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  1. 1. African Crop cience Conference Proceedings, Vol. 6. 113-118Printed in Uganda. All rights reservedISSN 1023-070X $ 4.00© 2003, African Crop Science Society Carry-over effect of thidiazuron in banana in vitro propagation at different culture cycles and incubation conditions A. M. MAKARA, P. R. RUBAIHAYO & M.J.S. MAGAMBO Department of Crop Science, Faculty of Agriculture, Makerere University, P. O. Box 7062, Kampala, UgandaAbstract: Thidiazuron (TDZ) is an active cytokinin that was shown to induce increased shoot proliferation and habituationin black walnut, Phaseolus lunatus and evergreen azalea but has not been widely investigated in bananas. In particular,the quality of transforming tissues from cytokinin dependence to cytokinin autonomy makes use of TDZ cost effective butthere is lack of information on this quality in banana micropropagation. A study was therefore conducted to investigate thecarry over effect of varying concentrations of TDZ and 22.2 µM BAP as control on proliferation of five banana cultivarson a hormone free medium under various incubation conditions. The results showed that TDZ had a carry-over effect thatenabled shoots to continue proliferating on a hormone free medium as the culture cycles increased and that this effect wassignificantly (P<0.05) higher than that of BAP. Accumulation of TDZ to high levels resulted in suppression of shootproliferation. The results further showed dark conditions enhanced higher proliferation rates than light conditions in somecultivars suggesting that in vitro proliferation is a photomorphogenically responsive process that is enhanced under darkconditions.Key words: TDZ, BAP, micropropagation, proliferation rates, recalcitrant, cultivars Introduction (BAP) and Zeatin (Talengera et al., 1994; Crouch et al., 1998). Diphenyl urea derivatives such as (TDZ) have not beenBananas and plantains (Musa spp) are a major starchy staple widely used in Musa species except in a few cases in whichfood in the equatorial belt of Africa stretching from East to relatively low concentrations (0.45-1.14mM) of (TDZ) haveWest (Hallam, 1995). The edible portion provides a rich been reported to increase proliferation rates (Arinaitwe etsource of easily digestible carbohydrates, minerals al., 2000). TDZ is the most potent of the urea-based(potassium, magnesium, phosphorus, calcium and iron) and compounds and the one that was first evaluated for use invitamin A (in plantains), B6 and C (in bananas) (Stover & plant tissue culture by Mok et al. (1982). Arinaitwe et al.Simmonds, 1987; Jeger et al., 1995). In Uganda, over 75% of (2000) further reported that TDZ was effective in breakingthe rural population and 70% of the urban population recalcitrance in some banana cultivars when cultured mediadepend on bananas as their staple food. It thus provides supplemented with adenine-based cytokinins. TDZ has alsoboth livelihood and income to its producers and traders been shown to induce habituation in some species(MAAIF, 2001). Despite the importance of bananas in (Huetteman & Preece, 1993). Mok et al. (1982) showed thatUganda, yields have declined from 8.4 tons/ha in 1970 to Phaseolus lunatus callus became cytokinin autonomous5.9 tons/ha in 2000 (MAAIF, 2001). The lack of clean when cultured on media containing various concentrationsplanting material is a serious production constraint of TDZ. Similar observations were also made by Neuman etresponsible for rapid decline of bananas and plantains in al. (1993) in tree species when TDZ was present in theUganda (Rubaihayo & Gold, 1993). Conventional clonal primary medium. This quality makes the use of TDZ inpropagation of bananas by suckers, the most practiced micropropagation cost effective. However, there is lack ofmethod, in addition to pest and pathogen dissemination information on this quality of TDZ in East African Highlandwithin the propagation units (suckers) is seriously limited bananas and plantains.by low multiplication rates (5-10 suckers per year) and non- The activity of any growth regulator when used in inuniformity of the crop stand (Vuylsteke et al., 1990). In vitro vitro is influenced by environmental conditions (Razdan;propagation offers a powerful tool for extending the 1993). Light intensities of 1500-3000 lux are used for in vitropotential of addressing the limitations of conventional plantlet incubation though higher or less ones may bemethods of propagation by particularly overcoming the required for some plant species (Vuylsteke, 1989). The 16problems low multiplication rates and pathogen hours daily cycle of light at an intensity of 1173 ± 42 lux isdissemination (Vuylsteke, 1998). used for routine micropropagation of East African highland Shoot proliferation in vitro largely depends on the bananas by (Talengera et al., (1994) but there has been noconcentration of cytokinin in the medium (Razdan, 1993; research on the effect of light intensity on the rates ofTrijilo & Garcia, 1996). However, although different proliferation in banana micro propagation. The mainmicropropagation protocols using different cytokinins have objective of this study was, therefore, to investigate thebeen used for several Musa species (Vuylsteke, 1989), those carry-over effect of TDZ supplemented medium on theemployed are mainly adenine based e.g., benzylaminopurine
  2. 2. proliferation of the selected banana cultivars in a hormone proliferation in cultivar Sukalindizi was generally highestfree medium under varying light incubation conditions. on hormone free medium after the basal cycle with 7.14 mM TDZ except at 16 hours light when it peaked at 9.14 in the Materials and methods third culture cycle (Fig. 2C). The results of proliferation rates of cultivar Gros Michel on hormone free MS medium atThe study was carried out in Plant Tissue Culture different subculture cycles and light incubation conditionsLaboratory located at Makerere University Agricultural after a 6-week exposure to various TDZ concentrations andResearch Institute Kabanyolo (MUARIK) in 2001-2002. 22.2mM BAP are presented in Figure 3. Shoot proliferationSword suckers and peepers of five cultivars from which the rates at 22.2 mM TDZ were significantly (P<0.05) lower thanexplants were excised were obtained from the field gene those at various TDZ concentrations. For the different TDZbank of East African Highland Bananas at MUARIK. The concentrations used in the basal cycle, the resultantmethods of explant excision, disinfection and inoculation proliferation rates on the hormone free medium were highestwere adopted from Talengera et al. (1994). After nine weeks at 7.14 mM TDZ. The results proliferation rates of cultivarof culture inoculation on modified Murashige & Skoog (1962) Bwara are presented in Figure 4. Unlike Kibuzi, Sukalindizibanana multiplication medium (Talengera et al.1994), multiple and Gros Michel, results for cultivar Bwara indicated thataxillary shoots that had formed on each explant were proliferation is best at 5.14 mM TDZ after which,separated and re-inoculated onto MS media supplemented proliferation gradually declines irrespective of the lightwith 5.14, 7.14, 9.14, 11.14, and 13.14 µM TDZ, with BAP at incubation conditions. The proliferation rates on 22.2mM22.2 µM (Crouch et al. 1998) used as control. The shoot TDZ were significantly (P<0.05) lower than TDZ, but highercultures were incubated for a basal cycle of six weeks under than for the other cultivars (>2.0) suggesting that the carried16 hours light of intensity 1773 ±42 lux and temperature of over BAP was enough to induce proliferation in this highly26±2oC. Again, the proliferated shoots were separated and prolific cultivar. Just like for the other cultivars, there was ainoculated on hormone-free MS medium. The shoots were general increase in proliferation rates with increase in cultureincubated at varying daily light incubation conditions of cycles on various TDZ concentrations used in the basaldark, 8 and 16 hours in the growth room. Subculturing on cycle. The results of proliferation rates of Kifuba on hormonehormone free-MS medium was done for three culture cycles free MS medium at different subculture cycles and lightto establish the carry-over effect of the TDZ concentrations incubation conditions after a 6-week exposure to variousand BAP used in the basal cycle in subsequent culture TDZ concentrations and 22.2mM BAP are presented incycles. At the end of each of the three culture cycles, shoots Figure 5. Proliferation rates in Kifuba, on 22.2 mM BAP usedper inoculated shoot were recorded and the data subjected in the basal cycle were much lower than in the rest of theto statistical analysis. cultivars suggesting that the carried over BAP was too low to induce proliferation in this highly recalcitrant cultivar. Results There was an increase in proliferation rates from shoots initially cultured on various TDZ concentrations up to 9.14The results indicated that mean shoot proliferation rates mM, there after, there was decline in proliferation rateswere significantly higher after the basal cycle with various irrespective of light incubation conditions. As in the rest ofTDZ concentrations than with 22.2 mM BAP in all the the cultivars, the highest proliferation rates were recordedcultivars and light incubation conditions (Figs 1-5). The in the third culture cycle with TDZ irrespective of the lightresults of proliferation rates of Kibuzi on hormone free MS incubation conditions. There was no clear trend to indicatemedium at different subculture cycles and light incubation the best light incubation condition, but in cultivarsconditions after a 6-week basal cycle exposure to various Sukalindizi, Gros Michel and Bwara, higher proliferationTDZ concentrations and 22.2mM BAP are presented in rates were recorded in the dark than at 8 and 16 hours ofFigure 1. Mean proliferation rates after the basal cycle of light when TDZ was used.22.2 mM BAP were significantly (P<0.05) lower than thosewhere the basal cycle medium was supplemented with TDZ Discussionirrespective of the light incubation conditions andsubculture cycles. Shoot proliferation was highest on 7.14 The results of this study indicate that the proliferation ratesmM TDZ, suggesting that this was the optimum of shoots originating from the basal cycle medium withconcentration for this cultivar. The results also showed a various TDZ concentrations were significantly (P<0.05)trend of increase in proliferation rates with increase in the higher than those from 22.2 µM BAP in all the five cultivarsnumber of culture cycles resulting from the residual effect (Figs 1-5). This finding suggests that TDZ had a high carryof TDZ treatment in the basal cycle. The results of over effect which enabled the shoots to continueproliferation rates of cultivar Sukalindizi are presented in proliferating on the hormone free medium. SimilarFigure 2. Just like Kibuzi, shoot proliferation of Sukalindizi observations were made by Neuman et al.(1993), on easternwas lower on 22.2 mM BAP than on TDZ irrespective of the black walnut (Juglans nigra ) cotyledon cultures in whichlight incubation conditions and subculture cycles. Shoot they found that when cultured on a primary medium
  3. 3. 10 cycle1 A 8 cycle2 6 4 cycle3 2 0 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 13.14 B A P in TDZ Kib 2 TDZ TDZ uM TDZ uM TDZ 10 10 B C 8 8 6 6 4 4 2 2 0 0 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 13.14 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 uM 13.14 uM BAP TDZ TDZ TDZ uM TDZ uM TDZ BAP TDZ TDZ TDZ TDZ TDZ Basal cytokinin concentrationsFigure 1: Proliferation rates of Kibuzi on Hormone free MS medium at different culture cycles and light incubationconditions (A-dark, B- 8 hours and C-16 hours light/d) after a basal cycle with various TDZ concentrations and 22.2 µMBAP (control) 12 10 A cycle1 8 cycle2 6 4 cycle3 2 0 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 uM 13.14 uM BAP TDZ TDZ TDZ TDZ TDZ 12 12 B C 10 10 8 8 6 6 4 4 2 2 0 0 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 uM 13.14 uM 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 13.14 BAP TDZ TDZ TDZ TDZ TDZ BAP TDZ TDZ TDZ uM TDZ uM TDZ Basal cytokinin concentrationsFigure 2: Proliferation rates of Sukalindizi on Hormone free MS medium at different culture cycles and light incubationconditions (A-dark, B- 8 hours and C-16 hours light/d) after a basal cycle with various TDZ concentrations and 22.2 µMBAP (control)
  4. 4. 10 A cycle1 8 cycle2 6 cycle3 4 2 0 22.2 uM 5 . 1 4 u M 7.14 uM 9.14 uM 11.14 13.14 BAP TDZ TDZ TDZ uM TDZ uM TDZ 10 10 B C 8 8 6 6 4 4 2 2 0 0 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 13.14 22.2 uM 5 . 1 4 u M 7.14 uM 9.14 uM 11.14 13.14 BAP TDZ TDZ TDZ uM TDZ uM TDZ BAP TDZ TDZ TDZ uM TDZ uM TDZ Cytokinin concentrationsFigure 3. Proliferation rates of Gros Michel on Hormone free MS medium at different culture cycles and light incubationconditions (A-dark, B- 8 hours and C-16 hours light/d) after a basal cycle with various TDZ concentrations and 22.2 µMBAP (control) 12 10 A cycle1 8 cycle2 6 cycle3 4 2 0 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 13.14 BAP TDZ TDZ TDZ uM TDZ uM TDZ 12 12 B C 10 10 8 8 6 6 4 4 2 2 0 0 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 13.14 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 13.14 BAP TDZ TDZ TDZ uM TDZ uM TDZ BAP TDZ TDZ TDZ uM TDZ uM TDZ Cytokinin concentrationsFigure 4 Proliferation rate of Bwara on Hormone free MS medium at different culture cycles and light incubationconditions (A-dark, B-8 hours light/d) after a basal cycle with various TDZ concentrations and 22.2µMBAP (control)
  5. 5. 12 10 A 8 cycle1 6 cycle2 4 cycle3 2 0 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 uM 13.14 uM BAP TDZ TDZ TDZ TDZ TDZ 12 12 B C 10 10 8 8 6 6 4 4 2 2 0 0 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 uM 13.14 uM 22.2 uM 5.14 uM 7.14 uM 9.14 uM 11.14 uM 13.14 uM BAP TDZ TDZ TDZ TDZ TDZ BAP TDZ TDZ TDZ TDZ TDZ Cytokinin concentrationsFigure 5. Proliferation rates of Kifuba on Hormone free MS medium at different culture cycles and light incubationconditions (A-dark, B- 8 and C-16 hours light/d) after a basal cycle with various TDZ concentrations and 22.2 µM BAP(control)containing TDZ, the cultures continued to grow even when inherent endogenous cytokinin levels in different cultivarscultured on secondary medium lacking growth regulators. therefore account for the expression of variations in cultivarSimilar findings were also reported by Gill and Oziaz-Akins shoot proliferation responses to different exogenous(1998) in peanut callus cultures while Christena et al. 1995 cytokinin concentrations (Pierik, 1987).demonstrated that 2-day exposure of Geranium cultures to Very high levels of TDZ beyond 9.14µM resulted in5 mM TDZ was sufficient to evoke higher embryogenic proliferation decline indicating that high TDZ levelsresponse than continuous exposure. TDZ therefore has the suppress shoot proliferation. Unlike adenine and purine-capacity of transforming cultured tissues from cytokinin based cytokinins like BAP, TDZ is resistant to cytokinin-dependence to cytokinin autonomy (Mok et al. 1987) degrading enzymes, and hence remains at relatively high The results also suggested that the carry-over effect of concentrations in the tissues and induces excessiveTDZ (manifested in shoot proliferation rates on hormone suppression of lateral buds, consequently resulting infree MS medium) on shoot proliferation rates was influenced reduced proliferation rates (Hueteman and Preece, 1993).by the concentration used in the basal cycle (Figs 1-5). Arinaitwe et al.(2000) reported that high TDZ concentrationsCultivar Bwara proliferated highest (7.6-9.9) at the lowest inhibit axillary shoot proliferation and stimulate formationTDZ concentration of 5.14 µM used in the basal cycle (Fig. of undistinguishable bulbous structures in bananas while4) suggesting that it is a highly prolific (highly non- Thomas and Katterman (1986) reported that high TDZrecalcitrant) cultivar as reported by Talengera et al. (1994). concentrations resulted in a higher number of extremelyIt has a high content of endogenous cytokinins hence stunted and undifferentiated shoots in tobacco cultures. Arequiring low TDZ concentrations for effective proliferation. similar observation was made in this study.In contrast, Kifuba proliferated highest (8.6-10.6) at a There was a general trend of increase in proliferation ratesrelatively high TDZ concentration of 9.14 µM (Fig. 5) with culture cycles on a hormone free medium after TDZsuggesting that as a recalcitrant cultivar (Talengera et al., had been used in the basal cycle, the highest being in the1994). It has less endogenous cytokinin content, hence third subculture cycle in all the cultivars (Figs 1-5)requiring higher exogenous cytokinins for effective suggesting that subculturing on a hormone free mediumproliferation. Cultivars Kibuzi, Sukalindizi and Gros Michel had a resultant reduction in the amount of TDZ carried overproliferated maximally at 7.14 µM TDZ suggesting that they hence releasing the dormant buds that result in increasedhave moderate endogenous cytokinin content (Fis 1-3). The shoot proliferation. Similar observations were made by
  6. 6. Christena et al . (1995) in somatic embryogenesis of Crouch, J.H., Vuylsteke, D.and Oritz, R. 1998. Perspectives ofGeranium in which embryogenic response of callus cultures application of biotechnology to assist genetic enhance-increased at every cycle of subculture on a secondary ment of banana (Musa spp.) Plant biotechn. 1(1), 1-12.medium devoid of hormones after an eight-day exposure to Hallam,G. 1995. Linguistic study of banana and plantain in5ìM TDZ. Africa. Leiden, Research School CNWS, The Netherlands. There was generally significant (P<0.05) effect of light Huetteman, S.C. & Preece, J.E. 1993. Thidiazuron; a potentincubation conditions on mean proliferation rates that cytokinin forAdepends on the type of cytokinin used. Light exerts its effects woody plant tissue culture. Plant cell, Tissue &Organ cul-through its influence on differential translocation of different ture. 33,105-119.growth regulators within the cultured tissues (Razdan, 1993). MAAIF, 2001. Agriculture Annual Report, 2001. Ministry ofIn Cultivars Sukalindizi, Gros Michel and Bwara had higher Agriculture Animal Industry and Fisheries (MAAIF),proliferation rates in the dark than in 8 and 16 hours light at Kampala Uganda, 2001.different TDZ concentrations suggesting that in vitro Mok, M.C. & Mok, D.E.S., 1982. The metabolism of [14 C] -proliferation is a photomorphogenically responsive process thidiazuron in callus cultures of Phaseolus lunatus L.that is enhanced under dark conditions. This could be true Physiology of plants., 65:427-432.since in vitro photosynthesis was found to be unnecessary Mok, M.C., Turner, J.E & Mujer, C.V. 1987. Biological andby Hartmann et al. (1990). Similar observations have also biochemical effects of Cytokinin-like phenyl urea deriva-been reported in other crops. For instance Pinker (2001) tives in tissue culture systems. Horticultural sience, 22;reported higher growth parameters of shoot proliferation (6): 1194-1197.rates and fresh weight in the dark than in chopper Murashige, T. & Skoog, 1962. A revised medium for rapid(intermittent) or continuous light in deciduous plant cultures growth bioassays with tobacco cultures. Physiologiawhile Rusli et al.(1998) reported similar observations when Plantarum., 15, 473-493.using dark and low irradiance in in vitro culture of Rosalia Neuman, M.C., Preece, J. E. Van Sambeek, J. W. & Gaffney, G.hybrida. R. 1993. Somatic embryogenesis and callus production from cotyledon explants of black walnut (Juglans nigra Conclusions and recommendations L.). Plant Cell. Tiss. Org. Cult. 32: 9-18 Radzan, M.K. 1993. Introduction to plant tissue culture. In-The study confirms that is deduced that TDZ has a carry- tercept, Hampshire, UK.over effect that transforms cultured tissues of East African Rubaihayo, P.R. & Gold, S.S. 1993. Rapid Rural Appraisal ofHighland bananas from exogenous cytokinin dependence Banana Production in Uganda. InfoMusa, 2(1): 15-16.to cytokinin autonomy. It is therefore, recommended that Rusli, I. & Debergh, P.C. 1998. Improvement of adventitiousfor cost-effective micropropagation, prolific, intermediate bud formation and plantlet regeneration from in vitro ex-and recalcitrant banana cultivars with different levels of plants of roses (Rosa hybrida L.). 4th Ph.D symposium.endogenous levels of cytokinin content be respectively Faculty of Agriculture and Applied Biological Sciences,cultured using TDZ at 5.14, 7.14 and 9.14 mM for a single Coupure Links, 653.cycle after which, subculturing should be done on a hormone Talengera, D. Magambo, M.J.S. and Rubaihayo, P.R. 1994.free medium for three or more cycles. It is also noted from Testing for a suitable medium for propagation of Eastthis study that dark conditions or low light intensities can African Highland bananas. African Crop Sci. J. 2 (1) 7-21.substitute for the relatively high light intensities Stover, R.H & Simmonds , N. W. 1987. Bananas ; 3rd Ed.conventionally used in the culture growth rooms. It is thus Longman, London. 486pp.recommended that in routine banana micropropagation, Thomas, J. C. & Katterman, F.R. 1986. Cytokinin activity in-cultures be incubated in the dark during multiplication and duced by Thidiazuron. Plant Physiology., 81: 681-683.provided with light at the rooting stage to reduce the Trijilo, I. & Garcia, E.1996. Strategies for obtaining somaclonalproduction costs per plantlet. variants resistant to yellow sigatoka (Mycospharella muscita). Infomusa. 5(2) 6-7. Acknowledgement Vuylsteke, D. 1989. Shoot tip culture for propagation, con- servation & exchange of Musa germplasm. Practical manu-The Rockefeller Foundation provided the funds for this als for handling crop germplasm in vitro. IBPGR, Rome,study through Forum Grant RF99005#52. Italy.2. pp56. Vuylsteke, D. 1998. Shoot tip culture for propagation and conservation of Musa germplasm. Tropical Agric . References (Trinidad) 62(4) 323-328 Vuylsteke , D. Swennen, R. & De Langhe, E. 1990. TissueArinaitwe, G., Rubaihayo, P.R. & Magambo, M.J.S. 2000. Pro- culture technology for improvement of African plantains. liferation rate effects of cytokinins on banana Musa spp. INIBAP workshop on sigatoka leaf spot disease of ba- cultivars. Scientia Horticulturae 86. 13-21pp. nanas. San Jose, Costa Rica. March 28-April 1st 1989. Pp316-337.