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Rice, one of the major crops in the world, is the staple diet for about 3 billions

of world population. In 2025 there will be 4.6 billion people depending on rice for

their daily nourishment, compared with three billion today. Hybrid rice breeding has

been successful in developing superior hybrids, which have brought about dramatic

increases in rice production (Yuan, 1992). In order to maintain the success and meet

the demands of ever increasing population, utilization of advanced technologies are to


be employed for yield enhancement. Conventional breeding methods take many years

as they involve selection within/between segregating populations over several

generations. Selection for certain traits is often made difficult by dominant recessive

relationships as recessive traits are masked by the dominance gene effects and it is

also difficult to separate the homozygous from heterozygous genotypes among the

plants having dominant traits.

In 1964, Guha and Maheswari reported the direct development of embryos

from microspores of Datura innoxia by culturing excised anthers. In case of rice,

haploids were first produced through anther culture by Niizeki and Oono (1968)

closely followed by Nishi and Mitsuoka (1969) in Japan. Doubled haploid breeding

through anther culture is a promising and convenient alternative to conventional

techniques for the production of homozygous recombinant lines at one step from a

cross combination. It is a friendly tool for plant breeders that provide a link between

conventional plant breeding and genomics.


2.1 Advantages of doubled haploid breeding

Anther culture is one of the best breeding methods with numerous advantages:

shortening breeding cycle by immediate fixation of homozygosity, increase in

selection efficiency, widening of genetic variability through the production of

gametoclonal variants and allowing early expression of recessive genes (Zapata

1992). Doubled haploid lines are homozygous, immortal and true breeding lines

which make them suitable for breeding as well as mapping purposes. Following are

some of the conditions that favor doubled haploid breeding over conventional


2.1.1 Rapid development of homozygous lines and shortening of breeding cycle

Doubled haploid breeding is an important technique for immediate fixation of

homozygosity and shortening the breeding cycle in varietal improvement Production

of haploids/doubled haploids through anther culture from Fi hybrid plants results in

true-breeding plants in less than one year, which otherwise takes 7 to 8 generations

through conventional methods (Gosal et al., 1996). Doubled haploid lines are useful

in breeding of rice (Oryza sativa L.), and many elite lines have been produced within

a short time by the anther culture of Fi plants (Zapata et al, 1991). In conventional

breeding programs like pedigree method, yield evaluation usually starts at Fe or F7

generation after the breeding lines attained a fairly homozygous state. However,

anther culture method requires only two years for first yield trial and moreover, the

tested doubled haploid lines were completely homozygous. This kind of elimination

of breeding generations through anther culture would greatly accelerate varietal

development programs. Theoretically, yield trial of doubled haploid lines could be

conducted in the second generation (Ai) plants derived from the anthers itself

resulting in saving time, money and labor. In the lines that were fixed through anther

culture from inter sub specific crosses; selection of desirable types would be more

reliable and time saving.

Anther culture would also be particularly useful for photoperiod-sensitive

rices since development of homozygous lines of these rices otherwise takes a veiy

long time. Senadhira et al, (2002) could identify salt tolerant anther culture derived

rice lines within a three year period. This system provides an unparalleled

opportunity to shorten the breeding cycle and fix agronomic traits in the homozygous

state, such as recessive genes for disease resistance (Datta. 2005). The obtention of

doubled haploid plants provides a,useful tool, enabling plant-breeding cycles to be

reduced in time (Devaux and Pickering, 2005).

2.1.2 Time saving advantages

In a changing world, breeders need to adapt speed and efficiency to develop

new cultivars which is important for the breeding industry. Time to market is essential

for cost calculations, thus efficient doubled haploid production for practical breeding

provides a significant competitive advantage and potentially bigger market shares.

The main advantage of using haploids in a breeding program is the production of

homozygous lines in a short time (Khush, 1997). The ability to produce homozygous

lines after a single round recombination saves a lot of time for the plant breeders. It

also takes less time to build up pure stocks of a new cultivar through doubled haploid

method. In the conventional system, stocks were usually derived from a single plant

of an advanced generation. Since double haploids were completely homozygous, all

stocks were identical and no purification system was required.

2.1.3 Efficiency and reliability of selection in doubled haploids

Doubled haploids have long been recognized as a valuable tool in plant

breeding since it not only offers the quickest method of advancing heterozygous

breeding lines to homozygosity, but also increases the selection efficiency over

conventional procedures due to better discrimination between genotypes within any

one generation. (Martinez et al, 1996; Yang, 1997; Chen et al, 1997). Snape (1989)

reported an increase in selection efficiency using doubled-haploid breeding compared

to conventional practices because of an increase in additive genetic variation, the

absence of dominance variation and segregation within-families, and a decrease in

environmental effects through greater replication possibilities. Thus, it should be

easier to identify superior genotypes in a cross and produce new cultivars through this

approach. In addition, anther culture could also be an effective vehicle for producing

variation as it allows early expression of recessive genes.

In conventional breeding, the early segregating-generation populations involve

variation attributable to both additive and non-additive genetic effects (Khush and

Virk, 2002), whereas doubled haploid (DH) lines exhibit variation only of additive

genetic nature, including additive x additive type of epitasis, which can be easily fixed

through a single cycle of selection. Thus, compared to the F2 population, fewer

doubled haploid plants would be required for purposes of selection of the desired

recombinants. It was estimated that about 150 pollen plants derived from Fi anthers

would be enough, instead of 4000-5000 F2 plants, for the purposes of selection of

desirable genotypes (Shen et al, 1983). Anther culture provides an easy to handle in

vitro selection for genetic improvisation of characters (Jahne et al, 1991) for superior

performance. Traditionally, breeders establish 3000-5000 plants in the F2 generation

alone while with anther culture, only 60-70 plants are required (Khush and Senadhira,

In conventional breeding, F3 or F4 generation exhibits genetic differences

between individuals within the plots, unlike doubled haploid plants where all

individuals are genetically identical. This makes visual selection of desirable lines

easier in early generations generated through double haploid approach compared to

conventional means. Thus, selection efficiency is increased in early generations in

doubled haloids compared to conventional populations. In the conventional method,

the recombinants with recessive characteristics could be identified only in the

homozygous state. As doubled haploids were completely homozygous, the selective

probability of these recombinants with recessive characteristics was two times higher

compared to conventional method. While screening for quantitative characters like

yield and quality, one could be sure that due to homozygosity, the recombinants with

desirable characters would not be lost, otherwise segregation from heterozygous loci

in subsequent generations might result in reduction of the selection effect.

2.1.4 Identification of superior crosses for more extensive exploitations

Anther culture was successfully used to alleviate the problems of high sterility

and skewed recombination in indicaljaponica hybrids. The doubled haploid (DH)

method might serve this purpose since it can provide a random sample of completely

homozygous lines in a relatively short time after crossing. It could be used to derive a

small sample of DH lines (about 20 lines) from many crosses for evaluating the

performance of different cross combinations. After the evaluation, the most promising

crosses could be evaluated extensively further either by using anther culture again or

by conventional method of generation advance and selection. The population size of

DH lines from selected crosses would be dependent on the number of genes

governing the target traits (Choo et al., 1985).

2.1.5 Mutation

Doubled haploid (DH) systems have many attractive features for inducing and

fixing mutations. The ability to fix mutations via doubled haploidy is of great

practical importance as induced mutations are predominantly recessive and cannot

normally be detected until the F2 generation at the earliest. The DH systems

themselves provide an opportunity to target haploid as well as doubled haploid cells

for mutation treatment and capture the mutation in a homozygous pure line. The

works that have been carried out to DH for rice improvement, such as better quality,

salt tolerance include development of NaCl-resistant DH lines from successive

cultures of anther derived calli in NaCl-supplemented media (Satish et al, 1997) that

revealed the different distribution pattern of K+/Na+ in callus and plant cells.

Schaeffer and Sharpe (1987) reported the mutant lines of increased lysine content

from successive cultures of anther derived calli in lysine analog-supplemented media.

Lee and Lee (2001) reported high frequency of rice mutants obtained from anther

culture treated with EMS, 10 and 20 days after anther inoculation. DH systems were

employed in the process of mutant induction and selection (Szarejko, 2003). Kyin et

al., (2005) obtained stable rice mutants flowering significantly earlier than the parent

cultivar Yar-2 by using DH mutant production and gamma rays as the mutagenic


2.2 Factors affecting doubled haploid breeding through anther culture

There are many factors that can affect the success of anther culture, such as the

physiological status of the donor plant (Afza et al, 2000; Jacquard et al, 2006),

genotypic variation (Raina 1997; Ramakrishnan et al..K 2005:He et al., 2006),

developmental stages of the microspore (Afza et al., 2000, Cha-um et al,2009), pre

treatment of anthers (Trejo-Tapia et al., 2002), culture media (Faruque et al, 1998;

Asaduzzaman et al, 2003), and growth conditions of the cultures (Raina and Irfan

1998). ?
2.2.1 Genotype

Among the factors reported so far that affect doubled haploid induction in

anther culture, genotype is a major one. It is very important to select genotypes that

are better to regenerate plants than that produce a larger number of calli. Guha-

Mukherjee (1973) reported that only 5 out of 18 cultivars showed pollen callusing and

in only four cases callus differentiation was observed. The differences among

genotypes may be due to the position of spikes in relation to the penultimate leaf and

the stage of the pollen development of anthers (Liang et al., 1982). Reviewing the

progress of anther culture in China, Hu (1984, 1985) had reported that although the

frequency of green plants was more than 10% (of the number of cultured anthers) in

japonica cultivars, it was only 1% in indica rices. Miah et al., (1985) reported that

anther culture response varied from 41% for a japonica cultivar to 0% for an indica

cultivar. Abe and Fustsuhara (1986) tested 66 indica and japonica cultivars and

reported that japonica varieties displayed a higher rate of callus induction and

regeneration than those of indica. Indica cultivars respond poorly to in-vitro

techniques (Abe and Futsuhara 1986; Hartke and Lorz 1989), and in consequence the

practical production of haploids from anther or microspore culture in rice breeding is

limited to Oryza sativa spp .japonica cultivars. The culture ability of hybrid rice that

possesses high extent of heterozygosity was higher than that of hybrids between

varieties (Chen, 1986). In the case of indica rice, early anther necrosis, poor callus

proliferation and albino plant regeneration were recognized as the major problems

(Chen et al, 1991). In general, indica cultivars of rice exhibit poorer androgenic

response than the japonica, Tongil rice (japonica x indica) and javanica cultivars

(Raina, 1997). Wen et al., (1991) reported that the callus induction rate was genotype

dependent, and production of calli was also dependent on medium composition, in

particular, plant hormones. Similarly, Lentini et al, (1995) reported that only one out

of 35 indica cultivars exhibited pollen callusing on Ng medium. Most of the in vitro

morphogenic responses were genotype-dependent (Shen et al., 1983; Bhojwani and

Razdan, 1996). Cultural variables and genotypes had an important role on successful

exploitation of anther culturability in indica rice (Mandal et al., 1995). Hartke and

Lorz (1989) tested 15 indica rice lines and found that seven of them produced

embryogenic calli and only four regenerated into plants. Genotypic variation in calli

induction and subsequent plant regeneration potential in rice was also reported by Abe

et al, 1985 and Khalequzzaman, M. et al., 2005. Genotypic specifcity to anther

culturability is a more serious problem in indica than japonica cultivars (Chung and

Sohn 1995). The genotype of donor plant plays an important role in callus induction

in anther culture of rice and other cereals crops (Bagheri et al, 2008).

2.2.2 Physiological status of donor plants

Varying temperature and light conditions during the growth of donor plants

affect anther response (Dunwell and Sunderland, 1973). Growth conditions,

particularly the physiological state of the anther donor plants at the time of anther

excision, were known to affect anther response in a number of plant species (Engvild 1

et al, 1972; Dunwell and Peny, 1973; Foroughi-Wehr and Mix, 19*79). Inflorescences

of rice plants treated with 2-chloroethylphosphonic acid for 48 h at 10C exhibited an

increased anther response (Wang et al, 1974). Hu et al, (1978) reported the influence

of temperature on callus induction frequency of Keng rice L; the frequency was high

when the temperature was 18.5-29.0C than when the temperature was 16-18 C. The

frequency of androgenesis was higher in anthers harvested at the beginning of the

flowering period and showed a gradual decline in relation to plant age (Maheswari et

at, 1980, 1982). It appeared that the disadvantageous temperature during the booting

stage of donor plants affected the normal development of microspore in vivo as well

as the androgenesis of microspore in vitro via its influence on the anther wall tissue

(Zhong and Liang, 1980). The variation in androgenetie response in rice anther

culture is dependent upon the donors plant growth environment (Chaleff and Stolarz,

1981). The frequency is also affected by environmental conditions and physiological

status of the donor plants, pre-treatment of the donor material (Lazar et al., 1985). In 7
the season with an unstable climate where plant to plant variations were known,

variations also existed among primary and secondary tillers which showed highest

response while tertiary and late tertiary tillers were poor in response (Hadi and Raina,
C .......... ' P

1987). Donor plants that came to flower during continuously cloudy/ rainy weather or

at very low temperature (16-18C), or at high temperature (26-30C) in a greenhouse

callused at a less frequency (Raina, 1989). Nitrogen supply must be sufficient to

maintain the photosynthetic activity of the plants and thus would ensure pollen

development and plays an important role in maintaining the anther donor plants in a

vigorous and healthy condition (Guha-Mukherjee, 1973; Tsav, 1981). Heenan (1984)

found that the effect of nitrogen in pollen development was most conspicuous at the

early microspore phase which is the stage receptive to anther culture.

Raina and Zapata (1997) observed that the plants grown in the field were

significantly superior to those grown in the glasshouse or pots. The frequency of

callus formation from anthers from the top part of the panicle was the lowest, while

those from the basal are the highest (Afza et al., 2000). The growth conditions of the

donor plant development regulate the physiology of the anthers in the spike and thus

greatly influence microspore response in anther culture (Cistue et al, 2003; Szarejko,


2.2.3 Developmental stage of Anther

In rice, anthers with the early or mid to late uni-nucleate pollen stage of

microspore development were found to be most responsive for culture. Several reports

indicated that the microspores in the late uni-nucleate stage were suitable for

inoculation (Guha-Mukherjee, 1973: Hsin-Ying et al, 1978). Oono (1975), Raina

(1977) and Genovesi (1978) conducted detailed studies and concluded that mid- to

late uni-nucleate pollen responds well. The rates of viable pollen grains were

relatively higher in anthers inoculated at early, mid and late uni-nucleate stages while

in anthers inoculated at the bi-nucleate stage, most of the pollen degenerated (Sun,

1978). Calli from late uni-nucleate pollen appeared to show less regeneration potential

and produced more albinos and very young microspores or tetrads were ineffective for

culture. Zapata et al, (1983) reported that the pollen between the mid to late uni

nucleate and early bi-nucleate stages showed better response. Anthers at the tetrad

stage do not respond at all and anther response falls sharply after the first pollen

mitosis. In these stages, starch deposition would begin, but no sporophytic

development and subsequently no macroscopic structures were formed in the

microspores. Pollen of this stage, when cold-pretreated or cultured, could deviate

from normal developmental programme and switch to sporophytic growth.

Two different pathways of androgenetic microspore development were known

in rice. The vegetative and/or generative nucleus starts to divide, resulting in multi

cellular microspores. As cell division continues, the pollen' grain ruptures eventually

and embryo ids are formed. The switch towards embryo id-callus development seemed

to occur more readily when the process of nuclear division had already been initiated

than when it had to be initiated in culture. The difficulties with the older stages of

pollen appear to be due to their commitment to differentiation into a male

gametophyte. For rice, the best stage has been described as uni-nucleate to early bi

nucleate stage (Jahne and Lorz, 1995). Therefore, determining the development stage

of pollen in the anthers is important to optimize the anther culture response of a given

rice genotype (Silva, 2010).

2.2.4 Pretreatments

Application of a variety of stresses, such as temperature pre-treatment, osmotic

shock and sugar starvation, during the labile developmental period of pollen grains is

known to be essential for the induction of androgenesis in several plants, including

cereals (Bhojwani and Razdan, 1996; Bhojwani et al., 1997). However, the type,

duration and the time of application of these pre-treatments may vary with the species

or even variety (Datta, 2001). Cold

Low temperature shock has been reported to enhance the androgenic response

in several species. Several researchers had reported a stimulatory effect of cold

pretreatment in rice. Chaleff et al, (1975) obtained a callusing frequency of 22.2%

from anthers cold pretreated at 6C for 5 days, compared to only a 10.5% frequency

from the untreated anthers. Hu et al., (1978) found a significant increase in response

when a cold treatment of 4-8 days at 10C was given immediately after inoculation of

rice anthers, Chen et al, (1980) found the response to cold pretreatment was

genotype-dependent. Zhao (1983) found that indica and japonica varieties differed in

their requirements for cold-shock treatment. Genovesi and Magill (1979) treated

anthers at 5 or 10C for 7 or 10 days or at 13C for 10 of 14 days, resulting in an

anther response of up to 73.6%, compared to a 17.9% response of untreated anthers.

Rush and Shao (1982) confirmed these results. Chaleff and Stolarz (1982) also

obtained increased anther response when anthers were pretreated at 7C for 3 days.

Low temperature treatment of anthers was considered to suppress the spontaneous

doubling of the chromosome number, which was in agreement with the observation of

Tsay and Chen (1984). Moon et al., (1989) reported that the proper treatment would

be 8-16 days in duration with slightly higher temperature for indica/japonica


Matsushima et al., (1988) had reported that a pre-treatment at 10 C for 10-30

days was necessary to induce sporophytic divisions in microspores of the japonica cv

Nipponbare. This was subsequently confirmed by several workers for japonica and

indica cultivars (Datta et al., 1990; Ogawa et al., 1992; Raina and Irfan, 1998).

Ogawa et al., (1995) observed that 28 days of pre-treatment at 10C was optimum for

the indica cv IR 24. Pande (1997) observed that cold pre-treatment was essential for

androgenesis in anther cultures of the indica cv IR43, and 10C for 10 days was most

suitable and pre-treatments longer than 11 days had resulted in albino production. The

positive effects of cold pretreatment on the induction of callus are due to the delay of

pollen or anther wall senescence; the increase of symmetric divisions of pollen grains;

and the release of substances necessary for androgenesis, mainly amino acids and

shock-thermic proteins (Xie et al, 1997; Kiviharju and Pehu, 1998). Heat

A temperature shock has been reported to improve the androgenetic response

in many plant species. Rice exposed to high temperatures at meiosis easily forms

albino plants, optimal temperature for regeneration appears to be genotype dependent

(Ouyang et al, 1983). High temperature pretreatment disrupts the normal integrated

development of somatic anther tissue and subsequently may synchronize the

physiological states of the two tissues, thereby stimulating the induction process

(Dunwell et al, 1983). Rice exposed to high temperatures at meiosis easily forms

albino plants, optimal temperature for regeneration appearing to be genotype

dependent (Ouyang et al, 1983). Heat pretreatment has been used to induce

embryogenesis from isolated anthers, as it disrupts the cytoskeleton in microspores in

the initial phase (Ferrie et al, 1995). Heat stress pretreatment has been an essential

factor to increase the efficiency of androgenesis in different species (Kumar et al,


2.2.5 Osmotic Stress

Osmotic stress is another pre-treatment which can substitute cold pretreatment

in androgenesis. Lai and Liu (1988) reported that the inclusion of mannitol, or the use

of a higher agar concentration in shoot regeneration medium, increased the plant

regeneration frequency in the jcponica rice variety TN5. The beneficial effect of

maltose on callus induction has been attributed mainly to a starvation effect on

microspores early in culture due :o slow hydrolysis of maltose and maintenance of a

relatively high and constant osmolality of maltose medium (Zhou et al, 1991).

Osmotic stress or reduction in cell water content influence the process of somatic

embryogenesis and plant regeneration. Wetherell (1984^-suggested that the osmotic

stress may disrupt the plasmodesmatal connections between the pre-embiyonic cells,

thus making the cells physiologically isolated and allowing a greater number of cells

to differentiate. Cistue et al, (1994) confirmed the importance of mannitol for the

induction process and found that application of 0.7M mannitol for 3.5 days improved

green plant regeneration significantly. Raina and Irfan (1998) had reported that

treatment of anthers in 0.4M mannitol solution was essential to induce androgenesis

in microspore cultures of indica and japonica cultivars. Ogawa et al, (1995) reported

that sugar starvation of the anthers of IR43 for 2 days at the beginning of culture

caused promotion of androgenesis (39%; 12 fold) which was more than any of the

mannitol pre-treatments. Hoekstra et al., (1993) who gave mannitol stress in

microspore culture found that yield of green plants increased from 50% to 97%. In the

absence of cold treatment, mannitol treatment promoted androgenesis in anther

cultures of cv. IR43 from 3% to 33.4% while with cold treatment no promotory effect

was seen (Pande, 1997).

2.2.6 Effect of Culture media

Culture medium is an important aspect in androgenesis of cereals. Culture

medium not only provides nutrition but also decides the fate of the microspores.

Progress has been made in the anther culture technique through modification of the

culture medium (Ghaemi et al, 1994). Ng medium has been most widely used in rice

anther culture (Raina, 1989, 1997). N6 (Chu, 1978) and MS (Murashige and Skoog,

1962) were the two most widely used media for anther culture of cereals. However,

by 1978, several media formulations were reported including N6 like Heh-2, Heh-5,

SK-3, SK-8, Szechuan medium, Medium V and Chaleffs R-2.

Low inorganic nitrogen had generally proved better for androgenesis in

cereals, particularly in form of NH4+. Chu et al, (1975) had demonstrated that the

level of ammonium nitrogen in the culture medium was critical for rice anther culture.

On this basis he developed the Ng medium which has been most widely used for rice

anther culture (Raina, 1989, 1997). However, it proved better for japonica cultivars

but indica varieties require even lower amount of NH4+. Raina (1989) reported that a

medium with high KNO3 and NH4+ ions completely replaced by 50 mg/1 casein

hydrolysate (CH) was significantly better than the original MSN medium for indica x

indica Fi hybrids, with regard to the frequency of green plant regeneration. This SK-1

medium was half as effective as Ng medium so far as the frequency of pollen

callusing is concerned but the calli formed on this medium produced twice as many

green plants as those on Ng medium. Raina and Zapata (1997) developed a new

medium called M 019 based on their detailed study on the medium requirement of the

indica cultivar IR43. This medium differs from SK-1 medium in substitution of CH

by a small amount of (NHLj^SC^ and the level of KH2PO4 increased from 170 mg/1

to 540 mg/1.

Sucrose has been used as a major carbohydrate source in the induction

medium. Reinert et al, (1977) suggested that sucrose level of 2 - 5% is good for rice

anther culture. Enhanced response of callus induction; multiple calli formation and

high green plant regeneration were achieved with potato-II medium (Rout et al,

1989). In cultures, sucrose rapidly breaks down to glucose and fructose, so that after 3

weeks of barley anther culture the medium did not contain any sucrose (Last and

Brettell, 1990). Wang et al, (1977) reported that sucrose concentration of above 6%

in the induction medium increased proportion of albino plants. On the other hand, the

hydrolysis of maltose during the same period was below the detectable level. The

substitution of sucrose with maltose substantially increased the formation of embiyo-

like structures in all genotypes (Navarro-Alvarez.eta/..J9.94a), Cp

Maltose has been reported to be a superior source of carbohydrate than

sucrose for androgenesis in several species, including cereals (Last and Brettel 1990,

Pande and Bhojwani 1999). Maltose increased somatic embryo formation and the

frequency of green plant regeneration in indica rice protoplast-derived tissues (Ghosh

Biswas and Zapata, 1993; Jain g^q^l995; Lentini et al, 1995). The toxicity of

sucrose for androgenesis is due to the sensitivity of microspores to fructose but not to

glucose. Maltose instead of other saccharides is superior in studies of anther culture of

cereal crops (Jahne and Lorz, 1995; Lentini et al, 1995; Raina, 1997). Potato extract

medium was said to be widely used in China for its low cost, simple production and

high efficiency. Potato extract (10-20%) in culture medium, was reported to greatly

enhance callusing of rice anthers (Chen et al, 1978). Inoue and Meada (1982)

reported that ABA could stimulate the differentiation of rice callus. Sandhu et al,

(1993) reported a high frequency regeneration of green plant in rice when lower

concentration of sucrose was used (3% w/v) for callus induction of anther.

Sorbitol also has been reported to enhance plant regeneration of rice when

applied to the regeneration medium (Ozawa and Komamme, 1989; Yoshida et al,

1994). Lentini et al, (1995) reported that with the addition of AgN03, the frequency

of green plant differentiation was also doubled. Several efforts has been made in the

recent times to improve the efficiency of rice anther culture such as improved

methods of culture techniques (Trejo-Tapia et al., 2002), formulation of new media

compositions (Cha-Um et al., 2009, Thomas 2008) and utilization of specialized

chemicals for callus induction (Datta et al., 2005). Research efforts on the

enhancement of response to anther culture have been confined mostly on

manipulation of callus induction and plant regeneration protocols (Balaehandran et

al., 1999).

Calcium in the medium is known to stimulate ethylene production in many

plant tissues. With AgNC>3, an anti-ethylene compound, the frequency of green plant

differentiation doubled (Lentini et al., 1995). It is also known to promote pollen

embiyo production in anther cultures of Brussels sprouts (Biddington et al., 1988).

Amino acids have been used as nitrogen source in in-vitro culture of various tissues,

including isolated microspores. Cho and Zapata (19881 reported that proline and

glutamine promoted callus formation in microspore cultures of a japonica cultivar.

These amino acids also induced a higher degree of plant regeneration and green plant

production than the medium containing alanine or no amino acid. Ogawa et al.,

(1995) also found glutamine to be effective for pollen callusing in microspore cultures

of an indica cultivar but alanine was far better than glutamine for plant regeneration

and green plant production.

2.2.7 Growth regulators

The growth regulators i.e. auxins and cytokinins are the most important

constituents in the rice anther culture medium. The effects of different combinations

of growth regulators on pollen callus initiation and plant regeneration was difficult to

assess because they depend on both concentration and combination and also

interaction with the plant genotype. Among auxins, 2, 4-D and NAA were the most

frequently used and equally effective for induction of callus from rice anthers. Auxins

have been essential plant growth regulators for the induction of callus from anthers of

cereals (Zhu et al., 1998). LAA and NAA may induce direct androgenesis, while 2; 4-

D promotes rapid cell proliferation and formation of non-embryogenic callus (Ball et

al, 1993). The rate of success can be enhanced by improving the composition of

tissue culture medium, especially by manipulating plant growth regulators (Mandal

and Gupta, 1995; Zhu et al., 1996). The media containing NH4+ below 5 milli

equivalent might be used for all indicia lines; however, the responses of different

genotypes to different basic media were different. Different genotypes require

different hormones. While some appropriate combinations of 2.4-D, NAA and kinetin

were effective for most genotypes, callus induction and plant regeneration of some

cultivars occurred only in the presence of zeatin.

A wide variety of growth-stimulating hormones had been tried, singly, and in

numerous combinations (Niizeki and Oono, 1968; Iyer and Raina, 1972), a

combination of auxins (LAA and 2, 4-D), a cytokinin (Kn), and complex growth

substances (CW, YE and/or CH) were used. Raina (1983) found from different

hormonal combinations, 2, 4-D (2 mg/litre) induced a high response. Higher

concentrations of auxins lowered the ability of callus to produce green plants in

japonica rice fYeh et aL-l-9S8V. The rate of regeneration of albino plants varied with

the type and concentration of auxin used during callus induction (Huang, 1985; Lenka

and Reddy, 1994). Among the group of auxins, 2, 4-D had shown good callusing

ability and when the callus was transferred to regeneration medium it was observed

the green plant production was more in 2, 4-D induced calluses than the calluses

induced on NAA. It was observed that 2, 4-D inhibited the organogenesis of calluses

whereas NAA gave rise to the formation of roots and sometimes to complete plants

(Comejo-Martin et al, 1981; Mandal and Gupta, 1995).

2.2.8 Gelling Agents

Agar is the most common gelling agent used to solidify media, being cheap

and easily available. The negative effect of agar is the release of impurities from agar

into the medium hampering growth and development. To avoid this problem agar

could be replaced by alternative gelling agents. Ficoll was first recommended as

buoyancy agent to prevent embryo ids from sinking in anther culture (Kao, 1981).

Ficoll is a non-ionic, synthetic polymer of sucrose which increases the surface

density. But, ficoll also changes medium osmotic potential. The ratio of green to

albino plantlets was also improved by the ficoll supplement, which was confirmed by

Lashermes (1992) ^s well.

2.2.9 Light and pH

Light is a limiting factor for tissue culture. Low light intensity during callus

induction and plant regeneration increases regeneration frequencies but decreases

green plant production (Ekiz and Konzak, 1993). High light intensity during plant

regeneration had significant positive effects on green plant regeneration from callus

compared to lower light intensify. The pH of the media also has a critical bearing on

anther culture.

2.2.10 Green plant regeneration

An optimal level of green plant regeneration was observed when calli were

transferred after 10-20 days (about 2mm size) of their emergence from the anthers.

Chaleff and Stolarz (1982) claimed high rate of regeneration (~ 70 %) using a

modified MS medium (R3) supplemented with NAA (2 mg/I) and Kn (0.3 mg/1) for

callusing as well as regeneration. Similarly, Zapata et al, (1983), using B 5 (Gambor -

et al, 1968) medium supplemented with NAA and Kn (1 mg/1 each), achieved plant

regeneration efficiency 40 %. The composition of both culture and regeneration media

is also critical in terms of green plant regeneration, especially considering the carbon

source (Scott and Lyne, 1994; Caredda et al, 1999; Naegeli et al, 1999;

Wojnarowiez et al, 2004). A number of studies have revealed that the composition

and physiochemical conditions of culture medium retain and revive the embryogenic

capability of rice callus and enhance green plant regeneration (Karim and Zapata,

1990; Shahjahan et al, 1992; Karim and Zapata, 1996; Mohiuddin et al, 2006; Geng

et al, 2008). Zhou (1996) and Rov and Mandal QOOST reported differences in the

regeneration ability through anther culture between genotypes and between sub

species. These' differences were due to gene segregation during meiosis in the

formation of microspores of Fi (Dewi et al, 2009).

2.2.11 Albino plant regeneration

Albinism in plants is characterized by lack of chlorophyll in normally green

tissues. Generation of albino plants is one of the major problems in anther culture of

rice (Chen et al, 1991) as well as other cereals. A serious drawback of androgenesis,

either in anther or microspore culture is the incidence of albinism breeding programs

where most or all regenerated plants have been found to be useless albinos (Machii et
Q ~----------------------

al, 1998; Holme et al, 1999; JAiti et al, 1999; Liu et al, 2002). Wang et al, (1977)

reported that sucrose concentration of above 6% in the induction medium increased

proportion of albino plants. In general, albino plants (e.g. wheat, barley and rice)

contain deleted forms of the plastid genome and they are devoid of 23S anid 16S

rRNA. Indica rice cultivars are more prone to this problem than japonica rice. Several

factors, including pretreatment, culture medium and the stage of the pollen affect the

frequency of albinos and the frequency varies from 5% to 100%. Albino plant

generation is specific to anther culture because only a few albino plants are generated

from somatic cells (Tsukahara et al, 1996).

Hie frequency of generation of albino plants during anther cultures in rice is

controlled by nuclear gene(s) (Quimio and Zapata, 1990: Sugimoto and Takeoka,

1998), and QILs controlling the albino generation have been identified: one QIL on

chromosome 10, which controls the frequency of albino plants among regenerated

plants (Yamagishi et al, 1998); and one QIL on chromosome 9, which affects albino

plant regeneration (He et al, 1998). Indica rice subspecies and japonica-indica

hybrids of rice often show distinct response to anther culture as compared with

japonica subspecies (Chen et al, 1991; Guiderdoni et al, 1992). Reports show that

the frequency of albino regeneration ranges from 10% (Genovesi and Magill 1979) to

100% (Tsay et al, 1982) in rice. Regeneration of albino plants from in-vitro anther or

callus culture from various species, including rice, has become a significant problem

^ (Babbar et al, 2000). Sun et al, (1979) reported that the basic cause of albinism in

rice is impairment of DNA in plastids or nuclei or in both of them. This study also

identified the absence of ribosome in ill-developed plastids as another cause of

albinism in rice. Albino seedlings do not survive for long and die soon after the food

reserves stored in the endosperm/cotyledons are exhausted (Mohanty et al, 2005). *\)

Some workers reported that 5-100% albinos found in their study for rice anther

culture (Miah et al, 1985; Talebi et al, 2007).

2.3 Ploidy analysis and genetic stability of anther derived lines

Rice is considered as an ancient aneuploid (Vandepoele et al, 2003), and

polyploidy resulted in tremendous evolutionary dominance both as far as yield and

stress resistance. Tetraploid rice was first discovered in 1933 (Nakamori, 1933) and

research on polyploid rice breeding was initiated in 1953 in China (Bao and Yan,

1956). Certain gene materials, such as dwarf rice resource and sterile line played

important roles in diploid hybrid rice breeding (Huang and Ying 1994). Regenerants

from rice anther calli consist of haploids, diploids, polyploids and mixoploids with

most of the plants (> 50%) being diploids (Iyer and Raina, 1972; Oono, 1975; Yin et

al.,., 1976; Chen and Li., 1978; Huang et al, 1978; Shen et al, 1983; Zapata, 1985;

Loo and Xu, 1986). Of the remaining, the majority were haploids. Huang et al,

(1978) examined a total of 2496 rice anther-derived plants. The frequencies of

haploids, diploids, polyploids, and mixoploids were found to be 35.3, 53.4, 5.2 and

6.0% respectively. Oono (1975) obtained 39.3% of haploids, 48.5% of diploids, 7.6%

of triploids and 1.5% tetraploids from rice anther culture. Generally, similar results

had been reported by most workers (Raina, 1989). Recently, Nakamura et al, (1994)

analyzed 386 plants from anther cultures of the japonica cv. Nipponbare and observed

a similar trend with haploids, diploids and triploids were obtained in 50.3%, 47.4%

and 2.3% proportion respectively.

Genetic investigations of the diploids had shown that over 90% of them were

stable for all major economic traits (Chen and Li, 1978; Sun et al, 1978; Tang, 1978;

Chu, 1982; Rush and Shao, 1982; Zhang, 1982). Tang (1978) studied 429 diploid

anther-derived plants. He found that 91.8% were uniform, 7.8% had slight variation,

and 0.7% exhibited significant variations. According to Loo and Xu (1986), among

more than 2000 pollen plants, only about 3% were found to be unstable. Zhang

(1982), Shen et al, (1983, 1984) and several others studied the stability of anther-

derived diploid plants over several generations. These investigations revealed that in

the majority of cases, the coefficient of variance did not increase with increasing

numbers of generations. Obviously, the diploids arose as a result of spontaneous

chromosome doubling of the haploid complement.

Xu et al, (1997) generated stable breeding lines from anther culture derived

variants of indica rice varieties Wagwag and IR64. Differences in some agronomic

traits were established for some variants. Other variants, however, were

morphologically and agronomicdly similar. Greater frequencies of distorted loci had

been found in anther-derived populations of rice (Xu et al, 1997). Afza et al, (2001)

worked on Random amplified polymorphic DNA analysis of the anther culture

derivatives in rice to determine the occurrence and extent of variation in rice (Oryza

sativa L.) Among 27 plants from nine anther culture-derived lines, variation was

detected in three plants from two lines by two primers. Apart from these, the

amplification products were monomorphic across all the regenerants from anther

culture-derived plants. Out of 40 tested primers, no difference in the banding pattern

was observed in three seed-derived plants. In rice, Guiderdoni (1991) monitored

segregation and recombination of heterozygous isozyme markers in anther-culture


derivatives and F2 plants, generated from japonica x japonica, japonica x indica Fi

hybrids, though some segregation distortions were found among AC-lines indicating

existence of in-vitro selection, but on an overall basis, there was neutrality of gametic

selection. In another study of rice, AC lines were compared with inbred lines

generated through SSD (Courto:s, 1993). Distribution parameters of 12 qualitative

traits were evaluated and in most cases, the means and variances were found to be

similar for SSD as well as the AC lines.

2.4 Cytogenetic characterization of anther derived plants

Cytogenetic characterization reveals that the anther derived plants are of

different ploidy levels. The plants derived from rice anther culture are not only

haploid but also diploid, and some plants become polyploids or aneuploids (Oono

1975, Chen et al., 1985). This ciploid is the doubled-haploid and the first doubled-

haploid rice lines were obtained by Nizzeki and Oono in 1968. Counting of

chromosome numbers directly by cytological observations is the most accurate way of

identification of ploidy level. Improved staining procedures have been used to identify

rice pollen nuclei (Gupta and Borthakur, 1987). The ploidy status of regenerated

plants was confirmed by counting chromosome number in root tip and in pollen

mother cells following usual acetocarmine staining technique (Khan, 1975). But this

method needs skilful cytological micro techniques and entails laborious work.

Therefore simple and indirect methods have been developed. Conversely, the nuclear

DNA.content determined being directly correlated with the jDbidy level (De Laat et

al, 1987; Dolez'el et al., 2007a: Galbraith 1984; Sharma et al., ^S^Sree Ramulu

and Dijkhuis 1986) and flow cytometry has arisen as a far more reliable methodology

for the determination of ploidy level. Various morphological attributes were also used

to verify the ploidy status as suggested by Sah and Niroula (2007).

Ploidy level in plants is often estimated by measuring the C-value (amount of

DNA in the unreplicated gametic nucleus) using flow cytometry (Clarindo et al.,

2008; Dart et al., 2004; Eaton et al., 2004; Grundt et al., 2005; Harbaugh, 2008).

Flow cytometry has been used to determine nuclear DNA content of plants (Galbraith

et at, 1983) and has become increasingly attractive compared to microscopic

techniques because sample preparation for flow cytometry is simple, any plant tissue

with nuclei can be studied, a large number of nuclei can be analyzed quickly, and

large quantities of data can be conveniently collected for statistical analysis

(Arumuganathan and Earle, 1991a, b).

In flow cytometry, a parameter is a measured property of the particles,

frequently assimilated to an optical chanmel, whereby a one-parameter histogram will

plot the subpopulation of cells that contain a given quantity of DNA or bind a given

number of antibody/fluorophore molecules (Robinson, 2006). Thus, such cell content

is assigned to one of several classes or channels represented on the x-axis, while the

number of cells assigned to each channel is named channel content, or simply count,

and is shown on the y-axis. So, all cells with about equal quantities of a given cell

content, say DNA, will form a peak. Therefore, for a typical DNA histogram of

euploid material, two peaks will be prodiced, one more prominent peak representing

the G1 phase (unreplicated, 2C) and the other (which will be formed at twice the

channel value) representing the G2/M phase (replicated, 4C) of the cell cycle (Ochatt

2008; Greilhuber and Dolezvel, 2009). The most frequently used staining methods for

chromosome counting are aceto-orcein cr aceto-carmine (Smith 1947), feulgen and

DAPI (Kapuscinski, 1995) staining, which stain only the chromosomes while the

cytoplasm remains clear. DAPI (4,6-diamidino-2-phenylindole) forms a fluorescent

complex by attaching in the minor grove of A-T rich sequences of DNA

(Kapuscinski, 1995; Ochatt, 2008) and, upon excitement under UV light, the AT-rich

sequences of chromosomes fluoresce bright blue. This method is thus much simpler

and faster than the feulgen technique but it requires a fluorescent microscope. DAPI

(AT-specific) is the most frequently used dye to determine ploidy level by flow

cytometry, while propidium iodide (PI), usually believed to be non-base pair specific

but which nevertheless exhibits a GC-preference (Vinogradov 1994), is preferred for

the determination of genome size. The distribution of nuclear DNA content within a

cell population is obtained by comparing the numbers of objects in the different peaks

or clusters, since the absolute DNA content is irrelevant. However, it is important to

include an internal standard, as a control, to analyze the ploidy level of Gl-cells by

comparing the positions of the major peak in profiles from different genotypes.

Pollen grain size was positively correlated with ploidy level. Higher ploidy

levels were associated with larger pollen grains, while the number of pollen grains
was not different between ploidy levels (Fukuhara. 200.01 A positive correlation

between pollen grain size and ploidy level was also reported in Arachis (Singsit and

Ozias-Akins, 1992) and pollen grain size (Singsit and 0zias-Akins,1992; Zonneveld

and van Iren 2001) have been utilized as an alternative convenient, rapid and reliable

method to identify the ploidy level of many plant species. Since then, a large number

of articles on this use of flow cytometry appeared, and the most frequently reported

use of flow cytometry in the literature has been to analyze the ploidy level of

individuals obtained following experiments of haplo-diploidization or chromosome

doubling (Dolezel et al, 2007b: Grewal et al, 2009; Ochatt, 2006, 2008; Ochatt et

al, 2005, 2009).

2.5 Application of doubled kaploids in hybrid rice breeding and genetics

Doubled haploidy (DH) has many applications in plant research and is

particularly attractive to plant breeders because it provides a rapid way to produce

large numbers of homozygous plants at any stage in a breeding programme,

significantly reducing the time to release new cultivars. The objective of using

doubled-haploid lines in cereals is to obtain complete homogeneity and complete

homozygosity within lines (Chu et al, 1985). Therefore, DH lines have the potential

for use in plant breeding, mutagenesis, in-vitro selection, molecular mapping and

plant transformation (Kasha et al., 1995). To date, pure line production in rice has

resulted in more than 20 rice varieties obtained through DH production worldwide

(Zepata-Arias. 2003). In Korea, haploid breeding has been elevated to a national rice

breeding program in 1977. Four rice varieties derived from anther culture have been

released for seven years (Chung, 1992). In China, a dozen important varieties and

more than 100 improved lines have been developed through 'anther culture breeding'

(Loo and Xu, 1990).

Anther culture is one such useful technique used to produce lines with

desirable combinations of required traits. It has been well integrated into rice breeding

programs especially in Japan and China, where a number of high yielding, disease

resistant and better quality rice cultivars have been selected from microspore derived

plants (Loo and Xu, 1991). Production of doubled haploid (DH) plants through

androgenesis induction is a promising and convenient alternative to conventional

selfing techniques for the generation of pure lines in breeding programs. Doubled-

haploid systems have been used in plant breeding programs (Khush and Virmani,

1996) or for genetic manipulation, as part of genetic transformation or quantitative

trait locus (QTL) analysis (Hyne et al, 1994; Tixier et al, 1998). Anther culture has

proved to be an efficient and useful technique for plant breeding and biotechnological

programme, providing a rapid production of doubled haploid plants (Suriyan, 2009).

Doubled haploid lines are used in practical breeding programmes. Haploids

are advantageous in transferring of quantitatively inherited traits from distant

genotypes to cultivars. DH production over inbred lines produced by successive self

pollination is that there is only one round of recombination, so that advantageous gene

combination found in the parent lines are more likely to be preserved. Doubled

haploids have featured strongly in the genetic analysis and breeding of crop plants

(Forster and Thomas, 2005).

2.5.1 Mapping quantitative trait loci (QTLS)

QTLs affect some important agronomic traits in cultivated rice. DH lines are

also useful for genetic analysis, particularly for that of quantitative traits (Snape et al,

1984), and quantitative trait loci (QTL) in rice have been mapped using DH lines

(Huang et al, 1996; Lu et al., 1996). Double haploid (DH) lines are useful for genetic

analysis, particularly quantitative traits because these lines are homozygous and can

be propagated without further segregation. As the quantitative trait loci (QTL) effects

are small and highly influenced by die environmental factors, accurate phenotyping

with replicated trials is needed. This is possible by doubled haploidy because of their

true breeding nature and convenience of producing in large numbers. Using DH

populations, 130 quantitative traits have been mapped in nine crop species. In total, 56

DH populations were used for QTL detection. Using DH populations, 130

quantitative traits have been mapped in nine crop species (Forster and Thomas, 2005).

In total, 56 DH populations were used for QTL detection (Maluszynski et al., 2003).

Similarly doubled haploid plants are used for QTL mapping of various traits in

several plant species, e.g., rice (Li et al., 2005; Ma et al., 2009), wheat (Dashti et al.,

2007; Zhang et al., 2008\japovica rice (Zichao et al., 2005), and barley (Ma et al,


2.5.2 Backcross breeding

In backcross breeding, there is a problem in identify the lines carrying the trait

of interest at each generation. The problem is particularly acute if the trait of interest

is recessive, as it will be present only in a heterozygous condition after each

backcross. The combination of doubled haploidy and molecular marker becomes

effective in solving this problem. In the back cross generation one itself a genotype

with the character of interest can be selected and converted into homozygous doubled

haploid genotype. Chen et al., (1994) used marker assisted backcross conversion with

doubled haploidy of BCi individuals to select stripe rust resistant lines in barley.

2.5.3 Bulked segregant analysis (BSA)

In bulked segregant analysis, a population is screened for a character of

interest and the genotypes at the two extreme ends form two bulks. Then the two

bulks are then tested for the presence or absence of molecular markers. Bulks indicate

the linkage between the marker and character of interest. BSA is dependent on

accurate phenotyping and the DH population has particular advantage in that they are

true breeding and can be tested repeatedly. DH populations are commonly used in

bulked segregant analysis, which is a popular method in marker assisted breeding.

2.5.4 Genomics

Double-haploid lines are ideal for genetic mapping because of their

homozygosity, uniformity and each line can be replicated indefinitely over several

locations (Tinker et al., 1996). Genetic maps are very important to understand the

structure and organization of genomes from which evolution patterns and syntenic

relationships between species can be deduced. It is possible to produce a genetic map

within two years of the initial cross regardless of the species. Map construction is

relatively easy using a DH population derived from a hybrid of two homozygous

parents as the expected segregation ratio is simple, i.e. 1:1. DH populations have now

been used to produce genetic maps of barley, rapeseed, rice, wheat, and pepper. DH

populations played a major role :n facilitating the generation of the molecular marker

maps in eight crop species.

Although QTL analysis has generated a vast amount of information on gene

locations and the magnitude of effects on many traits, the identification of the genes

involved has remained elusive to poor resolution of QTL analysis To overcome this

problem, a stable and fixed population i.e. DH population is used and genetic markers

are used to detect desired recombinant chromosome substitution lines in the target

region. Anther culture as a method of creating haploid strains has become greatly

useful in genetic studies and practical breeding (Yi, 1991). DH populations played a

major role in facilitating the generation of the molecular marker maps in eight crop

species (Maluszynski et al, 2003). In rice, molecular markers have been found to be

linked with major genes and QTLs for resistance to rice blast, bacterial blight, and

sheath blight in a map produced from a DH population (Wang et al., 2001). Many

commercial cultivars were originated from DH lines. In barley alone, over 100 DH

lines have been released as cultivars (Thomas et al, 2003).

2.5.5 Production of new cultivars

Traditional breeding methods are slow and take 10-15 years for cultivar

development. Another disadvantage is inefficiency of selection in early generations

because of heterozygosity. These two disadvantages can be overcome by DHs, and

more elite crosses can be evaluated and selected within less time. Uniformity is a

general requirement of cultivated line in most species, which can be easily obtained

through DH production. There are various ways in which DHs can be used in cultivar

production. The DH lines themselves can be released as cultivars; they may be used

as parents in hybrid cultivar production or more indirectly in the creation of breeders

lines and in germplasm conservation. Barley has over 100 direct DH cultivars.

According to published information there are currently around 300 DH derived

cultivars in 12 species worldwide.-Doubled haploidy already plays an important role

in hybrid cultivar production of vegetables, and the potential for ornamental

production is being vigorously examined. Another interesting development is that

fertile homozygous DH lines can be produced in species that have self-incompatibility

systems. Nearly about one hundred rice commercial varieties/improved lines had been

developed through anther culture in China with examples like Hua YU No.l, Xin Xiu,

and Hua Han Zao, Zhonghua No.8 and No.9 and Zhonj a No. 10. In South Korea,

the first cultivar bred by anther culture (Hwaseongbyeo) was commercially released

in 1985. The first salt tolerant rice cultivar IR51500-AC11-1 as PSBRcSO Bicol, an

anther culture-derived line from an indica/indica cross was recommended it for

commercial cultivation in salt-affected rice lands in the Philippines (Senadhira et al,


Varieties developed through anther culture yielded as high as 10.3 t/ha under

moderate fertility. The magnitude of hybrid vigour that could be realized in doubled

haploid lines (homozygous) derived through anther culture of hybrid rice can be

retained more or less the same le^el of vigour as the hybrids (Bong and Swaminathan,

1995). Lines derived from pollens of hybrids can perform more ideal than the hybrid

itself. In general, in China, yields from some lines were proved to be higher than those

of local standard cultivars (Niizeki, 1997). In India, promising lines with earliness and

high yield potential were selected from a large number of plants derived from anther

culture of several crosses. Parag 401, a semidwarf rice variety developed through

anther culture, was released for cultivation on irrigated vertisols of Maharastra State

of India (Patil et al., 1997).

At present improved rice varieties and lines derived from anther culture are

widely grown in China, Taiwan, South Korea, Japan, USA and India and in several

other countries (Misso et al, 1991; Zhang, 1992; Mia et al., 1996; Niizeki, 1997;

Raina and Zapata, 1997; Gupta, 1999). DHs are being routinely used in breeding

programs for new cultivar development in many crops (Veilleux, 1994). In India,

promising lines with earliness and high yield potential were selected from a large

number of plants derived from anther culture of several crosses. Similarly, the utility

of doubled haploid approach in indica rices was demonstrated for the first time in

India, through development and release of two varieties, Satya Krishna (CR Dhan 10)

and Phalguni. Satya Krishna (CR Dhan-10) has a duration of 135 days with erect,

non lodging, semi dwarf (105cm) plant type and has a yield potential is 5.0 t/ha in

wet season and 6.0 t/ha in dry season and was recommeded for cultivation in irrigated

and rainfed shallow low land ecologies (CRRI Annual Report 2007-2008). Phalguni

has a duration of 115-120 days with erect, non lodging, semi dwarf plant type and was

recommended for cultivation in irrigated as well as bunded upland ecologies with

productivity levels ranging from 4.0 -4.5 t/ha in wet season and 5-6 t/ha in dry season

(CRRI Annual Report 2009-2010).

2.5.6 Improving grain quality and nutritional quality

Rice is an essential food in the diet of one third of the worlds population. As

grain quality is an important aspect in the grain cereals especially in rice, rice breeders

are focusing on the improvement of rice grain quality. The most important challenge

in rice quality breeding was the development of good quality inbred varieties or

hybrid combinations in indica rices (Hanwei and Cunshan, 2001). Grain shape and

appearance, milling and head rice recovery, amylose content and alkali value are

some of the parameters that influence the cooking and eating quality of rice. Some

lines derived from "Koshihikari" anther culture had shown superior visual grain

qualities and excellent eating quality (Watanabe et al, 1998). Milyang-90, an anther

culture derived line which was having good grain quality with overall good

performance was isolated from more than 2000 lines (Chung, 1987). Zhu et al.,

(1990) reported that pollen clones obtained through anther culture in indica hybrid

rice possess wide and varied genotypes, and also wide characteristics of quality, so

the breeding of Guan-18, which was having good grain quality characteristics, was a

great achievement of anther culture quality breeding. Recently Nguyen and Long

(2003) by using four aromatic varieties like DS 20, IR 68144, Kloong Luang 1,

Suphan Buri for anther culture isolated several promising lines which showed good

performance and good quality. The two varieties developed at CRRI were also had

good grain quality. Both Satya Krishna and Phalguni possess excellent grain quality

traits like HRR, long slender grains, translucent kernels with high L/B ratio, high

KLAC, high elongation ratio after cooking and intermediate amylose content.

2.6 Use of anther culture in purification of parental lines for hybrid rice

Since the first complete set of three lines of hybrid rice was developed in early

1970s, they were used in production for more than 20 years. Because of gene drift,

mutation, artificial and biological confounding, and exposure to unfavorable natural

stresses, the purity of parental lines used in developing hybrid rice had strongly

decreased. Such a decrease during the long period of propagation and production

results in reduced yield potential and grain quality. It was therefore necessary to take

strict and effective measures to purify these parental lines. The conventional

technique of purification was elaborate and selection was made on phenotype which

cannot guarantee that selected materials would be genetically homozygous and stable.

Because anther culture could make genes highly homozygous, it was a more effective

method for purification (Zhu et al., 1998). In hybrid rice breeding, elite lines and

released varieties from varietal improvement programs were used as parental lines.

Besides narrow genetic diversity, the frequency of restorers and maintained among

these lines was 10-15 % only. The remaining genotypes (70-80%) cannot be directly

used in developing hybrids. Within the identified restorers and maintained, the

frequency of good combiners possessing desirable traits was still lower (Mishra et al,

2003). Therefore, improving parental lines must be an integral part of hybrid rice

breeding to develop heterotic hybrids and improve breeding efficiency. Anther culture

technique can be deployed to expedite the parental line improvement program (Liu et

al., 1998).

Xu et al., (1983) successfully purified CMS line V20A using anther culture by

generating different types with varying stages of mononucleate abortion. Investigating

Zhen Shan 97A purified via anther culture showed that the typical abortion rate and

sterility were 11.1% and 14%, respectively, higher than those of the donor. The

hybrid rice strain Shan You 6 developed from these purified lines yielded 19.1%

higher than the control (Ge et al, 1985). Bai et al, (1991) reported that hybrids

derived from restorer line anther culture purified Minghui 63 was significantly

improved in purity, seed-setting rate, yield potential, and resistance. Ge et al, (1989)

purified Zhen Shan 97 A, Zhen Shan 97 B, and Minghui 63 (a restorer line) via anther

culture. They reported that the purity of the hybrid increased when the parental lines

were purified via anther culture. Chen et al, (1994) obtained some normal sterile or

fertile pollen diploids through anther culture of the male sterile lines like II-32A, You

IA, and Xie Qing Zhao A and their respective maintainer lines and these purified

CMS lines were screened for use through test crosses, sterility tests, and heterosis

trials. At IRRI, 95 anther culture lines were regenerated from CMS line IR54752A

and from these, 10 lines were identified possessing 100% male sterility.

Zhu et al, (1993a) developed CMS restorer line 2374 with strong combining

ability by improving a pollen strain derived from Shan You 2 by the conventional

method. Zhu et al, (1992) bred a CMS restorer line (1044) via anther culture of

(Ce64 X 1050) Fj. Wang et al, (1994) adopted anther culture procedure to create new

restorer lines for CMS. Young panicles of Fi (Minghui63/Zi Gui) underwent anther

culture and 19 clusters of green plantlets were regenerated. From the anther culture

lines, restorer line Chuan Hui 802 having strong restorability was selected. Breeding

widely compatible restorers (WCRs) for indica CMS was attempted to utilize inter

subspecific heterosis. Some WCRs resulting in highly heterotic hybrids were

developed via the conventional technique, but this method requires a long process to

stabilize and subsequently test the WC and CMS restoring ability. By using anther

culture method, WCR lines we could be developed expeditiously (Zhu et al.; 1998).

Yin et al., (1994) obtained 20 WCRs and 3 intersubspecific hybrids with strong

heterosis from pollen-derived progenies of WCV 02428 and CPSLO 17 crossed with

CMS restorer lines.