OPTIMIZATION OF CONDITIONS FOR MICROPROPAGATION OF TWO COMMERCIAL SUGARCANE CULTIVARS CoPANT AND CoPANT 05224

Transcription

1 Journal of Cell and Tissue Research Vol. 13(3) (2013) (Available online at ISSN: ; E-ISSN: Original Article OPTIMIZATION OF CONDITIONS FOR MICROPROPAGATION OF TWO COMMERCIAL SUGARCANE CULTIVARS CoPANT AND CoPANT DOBHAL, U., SHARMA, M. D., SINGH, P., SHARMA, E., KUMAR, S. AND GAUR, A. K.? Molecular Biology and Genetic Engineering, College of Basic Science and Humanities, G.. B..Pant University of Agriculture and Technology, Pantnagar , Uttarakhand. E. Mail: Received: July 19, 2013; Accpted: August: 24, 2013 Abstract: A protocol for micropropagation of sugarcane through apical bud culture using two sugarcane varieties viz CoPant and CoPant was optimized. The apical buds used as explants were directly collected from field of N. E. Borlaug Crop Research Centre, Pantnagar in the month of September 2011 to April 2012.These apical buds were kept in MS medium fortified with different concentrations and combination of cytokinin benzyladenine purine BAP (1 mgl -1 to 5 mgl -1 ) and kinetin Kn (1 mgl -1 to 5 mgl -1 ) for contamination free establishment. After in vitro establishment, shooting was initiated within 15 days of growth period though shooting response was poor at an early stage, due to phenolic exudation. The problem of phenolic contamination was tackled to some extent by adding 0.2 % of insoluble polyvinylpyrrolidone (PVP) to culture medium. The shoots thus formed were transferred to multiplication medium containing 2 to 4 mgl -1 of BA, 1 mgl -1 of Kn and small quantity (0.5 mgl -1 ) of auxin indole -3 acetic acid (IAA). Maximum shoots were observed in medium supplemented with 2 mgl -1 of BA, 1 mgl -1 of Kn and 0.5 mgl -1 of IAA in both the varieties of sugarcane. MS medium (half strength) supplemented with different concentrations of auxins napthelene acetic acid (NAA) and Indole 3 butyric acid were used for in vitro root formation from proliferated shoots. Maximum roots were obtained in MS medium supplemented with 4 and 5 mgl -1 of NAA in both the varieties. The plantlets formed were successfully acclimatized and hardened in the month of April/May The response towards different hormonal combination was not different for these two varieties of sugarcane. Key words: Sugarcane cultivars, Copant 03220, Copant INTRODUCTION Sugarcane is an important cash crop that is cultivated for its stalks, which accumulate sucrose. It contributes 60% of the raw sugar produced worldwide, the remaining 40% coming from sugar beet [1] and has higher efficiency of converting solar energy into biomass than C3 plants, it is also one of the higher biomass yield crop. It is a major agricultural crop in tropical and sub-tropical regions of the world and important export product in many developing countries [2]. Sugarcane is commercially propagated from stem cutting or setts having two or three buds. This method requires large quantity of planting material, which is expensive as well as time consuming. After the development of clone/variety, major bottleneck in spreading of clone of or variety is slow propagation rate through conventional methods, which takes years [3]. Multiplication of desirable plants in vitro through biotechnological method is a very valuable approach in increasing plant productivity [4].Micropropagation is a reliable method 3921

2 J. Cell Tissue Research for rapid propagation and has been worked out by several workers [5-11]. This method has been successfully applied to breeding programs for rapid screening of clones for disease resistance, salt tolerance, drought tolerance, herbicide resistance and early maturity and high sugar contents. In sugarcane, micropropagation is important for rapid multiplication of elite genotypes/clones and for the quick spread of new varieties [12]. Micropropagtion is currently the only realistic means of achieving rapid, large scale production of disease free seed canes of newly developed varieties in order to speed up the breeding and commercialization process in sugarcane [13-17]. Plants raised through micropropagation are of uniform quality, pathogen free, can be produced much more rapidly as new cultivars could become commercially available within 2 to 3 years from development rather than 5 to 10 years needed using conventional propagation and can produce uniformly superior seeds and show improved vigour and quality. Two sugarcane varieties namely Isd-16 and Isd-28 from callus culture have been regenerated [18], the micropropagation of three elite sugarcane varieties by shoot tip culture has been reported [19] and commercial cultivar of sugarcane was regenerated using shoot tip culture [20]. Though sugarcane can also be propagated through callus culture and somatic embryogenesis [7,18,21-24], but it is not a reliable method of propagation because there are chances of mutation and somaclonal variation when plant are regenerated from somatic embryos [20]. Therefore present investigation was undertaken to establish a protocol for micropropagation of sugarcane using apical buds, which can be used further for mass propagation of improved variety for fast delivery to farmers, which in turn not allow pathogen attack on sugarcane, otherwise by the time sugarcane reach the farmers it becomes again susceptible to red rot or other diseases of sugarcane. The two important varieties of sugarcane CoPant and CoPant were used for the purpose, apical buds or shoot tips collected were used as explants as these are more responsive and fast growing part of the plant. MATERIAL AND METHODS Plant Material: The explants (apical buds) of two varieties CoPant and CoPant of sugarcane were collected from N.E Borlaug Crop Research Centre of G.B Pant University of Agriculture and Technology, Pantnagar. Establishment and in vitro shoot initiation: Physiologically younger tissue is generally much more responsive in vitro, therefore fresh and young apical buds of commercial sugarcane were used. These were washed thoroughly with tap water followed by a wash with 1 % (v/v) Labolene detergent for 15 minutes and then in running tap water for 30 minutes. Apical and axillary buds were surface sterilized with 70-90% ethyl alcohol for 30 seconds, followed by 0.1 % (w/v) HgCl 2 with two drops of Tween 80 per 100 ml. of solution for 1 minute. The explants were then rinsed 6 times with sterile double distilled water to remove all traces of HgCl 2 and blotted on sterile filter paper discs. The sterilized explants were inoculated on the MS medium fortified with varying range and combination of cytokinins (BA & Kn). All the cultures were incubated in an Environmental Chamber at 24 0 ± 2º C with 16: 8 hrs light: dark photoperiod controlled by clock timer and 60 % relative humidity. Shoot multiplication: The Shoots obtained from apical buds were transferred in multiplication medium fortified with cytokinin BA and Kn along with small quantity of auxin IAA (i.e. either 0.2 or 0.5 mgl -1 ). The multiple shoots thus excised were placed in rooting media. Root induction: The clumps of multiple shoots were separated and transferred to rooting medium supplemented with varying range of auxin(s) Naphthalene acetic acid and Indole Butyric acid. The rooted plantlets thus obtained were successfully transferred to the pots containing 1:1:1 ratio of sand, soil and compost for hardening. RESULTS Establishment and in vitro shoot initiation: The healthy apical buds of two varieties CoPant and CoPant were inoculated in MS medium supplemented with varying range and combination of cytokinin Benzyl Purine Adenine (BAP) and Kinetin (Kn). Initially the growth was very slow due to phenolic contamination in both the explants. The culture bottles get contaminated due to excess phenolic exudation. To combat this problem the media was fortified with 0.2% PVP. These chemicals solved 3922

3 Dobhal et al. Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig.1: Bud break in CoPant and Copant in MS medium fortified with BA and Kn after one week of growth period. Fig.2: Shooting response of CoPant in MS medium fortified with 2mgl 1 BA and 1mgl 1 of Kn. Fig. 3: Shooting response of CoPant in MS medium fortified with 2mgl 1 and 1mgl 1 of Kn. Fig. 4: Shooting response of CoPant in half strength MS medium fortified with 5mgl 1 of NAA Fig. 5 Fig. 5: Shooting response of CoPant in half strength MS medium fortified with 5mgl 1 of NAA 3923

4 J. Cell Tissue Research Table 1: Effect of different hormones on establishment of cultures. Values are mean of three replicates± S.D, Growth Regulators (mgl -1 ) No of shoot tips cultured CoPant 3220 CoPant No of established culture % of Established culture No.of shoot tips cultured Number of Established culture %of established culture 1BA:1Kn BA:1Kn BA BA Table 2: Effect of various concentrations and combination of plant growth regulators on shoot sugarcane varieties after 60 days of growth period. Values are mean of three replicates ± S.D differentiation in two Growth Regulators (mgl -1 ) Shoot Number Shoot length(cms) Shoot Number Shoot length (cms) CoPant CoPant BA : 1 Kn: 0.5 IAA ± ± ± ± 1.5 1BA: 1Kn:0.2 IAA ± ± ± ± BA:0.5Kn:0.5 IAA ± ± ± ± BA : 1 Kn ± ± ± ± 3.0 Table 3: Effect of different levels of auxins on root induction in two varieties of sugarcane. The Values are mean of three replicates ± S.D. Growth Regulators (mgl -1 ) Root Number Root length (cms) Root Number Root length (cms) CoPant CoPant NAA 6.66 ± ± ± ± 3.2 4NAA ± ± ± ± 1.5 5NAA ± ± ± ± IBA 7.33 ± ± ± ± IBA 3.66 ± ± ± ± 1.00 the problem of phenols, to some extent but cultures required frequent subculturing and the explants were subcultured every alternate day. Bud break occurs within fortnight and shoot appeared within few days of bud break. The shoots were excised from parent plant immediately, so as to protect it from phenolic contamination. The establishment percentage of the explants was very poor. The maximum 45 percent was obtained in CoPant 05224, in 1mgl -1 of BAP in combination with 1mgl -1 of Kn. In CoPant the maximum establishment (37.5%) was observed in combination of both the cytokinins BAP (2mgl -1 ) and Kn (1 mgl -1 ). Cytokinin BAP used alone was also able to initiate bud break and shooting but the response was very poor in comparison to BAP used in combination with Kn. Once established, the shoots were immediately transferred to multiplication media. The establishment percentage of CoPant was better in comparison to the CoPant variety of sugarcane. The combination of cytokinin(s) BAP and Kn was found to be better in comparison to the BAP used alone in both the varieties (Table 1, Fig 1). Shoot multiplication: The shoots one established were further subcultured for multiple shooting in MS medium fortified with varying range of Cytokinin BA and Kn along with auxin IAA. The highest number of shoots were obtained in MS media fortified with 2mgl -1 of BA in combination with 1 mgl -1 of Kn and 0.5 mgl -1 of IAA. In CoPant the mean numbers of shoots were with mean shoot length of 7 cms and that in CoPant was with mean shoot length 6.33, in the above mentioned combination in 60 days of study period. The shoot number and shoot length vary with varying combination (Table 2, Fig 2, 3) and combination of hormones but the response of two varieties was more or less the same in all the tried combinations of hormones. The shoots obtained were mostly healthy and easily separable. These shoots were then further subcultured. In all the combination 0.2% PVP was added in order to combat the problem of phenolic exudation, thus there was no problem of phenolic contamination and maximum number of cultured multiplication media showed good response and thus multiplication rate was high and resulted in well developed, healthy and easily separable plantlets. These plantlets thus obtained were placed in rooting medium Root proliferation: The plantlets or clumps of shoots obtained in multiplication media were separated and transferred to the rooting media. The half strength 3924

5 MS media was used as rooting media and was fortified with varying levels of auxins Naphthalene acetic acid (NAA) and Indole 3-Butyric acid (IBA) either alone or in combination. The lower concentrations (1-2 mgl -1 ) of both these auxins was not able to induce rooting in both the varieties, though higher level (3-5 mgl -1 ) of these auxins induced as well as established well differentiated and healthy roots within one week of growth period. Naphthalene acetic acid proved to be better hormone for root development as maximum number of roots (22) with root length 9 cms were obtained in Co Pant and 25 with 7 cms root in CoPant 05224, cultured in rooting media fortified with 5mgl-1 of NAA.Though the response in other concentration was also good in both the varieties (Table 3, Fig 4,5). The rooted plantlets thus obtained were successfully transferred to the pots containing 1:1:1 ratio of sand: soil: compost. DISCUSSION The apical buds of two cultivars CoPant and CoPant were inoculated with different concentrations and combinations of growth hormones for shoot initiations and establishment. The establishment percentage of the explants was very low due to the excessive exudation of phenols by these two varieties of sugarcane. The media get contaminated within few hours of inoculation due to the phenols produced by explants. Thus PVP was used in media for adsorbing these phenols and to protect the media from these phenols, this chemical solved the problem to some extent. The adsorption of phenols in the media prevents browning of tissues [5,8,9,15,28,35], the incorporation of activated charcoal, citric acid and PVP in medium helped in overcoming the problem of phenolic exudation in media [21,31,34]. In Sugarcane PVP was used to avoid phenolic compound in cultures [32, 33]. The two varieties studied showed more or less the same response towards different hormonal combination but phenolic contamination was more prevalent in CoPant 3220 in comparison to CoPant 5224.Thus the growth was slow in CoPant 3220, but eventually this variety showed good shooting and rooting response towards the hormonal combination in comparison to other variety. The MS media fortified with varying level of cytokinin BAP and Kinetin was used as shoot initiation media, cytokinin proved to be best for bud break and establishment of culture in both the varieties of sugarcane. Shoots of sugarcane Dobhal et al. cultivars on different BAP and kinetin concentrations have been obtained earlier [14], though other combinations have also been tried by other workers in different varieties of sugarcane, there is report of shoot induction and multiplication of sugarcane in 2 mgl -1 of BAP [3], 1 mgl -1 GA 3 and 1 mg/l Kn for organogenesis of sugarcane [29]. The maximum shoots were obtained in both the varieties in hormonal combination of cytokinin BAP & Kn and little quantity of auxin Indole Acetic Acid (IAA).The addition of IAA to the multiplication media proved to be synergetic in multiplication of shoots in the present study. The use of different auxins in small quantity along with cytokinin has been reported for inducing multiple shoots in sugarcane. There is a report of multiple shooting in sugarcane in MS media supplemented with 1 mgl -1 of BAP in combination with 0.1 mgl -1 of auxin GA 3 [19]. Multiplication of shoots in media supplemented with cytokinin BAP & Kn along with five quantity of auxin NAA [33], the use of cytokinin (BAP &Kn) combination for multiple shoot induction has also been stated [6]. Though shoot multiplication and elongation has also been reported in media devoid of cytokinin BAP [19], where multiple shoots were obtained in media comprising combination of cytokinin Kn with auxin IAA and IBA. There is a report that different genotypes give different response in different combinations of hormones [4]. The rooting was induced in half strength MS medium fortified with varying range of either NAA or IBA. The best rooting response with maximum number of roots as well as root length was obtained in 5 mgl-1 of NAA in both the varieties of sugarcane. Though roots were also obtained in media supplemented with IBA but response was best in NAA. Baksha et al. also reported NAA for best response of rooting in MS medium. He also reported rooting in combination of IBA with Bap but of poor quality. This is in agreement with our study. Root induction in 2mgl -1 of NAA [10] and rooting in liquid MS medium containing 5mgl -1 of NAA and 70 g/l of sucrose has been reported [3]. There is also an information of good rooting response in auxins IBA and IAA in different varieties of sugarcane. It was observed that IBA is superior over NAA in rooting response of three sugarcane variety HSF-240, CP and CPF-237 [20]. Best rooting was observed on media containing MS+ 1 mg/1 IBA + 6% sucrose [20] in mature sugarcane variety AEC But in our 3925

6 J. Cell Tissue Research study the use of higher concentration for NAA for best rooting response is recommended. Micropropagation can be used as best method for rapid multiplication of disease resistant variety. The disease resistant variety like red rot resistance as well as the variety with good agronomic traits like high ratooning, low water logging, high sugar contents and can be screened by marker assisted selection. These improved genotypes can be mass propagated through micropropagation. ACKNOWLEDGEMENTS This work is supported by Sugarcane Development Fund, Ministry of Food, Govt. of India. REFERENCES [1] Grivett, L. and Arruda, P.: Curr. Opin. Plant Biol., 5: (2002). [2] Heinz, D.M., Krisshnamurti, L., Nickel and Maretzki, A.: Applied and Fundamental Aspect of Plant Tissue and Organ Culture. Springer- Verlag, New York. pp (1977). [3] Cheema, K.L. and Hussain, M.: Inter. J. Agricul. Biol., 2: (2004). [4] Gupta, V., Sita, G.L., Shaila, M.S. and Jagannathan, V.: Plant Cell Rep., 12: (1993). [5] Anita, P., Jain, R.K., Schrawat, A.R. and Punia, A.: Indian sugar, 50: (2000). [6] Chattha, M.A., Imran, M.I., Abida, A., Muhammad, I. and Akhtar, A.: Pakistan Sugar J., 16: 2-6 (2001). [7] Gosal, S.S., Thind, K.L. and Dhaliwal, H.S.: Crop Improv., 2: (1998). [8] Hendre, R.R., Iyer, R.S., Kotwal, M., Khuppe, S.S and Mascarenheas, A.F.: Sugarcane, 1: 4-9 (1983). [9] Jadhav, A.B., Vaidya, E.R., Aher, V.B. and Pawar, A.M.: Indian J. Agric. Sci., 71: (2001). [10] Lal, N. and Krishna, R.: Indian sugar, 44: (1994). [11] Sauvaire, D. and Glozy, R.: CR Acad. Sci. Paris, Seri D, pp (1978). [12] Nickell, L.G. and Heinz, D.J.: N.Y. AM SRB Publications, 32: (1973). [13] Feldmann, P., Sapotille, J., Gredoire, P. and Rott, P.: Micropropagation of sugarcane. In: In vitro Culture of Tropical Plants. (Teisson, C. eds.). France: CIRAD: (1994). [14] Khan, S.A., Rashid, H., Chaudhary, F.M., Chaudhary, Z. and Afroz, A.: African J. Biotechnol., 7(13): (2008). [15] Lal, J., Pande, H.P. and Kawasthi, S.: A General Micropropagation Protocol for Sugarcane Varieties. New Botanist, 23: 1-19 (1996). [16] Lee, T.S.G.: Plant Cell Tiss. Org. Cult. 10: (1987). [17] Loranzo, J.C., Ojeda. E., Espinosa, A. and Borroto, C.: In vitro Cell Dev. Biol. Plan, 37: (2001). [18] Karim, M.Z., Alam, R., Baksha, R., Paul, S.K., Hossain, M.A and Rahman, A.B.M.M.: Pak. J. Biol. Sci., 5: (2002). [19] Khan, I.A., Dahot, M.U., Yasmin, S., Khatri, A., Nighat, S. and Naqvi, M.H.: Pak. J. Bot., 38(4): (2006). [20] Biradar, S., Biradar, D.P., Patil, V.C., Patil, S.S. and Kambar, S.S.: Karnataka, J. Agric. Sci., 22(1): (2009). [21] Bhansali, R.R. and Singh, K.: Phytomorphol, 32, (1984). [22] Islam, R., Haider, S.A., Alam, M.A. and Joarder, O.I.: Rice Biotech Quart., 25: 8 (1996). [23] Nadar, H.M., Soepraptop, S., Heinz, D.J. and Ldd, S.L.: Crop Sci., 18: (1978). [24] Shukla, R., Khan, A.Q. and Garg, G.K.: Sugarcane, 4: (1994). [25] Chawla, H.S.: Introduction to Plant Biotechnology. Science Publishers Inc., Enfield, New Hampshire., pp (2002). [26] Fridborg, G., Pedersen, M., Landstorm, L.E and Eriksson, T.: Physiol. Plant, 43: (1978). [27] George, E.F and Sherrington, P.D.: Plant propagation by tissue culture. Handbook and Directory of Commercial Laboratories. Exegenetics Ltd., Basingstoke, Hants, England, pp (1984). [28] Horner, M., McComb, J.A., McComb, A.J. and Street, H.E.: J. Exp. Bot., 28: (1977). [29] North, J.J., Ndakidemi, P.A. and Laubscher, C.P.: Plant Omics. J. 4(2): (2011). [30] Weatherhead, M.A., Burdon, J. and Henshaw, G.G.: Effects of activated charcoal as an additive to plant tissue culture media. Z. Pflanzenphysiol., 94: (1979). [31] Kumar, V., Radha, A. and Chitta, S.K.: Plant Cell Rep., 17(9): (1998). [32] Skirvin, R.M. and Chum, M.C.: Hort. Science, 14: (1979). [33] Siddiqui, S.H., Khatri, A., Javed, M.A., Khan, I.A. and Nizamani, G.S.: Pak. J.Agric. Res., 15 (1): (1994). [34] Singh, N., Kumar, A. and Garg, G.K.: Indian J. Biotechnol., (5): (2006). [35] Saini, N., Saini, M.L. and Jain, R.K.: Ind. J. Gent., 64: (2004). 3926