EFFECT OF COLD PRETREATMENT AND PHYTOHORMONES ON ANTHER CULTURE EFFICIENCY OF TWO INDICA RICE (Oryza sativa L.) HYBRIDS- AJAY AND RAJALAXMI

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1 , May ; Volume 1 (2) ISSN No EFFECT OF COLD PRETREATMENT AND PHYTOHORMONES ON ANTHER CULTURE EFFICIENCY OF TWO INDICA RICE (Oryza sativa L.) HYBRIDS- AJAY AND RAJALAXMI M. Rukmini, G J N Rao* and R N Rao Crop Improvement Division, Central Rice Research Institute, Cuttack , India. Received March 27, 2013; Revision April 04, 2013; Accepted May 04, 2013 Available Online May 7, 2013 KEYWORDS Anther culture Indica rice in vitro Cold pretreatment Phytohormones ABSTRACT Application of anther culture techniques for improvement of indica rices is a formidable task as they are known to be recalcitrant to culture unlike japonica rices. Since anther culture can effectively address the major problems associated with the adoption of hybrid rice in India, an effort was made to assess the influence of cold pretreatment and phyto hormones on the anther culture response of Rajalaxmi (CRHR 5) and Ajay (CRHR 7), two elite and popular indica rice hybrids. Cold pretreatment for 7-9 days at 10 o C was found to have a positive influence on the callus induction frequency irrespective of the media employed and prolonged treatment over the optimum proved to be inhibitory. Of the different auxins (2, 4-D, NAA) and cytokinins (Kinetin, BAP) and their combinations studied, a ratio of 1:4 for 2,4-D and NAA and 1:3:1 ratio of Kinetin: BAP: NAA ratio proved to be optimal for callus induction and green plant regeneration respectively. This information can be of use in the development of doubled haploids with superior yield and good grain quality from the elite hybrid cultivars. * Corresponding author gjnrao@gmail.com (G.J.N. Rao) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences. Production and Hosting by JEBAS ( All rights reserved.

2 70 Rukmini et al., 1. Introduction The development of anther culture techniques for production of rice was a major advance in the field of rice breeding in the last few decades and anther culture is one of the most intensively investigated areas of the in vitro culture methods of rice as doubled haploid approach can effectively reduce the time required for varietal improvement (Herath and Bandara 2011). However indica rice cultivars are generally recalcitrant to culture and also the green plant regeneration potential is limited. Since higher levels of callus induction and green plant regeneration is a pre-requisite for utilization of anther culture in breeding programs, detailed studies on the various factors that govern the culture response of anthers under in vitro condition of indica rices are crucial. To raise the yield ceiling in rice, which has become static after the green revolution, hybrid rice was considered as an option to increase productivity per unit area. However, despite its yield advantage over inbreds, the hybrid rice technology has not found favour with the farmers of India. The main reasons for its low adoption include the seed cost and the quality of the produce. Since doubled haploid approach can effectively address the problems associated with hybrid rice through production of high yielding doubled haploids with uniform grain quality, the basic studies on indica rice anther culture assumes great significance. Low temperature shock has been reported to enhance the androgenic response in several species including rice (Ogawa et al., 1995; Pande, 1997; Silva and Ratnayke, 2009;Gueye and Nidr, 2010; Sen et al., 2011). Some of the positive effects of cold pretreatment on callus induction include delay of anther wall senescence, increase of symmetric divisions of pollen grains and release of substances necessary for androgenesis, mainly amino acids and shock-thermic proteins (Kiviharju and Pehu, 1998). The growth regulators, mainly the auxins and cytokinins are known to control the dedifferentiation and differentiation processes in the in vitro cultures of crop plants. The rate of success can be enhanced by improving the composition of tissue culture medium, especially by manipulating plant growth regulators (Mandal and Gupta, 1995). Among the auxins, 2, 4-D and NAA were the most commonly used for induction of callus from rice anthers as auxins are the most essential growth regulators required for induction of callus from anthers of cereals (Zhu et al., 1998). The auxins, IAA and NAA may induce direct androgenesis while 2; 4- D promotes rapid cell proliferation and formation of callus (Ball et al., 1993). It was observed that for regeneration of plants, as both 2,4-D and NAA are not known to support regeneration, the use cytokinins like kinetin and BAP are required (Mandal and Gupta, 1995). This study was an attempt to evaluate the efficiency of the anthers from the elite indica rice hybrids using cold pretreatment and phytohormones in the culture media on both callus induction and green plantlet regeneration. 2. Materials and methods The experimental materials were Ajay (CRHR 7) and Rajalaxmi (CRHR 5), two elite indica hybrid rice varieties that were developed at Central Rice Research Institute, Cuttack. The boots (panicles along with the boot leaves in which they are still enclosed) were pulled out of the tillers in the morning hours from a healthy crop of the hybrids. The boots were wiped clean 2-3 times, with a clean muslin cloth moistened with 70% alcohol. The wrapped boots were placed in an incubator that was maintained at 10 o C for 8-10 days for cold pretreatment. The middle portion of the panicle from the pretreated spikelet was used for anther culturing. The spikelets were surface sterilized using 20% commercial bleach [bleach contains NaOCl 4% (W/V)] for 5 minutes and rinsed three times with sterile de-ionized water. Before culturing of anthers from a hybrid, cytological examination of microspore stages in the anthers was conducted and anthers with microspores at mid-uninucleate to early bi-nucleate stages were uniformly dusted over the surface of the media. For callus induction, two different media i.e. N 6 (Chu, 1978) and M O19 (Raina and Zapata, 1997), each supplemented with 2,4 -D (2mg/l), Kinetin (0.5 mg/l) and maltose (3%) were used. The ph of the medium was adjusted to 5.8 using 1N NaOH/1N HCl and solidified with agar-agar (0.8%) and the media were autoclaved at 121 o C (15psi) for 20 min. For callus regeneration, modified MS (Murashige and Skoog, 1962) medium supplemented with NAA (0.25mg/l),Kinetin (0.25mg/l), BAP (0.75mg/l) maltose/ sucrose (3% w/v) and agar-agar (0.8%) was used for solidification after adjusting the ph of the medium to 5.8. The pollen embryoids/calli from the responding anthers were transferred within one week of their formation onto the regeneration medium and the cultures were incubated under artificial light (~ 2000 lux) at 25±1 o C for callus regeneration. The green plantlets when they reached a size of around 1 cm in length were transferred to rooting medium for root formation. For root promotion in the plantlets generated from the calli, MS medium supplemented with NAA (1.0mg/l), Kn (0.1mgl -l ), maltose (5%) and agar-agar (1%) was used for solidification. The plants with well-formed roots were transferred to pots in the green house. Statistical analyses were carried out using CROPSTAT program (IRRI, 2008).

3 Effect of cold pretreatment and phytohormones on anther culture efficiency of two indica rice (Oryza sativa L.) hybrids- Ajay and Rajalaxmi Results 3.1 Effect of Cold pretreatment Of the pretreatment periods evaluated after (0-9days), both callus regeneration and green plant regeneration were observed to be significantly higher than the control (0 day treatment) when the anthers were pretreated at 10 o C for 7-9 days in CRHR 5 (Table 1) (Figure 1). On N 6 media, both callus induction (32.11%) and regeneration (36.22%) were higher after the 9 days cold pretreatment while in case of MO19 medium, the response was high for 7 days pretreatment and the response was moderate after 9 days. Figure 1: (A) Callus induction (B) Callus regeneration (C) Rooting (D) Albino plant regeneration. Table 1: Effect of cold pretreatment duration on culture response of CRHR-5 and CRHR-7. Media Days Callus induction (%) (%) Green Plant Reg (%) Albino Plant Reg(%) CRHR 5 N MO CRHR 7 N M O

4 72 Rukmini et al., In CRHR 7, the optimum levels of callus induction, callus regeneration and green plant regeneration were observed when the anthers were pretreated at 10 o C for 8 days on both N 6 (32.23%) and MO19 (28.42%). After 6 days of pretreatment, the albino frequency was high as compared to 8 days of pretreatment while after 10 days of treatment, the response was moderate [regeneration (31.24%); green plant regeneration (18.55%)] on N 6 while the response was low on M O19. After 12 days of treatment, the frequencies gradually decreased on both the media while the albino plant regeneration frequency was almost same in 10 and 12 days of treatment. Table 2: Analysis of variance of anther culture response of CRHR 5 and CRHR 7 after different cold pre-treatment periods. Character Source of variation Degree of freedom Mean Sum of Squares CV (%) Callus induction Callus Green Plant Albino Plant CRHR-7 Callus induction Callus Green Plant Albino Plant CRHR-5 Medium ** 0.3 Days ** Medium x Days 4 4.8** Error Medium ** 0.2 Days ** Medium x Days ** Error Medium ** 0.5 Days ** Medium x Days ** Error Medium ** 0.6 Days ** Medium x Days ** Error Medium ** 0.2 Days ** Medium x Days ** Error Medium ** 0.3 Days ** Medium x Days ** Error Medium ** 0.2 Days ** Medium x Days ** Error Medium ** 0.4 Days ** Medium x Days ** Error ** Significant at P=0.01 level

5 Effect of cold pretreatment and phytohormones on anther culture efficiency of two indica rice (Oryza sativa L.) hybrids- Ajay and Rajalaxmi. 73 Statistical analyses revealed highly significant differences (at 1% level) between media, duration of cold pretreatment and their interaction for all the four parameters tested (Table 2) in both the hybrids. The data showed that the critical pretreatment period was for 7-9 days and prolonging the treatment beyond 9 days proved to be inhibitory in both the media used. Similar trend was observed for regeneration and green plant regeneration frequencies also. 3.2 Influence of phytohormones on regeneration The experiments on anther callus regeneration that were conducted with callus generated on N 6 induction media using different combinations of phytohormones revealed that addition of auxin: cytokinin (NAA: Kn: BAP) in a ratio of 1:1:3 (0.25: 0.25: 0.75 mg/l) had stimulated highest regeneration ( %) in both the genotypes (Table 3). The positive influence of the same ratio (1:1:3) was evident even when the hormonal concentration was doubled (0.5:0.5:1.5 mg/l) but in the same proportion. Similar trend was observed in case of green plant regeneration ( %) in both the hybrids when the growth regulators were supplemented in the ratio of 1:1:3. While the incidence of albinos was also higher in both the genotypes in the media containing (NAA: Kn:BAP) in the ratio of 1:1:3. Callus regenerated on hormone free media had shown very less regeneration frequency. Addition of casein hydrolysate (CH) to the regeneration media had enhanced the regeneration frequency but in the media containing CH, necrosis of callus was observed. The analysis of variance revealed highly significant differences between the genotypes, phytohormonal combinations, and their interaction was evident for all the parameters observed (Table 4). Table 3: Influence of different hormonal combinations on the regeneration potential of CRHR 5 and CRHR 7 Variety Combinations (Kn:NAA:BAP) (all in mg -l ) Calli plated Calli regenerated (%) Green Plant Reg (%) Albino Reg (%) CRHR 5 0:0:0+ Maltose :0.25:0.75+ Maltose :0.25:0.75+Sucrose :0.5:1.5+ Maltose :0.5:1.5 + Sucrose :0.25:0.75+ Maltose CRHR 7 0:0:0+ Maltose :0.25:0.75+ Maltose :0.25:0.75+Sucrose :0.5:1.5+ Maltose :0.5:1.5 + Sucrose :0.25:0.75+ Maltose Table 4: Analysis of variance of different hormonal combinations on regeneration of CRHR 5 and CRHR 7. Character Source of variation Degree freedom Mean Sum of Squares CV (%) Callus Variety ** 0.2 Medium ** Green Albino Plant Plant Var x Med 5 4.5** Error Variety ** 0.4 Medium ** Var x Med Error Variety Medium ** Var x Med Error ** Significant at P= 0.01 level

6 74 Rukmini et al., 4. Discussion 4.1 Effect of Cold pretreatment The data suggests that the cold pretreatment was found to have a positive influence on the callus induction frequency irrespective of the media employed and a 7-9 day period of cold shock at 10 o C was observed to be optimal for the hybrids tested and any extended treatment over the optimum proved to be inhibitory. The treatment resulted in a 2-8 fold increase in callus induction but also resulted in early (~10-12 d) callus induction. The present results were in agreement with earlier studies (Lenka and Reddy, 1993) who also reported the inhibitory effect of prolonged period of pretreatment. Genotypic differences for culture response after treatment of different durations were recorded earlier with optimum periods ranging 7 d for Hsien/Keng hybrids (Liu et al.,1983), 5-8 d for IR53, IR58 (Alejar et al.,1995) and IR-50 (Krishnaraj and Shreerangaswamy,1993) and 3-7 d at 4 o C (Khatun et al., 2012). All the available reports support the view of Zapata- Arias (2003) who recommended a treatment at 8-10 o C for 8 days. The promotory effect of cold treatment was reported in other plant species like flax (Orbert et al., 2005) and rye (Roininen et al., 2005). Though Xie et al.(1995) proposed a cold pretreatment (4-13 o C) for 7 to 28 days for induction of callus, similar results including the present one do not support such a view. Albino plant production is one of the main problems in rice anther culture (Bishnoi et al., 2000; Grewal et al., 2009; Khatun et al., 2012).Though the albino formation did not follow any pattern in relation to the duration of cold shock in the present study, reports indicate that prolonged treatment has an adverse effect on green plant formation and significant increase in albino incidence (Gupta and Borthakur, 1987). Cold pretreatment is reported to enhance the stoppage of the gametophytic development of microspores during cold shock stress and guide the continuous division of the microspores into forming callus/embryo, i.e. a sporophyte during culture (Touraev et al.,1996; Heberle-Bors, 1996) and this shift from gametophytic mode of development to sporophytic mode may cause instability and the loss of chlorophyll is a manifestation of that shift. Of the two sugars i.e. sucrose and maltose, evaluated as the carbon sources, addition of maltose clearly enhanced callus production and regeneration efficiencies considerably. Enhanced anther culture efficiency and green plant regeneration was reported in indica rices when sucrose was replaced by maltose (Pande et al., 1999; Khatun et al., 2012).Maltose produces only glucose after degradation, but sucrose resolves into fructose and glucose in the medium. Fructose derived from sucrose may inhibit androgenesis. Likewise, maltose act as a superior carbon source for anther culture as it may result in higher ratio and the superiority of maltose might be associated with keeping a high percentage of swollen microspores and escalating their division rate (Xie et al.,1995). Besides, the beneficial effect of maltose on androgenesis is possibly due to the osmotic stabilization of culture medium, while sucrose plasmolize the microspores. 4.2 Influence of different phytohormonal combinations The most crucial constituents in the rice anther culture medium were auxins and cytokinins. Auxins have been essential plant growth regulators for the induction of callus from anthers of cereals (Zhu et al., 1998) and the type and level of the auxin present in culture medium regulates the formation of callus. Of the several phytohormonal combinations evaluated in the induction media, a ratio of 1:4 for Kn: 2, 4-D was proved to be beneficial for androgenesis while the phytohormonal combination of Kn: BAP: NAA in a ratio of 1:3:1 (0.25: 0.75:0.25 mg/l) has proved to be optimal for obtaining high green plant regeneration. The present study supports the view of Raina and Iyer (1974) that ideal phytohormonal combination was needed for ideal regeneration of 2,4-D induced calli. Trejo-Tapia et al. (2002) reported auxins were essential for the induction of callus from anthers and suggested that the type and concentration of auxins have influence on the induction. In conclusion, it can be said that since several physical and chemical factors influence the response of the genotypes to culture, the study examined two major factors: first, cold shock stress to the anthers prior to their culture and second, the combination of phytohormones in culture media. The study concludes that cold pretreatment has beneficial effects on callus induction and a pretreatment for 7-9 days at 10 o C was found to have a positive influence. On the callus induction frequency irrespective of the media employed and extended treatments over the optimum proved to have a negative influence.a combination of auxins and cytokinins (Kn: BAP: NAA in a ratio of 1:3:1) in the regeneration medium have a profound positive influence on the regeneration. Acknowledgements Authors are thankful to Dr. Ashutosh Mall (Senior scientist, IGFRI, Jhansi) for statistical analysis and Director, CRRI for the facilities and encouragement. References Anonymous (2008) CROPSTAT: a computer program for data management and basic statistical analysis. International Rice Research Institute, Los Banos, The Phillipines. Alejar MS, Zapata FJ, Senadhira D, Khush GS,Datta K (1995) Utilization of anther culture as a breeding tool in rice improvement. In: Terzi M, Cella R &Falavigna A (eds) Current Issues in Plant Molecular and Cellular Biology Kluwer Academic Publishers, Netherlands. Pp

7 Effect of cold pretreatment and phytohormones on anther culture efficiency of two indica rice (Oryza sativa L.) hybrids- Ajay and Rajalaxmi. 75 Ball ST, Zhou H, Konzak CF (1993) Influence of 2, 4-D, IAA, and duration of callus induction in anther cultures of spring wheat. Plant Science 90: Bishnoi U, Jain RK, Rohilla JS, Chowdhury VK, Gupta KR, Chowdhury JB (2000) Anther culture of recalcitrant indica x Basmati rice hybrids. Euphytica 114: Chu CC (1978) The N 6 medium and its applications to anther culture of cereal crops. Proc. Symp. Plant tissue culture, Beijing Grewal RK, Lulsdorf M,Croser J, OchattS,Vandenberg A,Warkentin TD (2009) Doubled haploid production in chickpea (Cicerarietinum L.): role of stress treatments. Plant Cell Reports 28: Gueye T,Ndir KN (2010) In vitro production of double haploid plants from two rice species (Oryza saliva L.and Oryza glaberrima Steudt.) for the rapid development of new breeding material. Scientific Research and Essays 5(7): Gupta HS, Borthakur DN (1987) Improved rate of callus induction from rice anther culture following microscopic staging of microspores in iron alum-haemotoxylin. Theoretical and Applied Genetics 74: Heberle-Bors E, Stöger E, Touraev A, Zarsky V, Vicente O (1996) In vitro pollen cultures: Progress and perspectives. In: Mohapatra SS and Knox RB (eds) Pollen Biotechnology. Gene Expression and Allergen Characterization. Chapman and Hall, New York Herath HMI, Bandara DC (2011) Anther culture performance in selected high yielding indica(of Sri Lanka) and japonica rice varieties and their inter sub-specific hybrids. Journal of National Science Foundation. 39: Khatun R, Shahinul Islam SM, Ara I, Tuteja T, Bari MA (2012) Effect of cold treatment and different media in improving anther culture response in rice (O. sativa L). in Bangladesh. Indian Journal of Biotechnology 11: Kiviharju E, Pehu E (1998) The effect cold and heat pretreatments on anther culture response of Avena sativa and A. sterilis. Plant Cell Tissue and Organ Culture 54: Krishnaraj S, Sreerangasamy SR (1993) In vitro salt tolerance screening in long-term anther culture of rice (Oryza sativa L.) variety IR 50. Journal of Plant Physiology 124: Lenka N, Reddy GM (1994) Role of media, plant growth regulators in callusing and plant regeneration from anthers of indica rice. Proceedings of the Indian National Academy of Sciences 60: Liu DY, Wang SD, Ding SL and Zhang MG (1983) The utilization of anther culture mcthod in the breeding of rice. In: J.H. Shen, Z.H, Zhang and S.D. Shi (Eds.), Studies on Anther Culture Breeding in Rice. pp Agricultural Press, Beijing. Mandal N, Gupta S (1995) Effect of and culture medium on androgenic callus formation and green plant regeneration in indica rice. Indian Journal of Experimental Biology 33: Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiologia Plantarum 15: Obert B, Dedieova B, Hricova A, Samaj J and Pretova A (2005) Flax anther culture: effect of genotype, cold treatment and media. Plant Cell Tissue and Organ Culture 79(2): Ogawa T, Fukuwa H, Ohakawa Y (1995) Plant regeneration through direct culture of isolated pollen grains in rice. Breeding Science 44: Pande H (1997) Androgenesis in anther cultures of an indica cultivar of Oryzasativa. Ph D Thesis.Delhi University. Delhi. Pande H,Bhojwani SS (1999) Promotion of androgenesis in rice anther culture by substitution of sucrose with maltose and mannitol. Biologia Plantarum. 41: Raina SK, Iyer RD (1974) A study of response to anther culture. Indian Journal of Genetics and Plant Breeding. 34 A: Raina SK, Zapata F (1997) Enhanced anther culture efficiency of indica rice (Oryzasativa L.) through modification of the culture media. Plant Breeding 116: Roininen IT, Tanhuanpaa P, Immonen S (2005) The effect of cold and heat treatments on the anther culture response of diverse rye genotypes. Euphytica 145: 1 9. Sen C, Singh RP, Singh MK, Singh HB (2011)Effect of Cold Pre treatment on Anther culture of Boro Rice Hybrids The International Journal of Plant Reproductive Biology 3: Silva TD,Ratnayake WJ (2009) Anther culture potential of indica rice varieties, KuruluThuda and BG 250.Tropical Agricultural Research and Extension 12: Touraev A, Indriato A, Wratschko I, Vicente O, Heberle- Bors E (1996) Efficient microspore embryogenesis in wheat

8 76 Rukmini et al., (Triticum aestivum L.) induced by starvation at high temperature. Sexual Plant Reproduction 9: Trejo-Tapia G, Amaya UM, Morales GS, Sanchez ADJ, Bonfil BM, Rodriguez-Monroy M and Jimenez-Aparicio A (2002) The effects of cold-pretreatment, auxins and carbon source on anther culture of rice. Plant Cell Tissue and Organ Culture 71: Xie JH, Gao M, Cai Q, Sheng X, Shen Y, Liang Z (1995) Improved isolated microspore culture efficiency in medium with maltose and optimized growth regulator combination in japonica rice (Oryza sativa). Plant Cell Tissue and Organ Culture 42: Zapata-Arias FJ (2003) Laboratory protocol for anther culture technique in rice. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants, a manual. Kluwer Academic Publishers, Dordrecht Zhu DY, Sun ZX, Pan XG, Ding XH, Shen XH, Won Y, Pan H, Yin JH, Alejar MS, Torrizo LB,Datta SK (1998) Use of anther culture in hybrid rice breeding. Proceedings of the 3rd International Symposium of Hybrid Rice Nov Hyderabad, India. In: Advances in Hybrid Rice Technology. International Rice Research Institute, Manila (Philippines). 21: )