Background

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.

Acknowledgements

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

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