Research Report

Anther Culture for Double Haploid Breeding in Rice-a Way Forward  

Swapan Kumar Tripathy
Department of Agricultural Biotechnology, College of Agriculture, OUAT, Bhubaneswar, India
Author    Correspondence author
Rice Genomics and Genetics, 2018, Vol. 9, No. 1   doi: 10.5376/rgg.2018.09.0001
Received: 27 Feb., 2018    Accepted: 22 Mar., 2018    Published: 19 Jun., 2018
© 2018 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Tripathy S.K., 2018, Anther culture for double haploid breeding in rice-a way forward, Rice Genomics and Genetics, 9(1): 1-6 (doi: 10.5376/rgg.2018.09.0001)


Anther culture is an important biotechnological tool for quick recovery of fixed breeding lines in rice. It is largely species and genotype-specific. O. glaberrima responds better to anther culture than O. sativa; and japonica sub-group is more responsive to microspore embryogenesis than indica types. Media recipes and culture techniques have been standardized for callus induction and green plantlet regeneration using indica and japonica varieties as well as their inter se hybrids. Besides, the genetic basis and scope of anther culture for double haploid breeding in rice have been discussed.

Anther culture; Double haploids; Genetic improvement; Rice


Creation of genetic variability is essential in any crop improvement programme. Breeders often resorted to hybridization or mutation breeding and even genetic transformation for genetic improvement of specific trait(s). Several conventional breeding methods are now available which take 8-10 years to develop a fixed (homozygous) breeding line. However, anther culture technique encompasses creation of usable genetic variability and increases the selection efficiency to obtain quick homozygosity from even F1. It provides fertile double haploids (DHs) with fixed derived character combination that might otherwise disappear in the course of an extended series of segregating generations in conventional breeding methods. It provides greater chance of recovery of desirable recessive genes compared to conventional breeding. Besides, the characters those expressing predominantly additive genetic effects, their values in DH lines could reach the heterotic level of the hybrid (Ba Bong and Swaminathan, 1995) which further pinpoint the prospect of anther culture technique in rice. The DH lines derived from anther culture of hybrids of genetically diverse parents are amenable for molecular mapping of valuable genes (simply inherited traits) and quantitative loci (QTLs) for agro-economic and quality traits in rice.


1 Anther Culture in Rice

Anther culture is a two-step process. First step is the initial development of callus that leads to second step, i.e. regeneration of green plants from callus. Gueye and Ndir (2010) reported recovery of a total of 93 regenerants out of which 79 were albinos. Tran and Vuong (2002) obtained frequency of 3.53% of callus induction in N6 medium and 1.12% in plant regeneration. Anther culture is affected by maturity of the donor plant (Afza et al., 2000), genotypic variation (He et al., 2006), microspore developmental stages (Afza et al., 2000), panicle pretreatment (Trejo-Tapia et al., 2002), temperature and duration of this pretreatment (Kiviharju and Pehu, 1998), culture media (Faruque et al., 1998; Asaduzzaman et al., 2003) and growth conditions (Raina and Irfan, 1998). Many researchers have reported genotypic specificity within indica subspecies by using improved media (Ratheika and Silva, 2007; Talebi et al., 2007). Cold pre-treatment led to anther wall senescence, increase symmetric division of pollen grains and release necessary substances for androgenesis, mainly amino acids and shock-thermic proteins (Kiviharju and Pehu, 1998). Besides, it stops the gametophytic development of microspores and shift to sporophytic mode of development. Substitution of cold treatment with mannitol enhanced androgenesis in anther culture of indica cultivar IR 43 from 3 to 33%. Higher concentration of nitrogen, phosphorus and potassium is reported to increase anther culture response in indica rice (Silva, 2010). Nirouli and Bimb (2009) reported higher callus induction frequency in N6 medium with 2,4-Dichlorophenoxy acetic acid (2.5 mg/L) + Kinetin (0.5 mg/L) than N6 + Naphthalene acetic acid (4 mg/l) + Kinetin (0.5 mg/l); but reverse was the case for green plant regeneration. Xa and Lang (2011) reported 5.13% to 9.27% callus induction and 6.17% to 14% regeneration from four crosses in MS (Murashige and Skoog) medium with combination 1 mg/L 6-benzylaminopurine + 2 mg/L Kinetin + 3% sucrose. He-2 medium produced highest callus induction, green plant regeneration and least albino plant development (Kaushal et al., 2014). The F1 hybrids are more responsive to anther culture than their parents. Herath et al. (2007) recorded highest callus induction frequency (29.4%) in N6 medium for F1 hybrid Hu Lo Tao × BG 90-2 with green plant regeneration frequency (41.0%) in MS medium from calli induced on N6 medium. However, positive relationship for both was noticed by Shahnewaz et al. (2004). Thuan et al. (2001) reported callus induction from anthers of F1 plants derived from four crosses of aromatic and improved rice cultivars cultured in N6 and MS media supplemented with 2,4-D (0.5 mg/L) + NAA (1.0 mg/L) + BAP (0.5 mg/L) showed better callus induction. Dash et al. (2014) reported a callus induction frequency of as high as 37.83 % from anther culture of a cross CRMS31B × CRMS24B when incubated at 26+1°C for 24 h.


Cytogenetic characterization has revealed that the anther derived plants have different ploidy levels (Sah and Niroula, 2007). Seventy percent of the regenerated plants are normally haploids while, rest accounts in vitro euploids (including spontaneous diploids) and aneuploid. The ploidy level in plants is estimated by chloroplast count in guard cells or chromosome count of root tip cells. However, recently measuring the C-value using flow cytometry seems to be a suitable approach (Ochatt et al., 2009).


2 Genetic Basis

O. glaberrima genotypes produced more callus than O. sativa genotypes. Indica cultivars under O. sativa showed low anther culturability (1.2% callus induction) whereas japonica cultivars had 20-fold higher (28.1%) anther culturability (Grewal et al., 2011). Ability of anther culture in rice is a quantitative trait controlled by the nuclear genome and it is inherited as a recessive character conditioned by a single block of genes and japonica appear to be a good combiner for callus induction (Miah et al., 1985). Grewal et al. (2011) used a set of 124 DH lines for SSR marker analysis generated from the japonica cultivar (IR69428) × indica variety (IR64). Simple sequence repeat analysis showed 1:1 ratio of indica and japonica alleles. Homozygosity was detected for all the marker loci in 124 DH lines and the genes for anther culturability are partially dominant.


Japonica types are more responsive to microspore embryogenesis than indica types in rice. Diverse genotype specificity of anther culture does exist within indica subspecies (Shahnewaz and Bari, 2004; He et al., 2006; Ratheika and Silva, 2007; Talebi et al., 2007). However, recent report shows that the “indica T23” rice genetic stock with wide compatible gene S5n (in chromosome 6) responds well to anther culture (Nguyen et al., 2016). The segregation distortion (SD) at S5 locus could be due to preferential selection of gametes with S5 indica allele for androgenesis (Yang et al., 2012). Callus induction from anthers and plant regeneration from the induced callus in rice are the two independent traits displaying quantitative inheritance (He et al., 2006; Bagheri and Jelodar, 2008). Additive effects seem to be more important than dominant effects of the genes concerned for callus induction while preponderance of dominant effects is reported for regeneration response (He et al., 2006). Selection of parents with high general combining ability (GCA) and most promising crosses revealing high specific combining ability (SCA) may pave the way for recovery of segregants with improved anther culture response.


Genes responsible for anther culture response are present on two specific chromosomal regions in rice. One in chromosome 1 control callus formation and another on chromosome 10 responsible for controlling the balance between albino/green plant regeneration capacities (Yamagishi et al., 1998). However, He et al. (1998) identified five QTLs on chromosomes 6, 7, 8, 10 and 12 responsible for callus induction and two QTLs on chromosomes 1 and 9 for green plant regeneration. They also detected a major QTL for albino plant differentiation on chromosome 9. Kwon et al. (2002a; 2002b) mapped the QTLs for green plant regeneration on chromosome 3 and 10 and molecular markers that co-segregate with these genes have been identified.


3 Double Haploid Breeding

Rice being highly self-pollinated, development and selection of pure breeding lines with desirable phenotype is the ultimate objective for genetic improvement. It normally needs 6-9 cycles of selfing followed by 3-5 years of field evaluation. In contrast, anther culture seems to be a suitable technique that significantly reduces the breeding period due to early fixation of homozygosity (Baisakh et al., 2001). In this context, doubled haploid lines derived from anther/pollen culture of F1 hybrids are promising tools to develop plant cultivars. 50-60% of haploids in culture undergo spontaneous chromosome doubling (endoreduplication) in rice (Germana, 2011). Thus, in rice, a specific step for chromosome doubling by the most common doubling agent “colchicine” (a mitotic inhibitor) may be omitted unless desired for increased DH production. Bishnoi et al. (2000) produced higher number of DH lines in indica rice (var. Gobind, HKR120, Basmati 370, and Taraori Basmati) and an indica-basmati hybrid (Bishnoi et al., 2000) via colchicine treatment (0.1%, w/v). To reduce chances of mixoploids and polyploids resulting chimeric plants and low seed set, colchicine treatment at whole plant level is normally avoided in rice. However, the application of colchicine during early stages of androgenesis could alleviate above problems (Castillo et al., 2009) and increased the frequency of DH development (Alemanno et al., 1994). Supplementation of 0.2 g/L-0.5 g/L colchicine for 24-48 h incubation in LS media followed by transfer to colchicine-free medium could induce as high as 65-70% viable DH plantlets and it was twice compared with conventional anther culture without colchicines (Alemanno et al., 1994; Premvaranon et al., 2011). This new technique can therefore be applied to rice in order in shorten time to produce higher number of double haploid plantlets.


Anther culture ensures production of stable desirable recombinants with high efficiency stacking of specific target genes (without masking effects) in homozygous state. Variation among anther culture derived DHs is due to unlocking new genetic variation. Inter-varietal/interspecific heterotic hybrids may be amenable for genetic improvement through anther culture. Genetic analysis and validation of quantitative trait loci associated with reproductive-growth traits and grain yield under drought stress in a doubled haploid line population of rice (Oryza sativa L.) was studied by Sellamuthu et al. (2011). At present, more than 200 crop varieties have been developed by utilizing a doubled haploid approach (Thomas et al., 2003). DH lines produced from anther culture have many advantages like improved grain quality, resistance to diseases, tolerance to biotic and abiotic stress, superior performance for some agronomic traits over the parents and/or lines used as checks (Winzeler et al., 1987). Xa and Lang (2011) recovered 133 DH lines out of which 22 outstanding DH lines were selected for yield and grain quality. Purwoko et al. (2010) recovered 92 doubled haploid lines from 13 crosses out of which 24 lines had seed yield more than 26 g per hill (transplanted single seedling per hill) with tolerance to biotic and abiotic stresses. Several rice varieties have been developed by anther culture in China (Tanfeng 1; Bua Yu 2; Hua Yu 1; Hua 03; Xin Xiu; Xhongua 8; Ta Be 78 and Guan 18), Argentina (Patei and Moccoi) and Hungary (Dama).


Limited morphogenetic potential of anther derived calli and a higher percent of regenerated albino plants (Roy and Mandal, 2005) seem to be major constraints for double haploid breeding. Green plant regeneration is reported to be enhanced by six days of cold pretreatment to the panicles (Sen et al., 2011). Field grown plants show superiority over to those grown in the glasshouse or pots (Veeraraghavan, 2007). Supplementation of organic adjuvants like yeast extracts, casein hydrolysate and coconut water to N6 media show enhanced androgenic callus induction in indica rice varieties (Roy and Mandal, 2005). High frequency of callus induction was also obtained from incorporation of 2,4-D, NAA and kinetin to the anther culture media (Lal et al., 2014; Mukherjee et al., 2015). Profuse micro tillering of androgenetic plantlets was found in elite indica rice variety IR 72 which can be further depressed by higher concentration of BAP (Roy and Mandal, 2011). Mohiuddin et al. (2014) recovered genetically uniform dwarf DHs from anther culture of an advance breeding line BR802-78-2-1-1. Few of these DHs revealed high fertility status of spikelet with long-bold and long-slender grain. Anitha and Reddy (1997) could successfully recover salt tolerant plantlets from anther culture derived calli of Pokkali and Korgut subjected to 100 mM NaCl stress. Genomic approaches such as association or QTL mapping benefit greatly from the use of DH populations. In India, Satyakrishna (CR Dhan 10) and Phalguni (CR Dhan 801) are the first released varieties from DH lines (CRRI Annual Report, 2008; 2010). Besides, a rice variety “Parag 401” has been bred through double haploid breeding.


4 Epilogue

Several breeding methods are now available to broaden genetic variation and recovery of desirable gene combinations. In each case, the breeding population is derived from single zygotes. In contrast, each anther of an intervarietal/interspecific hybrid carries thousands of pollen grains with different genotype and these can be induced to form stable double haploid plantlets within short time using anther culture. The DH populations may be amenable for molecular mapping of valuable genes and isolation of plant types with high yield, disease resistance and improved quality traits. Besides, a significant proportion of haploids regenerated through anther culture may be used to detect and fix (by colchicine) desirable recessive traits induced through mutation or spontaneous gameto clonal variation.



I sincerely acknowledge and thankful to all researchers for their valuable contributions included in this pursuit as references.



Alemanno L., and Guiderdoni E., 1994, Increased doubled haploid plant regeneration from rice (Oryza sativa L.) anthers cultured on colchicine supplemented media, Plant Cell Rep., 13: 432-436


Afza R., Shen M., Zapata-Arias F.J., Xie J., Fundi H.K., Lee K.S., Bobadilla-Mucino E., Kodym A., 2000, Effect of spikelet position on rice anther culture efficiency, Plant Sci., 153: 155-159


Anitha S.K., and Reddy G.M., 1997, Anther culture studies and selection of salt tolerant cell lines from indica rice, Procced. Ind Nat Sci Acad., B63(1&2): 99-106


Asaduzzaman M., Bari M.A., Rahman M.H., Khatun N., Islam M.A., and Rahman M., 2003, In vitro plant regeneration through anther culture of five rice varieties, J Biol Sci., 3: 167-171


Ba Bong and Swaminathan M.S., 1995, A text book of rice science, ed Chandrasekhar B, Annadurai K and Kavimani R

PMCid: PMC230404


Bagheri N., and Jelodar N.B., 2008, Combining ability and heritability of callus induction and green plant regeneration in rice anther culture, Biotechnology, 7(2): 287-292


Baisakh N., Datta K., Oliva N., Ona I., Rao G.J.N., Mew T.W., and Datta S.K., 2001, Rapid development of homozygous transgenic rice using anther culture harboring rice chitinase gene for enhanced sheath blight resistance, Plant Biotechnol, 18(2): 101-108


Bishnoi U., Jain R.K., Rohilla J.S., Chowdhury V.K., Gupta K.R., and Chowdhury J.B., 2000, Anther culture of recalcitrant indica x basmati rice hybrids, Euphytica, 114: 93-101


Castillo A.M., Cistué L., Vallés M.P., and Soriano M., 2009, Chromosome doubling in monocots, Forster BP. Edited by: Touraev A. Mohan Jain S (ed) Advances in haploid production in higher plants, Springer pub., pp.329-338


Dash A.K., Rao J.G.N., and Rao R.N., 2014, Effect of genotype on anther culture response in indica rice hybrids of maintainer lines, Oryza, 51(2): 165-167


Faruque M.O., Farzana T., Seraj Z.I., Sarker R.H., and Khatun A.A., 1998, Variations in green plant regeneration response from anthers of indica rice and their hybrids with japonica cv, Taipei 309, Plant Cell Tissue Organ Cult, 54: 191-195


Germana M.A., 2011, Anther culture for haploid and doubled haploid production, Plant Cell Tiss Organ Cult., 104: 283-300


Grewal D., Manito C., and 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 Sci. Soc. of America, 51(6): 2544-2553


Gueye T., and Ndir K.N., 2010, In vitro production of double haploid plants from two rice species (Oryza sativa L. and Oryza glaberrima Steudt.) for the rapid development of new breeding material, Sci Res and Essays, 5(7): 709-713


He P., Shen L.S., Lu C.F., Chen Y., and Zhu L.H., 1998, Analysis of quantitative trail loci which contribute to anther culturability in rice (Oryza sativa L.), Mol Breed, 4: 165-172


He T., Yang Y., Tu S.B., Yu M.Q., and Li X.F., 2006, Selection of interspecific hybrids for anther culture of indica rice, Plant Cell Tissue Organ Cult., 86: 271-277


Herath H.M.I., Bandara D.C., and Samarajeewa P.K., 2007, Effect of culture media for anther culture of indica rice varieties and hybrids of indica and japonica, Tropical Agricultural Res & Extn, 10: 17-22


Kaushal L., Balachandran S.M., Ulaganathan K., and Shenoy V., 2014, Effect of culture media on improving anther culture response of rice (Oryza sativa L.), Int J Agric Innov & Res, 3(1): 218-224


Kiviharju E., and Pehu E., 1998, The effect cold and heat pretreatments on anther culture response of Avena sativa and A. sterilis, Plant Cell Tiss Organ Cult., 54: 97-104


Kwon Y.S., Kim K.M., Eun M.Y., and Sohn J.K., 2002a, QTL mapping and associated marker selection for the efficacy of green plant regeneration in anther culture of rice, Plant Breed, 121: 10-16


Kwon Y.S., Kim K.M., Kim D.H., Eun M.Y., and Sohn J.K., 2002b, Marker assisted introgression of quantitative trait loci associated with plant regeneration ability in anther culture of rice (Oryza sativa L.), Mol Cells, 14: 24-28

PMid: 12243348


Lal D., Shashidhar H.E., Ramanjini Godwa P.H., and Ashok T.H., 2014, Callus induction and regeneration from in vitro anther culture of rice (Oryza sativa L.), Int J Agric Environ & Biotechnol., 7(2): 213-218


Miah M.A.A., Earle E.D., and Khush G.S., 1985, Inheritance of callus formation ability in anther cultures of rice (Oryza sativa L.), Theor Appl Genet., 70: 113-116

PMid: 24254166


Mohiuddin A.K.M., Karim N.H., and Sultana S., 2014, Development of improved doubled-haploids through anther culture of indica rice (Oryza sativa L.), Ann Biol Res., 5 (10): 6-13


Mukherjee A., Islam M.R., Nasiruddin K.M., and Banerjee P., 2015, Study on callus initiation and plantlet regeneration ability of some rice genotypes, Int J Sci & Tech Res., 4(10): 354-361


Nguyen H., Chen X.Y., Jiang M., Wang Q., Deng L., Zhang W.Z., and Shu Q.Y., 2016, Development and molecular characterization of a doubled haploid population derived from a hybrid between japonica rice and wide compatible indica rice, Breed Sci., 66: 552–559
PMid: 27795680

PMCid: PMC5010298


Niroula R.K., and Bimb H.P., 2009, Effect of genotype and callus induction medium on green plant regeneration from anthers of Nepalese rice cultivars, Asian J Plant Sci., pp.1-7


Ochatt S., Pech C., Grewal R., Conreux C., Lulsdorf M., and Jacas L., 2009, Abiotic stress enhances androgenesis from isolated microspores of some legume species (Fabaceae), J Plant Physiol., 166(15): 1314-1328
PMid: 19324457


Premvaranon P., Vearasilp S., Thanapornpoonpong S., Karladee1 D., and Gorinstein S., 2011, In vitro studies to produce double haploid in Indica hybrid rice. Biologia, 66(6): 1074-1081


Purwoko B.S., Dewi I.S., and Khumaida K., 2010, Rice anther culture to obtain doubled-haploids with multiple tolerances, As. Pac. J. Mol. Biol. Biotechnol., 18(1): 55-57


Raina S.K., and Ifran S.T., 1998, High-frequency embryogenesis and plantlet regeneration from isolated microspores of indica rice, Plant Cell Rep., 17: 957-962


Ratheika V., and Silva T.D., 2007, A study on the anther culture response in different varieties of rice (Oryza sativa L.) subspecies indica, Proceedings of the 27th Annual Sessions of the Institute of Biology, Sri Lanka, pp.28


Roy B., and Mandal A.B., 2005, Anther culture response in indica rice and variations in major agronomic characters among androclones of a scented cultivar, Karna local. Afr. J. Biotechnol., 4(3): 235-240


Roy B., and Mandal A.B., 2011, Profuse microtillering of androgenic plantlets of elite indica rice variety IR 72, Asian J. Biotechnol., 3: 165-176


Shahnewaz S., and Bari M.A., 2004, Effect of concentration of sucrose on the frequency of callus induction and plant regeneration in anther culture of rice (Oryza sativa L.), Plant Tissue Cult, 14(1): 37-43


Shahnewaz S., Bari M.A., Siddique N.A., and Rahman M.H., 2004, Effects of genotype on induction of callus and plant regeneration potential in vitro anther culture of rice (Oryza sativa L.), Pak J Biol Sci., 7: 235-237


Sah B.P., and Niroula R.K., 2007, Successful regeneration and characterization of anther derived rice hybrid plants from O. sativa L. × O. Rufipogon Griff., Sci World, 5(5): 14-18


Sellamuthu R., Liu G.F., Ranganathan C.B., and Serraj R., 2011, Genetic analysis and validation of quantitative trait loci associated with reproductive-growth traits and grain yield under drought stress in a doubled haploid line population of rice (Oryza sativa L.), Field Crops Res., 124: 46-58


Sen C., Singh R.P., Singh M.K., and Singh H.B., 2011, Effect of cold pretreatment on anther culture of boro rice hybrids, Int J Plant Rep Biol., 3: 69-73


Silva T.D., 2010, Indica rice anther culture: Can the impasse be surpassed? Plant Cell Tiss Organ Cult, 100(1): 1-11


Talebi R., Rahemi M.R., Arefi H., Nourozi M., and Bagheri N., 2007, In vitro plant regeneration through anther culture of some Iranian local rice (Oryza sativa L.) cultivars, Pak J Bio Sci., 10(12): 2056-2060
PMid: 19093446


Thomas W.T.B., Foster B.P., and Gertsson B., 2003, Double haploids in breeding, In: Doubled haploid production in crop plants, (eds. M.Maluszynski, K Kasha, BP Foster and I Szarejko), Klewer Academic Publishers, pp.337-350


Trejo-Tapia G., Amaya U.M., Morales G.S., Sanchez A.D.J., Bonfil B.M., Rodriguez-Monroy M., and Jimenez-Aparicio A., 2002, The effects of cold-pretreatment, auxins and carbon source on anther culture of rice, Plant Cell Tissue Organ Cult, 71: 41-46


Thuan O.T., Tuan V.D., and Ba Bong B., 2001, Study on anther culture of F1 plants from crosses between aromatic and improved rice cultivars, Omonrice, 9: 41-45


Tran D.G., and Vuong D.T., 2002, Effect of different media and genotypes on anther culture efficiency of F1 plants derived from crosses between IR64 and new plant type rice cultivars, Omonrice, 10: 107-109


Veeraraghavan R., 2007, A study on the comparison of anther culture response in different varieties of rice (Oryza sativa L.) subspecies indica, University of Colombo, Colombo, Sri Lanka

PMCid: PMC1891355


Winzeler H., Schmid J., and Fried P.M., 1987, Field performance of androgenetic, doubled haploid spring wheat lines in comparison with lines selected by the pedigree system, Plant Breed, 99: 41-48


Xa T.T., and Lang N.T., 2011, Rice breeding for high grain quality through anther culture, Omonrice, 18: 68-72


Yang J.Y., Zhao X.B., Cheng K., Du H.Y., Ouyang Y.D., Chen J.J., Qiu S.Q., Huang J.Y., Jiang Y.H., Jiang L.W., Ding J.H., Wang J., Xu C.G., Li X.H., and Zhang Q.F., 2012, A killer-protector system regulates both hybrid sterility and segregation distortion in rice, Science, 337: 1336-1340
PMid: 22984070


Yamagishi M., Otani M., Higashi M., Fukuta Y., Fukui K., Yano M., and Shimada T., 1998, Chromosomal regions controlling anther culturability in rice (Oryza sativa L.), Euphytica, 103: 227-234


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