Sarhad J. Agric. Vol.24, No.4, 2008 SOMATIC EMBRYOGENESIS AND PLANTLET FORMATION IN DIFFERENT VARIETIES OF SUGARCANE (Sacchrum officinarum L.) HSF-243 AND HSF-245 SHAGUFTA NAZ, AMIR ALI and AQSA SIDDIQUE Botany Department, Lahore College for Women University, Lahore Pakistan Email : drsnaz 31 @hotmail.com ABSTRACT An efficient protocol was developed for the induction of callus, somatic embryogenesis and regeneration response of two sugarcane varieties: HSF-243 and HSF-245, in this study. Callus was induced on young leaf explants cultured on Murashige and Skoog s medium supplemented with different concentration of 2, 4-D singly or in combination with BAP. The highest percentage of callus induction was observed in the medium containing 3mg/l of 2, 4-D. Callus was sub-cultured on MS medium supplemented with different growth regulators for the induction of somatic embryogenesis. Direct and indirect somatic embryogenesis was observed from both varieties. The best performance, with the different concentrations, was observed on MS medium supplemented with 2, 4-D 2.0 mg/l in HSF-243. Regeneration through embryogenic callus was observed on MS medium along with 2.0 mg/l 2, 4-D and 1.0 mg/l BAP, after 6-8 weeks where the regeneration percentage was 85. The maximum plantlets formation was achieved when embryogenic calluses were shifted to basal media. Key Words: 2, 4-D (2, 4 Dichlorophenoxyacetic Acid), Bap (Benzylaminopurine), IAA (Indole Acetic Acid), MS (Murashige And Skoog S Medium), Sugarcane (Saccharum officinarum), Callogenesis, Somatic Embryogenesis, Leaf Whorls As Explants Citation: Naz, S., A. Ali and A. Siddique. 2008. Somatic embryogenesis and plantlet formation in different varieties of sugarcane (Sacchrum officinarum L.) HSF-243 and HSF-245. Sarhad J. Agric. 24(4): 593-598. INTRODUCTION Tissue culture of sugarcane has received considerable research attention because of its economic importance as a cash crop. Sugarcane has been grown in different parts of the world, from the tropics to subtropics, and accounts for around 60% of the world s sugar. It is also one of the important cash crops in many developing and developed countries, with a high trade value. The importance of sugarcane has increased in recent years because cane is an important industrial raw material for sugar industries and allied industries producing alcohol, acetic acid, butanol, paper, plywood, industrial enzymes and animal feed. Another important use of sugarcane is the production of press mud, which is used as a source of organic matter and nutrients for crop production (Raja, 2006). Sugarcane is one of the most important cash crops of Pakistan. Pakistan lies outside of the tropical zone in which sugarcane thrives. Although Pakistan is the fifth largest sugarcane growing country in the world,but its per acre yield is lowest in the world. Average production of sugarcane in different countries of the world is about 800-00 mound per acre while in Pakistan it is only 458 mound per acre and sugar recovery of Pakistan was around 8.5%, which is the real cause of the high cost of production (Imam, 2001). There are several reasons for low yields of sugarcane and low sucrose recovery from the cane. About twothirds of the sugarcane area remains under ratoon crops, which reduces output, particularly in the northern areas where frost is prevalent in winter. Among the several possible reasons, the most important one is non-availability of disease free elite stock for seeding (Ali et al. 2008). Lack of advanced technologies in sugarcane propagation, and lack of multiplication procedure has long been a serious problem in sugarcane breeding program. Usually to 15 years of work is required to complete a selection cycle, and an improved variety can be planted commercially several years later, when enough seed canes will have been produced (Ali et al. 2008). Currently, the use of in vitro technology for improvement of sugarcane is an alternative to the conventional methods, because it is rapid, reliable and sustainable option (Khan et al., 2004). Sugarcane tissue culture has received considerable research attention because of sugarcane s economic importance as a cash crop. Plant regeneration through tissue culture technique would be a viable alternative for improving the quality and production of sugarcane. Initial attempts to regenerate plants through in vitro technique were made on sugarcane by Nickell (1964) and Heinz and Mee (1969).
Shagufta Naz et al. Somatic embryogenesis and plantlet formation in sugarcane varieties 594 Somatic embryogenesis technique has the potential to improve plants by producing a high frequency of somaclonal variants. It is also an important pathway for regeneration of plants from cell culture system. It is also a method commonly used in large scale production of plants and synthetic seeds (Philips and Gamborg, 2005). In somatic embryo, somatic cells develop by rapid division to form complete embryo similar to that of zygotic embryo. Both embryos undergo basically the same stages of development, namely globular, heart shaped, torpedo, cotyledonary and mature stages (Pareek, 2005). Somatic embryogenesis is an efficient and high volume propagation system for the production of large number of plants within a short period (Arencibia, 1998). Plant regeneration through somatic embryogenesis has been reported in sugarcane using young leaf rolls and immature inflorescences (Guiderdoni et,al 1995). In this and other studies, embryogenic callus was induced in the presence of 2, 4 dichlorophenoxyacetic acid (2, 4-D) as reported by Aamir et al. (2007) Direct somatic embryogenesis offers several advantages in crop improvement; cost-effectiveness and largescale clonal propagation is possible using bioreactors, which ultimately leads to the automation of somatic seed production and development of artificial seeds (Vasil, 1987; Philips and Gamborg, 2005). Recently, Eudes et al. (2003) reported the induction of direct somatic embryogenesis and regeneration of fertile plants in cereals. In sugarcane, direct somatic embryogenesis has only been reported using young leaf rolls and the culture system has been employed for genetic transformation (Snyman, et al., 2000). The present study was conducted to establish the callogenesis response and induction of somatic embryogenesis in HSF-243 and HSF-245 and to develop a reproducible protocol for efficient regeneration of these varieties through somatic embryogenesis. MATERIALS AND METHODS Plant Materials The explants of two different varieties, HSF-243 and HSF-245, were obtained from Ayub Agriculture Research Centre, Faisalabad. Leaf tissues were used as explants. The field collected plant material was washed several times with tap water with a few drops of liquid soap. The outer old leaf-base coverings were removed carefully without damaging the inner young and delicate tissue. The explant was then surface sterilized with ethanol to avoid contamination. Callus Induction Leaf tissues of 3.0-6.0 mm were excised and inoculated on MS (Murashige and Skoog, 1962) medium supplemented with different ratios of auxins and cytokinins either singly or in combinations to identify the appropriate media combinations for regeneration of sugarcane through callogenesis and somatic embryogenesis as mentioned in Table I. MS media consisted of MS basal salts (Macro, micronutrients, Iron EDTA and vitamins) with 3% sucrose, ph was adjusted to 5.5-5.7 and autoclaved at 120 C for 20 min at 5 kpa. Difco bacto agar was used as gelling agent. Explants were incubated at 23±2 C under 16-18 h light period (from fluorescent light tubes) and 6-8 h dark with light intensity of 2000-3000 lux or in dark. Induction of Somatic Embryo Embryogenic response was observed by the presence of direct somatic embryos on the cultured explant after four week culture period and the number of regenerated plants were recorded per explant after six-week culture period in both varieties. However, indirect somatic embryogenesis and number of regenerated plants were observed after shifting on selected media (different concentrations of auxins and cytokinin as mentioned in Tables II and III). Regeneration of Plantlets from Somatic Embryos For obtaining maximum proliferation of plantlets, regenerated plants were shifted to basal media and combinations of BAP and IAA for further growth (Table IV). RESULTS AND DISCUSSION Callus Formation in HSF-243 and HSF-245 Callus induction was observed within three-weeks after inoculation from the leaf sheath explants on modified MS medium containing different concentrations of 2,4-D and combinations of 2,4-D and BAP (Table I). It
Sarhad J. Agric. Vol.24, No.4, 2008 595 was evident that the callus induction was triggered on all concentrations, but the best callus induction was observed on MS medium supplemented with 2, 4-D 3 mg/l from leaf explant of both varieties (HSF-243 and HSF-245). On this medium composition, the explant of HSF-243 produced pale yellow color, less compact, nodular callus and on the other hand explants of HSF-245 produced golden yellow, compact, friable callus. The percentage of callus induction was 95% and 98% respectively (Table I, Fig. 1B). Different combinations of auxin-cytokinin interaction were also used for callus induction and proliferation. The variety HSF-243 showed good response on MS6 medium i.e. MS medium supplemented with 2mg/l of 2, 4-D and 1mg/l of BAP, which was 80%. However, HSF-245 responded on MS5 medium with 85% callus induction i.e. MS medium supplemented with 1 mg/l of 2, 4-D and 1 mg/l of BAP. Therefore, it was concluded that 2, 4-D alone showed best results for callus growth and proliferation in both varieties. Similar results were also reported by Alam et al., (2003), where they observed that callus formation from leaf sheath explants of sugarcane (Sacchrum officinarum) cultivars; Isd 18, Isd 20 and B34-4 was observed within 3 weeks of incubation on modified MS medium supplemented with 2,4-D ranging from 1.0 to 4.0 mg/l and coconut milk. Effective calluses were formed on the medium supplemented with 3.0 mg/l 2, 4-D and % coconut milk. Hao et al., (2001) reported that the MS medium with 2, 4-D at 1.0-3.0 mg/liter was better for callus induction. Lal (2003) studied a procedure for obtaining high frequency plant regeneration from the callus of sugarcane cv. B.O.91. Subapical slices and leaf roll explants of sugarcane formed callus on MS medium supplemented with 2,4-D at 5 mg/l. Dry matter content of callus was highest (12%) at 5mg/l 2,4-D level. Callus was maintained on MS medium with 2, 4-D at 3 mg/l. Somatic Embryogenesis in HSF-243 AND HSF-245 Direct somatic embryogenesis without intervening callus phase and indirect somatic embryogenesis with callus phase has also been reported in sugarcane. In this study, explants and proper combinations of growth regulators were used for the induction of direct and indirect somatic embryogenesis in HSF-243 and HSF- 245. Among different concentrations and combinations for direct somatic embryogenesis, best performance was noticed on MS medium supplemented with 2, 4-D 2.0 mg/l and BAP 1mg/l in HSF-243. (Fig. 1b) On this combination, the percentage of induction of direct somatic embryogenesis was 85% (Table II) and the rate of regeneration was 75%. However,,the best medium for the induction of indirect somatic embryogenesis was MS supplemented with 2, 4-D 3mg/l. The result for the induction of indirect somatic embryogenesis was 80% and the rate of regeneration was 70% (Table III). Different concentrations of 2, 4-D from 1 mg/l to 4 mg/l were used for the induction of somatic embryogenesis in HSF-245. Among different combinations for direct somatic embryogenesis, the best performance was observed on MS medium supplemented with 2, 4-D 1.0 mg/l and BAP 1mg/l in HSF-245. On this combination the percentage of induction of somatic embryogenesis was 90% and the rate of regeneration was 85% (Table II). However, maximum responsive media for the induction of indirect somatic embryogenesis were MS2 medium MS medium supplemented with 2, 4-D 2mg/l and MS6 medium i.e. with 2mg/l of 2, 4-D and 1mg/l of BAP(Fig. C, D) The percentage of induction of indirect somatic embryogenesis was 85% and the rate of plant regeneration via indirect somatic embryogenesis was 60% from MS2 medium For MS6 medium result for the induction of indirect somatic embryogenesis was 80% and the rate of regeneration was 75% (Table III).Various researchers have also reported similar results of somatic embryogenesis on different varieties of sugarcane. Manickavasagam and Ganapathi (1998) reported direct somatic embryogenesis and plant regeneration from leaf explant of sugarcane cultured on MS medium supplemented with 1,2,3 or 4 mg, 4-D/l alone or with 0.5,1 or 2mg BAP/l. Somatic embryos were formed in most treatments within 12-15days without a callus interphase. Best results were obtained with 2mg 2, 4-D+1mg BAP. Kaur et al., (2001) reported that the medium MS + 2, 4-D + BAP (0.5 mg/l) induced excellent callusing in the spindle explants. The calluses maintained through sub culturing on MS medium supplemented with proline resulted in good somatic embryogenesis. Geetha and Padamanabhan (2001) reported the effect of hormones on direct somatic embryogenesis in sugarcane. The effects of 2, 4-D (2, 3 and 4 mg/l) alone or in combination with BA (0.5 and 1.0 mg/l) on the somatic embryogenesis of sugarcane.was observed. The treatment with 3 mg 2, 4-D + 0.5 mg BA produced the highest number of somatic embryos (48). Gill et al., (2004) reported factors affecting somatic embryogenesis and subsequent plant regeneration in vitro in sugarcane. The cultures derived from different sugarcane varieties were
Shagufta Naz et al. Somatic embryogenesis and plantlet formation in sugarcane varieties 596 highly genotype specific. They were affected by medium constitution, auxins, sugars, amino acids, growth retardants, and desiccation among others. Fig.1 Somatic embryogenesis and plantlet formation in different varieties of Sugarcane : A) Induction of direct embryoids on the cut surface of explant on medium containing 2.0 mg/l 2,4-D (6x) B) Embryogenic callus grown with 1.0 mg/l BAP.+2.0mg/l2,4-D (2x) C) Somatic embryos on globular stage on medium supplemented with 3.0 mg/l 2,4-D( 40x) D)Heart shaped embryos after four weeks of incubation on same medium (40x) E) Conversion of embryos in to multiple shoots aon medium 2.0mg/l BAP +1.0mg/l IAA (2x) F)Elongation and shoot mass propagation after 8 weeks (6x) G) Root formation within -15 days after transferred to the root induction medium (x). Table I. Effect of Different Concentrations of Hormonal Combinations on Callogenesis from Leaf Explants in HSF-243 and HSF-245
Sarhad J. Agric. Vol.24, No.4, 2008 597 Table II. Effect of different concentration of hormones and hormonal combinations on direct somatic embryogenesis in HSF-243 and HSF-245 Table III. Effect of different concentration of hormones and hormonal combinations on indirect somatic embryogenesis in HSF-243 and HSF-245 Table IV. Plantlets formation in HSF-243 and HSF-245 Treatment no. Media Conc. mg/l No. of cultures examined Age of culture Plant formation HSF-243 HSF-245 (Weeks) (%) HSF-243 HSF-245 MS9 Basal 12 90 95 MS MS11 BAP + IAA 1+1 2+1 12 12 55 75 65 80 MS12 2+2 12 40 50 Effect of Media Composition on Plantlets Proliferation For plantlet multiplication, basal medium was used along with different combinations of IAA and BAP at different concentrations. The number of regenerated plants was recorded per explant after twelve-week of culture period. The best results for regeneration were found on basal media, which was 90% for HSF-243 and 95% for HSF- 245. Among different combinations of IAA and BAP (MS-MS11), better response was observed with MS11 medium i.e. MS medium supplemented with 2mg/l of BAP and 1mg/l of IAA. The result for regeneration in HSF- 243 was 75 %. (Table IV, Fig. 1E). However, 80% regeneration was shown by HSF-245 in MS11 medium i.e. MS medium supplemented with 2mg/l of BAP and 1mg/l of IAA (Table IV, Fig. 1 F, G). Pawar et al., (2002) reported rapid multiplication of sugarcane cultivars Co-86032, Co-8014 and CO-740 on MS medium supplemented with 0.5 mg IAA, BAP, and kinetin/liter. Naz (2004) reported the rapid multiplication and acclimatization response of 17
Shagufta Naz et al. Somatic embryogenesis and plantlet formation in sugarcane varieties 598 different varieties of sugarcane. Apical meristems of these varieties were cultured on MS medium supplemented with combinations of BAP with KIN and NAA. Lower concentration of BAP showed higher rate of multiple shoot formation. Half strength MS medium with 5mg/l NAA exhibited the high rate of root formation within -15 days. CONCLUSION Summarizing the main findings it is concluded that 2,4-D is more potent to callus initiation as compared to other hormonal combinations.callus was subcultured in different media for induction of somatic embryogenesis.the present study reported the high percentage of plantlet formation from leaf tissue in both varieties of sugarcane, HSF- 243 and HSF-245.The present established protocol can be used further for genetic improvement of sugarcane. REFERENCES Ali, A., S. Naz.and J. Iqbal. 2007. Effect of different explants and media compositions for efficient somatic embryogenesis in sugarcane. (Sacchrum officinarum). Pak. J. Bot. 39(6): 1961-1971. Ali, A., S. Naz, F.A. Siddique and J. Iqbal. 2008. Rapid clonal multiplication of sugarcane through callogenesis and organogenesis. Pak. J. Bot. 40(1) :123-138. Ali, A., S. Naz, F.A. Siddique and J. Iqbal. 2008. An efficient protocol for large scale production of sugarcane through micropropagation. Pak. J. Bot. 40(1) :139-149. Alam, R., S.A. Mannan, Z. Karim and M.N. Amin. 2003. Regeneration of sugarcane (Sacchrum officinarum) plantlet from callus. Pak. Sugar J. 18, 15-19. Arencibia, A. 1998. Gene transfer in sugarcane. In: Biotechnology of Food Crops in Developing Countries, Springer-Verlag, New York. pp. 79. Eudes, F., S. Acharya, L.B. Selinger and K.J. Cheng. 2003. A novel method to induce direct somatic embryogenesis, secondary embryogenesis and regeneration of fertile green cereal plants. Plant Cell Tissue Org. Cult. 73, 147 157. Geetha, S. and D. Padmanabhan. 2001. Effect of hormones on direct somatic embryogenesis in sugarcane. Sugar Technol. 3,120-121. Gill, N.K., R. Gill and S.S. Gosal. 2004. Factors enhancing somatic embryogenesis and plant regeneration in sugarcane (Saccharum officinarum L.). Ind. J. Biotechnol. 3, 119-123. Guiderdoni, E., B. Merot, P.F. Klsomtramage, P. Feldman and J.C. Glaszman. 1995. Somatic embryogenesis in sugarcane. In Biotechnol. in Agric. and Forest. (ed. Y.P.S. Bajaj), Springer- Verlag, Berlin. pp. 92 113. Hao, T., W. Zi Lin, L.H. Ming, P.S. Ming, Q.W. Yong and Z.D. Huo. 2001. Rapid propagation techniques for tissue culture of chewing cane. Fuji. J. Agric. Sci. 16, 49-52. Heinz, D.J. and G.W. Mee. 1969. Plant differentiation from callus tissue of Saccharum species. Crop Sci. 9: 346-348. Imam, P. 2002. Sugar Industry: Problems, Prospects. The Daily Dawn Econ. and Business Rev. Feb. 4-, 2002. Kaur, A., S.S. Goasal, R. Gill and K.S. Thind. 2001. Induction of plant regeneration and somaclonal variation for some agronomic traits in sugarcane (Sacchrum officinarum L.). Crop Improv. 28: 167-172. Khan,I.A., A. Khatri, M. Ahmed, S. Raza, N. Seema, G.S. Nizamani, Khanzada, and M.H. Naqvi. 2004. Syudy of genetic variability in regenerated sugarcane plantlets derived from different auxin concentration. Pak. Sugar J. 19(6):35-38. Lal, N. 2003. High frequency plant regeneration from sugarcane callus. Sugar Technol. 5: 89-91. Manickavasagam, M.and A. Ganapathi. 1998. Direct somatic embryogenesis and plant regeneration from leaf explants of sugarcane. Ind. J. Exp. Bio. 36:832-835. Murashige,T. and F. Skoog. 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15:473-497. Naz, S. 2004. Micropropagation of promising varieties of sugarcane and their acclimatization response. Pak. Sugar J. 19: 58-64. Nickell, L.G. 1964. Tissue culture of sugarcane, another research tools. Hawaiian Planters Res., 57, 223-229. Pareek.L.K. 2005. Trends in Plant Tissue Culture and Biotechnology. Agrobios. 11:334. Philips, G, C. and O.L. Gamborg. 2005. Plant cell tissue and organ culture. pp. 91-93. Published by N.K. Mehra.Narosa publishing house 6, community centre.new Delhi, India. Parrott, W. A., S.A. Merkel and E.G.Williams. 1991. Somatic embryogenesis: Potential for use in propagation and gene transfer systems. In Adv. Methods in Plant Breed. and Biotechnol. (ed. D.R. Murray). pp. 158 200. Pawar, S.V., S.C. Patil, V.M. Jambhale, R.M. Naik and S.S. Mehetre. 2002. Rapid multiplication of commercial sugarcane varieties through tissue culture. Indian Sugar. 52: 183-186. Raja, N. and H. Abbas. 2006. Sugarcane Overview, Http://www.pakistan/com/English/allabout/crop/sugarcane.shtml (Verified Aug. 26, 2006). Snyman, S.J., M.P. Watt, F.C. Huckett and B.I. Botha. 2000. Direct somatic embryogenesis for rapid, cost-effective production of transgenic sugarcane (Saccharum spp. hybrids). Proc. South African Sugar Tech. Assoc. 74: 186 187. Vasil, I. K. 1987. Developing cell and tissue culture systems for the improvement of cereals and grass crops. J. Plant Physiol. 128: 192 218.