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International Journal of Agriculture and Crop Sciences.

Available online at www.ijagcs.com


IJACS/2015/8-1/15-26.
ISSN 2227-670X ©2015 IJACS Journal

Auxin to improve green plant regeneration of rice


anther culture
Lovelin Kaushal1*, SM Balachandran2, K Ulaganathan3, Akhilesh Kumar Singh1, Rahul
Priyadarshi1 , Vinay Shenoy1
1 . Barwale Foundation, Himayathnagar, Hyderabad, Andhra Pradesh, India.
2. Directorate of Rice Research (DRR), Hyderabad, India.
3. Centre for Plant Molecular Biology (CPMB), Osmania University, Hyderabad.

Corresponding authors email: lovelinkaushal@barwalefoundation.org

ABSTRACT: Anther culture is a useful technique to quick fix lines with desirable combinations of the
required traits. The effect of auxin source (Dichlorophenoxyacetic acid : 4-amino-3,5,6-
trichloropicolinic acid) to overcome the lower regeneration ability was investigated on 3 different
media. A significant and negative heterosis for the in vitro characters in terms of mid parental values
was observed in the F1 hybrid (IR58025eB x Dular). Auxins were essential for the induction of callus,
and type and concentration of auxins also influenced this process. Substitution of 2,4-D by Picloram
in the callus induction media had positive influence on improved regeneration capability. Increased
auxin concentration could be beneficial for callus induction, but detrimental for green plant
regeneration. There appeared to be a relationship between increased albino development with
increase in level of auxins. G x M x A x C synergistically contribute to enhanced callus induction and
regeneration of androgenic plants, especially for F1. Recurrent parent IR58025eB proved to be the
best genotype and He2 medium with Picloram (1 mg/L) concentration desirous for overall efficiency
of green plant regeneration from induced callus.

INTRODUCTION

Rice is one of the world’s most important cereal crop represents the staple food for more than half of
the global populations. To meet dramatic increase in cereal demand worldwide, considerable efforts are being
directed towards improvement of existing cultivars for combined tolerance to biotic and abiotic stress which
would substantially increase rice productivity. One way this is can be achieved by gene stacking and
pyramiding. The employment of doubled haploidy and Marker Assisted selection (MAS) can enhance the
stacking process. The production of doubled haploids via anther culture represents an alternate to traditional
crop improvement programs. Doubled haploid technology platform offers a rapid mode of truly homozygous line
production that helps to expedite crop breeding programs where homogeneity is an absolutely essential
parameter for rapid crop development (Basu et al., 2011). Anther culture is a proven useful technology to
expedite the breeding and release of new rice cultivars (Khush and Virmani, 1996). In vitro culture of anthers
from a heterozygous F1 hybrid fixes desirable gene combinations in only one generation by spontaneous or
chemically induced production of homozygous fertile plants, which are referred to as doubled haploids (DH).
Thus it has become handy to improve parental lines or evolve varieties in the shortest period with less effort.
Using conventional breeding methods, the production of homozygous lines is a long procedure of 5-6
generations (Darvey and Zhao, 2007). In contrast, the production of DH lines from heterozygous parent in a
single generation and leads rapidly to homozygous lines (Snape, 1989). Anther culture has been well
integrated into rice breeding program, especially in China and Korea, and a number of high yielding, disease
resistant and better quality rice varieties have been selected from anther culture derived plants (Hu, 1985;
Chung, 1992). However, the genotype dependency, low callusing and regeneration potential, occurrence of
albino or haploid plants etc. are important bottlenecks to the application of this non conventional breeding
method (Gioi and Tuan, 2002).
In rice, the inheritance of callus induction and plant regeneration is transmitted by blocks of genes with
predominantly additive effects, with the occurrence of partial dominance and with no indication of inter-allelic
interaction (Miahet al., 1985; Quimio and Zapata, 1990). Callus induction and plant regeneration are influenced
by many factors like genotype, pretreament, culture media composition etc. Genotype is the main contributor to
the in vitro response of cereal tissue culture (Hartmann et al., 1989). A relationship between genotypes and in
vitro response has been reported in several cereals, such as maize (Green and Philips, 1975), oats (Cummings
Intl J Agri Crop Sci. Vol., 8 (1), 15-26, 2015

et al., 1976), barley (Dale and Deambrogio, 1979), wheat (Sears and Deckard, 1982) and rice (Chung, 1982).
Though genotype is the deciding factor for anther response, modification of nutrient composition and
manipulating plant growth regulators can enhance the anther response (Mandal and Gupta, 1995). Nutrient
composition is regarded as the major sources of variation in in vitro culture. The indica cultivars require even
+ - +
lower level of (NH4) ions. The ratio of NO3 : NH4 has been observed to be an important determinant for
success of another culture in indica rice (Grim and Hodges, 1990). Organic nitrogen supplement such as
casein hydrolysate (CH) which is a sources of calcium, several micronutrient, vitamins and amino acids added
to the medium has been particularly beneficial for positive anther culture response (Roy and Mandal, 2005),
although a few reports suggest otherwise (Raina and Zapata, 1997; Lentini et al., 1995). Silva (2009) reported
that low responsiveness of indica genotype is due to early senescence of cultured anthers and the senescence
is likely to be accelerated with in vitro generation and accumulation of the ethylene in sealed culture vessels.
Thus the use of AgNO3 as inhibitor of ethylene biosynthesis is likely to be advantageous in rice anther culture.
The effects of plant growth regulators have been widely investigated in another culture. It is regarded
as important parameter in determining the success of induction of calli and plant regeneration. The
combinations of hormone type and concentration of hormones can greatly affect the development of
microspores and impact the morphogenetic proceed leading to the production of the plants ( Trejo-Tapia et al.,
2002). Ball (1993) stated that the type and concentration of growth regulators as well as their interactive
presence can be the deciding factors that would influence pollen embryogenesis. Numerous detailed and
extensive studies were undertaken to analyze a broad spectrum of type of auxins and cytokinin for culture
initiation. Both auxin and cytokinin are crucial constituents in rice anther culture medium, control the
dedifferentiation and dedifferentiation processes in the in vitro cultures. 2,4-D is the most widely used growth
regulator irrespective of the explants in all cereal crop species. Previous studies have proposed that 2,4-D as
only hormone for in vitro development of rice microspores and hence it was always included in the culture
medium either singly or in combination with a cytokinin and/or with other auxins (Niizeki and Oono, 1968; Guha
et al., 1970; Iyre and Raina, 1972; Wang et al., 1974). Among the synthetic auxins sources 2,4-
Dichlorophenoxyacetic acid (2,4-D) and Napthalin acetic acid (NAA) were commonly used for callus induction
from rice anthers as auxins are the most essential growth regulators required for induction of callus from
anthers of cereals (Zhu et al., 1998). Nevertheless, prolonged exposures of cell cultures to high concentration
of 2,4-D affects the frequency of regenerated plants and causes chromosomal abnormalities (Deambrogio and
Dale, 1980; Ziauddin and Kasha, 1990). Previous investigation has revealed the suitability of 2,4-D for callus
induction (Zhu et al., 1998) and for green plant regeneration (Bregitzer et al., 1995). Much work had been done
in order to replace the 2,4-D with other synthetic auxin specially, in cereals. 4-amino-3, 5, 6-trichloropiclorinic
acid (Picloram) is an alternate synthetic auxin used in anther culture of many cereal crops. A comparative
investigation of 2,4-D and Picloram on anther culture response revealed that a similar level of embryogenesis
induction observed from inflorescence of wheat, barley and tritordeum (Hordeum chilense x Triticum turgidum
conv durum). Surprisingly, regeneration from cultures derived from Picloram containing medium was almost
twice as efficient as regeneration from cultures induced on 2,4-D (Barro et al., 1999). Mondoza and Kaeppler
(2002) and Sharma et al. (2005c) reported that a significant increase in the frequency of plant regeneration was
caused by Picloram was confirmed for embryo derived culture. With ability to increase the green plant
regeneration capabilities in rice, the doubled haploidy technology may play an ever increasing role in practical
breeding. The specific objective of this study was to evaluate the effect of auxin source [Dichlorophenoxyacetic
acid (2,4-D): 4-amino-3,5,6-trichloropicolinicacid (Picloram) to triumph over the lower regeneration efficiency in
the experimental genotypes.

MATERIAL AND METHODS

MATERIAL

The material consist of Dular (donor for wide compatibility trait), IR58025eB (recurrent parent with eui
gene) and their F1 and BC1F1 of rice (Oryza sative L.). The mother plants were grown at Barwale Foundation
farm providing all the standard agronomic practices (Table 1).

METHODS

Three basal media viz. B5 (Gamborg et al., 1968), He2 (Huang et al., 1978) and SK1 (Raina and
Zapata, 1997) with certain modifications based on available literature were used for evaluation of anther culture
response (Kaushal et al., 2014b). The major modifications made are shown in the Table 2. Every medium was
supplemented with different level (1 mg/L or 2 mg/L)of hormones (2,4-D/ Picloram) along with other hormones
(NAA and Kinetin), natural additives (CH) and AgNO3 as ethylene inhibiting agent. MS (Murashige and Skoog,
1962) medium supplemented with 1.5 mg/1 BAP, 1 mg/l Kinetin, 1 mg/l NAA was used for regeneration. The

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pH was adjusted to 5.8 with 1N NaOH before the addition of Gel Right, after which the media was autoclaved at
0
121 C for 15 minutes.

Anther culture
Panicles were sampled at appropriate stage from the primary tillers of the each genotype in the
morning (9 am to 10 am). Boots were sampled when the distance between flag leaf and penultimate leaf was 5-
7 cm (Croughan, 1998). Boots were wrapped in muslin cloth, sealed in the polythene bags and refrigerated at
12° C for 5 days (Kaushal et al., 2014a). Subsequently, panicles were surface sterilized in 0.1% mercuric
chloride and rinsed thoroughly with sterilized distilled water under aseptic condition. Selection of the spikelet
was based on cytological observations and position of the anthers in the spikelet. Anthers occupying 1/3 to 1/2
of the spikelet length was suitable for anther culture (Lapitan and Villegas, 1999). Individual spikelet was cut at
the base while holding from the tip, to detach the anther lobes from the filaments. The released anthers were
dropped onto Gel Right solidified media contained in petri dishes. Anthers were made to spread evenly on the
surface by rotating the petri dish during anther plating. Approximately 100 anthers were inoculated onto each
medium for callus induction. The cultures were incubated in complete darkness at 25±10C for 4-5 weeks for
callus induction. The cultured plates were examined periodically at weekly intervals to observe the progress in
respect of callus formation. Data on percentage of callus induction was recorded. Calli of at least 1- 2 mm
diameter were transferred from callus induction medium to regeneration media and kept under 16 h
photoperiod (2000 lux) at the same temperature. The cultures were examined weekly and data on percentage
of calli regenerating green and/or albino plants was recorded. In regeneration medium some microspore calli
lost their ability to produce plants and turned brown while others differentiated into green or albino plants. The
unrooted green shoots were transferred into rooting medium (half strength MS medium without hormone).
Finally green plantlets with adequate root formation were transferred to green house for acclimatization under
standard agronomic practices.
Data related to callus induction was recorded out during 60-80 days after plating. The frequencies of
callus induction and regeneration were estimated as follows: callus induction frequency (%) = number of
anthers producing calli/number of anthers plated x 100; green or albino plant frequency (%) = number of green
or albino regenerating calli/ number of calli transferred x 100. Statistical analyses were carried out using
CROPSTAT program version 7.2.2007.3 (IRRI, 2008). Analysis of variance (ANOVA) was done in order to
study the effects of genotype, medium, hormone type, concentration and their interaction. Heterosis (mid
parent) percentage was calculated by using formula (F1-MP/MP) x 100 (Matzinger et al., 1962).

RESULTS

Callus induction commenced three weeks from the culture. It was noticed that calli produced in the
experiment were creamy white in colour and compact, except for F1 with friable texture (Figure 1a). Calli of 1-2
mm in size were transferred to MS regeneration medium. Within 5-15 days, green spots were observed
developing on the callus surface (Figure 1b) and subsequently into green shoots (Figure 1c) or albino plants
(Figure 1d). The regenerated plantlets were found to be rootless indicating their androgenic development.
When such shootlets were transferred on to MS liquid media for root development (Figure 1e) they put forth
prolific roots in a short time. Well developed plants with profuse roots were acclimatized in green house under
controlled temperature and high humidity (Figure 1f).
Analysis of the variance (ANOVA) suggested that it is neither genotype, medium, auxin type or its
concentration, but their interaction had significant effects on callus induction and regeneration (p<0.01). Calli
was induced in all the genotypes in all treatments. Among the genotype evaluated, highest callus induction was
observed in IR58025eB (32.26-40.45%), followed by BC1F1 (24.72-36.79%), F1 (21.29-33.00%) and least in
Dular (17.03-28.11%). Thus, genotypes followed a pattern of callus induction (IR58025eB> BC1F1> F1> Dular).
Similarly, significant interaction effect was noted for regeneration as well. Even similar to callus induction
greater genotypic differences were found in the efficiency of generation of green plants. Green spots formation
followed a similar pattern of development (IR58025eB> BC1F1> F1> Dular). Among the tested genotypes,
IR58025eB was highly responsive to green spots formation (18.18-43.64%), followed by BC1F1 (4.27-21.21%),
Dular (3.88-21.06%) and F1 (2.88-15.38%). The data reflect that the recurrent parent IR58025eB showed best
androgenic response in terms of both callus induction and green plant formation (Figure 2).
Our results provided a clear picture about the effect of different medium composition on anther
response. Of the three media evaluated, the SK1 medium produced least frequency of callus ranging from
17.3-36.26%, followed by B5 medium (20.14-40.18%). However, He2 medium promoted highest callus
induction percentage varying from 23.05-40.46%. Both callus induction and green plant regeneration followed
same pattern of development (SK1< B5< He2). SK1 medium produced less frequency of callus regeneration
(3.13-35.09%) followed by B5 medium (2.88 to 39.35%). However, He2 medium promoted highest levels of
green plant regeneration ranging from 4.79% to 43.64%. A highly significant effect of auxin type on both callus

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induction and green plant regeneration was detected, though interaction was also observed. Among the two
auxin evaluated, with 2,4-D the callus induction frequency was relatively higher (19.33-40.46%), compared to
17.03-40.16% for Picloram. This indicates that 2,4-D is suitable for callus induction (2,4-D> Picloram).
Interestingly, the rate of green plant increased significantly in the presence of Picloram. Lower regeneration
capabilities observed in the presence of 2,4-D ranged from 2.88 to 38.37%. Addition of Picloram instead the
former, increased the green plant regeneration ability from 6.20 to 43.65%. Again, among the two hormone
concentrations evaluated, 2 mg/L of hormone showed upward callus induction rate (19.48-40.46%), compared
to 1 mg/L (17.03-38.37%). In turn, it impact negatively towards regeneration. The regeneration frequency was
better and varied from 4.47-43.65% with 1 mg/L, compared to 2 mg/L (2.88-33.50%). This indicates that 1 mg/L
was more effective than 2 mg/L.
Depending on genotype with their interactive effects, the frequency of callus formation varied from
17.03% for Dular in SK1+ Picloram (1 mg/L) to 40.46 % for IR58025eB in He2+ 2,4-D (2 mg/L). Whereas, the
frequency of green spots formation varied from 2.88 % for F1 in B5+2,4-D (2 mg/L) to 43.64 % for IR58025eB in
He2+ Picloram (1 mg/L). Albinism is counted a major problem in rice anther culture, especially in indica rice
which restricts the use of this breeding technique. The frequency of albino plants developed was lower across
the genotypes and media as compared to the frequency of green plants. The highest albino formation was
observed for IR58025eB (4.42-20.46%), followed by F1 (3.04-12.51%), BC1F1 (2.70-8.05%) and Dular (3.01-
7.77%). The albinism followed a trend contrary to green spots formation (He2< SK1< B5). Lowest frequency of
albino formation was observed in He2 (3.01-11.66%), followed by SK1 (3.34-18.56%) and B5 (2.70-20.46%).
Again, 2,4-D promoted higher albino development (3.23-18.56%) compared to Picloram (2.70-17.03%).
Analysis of variance revealed that frequencies of albinos increased with increased auxin concentration, like 1
mg/L regenerate 2.70-15.14% of albinos, whereas 2 mg/L further increased the frequencies varying from 5.48-
18.56%. The frequency of total regeneration with high green plant and least albino plant recovery was highest
in He2 medium.

DISCUSSION

The production of DH plants is valuable in crop breeding because it enables breeders to obtain
homozygous lines or plants directly from hybrid individual (Teparkum and Veilleux, 1998). Analysis of variance
showed very highly significant difference between genotypes, media, auxin source, auxin level and their
interaction effects (p< 0.001) on the capacity of androgenesis. Our results provided a clear picture on
interaction effect is presented in Table 3.
As expected, the genotype had a very highly significant impact (p< 0.001) on both callus induction and
green plant regeneration. The present study revealed that plant genotype is one of the major factors influencing
the capacity of androgenesis. In the conducted experiment the observations definitely confirmed the previous
findings (Kaushal et al., 2014a, 2014b; Silva and Ratnayaka, 2009; Talebi et al., 2007). Genotypic differences
existed for anther response as evidenced by the significant variation in term of their ability to form calli and the
efficiency of regeneration of green and albino plants. All the four rice genotypes analyzed provided good levels
of callus induction but diverse with respect to level of regeneration, revealing the presence of genetic diversity
in the material used. Miah et al. (1985) reported that a considerable variation of pollen callusing and plant
regeneration was noted among indica cultivars. Similar observation has been noticed in the present study. The
results revealed that recurrent parent (IR58025eB) consistently was the most responsive cultivar showing by far
the best induction and green plant regeneration capacity, followed by BC1F1, F1 and Dular. Both traits followed
same pattern of genotypic response (IR58025eB> BC1F1> F1> Dular). The statistical analysis revealed that a
significant negative heterosis for the in vitro culture response in rice was observed in F1 hybrid for both callus
induction (-13.33%) and green plant regeneration (-68.89%). Compared to mid parent heterosis, F1 showed
lower anther response to both callus induction and green plant formation. Also, the value implies that heterosis
has strong negative impact on green plant regeneration compared to callus induction. The results of the
present study are in accordance with the previous reports. Hennawy et al. (2011) stated that a significant and
negative better parent heterosis was observed in five out of 15hybrids for callus induction. Similarly, Kaushal et
al. (2014a; 2014b) found either intermediate response between parents or lower response than two parents in
the experimental genotypes for both callus induction and green plant regeneration. In contrast to this, there
have been several reports on positive heterosis for anther culture response. A significant positive heterosis was
observed in callus induction in rice (Herath and Bandara, 2011; Yan et al., 1996) and in wheat (Hennawy et al.,
2011). Picard et al. (1990) stated that in vitro anther culture response of a hybrid was affected by both parents.
Callus induction was mainly controlled by additive genetic effects with less effect of the maternal effects.
Whereas, green plant regeneration was mainly controlled by gametic additive, maternal and cytoplasmic
effects. Yan et al. (1996) and Zhang and Qifeng (1993) reported that considerable genetic gain can be made
for callus induction trait by hybridization and selection of higher responsive cultivar and recalcitrant indica types,
but achievement in the transfer to green plant regeneration ability may not be of comparable magnitude since

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the gene controlling the trait show less additive effects and relatively low heritability. Masojc et al. (1993) stated
that one of the high responding parents could be used to generate responding F1 hybrids, although there is no
guarantee of a high response in the hybrids because the inheritance of anther culture response may be more
complicated. Genetic additives effect was reported to be significant for callus induction, green plant
regeneration and culture efficiency (Yan et al., 1996). Ping et al. (1997) reported that callus induction process is
under gametophytic control and positive alleles would be strongly selected during the anther culture of F1
hybrids. Both additive and non additive effects of gene were disclosed probably functioning in directing plant
regeneration in rice (Peng and Hodge, 1989). Compared to F1, BC1F1 showed improved callus induction and
green plant regeneration. The callus inducing ability of F1 ranged from 21.30-33.00%, whereas for BC1F1 it
varied from 24.72-36.79%. Similarly, the green plant regeneration capacity of F1 ranged from (2.88-15.39%)
and 4.28-21.22% for BC1F1. As BC1F1 genome contain ¾ proportion of IR58025eB back ground and rest 1/4 of
donor, the better anther response might be due to transfer of anther culture enhancing traits from IR58025eB
(recurrent parent) in to BC1F1.
Influence of different media on anther culture response was statistically significant in the present study.
Huang et al. (1978) reported that indica genotype requires low NH4 + just half of the concentration required by
japonica. The good response of He2, B5 and SK1 seems to be due to lower level NH4 + in induction media in
indica rice (Lentini et al., 1995). Both callus induction and green spots formation followed the same pattern
(SK1<B5<He2). Similar observation was reported by Kaushal et al. (2014b). Overall, He2 proved to be superior
medium with enhanced callus induction and green spots formation with least albino formation. Silva (2009)
demonstrated that the induction medium plays a vital role in determining the successes of green plant
regeneration. Again the effect of media was dependant on the type of auxins used.
Hormones played an important role in androgenesis is evident from the present study. Despite high
callus induction activity of 2,4-D, the plant regeneration capabilities of calli were generally low for F1. Similar
observation was reported from the previous studies. Chen et al. (1991), reported that callus forming ability from
anthers of rice was high in medium supplemented with 2,4-D, but the regeneration ability from these calli was
quite low as compared to calli formed on medium supplemented with NAA. However, replacement of 2,4-D with
Picloram in the callus induction medium appeared to have a beneficial effect on green plant regeneration. A
significant increase in anther culture efficiency and green plant formation was reported in rye (Secale cereal)
anther culture when 2,4-D was replaced by Picloram (Daniel, 2006). These corroborate the findings of the
present study. However, in a few reports it is mentioned that Picloram is ineffective for anther culture in non
cereals crops like coconut (Perera et al., 2009). Also, Bishnoi et al. (2000) stated that reduction of 2,4-D to 0.5
mg/L ( and omission of Picloram altogether) in the callus induction medium appeared eventually to be the most
effective for anther response. However, the above conclusion may not applicable to all rice cultivars, as
genotypic difference in hormone requirement have been reported (Liang, 1978). Many reports have been
described on the successful regeneration of anther derived callus from medium supplemented with 2,4-D.
Kaushal et al. (2014b) reported that IR58025eB produced a maximum of 40.99% regeneration in He2
medium with 2,4-D, whereas IR58025eB x Dular produced least regeneration (6%) in the same He2 medium.
These indicate that hormonal requirement is genotype specific and different genotypes are reported to respond
differently under different hormone concentrations.
Raising the level of hormone from 1 mg/L to 2 mg/ L apparently encouraged callus yield, but inhibited
green plant regeneration (Figure 3a & 3b; Figure 4a & 4b). The higher callus induction and low rate of
regeneration observed in this study might be attributed to relatively higher doses of auxin sources used in the
callus induction media, or may be due to poor quality of callus produced in the induction phase or genotypic
difference in hormone requirement. These findings are consistent with previous reports. Many reports
mentioned on detrimental side effects of higher auxin levels. Auxin in high concentrations will prevent green
plant regeneration (Mandal and Gupta, 1995). Raina and Zapata (1997) reported that 2,4-D has proven to be a
potent auxins for callus induction from cultured anthers, but the regeneration ability of callus induced under high
2,4-D levels is poor, especially for indica rice, in comparison to callus induced on medium with lower 2,4D
levels. The results of the present study are in accordance with previous reports. Sohn et al. (1997) reported
that the ability of callus formation and plant regeneration was higher on the Picloram (1 mg/L) added medium
than that of 2,4-D (1mg/L) from unpollinated ovary culture of rice. Daniel (2006) reported on the positive green
plant regeneration response of Picloram (1 mg/L). However, it does not support the statement of Picloram has
a positive callus induction response. The callus induced during the present experiment was yellowish and
compact in texture, except for F1 from 2,4-D supplemented media. The poor quality of callus (friable texture) in
F1 might be one of the reasons behind the poor green plant regeneration. The present results were in
agreement with earlier studies stating that the friable callus considered to be of poor quality with a low potential
for regeneration in wheat (Moris and DeMacon, 1994). Kaushal et al. (2014a; 2014b) reported that friable type
with very low regeneration ability was produced in IR58025eB x Dular, when callus induction medium was
supplemented with 2,4-D. Bishnoi et al. ( 2000) reported that reduction of the level of 2,4-D to 0.5 mg/L (and
omission of Picloram altogether) in the callus induction medium appeared eventually to be the most effective for

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anther culture response, even though confounding effects from modifications made to various other culture
media components did not allow a clear and effective relationship to be established between growth regulators
and anther response.
A further disadvantage of anther culture in rice breeding is the high rate of albino plants. Albino plants
which are frequently regenerated from pollen derived calli, are often a limiting factor in rice anther culture (Islam
et al., 2012; Grewal et al., 2009; Yamagishi, 2002). The present investigations revealed that increased
frequency of albino development was observed with 2 mg/L concentration. Again, with increase in albino plant
production, a simultaneous reduction in green plant formation was observed. This implies that increased
hormone concentration caused a positive effect on albino formation, but in turn showed opposite effect on
green plant regeneration. This also implies that 2 mg/L hormone was detrimental to anther response. Similar
observation was reported in the previous studies. Sah (2008) reported that higher rate of albino plant
production might be attributed to higher rates of 2,4-D in the media. Sohn et al. (1997) reported that the high
concentration of Picloram increased markedly the frequency of albino plant from unpollinated ovary derived
callus. Hormone at low concentration in the callus induction medium appeared eventually to be the most
effective for anther response by increasing green plant regeneration and reduced albino formation. Even
though albinism is genetically determined, the trait is amenable for manipulation, at least to some extent, by
reducing the hormone concentration.
Analysis of the effect of synthetic auxin on callus induction and subsequent regeneration in the tested
genotypes revealed that the strongest stimulation of this process was provided by He2 medium with Picloram
(1 mg/L). Therefore, media modifications must consider the type of growth hormone along with its concentration
in order to achieve high callus induction with good regeneration ability rather than simply inducing prolific
callusing, from which regeneration would not be possible (Silva, 2010). Addition of AgNO3 as an anti ethylene
agent to delay anther senescence has positive impact in anthers response in the present study. It was
speculated that AgNO3 had positive effect on embryogenesis by blocking the inhibitory effect of endogenously
produced ethylene in-culture vessels. Lentini et al. (1995) reported that addition of 10 mg/L of AgNO 3, anti
ethylene compound to callus induction medium promoted 2 fold increase in pollen callusing frequency (10.1%
to 20.06%) and green plant regeneration. Similar positive effect of AgNO3 was reported in anther culture of
wheat and brassica (Ghameni et al., 1994; Williams et al., 1990). Addition of organic nitrogen supplement such
as casein hydrolysate (CH) in the medium seemed to be beneficial for positive anther response. The present
results indicate that both androgenic response and regeneration ability were greatly genotype dependant and
their response to different kind of treatment is not the same.

CONCLUSION

The genotypic differences, media composition, type of auxin and their interaction clearly influence the
anther culture ability of rice. As suspected, investigated cultivars displayed a diverse reaction to the applied
combinations of growth regulators. Interestingly, Picloram showed positive green plant regeneration efficiency
for rice in addition to earlier reports on improved regeneration in other cereals. Cultivar IR58025eB, proved to
be the best genotypes and He2 medium with Picloram (1 mg/L) to be the best medium in terms of their
regeneration ability specially for F1 from a cross between IR58025eB and Dular.

ACKNOWLEDGEMENTS

Authors thankful to The Executive Director and Usha B. Zehr Director, Barwale Foundation, for
providing facilities for the execution of this research work.

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Figure2. Effect of genotype on anther culture response

Figure3a. Effect of picloram (1 mg/L) on anther culture

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Figue3b . Effect of 2, 4-D (1mg/L) on anther culture response

Figure4a . Effect of Picloram (2mg/L) on anther culture response

Figure4b . Effect of 2, 4-D (2mg/L) on anther culture response

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a b c

e f
d

Figure 5. (a) Callus induction (b) Green plantlet formation (c) Albino shoot regeneration (d)
Plantlet development (e) Root development (f) Acclimatization

Table 1. Rice
Genotype Characteristic
Dular Donor of Wide Compatibility (WC) gene
IR58025eB (25eB) Recurrent parent with uppermost internode elongation (eui) gene
25eB x Dular F1 with eui x WC gene
25eB x Dular x 25eB BC1F1 with eui x WC gene
genotypes used in the study

Table 2. Composition of callus induction media used

Components B5 He2 SK1

(mg/L)
(NH4)2SO4 134 231 231
KNO3 3125 3181 3180
KH2PO4 0 800 540
NaH2PO4.H2O 150 0 0
MgSO4.7H2O 250 3.5 185
CaCl2.2H2O 150 166 440
H3BO3 3 1.6 6.2
MnSO4.4H2O 10 22.3 22.3
ZnSO4.7H2O 2 1.5 1.5
Na2MoO4.2H2O 0.25 0.25 0.25
KI 0.8 0.8 0.8
CuSO4.5H2O 0.025 0.025 0.025
CoCl2.6H2O 0.025 0.025 0.025
FeSO4.7H2O 27.8 27.8 27.8
Na2EDTA 37.5 37.5 37.5
Thiamine-HCl 10 10 2.5
Nicotinic acid 1 0.5 2.5
Pyridoxine-HCl 1 0.5 2.5
Glycine 0 2 2
Inositol 100 100 100
NAA 1 1 1
Kinetin 0.5 0.5 0.5
Maltose 30 30 30
Gel Right 2,700 2,700 2,700
Casein Hydrolysate 200 200 200
AgNo3 8 8 8
pH 5.8 5.8 5.8

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Table 3. Interaction effect of Genotype, Medium, Hormone and its concentration on anther culture efficiency in rice.

Genotype Medium Hormone Conc (mg/L) CI % RG % ABN %


1 20.14 17.82 4.46
2,4-D
2 25.05 6.96 3.48
B5
1 21.56 19.05 3.81
Picloram
2 24.44 9.92 6.61
1 23.77 20.01 3.34
2,4-D
2 28.11 11.61 5.80
Dular He2
1 23.05 21.07 3.01
Picloram
2 25.67 17.40 6.96
1 19.33 16.47 5.49
2,4-D
2 22.44 3.89 7.77
SK1
1 17.03 15.59 5.20
Picloram
2 19.48 7.62 7.62
1 35.88 33.36 18.23
2,4-D
2 40.18 18.19 20.46
B5
1 34.40 39.35 13.12
Picloram
2 37.60 30.31 17.03
1 38.37 38.77 6.75
2,4-D
2 40.46 23.78 11.66
25eB
1 36.86 43.65 4.42
Picloram
He2 2 40.16 33.50 7.77
1 32.86 30.32 15.16
2,4-D
2 36.26 20.62 18.56
1 32.26 35.09 11.70
Picloram
SK1 2 33.77 26.67 15.56
1 26.24 4.47 3.61
2,4-D
2 30.62 2.88 8.65
1 23.82 12.07 3.45
Picloram
B5 2 26.06 6.20 6.20
1 29.59 6.45 3.23
2,4-D
2 33.00 4.79 7.19
F1 He2
1 24.07 15.39 3.08
Picloram
2 26.69 8.22 8.22
1 23.69 4.60 9.20
2,4-D
2 27.82 3.13 12.51
SK1
1 21.30 6.96 3.48
Picloram
2 24.59 6.73 6.73
1 31.53 13.71 5.48
2,4-D
2 34.92 4.28 6.42
B5
1 28.08 18.23 2.70
Picloram
2 30.60 13.07 5.23
1 34.32 19.88 4.97
2,4-D
2 36.79 9.04 6.78
BC1F1 He2
1 29.27 21.21 3.03
Picloram
2 31.18 17.88 6.70
1 28.25 13.37 3.34
2,4-D
2 30.47 5.37 8.05
SK1
1 24.72 14.77 3.48
Picloram
1 29.35 7.64 7.64
C.V. % 1.6 3.8 2.6
LSD % 0.66 0.85 0.27

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Table 4. ANOVA table for anther culture efficiency

Source of
Variant DF MSS F ratio PROB CV
variation
Genotype (G) 3 1750.22 779771.27 0.00
Media (M) 2 383.97 1710.68 0.00
Auxin (A) 1 341.82 1522.90 0.00
Concentraion
(C) 1 472.78 2106.38 0.00
G*M 6 2.05 9.14 0.00
Callus G*H 3 26.80 119.40 0.00
1.6
induction G*C 3 0.85 3.80 0.01
M*H 2 7.01 31.25 0.00
M*C 2 1.62 7.23 0.00
H*C 1 6.76 30.10 0.00
G*M*H 6 7.80 34.76 0.00
M*H*C 2 1.89 8.42 0.00
G*M*H*C 15 1.54 6.88 0.00
Genotype (G) 3 5198.86 13928.66 0.00
Media (M) 2 570.94 1529.66 0.00
Auxin (A) 1 1079.53 2892.25 0.00
Concentraion
(C) 1 2758.78 7391.26 0.00
G*M 6 11.78 31.57 0.00
Green plant G*H 3 49.12 131.60 0.00
3.8
regeneration G*C 3 143.61 384.75 0.00
M*H 2 40.52 108.56 0.00
M*C 2 7.25 19.43 0.00
H*C 1 62.83 168.32 0.00
G*M*H 6 4.12 11.03 0.00
M*H*C 2 1.11 2.97 0.05
G*M*H*C 15 17.24 46.20 0.00
Genotype (G) 3 727.37 1841.15 0.00
Media (M) 2 159.07 402.65 0.00
Auxin (A) 1 119.21 301.74 0.00
Concentraion
(C) 1 479.97 1214.90 0.00
G*M 6 100.90 255.41 0.00
Albino plant G*H 3 34.95 884.78 0.00
2.6
development G*C 3 7.22 182.68 0.00
M*H 2 9.24 233.94 0.00
M*C 2 7.14 180.63 0.00
H*C 1 4.98 125.94 0.00
G*M*H 6 12.91 326.79 0.00
M*H*C 2 1.45 36.65 0.00
G*M*H*C 15 2.93 74.17 0.00

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