Subscribe Now Subscribe Today
Research Article

Anther Culture Response in Boro Rice Hybrids

Chaitali Sen and R.P. Singh
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail

The objective of the present study was to determine the effect of genotype and culture media in anther culture of boro rice hybrids. Anthers from three f1 hybrids of boroxhigh yielding indica rice varieties GautamxBPT 5204, Krishna HansaxNDR 359 and IR64xNDR 359 were cultured on modified SK media and modified N6 media. Modified SK media was found to be more suitable for callus formation in all the f1 hybrids. The cross GautamxBPT 5204 showed maximum callus induction frequency followed by Krishna HansaxNDR 359. Only the cross of Krishna HansaxNDR 359 was responsive to green plant regeneration while IR64xNDR359 and GautamxBPT 5204 resulted in only albino plants. Anther culture response is influenced by the genotype and media composition in indica rice hybrids.

Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

  How to cite this article:

Chaitali Sen and R.P. Singh, 2011. Anther Culture Response in Boro Rice Hybrids. Asian Journal of Biotechnology, 3: 470-477.

DOI: 10.3923/ajbkr.2011.470.477

Received: December 14, 2010; Accepted: May 17, 2011; Published: July 09, 2011


Traditional boro rice is a unique type of Oryza sativa grown during the winter season (minimum temperature around 4-10°C) in the depressions around rivers and lakes (Singh et al., 2003). It makes a significant contribution in enhancing the overall rice production in eastern India. Genetic improvement in boro rice can be made by the application of biotechnological tools like anther culture in superior hybrids of boro rice.

Androgenesis in flowering plants provides an understanding of the biological basis of single cell microspore embryogenesis to the production of a doubled haploid plant. This system provides an excellent opportunity to shorten the breeding cycle for rapid production of doubled haploids and fix agronomic traits (Suriyan et al., 2009). There are many factors that can affect the success of anther culture, such as the maturity of the donor plant (Afza et al., 2000; Jacquard et al., 2006), panicle pretreatment (Trejo-Tapia et al., 2002), microspore developmental stages (Afza et al., 2000; Cha-Um et al., 2009). In almost all species genotype no doubt is a deciding factor in achieving success in anther culture response (Ramakrishnan et al., 2005; He et al., 2006). The genetic makeup of indica subspecies makes them recalcitrant to in vitro anther culture.

However, components of tissue culture media are also important (Faruque et al., 1998; Asaduzzaman et al., 2003) and have been demonstrated to have a crucial role in coaxing an in vitro response from cultured anthers of otherwise recalcitrant genotype. Monirul et al. (2004) observed that addition of 1 mg L-1 2, 4-D and 1 mg L-1 kinetin to callus induction medium improved the callus induction and regeneration potential of the responsive hybrid rice line IR-69690. Hassan et al. (2001) observed direct regeneration of maize hybrid M95xS 95 and inbred S2-9 on N6 medium supplemented with 2, 4-D (2 mg L-1) and kinetin (1.5 mg L-1).

Genotype may differ widely in their basal media requirement for dedifferentiation and redifferentiation. Therefore, in the present investigation, efforts were made to analyze the effect of genotype and different media formulations on callus induction and plant regeneration in three boro rice hybrids of GautamxBPT 5204, Krishna HansaxNDR 359 and IR 64xNDR359.


Hybridization and anther culture experiments were carried out in three f1 hybrids Gautamx BPT 5204, Krishna HansaxNDR 359 and IR 64xNDR 359, in 2008-2009. Crosses were made between boro rice varieties Gautam, Krishna Hansa and IR64 (females) possessing cold tolerance and high yielding rice varieties BPT 5204 and NDR 359 (males) in Agriculture Research Farm, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi. Boots (Panicles) were collected from the primary tillers of the selected hybrids in morning hours (7.00, 8.00 a.m.) when the middle florets of the panicles contain uninucleate microspores in their anthers. Boots were wrapped in moist tissue paper covered with aluminum foil and were kept in refrigerator for 8-10 days for cold pretreatment. Sterilization of panicles was carried out by dipping intact boots in 70% alcohol for 2 min. Panicles were surface sterilized with 0.1% aqueous HgCl2 solution for 8 min and rinsed several times with distilled water. The outer covering of the panicle was removed with sharp scalpel and spikelet was cut at the base to excise the anthers. Anthers from f1 hybrids were inoculated for callus induction on modified N6 and modified SK media (Table 1).

Table 1: Media composition for callus induction
The-sign in the blank boxes indicate the absence of that particular compound in the media composition. N6 Media by Chu et al. (1975) SK media by Raina and Zapata (1997)

Thirty anthers were inoculated in each culture tube containing 10.0 mL medium. There were ten test tubes per replication and three replications were used for a particular media concentration for a particular hybrid. The cultures were incubated in complete darkness at 25±1°C for 4-5 weeks for callus induction. The culture tubes were examined periodically at weekly intervals to observe the progress in respect of callus formation. Data on percentage of callus regenerating was recorded. Calli of at least 2 mm diameter were transferred to culture tubes containing 20 mL regeneration medium consisting of MS media supplemented with 2 mg L-1 BAP, 1 mg L-1 Kinetin, 1 mg L-l NAA. The pH of the medium was adjusted to 5.8 with 1 N HCl or 1 N NaOH before adding agar and autoclaving. The culture tubes were plugged with non-absorbent cotton wrapped in cheese cloth. Cultures were inoculated for four weeks under 16/8 light/dark at 25±2°C. Green and albino plantlet regeneration percentage were observed and recorded. The unrooted green shoots of transferable sizes were transferred into rooting medium (half strength MS medium supplemented with 2 of NAA and 0.1 mg L-1 kinetin). The cultures were kept in growth chamber at 4000-lux1 cool fluorescent light at 25±1°C for plantlet (shoot with root) regeneration.


The responsive anthers showed slight swelling around it and subsequently started callusing asynchronously after 3-4 weeks (Fig. 1a, b). Table 2 exhibits the callus induction frequencies. Callus induction was observed in all the three f1 hybrids however, induction frequency varied with the genotype and medium. The anther culture response was better on modified SK medium as compared to modified N6 media for all the rice hybrids studied. Out of three crosses evaluated, maximum callus induction frequency from anthers was observed in the cross GautamxBPT 5204 (5.77) on modified SK media and minimum was found in IR64xNDR 359 (0.22) on modified N6 medium.

Calli derived from the anthers of three rice hybrids were transferred to regeneration (MS) media for shoot regeneration. Anther calli on differentiation gave rise to shoots in all the genotypes under study but shoot differentiation varied with the genotype (Fig. 1c, d). It is evident from Table 3 that green shoot regeneration was observed only in Krishna HansaxNDR 359 (8.95%) (Fig. 1e), Maximum shoot regeneration was observed in GautamxBPT 5204 but all the shoots were albino. Minimum shoot regeneration was observed in the cross IR 64xNDR 359 however, shoots devoid of chlorophyll (albino) regenerated in all the three crosses (Fig. 1f). Initiation of roots in rooting media is shown in Fig. 1g and hardening and acclimitazation of green plants is shown in Fig. 1h.

Table 2: Callus induction frequency
1The lux (symbol: lx) is the SI unit of illuminance and luminous emittance measuring luminous power per area. It is used in photometry as a measure of the intensity, as perceived by the human eye, of light that hits or passes through a surface. One lux is equal to one lumen per square metre: 1 lx = 1 l m/m2 = 1 cd·sr·m-2

Table 3: Green and albino shoot regeneration frequency
The blank (-) represents no regeneration of plants from calli in that particular hybrid

Fig. 1(a-h): Callus induction from anthers ,regeneration, rooting and hardening of boro rice hybrids (a) Callus induction, (b) Callus proliferation, (c) Initiation of green shoots in Krishna HansaxNDR 359, (d) Initiation of albino shoots, (e) Elongation of green shoots in Krishna HansaxNDR 359, (f) Elongation of albino shoots, (g) Initiation of roots in rooting media, (h) Hardening and acclimitazation of green plants


Genotype and nutrient composition of the culture media are the major sources of variation in in vitro culture and regeneration (Bishnoi et al., 2000; Talebi et al., 2007). The presence of significant variation in callus induction due to genotype, media composition and genotypexmedia interaction was observed by Bagheri and Jelodar (2008). Niroula and Bimb (2009) demonstrated the possibility of enhancing androgenic response by manipulating media composition using responsive genotypes of Nepalese rice cultivars.

N6 medium (Chu et al., 1975) which has been widely used for anther culture was found less suitable for indica rice anther culture (Gosal et al., 1997). The indica cultivars require lower NH4+ ions therefore in the modified SK media amount of (NH4)2 SO4 was reduced.

In the present study the presence of maltose as a carbon source along with AgNO3 and Casein Hydrolysate (CH) in the modified SK media, have enhanced the androgenic potential in boro rice hybrids. The callus induction frequency was enhanced in all the hybrids by replacing sucrose with maltose in the SK medium. This result of increase in the callus induction frequency by the use of maltose have been supported by the study with barley microscope culture (Hunter, 1987) wheat (Mejza et al., 1993) and rice (Xie et al., 1995). One mechanism by which maltose may influence androgenic response is through differences in the breakdown products of the two disaccharides. Sucrose is hydrolyzed to glucose and fructose by invertase, both of these monosaccharides have been found to inhibit anther culture response.

The positive effect of maltose was associated with keeping a high proportion of swollen microspores and increasing their division rate whereas sucrose plasmolyze the microspores. Kasha et al. (1990) reported plasmolysis followed by dying of microspores at 3% sucrose containing media. Further maltose stabilizes the culture medium osmotically (Kuhlmann and Foroughi-Wher, 1989) and it releases glucose at a slow rate after degradation (Last and Brettell, 1990).

The 8 mg L-1 of silver nitrate which acts as an ethylene antagonist was added in, modified SK media. It has been reported that the presence of ethylene in the culture medium inhibits somatic embryogenesis and shoot regeneration (Biddington, 1992; Vain et al., 1989). Ethylene is produced by plant cells in closed culture vessel (Gamborg and LaRue, 1971) and the gelling agent like agar (Mensuari et al., 1992). Some studies showed that the presence of auxin particularly 2, 4-D stimulates ethylene production (Yang and Hoffman, 1984). AgNO3 has been employed in tissue culture studies as it interferes in the ethylene perception mechanism. Further it is easily soluble in water and is not phytotoxic at effective concentrations (Beyer, 1976). Casein hydrolysate which is a source of calcium, several micronutrients, vitamins and amino acids, was added in modified SK media. Improvement in callus induction and growth by the addition of casein hydrolysate was also reported by Khaleda and Al-Forkan (2006) in deepwater rice.They also stated that callus formation and plant regeneration was influenced by interaction of media components.

Differential response for callus induction was found in the three f1 hybrids with different media. Callus induction frequency as determined by anthers forming calli varied between 0.22 to 5.77% depending upon the culture medium and the genotype. Shahnewaz et al. (2004) also observed a callus induction frequency ranging from 1.42 to 8.06%.

Maximum callus induction was observed in the cross of GautamxBPT 5204 but it lacked the ability to produce green plants. Krishna HansaxNDR 359 was the only hybrid that exhibited green plant regeneration potential. This indicates that genotype also acts as an important factor for the production of green plants. This is in accordance with the studies reported by Raina and Zapata (1997), Zhu et al. (1991) and Xie et al. (1995) that medium used for callus induction and genotype of the donor significantly affected green plant regeneration potential. Plant regeneration was observed in MS media with different combination and concentration of BAP, Kinetin and NAA by (Jubair et al., 2008). In contradiction to the present study, Roy and Mandal (2011) clearly indicated that higher concentration of BAP had inhibitory effects on microtillering of androgenic plantlets of rice variety IR 72.

The recovery of albino plants from microspore derived calli in rice especially in indica rice varieties has been a formidable obstacle to the utilization of rice anther culture for indica rice improvement (Chen et al., 1991; Raina and Zapata, 1997; Sripichitt et al., 2000; Chowdhury and Mandal, 2001).

Occurrence of albino plant regeneration seems to be a common phenomenon in rice anther culture, as the albino shoot regeneration frequencies were more in all the crosses as compared to the green shoot regeneration frequencies in the present study. In general, albino plants (e.g., wheat, barley and rice) contain deleted forms of the plastid genome (Day and Ellis, 1985; Harada et al., 1991; Zubko and Day, 2002).


The present study indicate that the effect of genotype and media composition significantly affected callus induction frequency in the f1 hybrids. Nevertheless the modified SK media can be considered more favorable for induction of callus in the f1 hybrids of boroxhigh yielding rice varieties. The cross of Krishna HansaxNDR 359 was observed to be most responsive to anther culture for the production of green shoots and therefore this cross can be further exploited for the production of doubled haploids.

1:  Afza, R., M. Shen, F.J. Zapata-Arias, J. Xie and H.K. Fundi et al., 2000. Effect of spikelet position on rice anther culture efficiency. Plant Sci., 153: 155-159.
CrossRef  |  

2:  Asaduzzaman, M., M.A. Bari, M.H. Rahman, N. Khatun, M.A. Islam and M. Rahman, 2003. In vitro plant regeneration through anther culture of five rice varieties. J. Biol. Sci., 3: 167-171.
Direct Link  |  

3:  Bagheri, N. and N.B. Jelodar, 2008. Combining ability and heritability of callus induction and green-plant regeneration in rice anther culture. Biotechnology, 7: 287-292.
CrossRef  |  Direct Link  |  

4:  Beyer, E.M., 1976. Silver ion: A potent anti-ethylene agent in cucumber and tomato. HortScience, 11: 175-196.

5:  Biddington, N.L., 1992. The influence of ethylene in plant tissue culture. Plant Growth Regul., 11: 173-178.
CrossRef  |  Direct Link  |  

6:  Bishnoi, U.S., R.K. Jain, K.R. Gupta, V.K. Chowdhury and J.B. Chowdhury, 2000. High frequency androgenesis in indica x basmati rice hybrids using liquid culture media. Plant Cell Tiss. Org. Cult., 61: 153-159.
Direct Link  |  

7:  Cha-um, S., B. Srianan, A. Pichakum and C. Kirdmanee, 2009. An efficient procedure for embryogenic callus induction and double haploid plant regeneration through anther culture of Thai aromatic rice (Oryza sativa L. subsp. indica). In vitro Cell. Dev. Biol. Plant, 45: 171-179.
CrossRef  |  Direct Link  |  

8:  Chen, C.C., H.S. Tsay and C.R. Huang, 1991. Factors Affecting Androgenesis in Rice (O. sativa L.). In: Biotechnology in Agriculture and Forestry Rice, Bajaj, Y.P.S. (Ed.). Vol. 14, Springer Verlag, Berlin, ISBN: 354051810X, pp: 193-215.

9:  Chowdhury, B. and A.B. Mandal, 2001. Microspore embryogenesis and fertile plantlet regeneration in a salt susceptible x salt tolerant rice hybrid. Plant Cell Tiss. Org. Cult., 65: 141-147.
CrossRef  |  

10:  Chu, C.C., C.C. Wang, C.S. Sun, K.C. Hsu, K.C. Yin, C.Y. Chu and F.Y. Bi, 1975. Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Scient. Sin., 18: 659-668.
Direct Link  |  

11:  Day, A. and T.H.N. Ellis, 1985. Deleted forms of plastid DNA in albino plants from cereal anther culture. Curr. Genet., 9: 671-678.
Direct Link  |  

12:  Faruque, M.O., T. Farzana, Z.I. Seraj, R.H. Sarker and S.A.A. Khatun, 1998. Variations in green plant regeneration response from anthers of indica rice and their hybrids with japonica cv. Taipei 309. Plant Cell Tissue Organ Cult., 54: 191-195.
CrossRef  |  Direct Link  |  

13:  Gamborg, O.L. and T.A.G. LaRue, 1971. Ethylene production by plant cell culture. Physiol. Plant, 48: 394-401.
Direct Link  |  

14:  Gosal, S.S., A.S. Sindhu, J.S. Sandhu, R. Sandhu-Gill, B. Singh, G.S. Khera and H.S. Dhaliwal, 1997. Haploidy in Rice. In: Cereals. In vitro Haploid Production in Higher Plants, Jain, S.M., S.K. Sopory and R E. Veilleux (Eds.). Vol. 4, Kluwer Academic Publishers, Dordrecht, pp: 1-35.

15:  Harada, T., T. Sato, D. Asaka and I. Matsukawa, 1991. Large-scale deletions of rice plastid DNA in anther culture. Theor. Applied Genet., 81: 157-161.
CrossRef  |  

16:  Hassan, L., S.D. Ahmad and A. Okumus, 2001. The direct regeneration of maize haploids through anther culture. J. Biological Sci., 1: 900-901.
CrossRef  |  Direct Link  |  

17:  He, T., Y. Yang, S.B. Tu, M.Q. Yu and X.F. Li, 2006. Selection of interspecific hybrids for anther culture of indica rice. Plant Cell. Tissue Org. Cult., 86: 271-277.
Direct Link  |  

18:  Hunter, C.P., 1987. Plant regeneration method. European Patent Application No. 0245 898 A2. pp: 1-8.

19:  Jacquard, C., R. Asakaviciute, A.M. Hamalian, R.S. Sangwan, P. Devaux and C. Clement, 2006. Barley anther culture: Effects of annual cycle and spike position on microspore embryogenesis and albinism. Plant Cell Rep., 25: 375-381.
CrossRef  |  PubMed  |  Direct Link  |  

20:  Jubair, T.A., U. Salma, N. Haque, F. Aktar, I.J. Mukti, A.K.M.F. Haque and M.R. Ali, 2008. Callus induction and regeneration of local rice (Oryza sativa L.) variety topa. Asian J. Plant Sci., 7: 514-517.
CrossRef  |  Direct Link  |  

21:  Kasha, K.J., A. Ziauddin and E. Simion, 1990. Barley and wheat microspore culture. Abstr. 7th Int. Congress on Plant Cell Tissue Culture, Amsterdam, pp: 187.

22:  Khaleda, L. and M. Al-Forkan, 2006. Stimulatory effects of casein hydrolysate and proline in in vitro callus induction and plant regeneration from five deepwater rice (Oryza sativa L.). Biotechnology, 5: 379-384.
CrossRef  |  Direct Link  |  

23:  Kuhlmann, U. and B. Foroughi-Wher, 1989. Production of doubled haploid lines in frequencies sufficient for barley breeding programs. Plant Cell Rep., 8: 78-81.
CrossRef  |  

24:  Last, D.I. and R.I.S. Brettell, 1990. Embryoid yield in wheat anther culture is influenced by the choice of sugar in the culture medium. Plant Cell Rep., 9: 14-16.

25:  Islam, M.M., S.K. Adhikary, P. Gain, M.M. Rahman and N.A. Siddique, 2004. Effect of plant growth regulators on callus induction and plant regeneration in anther culture of rice. Pak. J. Biol. Sci., 7: 331-334.
CrossRef  |  Direct Link  |  

26:  Mensuari, S.A., M. Panizza and E. Tognoni, 1992. Quantification of ethylene losses in different container seal systems and comparison of biotic and abiotic contributions to ethylene accumulation in cultured tissues. Physiol. Plant., 84: 472-476.
CrossRef  |  

27:  Mejza, S.J., V. Morgant, D.E. DiBona and J.R. Wong, 1993. Plant regeneration from isolated microspores of Triticum aestivum. Plant Cell Rep., 12: 149-153.
CrossRef  |  

28:  Niroula, R.K. and H.P. Bimb, 2009. Effect of genotype and callus induction medium on green plant regeneration from anther of Nepalese rice cultivars. Asian J. Plant Sci., 8: 368-374.
CrossRef  |  Direct Link  |  

29:  Raina, S.K. and F.J. Zapata, 1997. Enhanced anther culture efficiency of indica rice (Oryza sativa L.) through modification of the culture media. Plant Breed., 116: 305-315.
Direct Link  |  

30:  Ramakrishnan, S.H., S. Saravanan, C.R. Anandakumar and J.R. Kannanbapu, 2005. In vitro androgenesis in rice (Oryza sativa L.). Asian J. Plant Sci., 4: 600-602.
CrossRef  |  Direct Link  |  

31:  Singh, R.K., H. Mahabub and R. Thakur, 2003. Boro Rice. Fine Grains Pvt Ltd., IRRI, New Delhi, pp: 1-3.

32:  Roy, B. and A.B. Mandal, 2011. Profuse microtillering of androgenic plantlets of elite indica rice variety IR 72. Asian J. Biotechnol., 3: 165-176.
CrossRef  |  Direct Link  |  

33:  Shahnewaz, S., M.A. Bari, N.A. Siddique and M.H. Rahman, 2004. Effects of genotype on induction of callus and plant regeneration potential in vitro anther culture of rice (Oryza sativa L.). Pak. J. Biol. Sci., 7: 235-237.
CrossRef  |  Direct Link  |  

34:  Sripichitt, P., T. Ozawa, M. Otani and T. Shimada, 2000. Improved method for anther culture of an indica rice cultivar of Thailand. Plant Prod. Sci., 3: 254-256.
Direct Link  |  

35:  Suriyan, C., S. Bootsaya, P. Aussanee and K. Chalermpol, 2009. An efficient procedure for embryogenic callus induction and doubled haploid plant regeneration through anther culture of Thai aromatic rice (Oryza sativa L. subsp. indica). In vitro Cell. Dev. Biol. Plant, 45: 171-179.

36:  Talebi, R., M.R. Rahemi, H. Arefi, M. Nourozi and N. Bagheri, 2007. In vitro plant regeneraton through anther culture of some Iranian local rice (Oryza sativa) cultivars. Pak. J. Biol. Sci., 10: 2056-2060.
PubMed  |  

37:  Trejo-Tapia, G., U.M. Amaya, G.S. Morales, A.D.J. Sanchez and B.M. Bonfil et al., 2002. The effects of cold-pretreatment, auxins and carbon source on anther culture of rice. Plant Cell Tissue Org. Cult., 71: 41-46.
CrossRef  |  

38:  Vain, P., P. Flament and P. Soudain, 1989. Role of ethylene in embryogenic callus initiation and regeneration in Zea mays L. J. Plant Physiol., 146: 537-540.
Direct Link  |  

39:  Xie, J., M. Gao, Q. Cai, X. Cheng, Y. Shen and Z. Liang, 1995. Improved isolated microspore culture efficiency in medium with maltose and optimized growth regulator combination in japonica rice (Oryza sativa). Plant Cell Tissue Org. Cult., 42: 245-250.
CrossRef  |  Direct Link  |  

40:  Yang, S.F. and N.E. Hoffman, 1984. Ethylene biosynthesis and its regulation in higher plants. Ann. Rev. Plant Physiol., 35: 155-189.
CrossRef  |  Direct Link  |  

41:  Zhu, D.Y., X.H. Ding and J.H. Yi, 1991. Anther Culture and Breeding in Indica Rice. In: Tissue Culture of Field Crops, Yan, C.J. (Ed.). Shanhai Press, Shanghai, pp: 153-169.

42:  Zubko, M.K. and A. Day, 2002. Differential regulation of genes transcribed by nucleus-encoded plastid RNA polymerase and DNA amplification, within ribosome-deficient plastids in stable phenocopies of cereal albino mutants. Mol. Gen. Genomics, 267: 27-37.
CrossRef  |  

©  2021 Science Alert. All Rights Reserved