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Rice is one of the most important cereal crops

Rice is one of the most important cereal crops, providing carbohydrate sources for over half of the world’s population (Cassman, 1999; Khush, 2005). It has a high amount of calories necessary for human diet; and in Asian countries, it makes up around 40-80% of the normal diet. Although Asian countries have the vital climate to produce up to 90% of the world’s rice yields, it has not been achieved so far for many reasons. Presently, the rice production in the world has been raised to 650 million tonnes with a cultivable area of around 156 million hectares. Rice is recognized as the third most economical crop in the world (Abdullah et al., 2008) and Asian countries are the biggest consumers of rice with over 75% of rice yields. The rate of rice consumption has increased by 1.8% per year while, the rate of rice production has decreased (Hussain et al., 2010).

Although rice production has increased almost three folds over the past three decades, continued increase in yield potential and the number of high yielding varieties are necessary to meet the ever growing demand of the global population. Rice biotechnology has already contributed to the production of improved varieties and the wide hybridization, including use of embryo rescue technique has allowed the transfer of useful genes from the wild Oryza species to the elite cultivars. With gradual reduction in crop land area, it is imperative to increase productivity levels per unit area. Hybrid rice, which can offer significant yield advantages over inbreds, has revolutionized the rice productivity and production in the world. However, the hybrid rice production technology is bit tedious and costly and therefore, the cost of seed material is very high and many a times, the farmers may not offer to buy the seeds (as always the F1 hybrid seeds are to be used). In addition, poor grain cooking quality limits its popularity further among farmers (Mishra and Rao, 2016; Naik et al., 2017). Therefore, other suitable techniques/ approaches are required to produce the high productive and quality seed material besides completely homozygous lines for further use in crop improvement programmes.

Tissue culture techniques coupled with recent developments in molecular biology have opened a new vista for broadening crop gene pools contributing to increasing the efficiency of conventional plant breeding methods. Many new rice cultivars have been developed through biotechnological techniques like anther culture, embryo rescue and somaclonal variation (Brown and Thorpe, 1995; Zapata et al., 2004). One of the most striking examples of cellular totipotency is a phenomena known as microspore or pollen embryogenesis (also referred to as androgenesis) where immature pollen or microspores are induced for the complete reprogramming of the developmental plan to embryo with the haploid (gametic) number of chromosomes rather than pollen grain formation (Reynold, 1997).

Anther culture, an innovative method for hastening the generation of homozygous doubled haploid (DH) can be used to accelerate the varietal improvement programs in rice (Herawati et al., 2010). In vitro anther culture provides a rapid method for inducing homozygosity and developing true breeding lines in the immediate generation from any segregating population thereby, contributes in shortening the breeding cycle of new varieties.

Haploid plants are recognized by the existence of only one set of chromosomes in their cells and in nature, haploids occur as an irregularity when the haploid egg forms an embryo without fertilization. Haploids are sexually sterile and therefore, doubling of chromosomes is required to produce fertile plants which are called doubled haploids or homozygous diploids. Several scientists have successfully produced callus and haploid plants through anther culture and isolated pollen/anther from targeted plants (Davey and Anthony, 2001). The technique of anther culture can be allied in plant breeding to accelerate the process of obtaining pure lines with numerous advantages. Of them, shortening breeding cycle by immediate fixation of homozygosity which allows an easy selection of phenotypes for quantitative characters, widening of genetic variability through the production of gametoclonal variants, increased selection efficiency and allowing early expression of recessive genes are important. In addition, screening of haploid cells against cold tolerance, salinity, pathotoxins and other biotic and abiotic factors before plant regeneration also becomes possible through this technique.

Anther culture technique also offers great opportunities for improving grain quality of rice. Development of rice varieties using anther culture techniques has been reported in several countries. Most of the anther culture derived varieties are of the Japonica type. The Indica type rice is generally recalcitrant to culturability compared to Japonica and needs improvement through basic research of culturability. In vitro response of anthers of both japonica and indica are genotype specific and affected by too many factors. Thus, each genotype needs standardization.

Enormous advances have been made throughout the world since the invaluable discovery of androgenic haploidy by Guha and Maheshwari (1964) and production of rice haploids by Niizeki and Oono (1968). Though anther culture has been recognized as a valuable tool in plant breeding programs, its application is limited due to difficulties in the induction of embryogenic calli from some genotypes (Lee and Lee, 2002). In general, indica rice has low anther culturability (Dewi et al., 2009) and the recalcitrance of them relates to early anther necrosis, poor callusing ability, low plant regeneration and frequent regeneration of albino plants (Grewal et al., 2009). In this regard, several attempts at anther culture of indica rice have met with limited success. Therefore, an efficient and high throughput technique is required to improve the callusing and regeneration ability of the androgenesis.

The haploid induction and subsequent regeneration of embryos depends on many factors, which can be limiting. These include plant genotype (Shen et al., 1982), the microspore developmental stage (Chen, 1976), cold pretreatment of the panicle (Sunderland, 1978; Chaleff and Stolarz, 1981; Chen et al., 1982; Zapata et al., 1982), growth conditions of the donor plants (e.g. photoperiod and light intensity; Lee et al., 1988), the orientation of the plated anthers (Mercy and Zapata, 1987), the nitrogen source of the callus-induction medium (Chen et al., 1982, Tsay et al., 1982) and the carbon source, composition of the culture medium (including culture on “starvation” medium low with carbohydrates and/or macro elements followed by transfer to normal regeneration medium specific to the species), physical factors during tissue culture (light, temperature). Therefore, it is highly imperative to come out with a protocol to produce haploids in each of the genotypes in question. Further, it is also equally important to standardize the protocol for doubling the chromosome of haploid plants and confirm their ploidy through cytological studies or through other known approaches.

With this background, the present study was taken up with an overall goal of producing the doubled haploid (DH) lines from anthers of KRH4 rice hybrids and further characterizes them for yield potential besides the cooking quality. The objectives set to achieve the goal are as follows,
– Standardization of anther culture protocol for generation of haploids and doubled haploids from KRH4 rice hybrid
– Identification of true haploids and doubled haploids through markers and cytological studies
– Characterization of the developed doubled haploid plants for
i. Growth and productivity
ii. Cooking properties