Professional Documents
Culture Documents
REVIEW OF LITERATURE
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
be employed for yield enhancement. Conventional breeding methods take many years
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
haploids were first produced through anther culture by Niizeki and Oono (1968)
closely followed by Nishi and Mitsuoka (1969) in Japan. Doubled haploid breeding
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
Anther culture is one of the best breeding methods with numerous advantages:
13
selection efficiency, widening of genetic variability through the production of
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
breeding.
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
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
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
14
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
%
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
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
breeding since it not only offers the quickest method of advancing heterozygous
breeding lines to homozygosity, but also increases the selection efficiency over
15
conventional procedures due to better discrimination between genotypes within any
one generation. (Martinez et al, 1996; Yang, 1997; Chen et al, 1997). Snape (1989)
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 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
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
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
alone while with anther culture, only 60-70 plants are required (Khush and Senadhira,
<r
between individuals within the plots, unlike doubled haploid plants where all
16
individuals are genetically identical. This makes visual selection of desirable lines
probability of these recombinants with recessive characteristics was two times higher
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
Anther culture was successfully used to alleviate the problems of high sterility
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
17
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
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,
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
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
treatment.
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),
developmental stages of the microspore (Afza et al., 2000, Cha-um et al,2009), pre
18
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
techniques (Abe and Futsuhara 1986; Hartke and Lorz 1989), and in consequence the
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
19
(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
particular, plant hormones. Similarly, Lentini et al, (1995) reported that only one out
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
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).
Varying temperature and light conditions during the growth of donor plants
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
increased anther response (Wang et al, 1974). Hu et al, (1978) reported the influence
20
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
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,
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
V
1987). Donor plants that came to flower during continuously cloudy/ rainy weather or
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
Raina and Zapata (1997) observed that the plants grown in the field were
21
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,
2003).
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
(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
22
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
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
2.2.4 Pretreatments
shock and sugar starvation, during the labile developmental period of pollen grains is
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
2.2.4.1 Cold
Low temperature shock has been reported to enhance the androgenic response
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
23
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
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.
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
varieties.
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
k
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;
24
and the release of substances necessary for androgenesis, mainly amino acids and
2.2.4.2 Heat
in many plant species. Rice exposed to high temperatures at meiosis easily forms
(Ouyang et al, 1983). High temperature pretreatment disrupts the normal integrated
physiological states of the two tissues, thereby stimulating the induction process
(Dunwell et al, 1983). Rice exposed to high temperatures at meiosis easily forms
dependent (Ouyang et al, 1983). Heat pretreatment has been used to induce
the initial phase (Ferrie et al, 1995). Heat stress pretreatment has been an essential
2003).
in androgenesis. Lai and Liu (1988) reported that the inclusion of mannitol, or the use
regeneration frequency in the jcponica rice variety TN5. The beneficial effect of
relatively high and constant osmolality of maltose medium (Zhou et al, 1991).
25
Osmotic stress or reduction in cell water content influence the process of somatic
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
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
microspore culture found that yield of green plants increased from 50% to 97%. In the
cultures of cv. IR43 from 3% to 33.4% while with cold treatment no promotory effect
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,
26
by 1978, several media formulations were reported including N6 like Heh-2, Heh-5,
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
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.
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
27
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
sucrose for androgenesis in several species, including cereals (Last and Brettel 1990,
Pande and Bhojwani 1999). Maltose increased somatic embryo formation and the
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
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
28
methods of culture techniques (Trejo-Tapia et al., 2002), formulation of new media
chemicals for callus induction (Datta et al., 2005). Research efforts on the
al., 1999).
plant tissues. With AgNC>3, an anti-ethylene compound, the frequency of green plant
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
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
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
29
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-
al, 1993). The rate of success can be enhanced by improving the composition of
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
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
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
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
30
whereas NAA gave rise to the formation of roots and sometimes to complete plants
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
buoyancy agent to prevent embryo ids from sinking in anther culture (Kao, 1981).
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
Light is a limiting factor for tissue culture. Low light intensity during callus
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.
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.
modified MS medium (R3) supplemented with NAA (2 mg/I) and Kn (0.3 mg/1) for
31
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
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
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,
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)
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
32
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
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
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
33
2.3 Ploidy analysis and genetic stability of anther derived lines
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,
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%
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
34
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
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
been found in anther-derived populations of rice (Xu et al, 1997). Afza et al, (2001)
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
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
35
traits were evaluated and in most cases, the means and variances were found to be
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
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
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
36
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
plot the subpopulation of cells that contain a given quantity of DNA or bind a given
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
DAPI (Kapuscinski, 1995) staining, which stain only the chromosomes while the
(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
37
(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
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
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
9
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
doubling (Dolezel et al, 2007b: Grewal et al, 2009; Ochatt, 2006, 2008; Ochatt et
38
significantly reducing the time to release new cultivars. The objective of using
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
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'
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
selfing techniques for the generation of pure lines in breeding programs. Doubled-
haploid systems have been used in plant breeding programs (Khush and Virmani,
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
39
Doubled haploid lines are used in practical breeding programmes. Haploids
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
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
populations, 130 quantitative traits have been mapped in nine crop species. In total, 56
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.,
40
2007; Zhang et al., 2008\japovica rice (Zichao et al., 2005), and barley (Ma et al,
2004).
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
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.
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
2.5.4 Genomics
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
41
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
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
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
Traditional breeding methods are slow and take 10-15 years for cultivar
42
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
lines and in germplasm conservation. Barley has over 100 direct DH cultivars.
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
2002).
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
43
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
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
productivity levels ranging from 4.0 -4.5 t/ha in wet season and 5-6 t/ha in dry season
44
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.
45
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,
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
Zhen Shan 97A purified via anther culture showed that the typical abortion rate and
46
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
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
47
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
48