Plant Cell, Tissue and Organ Culture 10:47-55 (1987) Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands 47 Short communication Micropropagation of sugarcane (Saccharum spp.) TSENG SHENG GERALD LEE IAA/PLANALSUCAR, Caixa Postal 153, 13.600, Araras-SP, Brazil Received 22 May 1986; accepted in revised form 24 March 1987 Key words: Saccharum spp., sugarcane, micropropagation, shoot tip culture, tissue culture Abstract. Micropropagation of sugarcane (Saccharum spp.) was studied using two procedures: (1) shoot tip culture; (2) indirect somatic embryogenesis from callus. Shoot tip culture was considered a better method for micropropagation, since it produced plants phenotypically similar to the mother plant and gave a much more rapid multiplication rate when compared to the other procedure. Introduction The lack of multiplication procedures has long been a serious problem in sugarcane breeding programs. Usually after 10 to 15 years of work to complete a selection cycle, an improved variety can be planted commercially only several years later, when enough seedcanes have been produced. The time spent for this multiplication is considered a serious economic problem, mainly in view of the higher yields that would be obtained by planting the new variety earlier on a large commercial scale. Furthermore, it is also quite possible that the new variety could enter in its degenerative cycle earlier, because of the continued contamination by systemic diseases which quite often happens during the multiplication stage in the open field. Tissue culture techniques have been used for sugarcane multiplication. Barba et al. [1] reported on a callus culture method which, within 931 months, produced planting material from a single spindle sufficient to plant a hectare. Sauvaire and Galzy [9] described a method using axillary buds for micropropagation of sugarcane. Recently, shoot tip culture was also reported for sugarcane mass propagation [3,4,6]. The work described here aimed at comparing two methods of sugarcane micropropagation.
- Kinetin - arginine 48 Materials and methods Sugarcane plants, variety RB735275, were obtained from the Experimental Station of PLANALSUCAR (Institute of Sugar and Alcohol-IAA) in Araras-SP, Brazil. Single buds from cane plant were sterilized in a 20% commercial hypochlorite solution for 40 minutes, washed with water and planted in vermiculite in a growth chamber at 37 C for 15 days. Two procedures were used for sugarcane micropropagation: Procedure 1: shoot tip culture and multiplication in MP II medium; Procedure 2: callus culture and multiplication in MC (callus proliferation) and MP (tillering) media. When the seedlings were about 20-25 cm tall, stem segments of about 4 cm, together with meristem tip, were excised. They were sterilized for 1 minute in 70% alcohol and 20 minutes in 20% hypochlorite solution. The segments were then washed 3 times with water under sterile conditions and shoot tip was dissected as described before [4, 6]. A spindle leaf near the meristem tip, 0.5-1.0 cm in length, was used for callus culture. Media used for callus culture were MC for callus induction and proliferation and MP for plant regeneration. A half-strength medium of Murashige and Skoog salts (1/2 MS) was also used, mainly for growth of plantlets. MP Table 1. Media used for callus induction (MC), shoot differentiation (MP) and shoot tip culture (MP II). Ingredients Callus Shoot Shoot tip induction differentiation culture (mg/1 ) (mg/1 ) (mg/1 ) 1. Macronutrients MS MS MS 2. Micronutrients MS MS MS 3. Vitamins and Hormones - Myo-inositol 100 100 100 - Thiamine-HCl 1 1 1-2,4-D 3 - - 1 0.1 - NAA - 1 - - BAP - - 0.2 4. Other supplements coconut milk - 10% (v/v) - 5. Amino acid 60 - - 6. Sucrose 20.000 20.000 20.000 7. Agar 8.000 8.000 - ph before autoclaving 5.8 5.8 5.8
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II was used for shoot tip culture. The composition of these media is shown in Table 1. Tissue culture was conducted in a growth room with a 16 hour photoperiod and 37.5 #E.S -1.M -2 of light intensity measured 20 cm above the cultures. The temperature in this room was kept between 25-30 C. The size of the experiment was described under each table. Each experiment was repeated at least twice. The resulting plantlets were counted and transplanted into polystyrene wells containing vermiculite and transferred to either a greenhouse or a nursery bed covered with plastic sheets. The plants were transfered to the field after being acclimatized for 2 months as described elsewhere [5]. Judgment of the variation of the plants were done by visual observation. 51 Results and discussion Tables 2 and 3 show the multipliction rate attained for each procedure. Shoot tip culture was apparently a better technique for propagation purposes. In 3 month, 78,408 plantlets were produced while by the callus culture method only 896 plantlets were obtained after 5 months in culture (Figs 1, 2, 3, 4). After the second passage of plantlets in MP, they can be cultured in 1/2 MS. The plantlets grew much faster in the medium, but the multiplication rate was impeded. Fig. 1. Shoot tip development of sugarcane in MP II medium. (a) 0 days; (b) 3 days; (c) 18 days; (d) 25 days.
52 Fig. 2. Multiplication of plantlets obtained from shoot tip in MP II medium. Procedure 1 produced normal plants during 7 cycles of multiplication. After this, a green mass started to appear at the base of the formed shoots. Procedure 2 started to produce growable plantlets in MP 3 (the 3rd subculture in MP) and in MP 4 some albino plants were observed. The variability of plants produced by procedures 1 and 2 was compared Fig. 3. Shoot regeneration on spindle leaf callus in MP medium.
53 Fig. 4. Fully developed shoots obtained from shoot tip culture in MP II medium (left) and fully differentiated plantlets obtained from callus culture in MP medium (right). Both have been cultured in the laboratory for 5 months, period starting from the explant stage, with that of their mother-plants after one year growing in the field, by visual observation. Procedure l apparently produced normal plants but they were usually more vigorous than their mother-plants. It is likely that these plants were healthier than the mother-plants. On the other hand, some variability Fig. 5. Plants obtained from shoot tip culture under acclimatization.
54 Fig. 6. Plants obtained from shoot tip culture growing in the field (right) together with the normal mother-plants (left). was constantly observed among plants produced by procedure 2. This procedure has been used as routine method for producing somaclonal variant plants at PLANALSUCAR'S laboratory, as well as in many other laboratories [2, 7, 8]. Since theoretically more callus should produce more plantlets an attempt was made to repeatedly subculture callus in MC in order to increase its production. Unfortunately, as the number of passages in MC increased, less plantlets were regenerated in MP. With regard to the two methods tested, only procedure 1 seems to be
viable. As may be seen from Table 2, before any abnormal shoots appeared, enough material had been obtained. The 78,408 plantlets produced in three months, with a further two months of acclimatization, could be used for planting about four hectares (Figs. 5, 6). Any laboratory with a culture room of 3 x 7 m could easily produce this amount. The multiplication rate achieved by using MP II as the medium varied according to the variety. Since each multiplication cycle takes only 15 days, the difference in number could be easily achieved by doing one or two more cycles. Thus, the procedure could be a viable method for propagation of newly released varieties or promising clones. 55 Acknowledgements The author wishes to express his thanks to Dr. L. Sodek, Dr. S. Matsuoka, and Dr. Y. Masuda for their suggestions and revision of this manuscript. Thanks are also due to Mrs. S. Perissatto Maneghin and Miss L.T. Picollo for their technical assistance. The cooperation of mrs. M.L.F. Mazetto is also appreciated. References 1. Barba RC, Zamora AB, Mallion AK, Linga CK (1978) Sugarcane tissue culture research. Proc Int Soc Sug Cane Technol 16:1843-1863 2. Heinz D J, Krishnamurthy M, Nickel LG, Maretzki A (1977) Cell, tissue and organ culture in sugarcane improvement. In: Reinert J, Bajaj YPS (eds) Plant Cell, Tissue and Organ Culture. Berlin: Springer-Verlag, pp. 3-17 3. Hendre RR, Iyer RS, Kotwal M, Khuspe SS, Mascarenhas AF (1983) Rapid multiplication of sugar cane by tissue culture. Sugarcane 1:5-8 4. Lee TSG (1984) Micropropaga~ao de cana-de-aqticar atrav6s de cultura de meristema apical. Saccharum APC 7:36-39 5. Lee TSG, Bacchi OOS (1984) Improved rooting of differentiated shoots from sugarcane callus tissue. Turrialba 34:481-484 6. Lee TSG (1986) Multiplication of sugarcane by apex culture. Turrialba 36:231-235 7. Liu MC (1981) In vitro methods applied to sugar cane improvement. In: Thorpe TA (ed) Plant Tissue Culture; Methods and Applications in Agriculture. New York: Academic Press, pp 299-323 8. Krishnamurthy M, Tlaskal J (1974) Fiji disease resistant Saccharum officinarum var. Pindar sub-clones from tissue cultures. Proc Congr Int Soc Sug Cane Technol 15:130-137 9. Sauvaire D, Galzy R (1978) Multiplication v6g&ative de cantle h sucre (Saccharum sp.) par bouturage in vitro. CR Acad Sc Paris, S6rie D: 467-470