cytometry
allowed rapid and reliable determination of ploidy level in anther culture
derived plant (Martínezet al., 1994). Microspore-derived plants are
analyzed by flow cytometry to check their DNA contents and by comparison with a
known diploid individual, infer their ploidy level. Thus, in parallel to
microspore derived plants, diploid donor plants are analyzed as control for the
2C DNA content to compare with flow cytometry. SSR (simple sequence repeat)
markers are considered as the key choice for genotyping. They provide highly
reproducible results as compared to the other PCR-based markers (Song et al.,
2019). Simple sequence repeats (SSRs)
are abundant and are distributed throughout the rice genome. They can be
suitably used for understanding the source of origin of callus i.e. originated
from the microspore mother cells or embryogenesis from diploid somatic tissue.
SSRs have been successfully used to identify homozygous spontaneous-doubled
haploids in rice hybrids (Samantaray et al., 2021).
Significance of double haploid line
haploid and double haploid line development have been carried out at very few
places in India and the work is abandoned in remaining parts of the country for
commercialization to the farmer community. There is continually a prerequisite
to carry intensive and extensive studies on this aspect using in-vitro
laboratory conditions. It will help in the generation of variable germplasm to
meet the requirement of food in the coming days of increasing population in
India and worldwide.
Ahmadi B, & Ebrahimzadeh H (2020)
In-vitro androgenesis: Spontaneous vs. artificial genome doubling and
characterization of regenerants. Plant
cell reports, 39(3):299-316.
Asia society (2023). India
Accessed on 23-05-2023.
Bajaj Y P S (1990) In-vitro
production of haploids and their use in cell genetics and plant breeding.
In Haploids in crop improvement (pp.
3-44). Springer, Berlin, Heidelberg.
Blakeslee A F, Belling J, Farnham
ME, & Bergner, AD (1922) A haploid mutant in the jimson weed," Datura stramonium". Science, 55(1433): 646-647.
Calabuig-Serna A, Porcel R,
Corral-Martínez P, & Seguí-Simarro JM (2020) Anther culture in eggplant (Solanum melongena L.). In Plant
Embryogenesis (pp. 283-293). Humana, New York, NY.
Chen CC, Tsay HS & Huang CR (1991)
Factors affecting androgenesis in rice (Oryza sativa L.). In: Bajaj Y P
S. Biotechnology in Agriculture and Forestry. Berlin Heidelberg: Springer:
Department of Agriculture &
Farmers Welfare Ministry of Agriculture & Farmers Welfare Government of
India. Annual Report 2021-22.
Dwivedi SL, Britt AB,Tripathi L,bSharma
S,Upadhyaya HD, & Ortiz R (2015) Haploids: Constraints and opportunities in
plant breeding. Biotechnology Advances; 33: 812–829.
FAO Trade and Markets Division
(2017) Food Outlook: Biannual report on global food markets, November 2017.
Food outlook, food and agriculture organization of the United Nations.
Grewal D, Manito C &
Bartolome V (2011) Doubled haploids generated through anther culture from
crosses of elite Indica and Japonica cultivars and/or lines of rice:
large-scale production, agronomic performance, and molecular characterization. Crop
Guha S, & Maheshwari SC
(1964) In-vitro production of embryos from anthers of Datura. Nature; 204(4957): 497-497.
Humphreys DG, & Knox RE
(2015) Doubled haploid breeding in cereals. In Advances in plant breeding strategies: Breeding, biotechnology and
molecular tools (pp. 241-290). Springer, Cham.
International Rice Genome
Sequencing Project.(2005). The map-based sequence of the rice genome. Nature;436(7052):793-800.
Kurata N, Nagamura Y, Yamamoto K,
Harushima Y, Sue N, Wu J, Antonio BA, Shomura A, Shimizu T, Lin SY, et al.
(1994) A 300 kilobase interval genetic map of rice including 883 expressed
sequences. Nat Genet; 8(4):365-72.
Kurtar ES & Balkaya A (2010)
Production of in-vitro haploid plants from in situ induced
haploid embryos in winter squash (Cucbita
maximaDuchensne ex Lam) via irradiated pollen. Plant cell tissue Org. Cult;102:
Martínez R, Cesar P, Arumuganathan
K,Kikuchi H & Earle E (1994) Nuclear DNA
content of ten rice species as determined by flow cytometry. The Japanese
Journal of Genetics; 69:513-523.
Mohammadi PP, Moieni A &
Javaran MJ (2007) Colchicine induced embryogenesis and doubled haploid production
in maize (Zea mays L.) Anther
culture. Iranian Journal of Biotechnology; 5(3).
Molina J, Sikora M, Garud N,
Flowers JM, Rubinstein S, Reynolds A, Huang P, Jackson S, Schaal BA, Bustamante
CD, Boyko AR & Purugganan MD (2011) Molecular evidence for a single
evolutionary origin of domesticated rice. Proc Natl Acad Sci U S A. 2011 May
Niizeki H & Oono K (1968) Induction
of haploid rice plant from anther culture. Proc
JpnAcad; 44: 554–557.
Samantaray S et al. (2021)
Doubled Haploids in Rice Improvement: Approaches, Applications, and Future
Prospects. In: Ali, J., Wani, S.H. (eds) Rice Improvement. Springer, Cham.
Silva TD & Ratnayake WJ (2009)
Anther culture potential of Indica rice varieties, KuruluThuda and
BG250. Trop Agric Res Ext; 12(2): 53–56.
Song S, Tian D, Zhang Z, Hu S &Yu
J (2019) Rice genomics: over the past two decades and into the future. Genom
Yang Y, Zhu K, Xia H, Chen L &
Chen K. (2014) Comparative proteomic analysis of Indica and Japonica rice
varieties. Genet Mol Biol;37(4):652-61.