This study investigated the carry-over effect of thidiazuron (TDZ) on banana shoot proliferation under different culture cycles and light conditions. Shoots were initially cultured for 6 weeks on media supplemented with varying TDZ concentrations or benzylaminopurine (BAP, control). Shoots were then transferred to hormone-free media and subcultured for 3 cycles under dark, 8 hr, or 16 hr light. Results showed TDZ had a stronger carry-over effect than BAP, enabling continued shoot proliferation without hormones. Proliferation generally increased with culture cycles and was higher under dark conditions for some cultivars. TDZ concentrations of 7.14 mM produced the highest proliferation for most cultivars

1. African Crop cience Conference Proceedings, Vol. 6. 113-118
Printed in Uganda. All rights reserved
ISSN 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, Uganda
Abstract: Thidiazuron (TDZ) is an active cytokinin that was shown to induce increased shoot proliferation and habituation
in 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 but
there is lack of information on this quality in banana micropropagation. A study was therefore conducted to investigate the
carry over effect of varying concentrations of TDZ and 22.2 µM BAP as control on proliferation of five banana cultivars
on a hormone free medium under various incubation conditions. The results showed that TDZ had a carry-over effect that
enabled shoots to continue proliferating on a hormone free medium as the culture cycles increased and that this effect was
significantly (P<0.05) higher than that of BAP. Accumulation of TDZ to high levels resulted in suppression of shoot
proliferation. The results further showed dark conditions enhanced higher proliferation rates than light conditions in some
cultivars suggesting that in vitro proliferation is a photomorphogenically responsive process that is enhanced under dark
conditions.
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 been
Bananas and plantains (Musa spp) are a major starchy staple widely used in Musa species except in a few cases in which
food in the equatorial belt of Africa stretching from East to relatively low concentrations (0.45-1.14mM) of (TDZ) have
West (Hallam, 1995). The edible portion provides a rich been reported to increase proliferation rates (Arinaitwe et
source 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 in
vitamin 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 breaking
the rural population and 70% of the urban population recalcitrance in some banana cultivars when cultured media
depend on bananas as their staple food. It thus provides supplemented with adenine-based cytokinins. TDZ has also
both 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 that
Uganda, yields have declined from 8.4 tons/ha in 1970 to Phaseolus lunatus callus became cytokinin autonomous
5.9 tons/ha in 2000 (MAAIF, 2001). The lack of clean when cultured on media containing various concentrations
planting material is a serious production constraint of TDZ. Similar observations were also made by Neuman et
responsible for rapid decline of bananas and plantains in al. (1993) in tree species when TDZ was present in the
Uganda (Rubaihayo & Gold, 1993). Conventional clonal primary medium. This quality makes the use of TDZ in
propagation of bananas by suckers, the most practiced micropropagation cost effective. However, there is lack of
method, in addition to pest and pathogen dissemination information on this quality of TDZ in East African Highland
within 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 in
uniformity 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 vitro
potential of addressing the limitations of conventional plantlet incubation though higher or less ones may be
methods of propagation by particularly overcoming the required for some plant species (Vuylsteke, 1989). The 16
problems low multiplication rates and pathogen hours daily cycle of light at an intensity of 1173 ± 42 lux is
dissemination (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 no
concentration of cytokinin in the medium (Razdan, 1993; research on the effect of light intensity on the rates of
Trijilo & Garcia, 1996). However, although different proliferation in banana micro propagation. The main
micropropagation protocols using different cytokinins have objective of this study was, therefore, to investigate the
been used for several Musa species (Vuylsteke, 1989), those carry-over effect of TDZ supplemented medium on the
employed are mainly adenine based e.g., benzylaminopurine

2. proliferation of the selected banana cultivars in a hormone proliferation in cultivar Sukalindizi was generally highest
free 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 at
The study was carried out in Plant Tissue Culture different subculture cycles and light incubation conditions
Laboratory located at Makerere University Agricultural after a 6-week exposure to various TDZ concentrations and
Research Institute Kabanyolo (MUARIK) in 2001-2002. 22.2mM BAP are presented in Figure 3. Shoot proliferation
Sword suckers and peepers of five cultivars from which the rates at 22.2 mM TDZ were significantly (P<0.05) lower than
explants were excised were obtained from the field gene those at various TDZ concentrations. For the different TDZ
bank of East African Highland Bananas at MUARIK. The concentrations used in the basal cycle, the resultant
methods of explant excision, disinfection and inoculation proliferation rates on the hormone free medium were highest
were adopted from Talengera et al. (1994). After nine weeks at 7.14 mM TDZ. The results proliferation rates of cultivar
of culture inoculation on modified Murashige & Skoog (1962) Bwara are presented in Figure 4. Unlike Kibuzi, Sukalindizi
banana multiplication medium (Talengera et al.1994), multiple and Gros Michel, results for cultivar Bwara indicated that
axillary 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 light
with 5.14, 7.14, 9.14, 11.14, and 13.14 µM TDZ, with BAP at incubation conditions. The proliferation rates on 22.2mM
22.2 µM (Crouch et al. 1998) used as control. The shoot TDZ were significantly (P<0.05) lower than TDZ, but higher
cultures were incubated for a basal cycle of six weeks under than for the other cultivars (>2.0) suggesting that the carried
16 hours light of intensity 1773 ±42 lux and temperature of over BAP was enough to induce proliferation in this highly
26±2oC. Again, the proliferated shoots were separated and prolific cultivar. Just like for the other cultivars, there was a
inoculated on hormone-free MS medium. The shoots were general increase in proliferation rates with increase in culture
incubated at varying daily light incubation conditions of cycles on various TDZ concentrations used in the basal
dark, 8 and 16 hours in the growth room. Subculturing on cycle. The results of proliferation rates of Kifuba on hormone
hormone free-MS medium was done for three culture cycles free MS medium at different subculture cycles and light
to establish the carry-over effect of the TDZ concentrations incubation conditions after a 6-week exposure to various
and BAP used in the basal cycle in subsequent culture TDZ concentrations and 22.2mM BAP are presented in
cycles. At the end of each of the three culture cycles, shoots Figure 5. Proliferation rates in Kifuba, on 22.2 mM BAP used
per inoculated shoot were recorded and the data subjected in the basal cycle were much lower than in the rest of the
to 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 9.14
The results indicated that mean shoot proliferation rates mM, there after, there was decline in proliferation rates
were significantly higher after the basal cycle with various irrespective of light incubation conditions. As in the rest of
TDZ concentrations than with 22.2 mM BAP in all the the cultivars, the highest proliferation rates were recorded
cultivars and light incubation conditions (Figs 1-5). The in the third culture cycle with TDZ irrespective of the light
results of proliferation rates of Kibuzi on hormone free MS incubation conditions. There was no clear trend to indicate
medium at different subculture cycles and light incubation the best light incubation condition, but in cultivars
conditions after a 6-week basal cycle exposure to various Sukalindizi, Gros Michel and Bwara, higher proliferation
TDZ concentrations and 22.2mM BAP are presented in rates were recorded in the dark than at 8 and 16 hours of
Figure 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 those
where the basal cycle medium was supplemented with TDZ Discussion
irrespective of the light incubation conditions and
subculture cycles. Shoot proliferation was highest on 7.14 The results of this study indicate that the proliferation rates
mM TDZ, suggesting that this was the optimum of shoots originating from the basal cycle medium with
concentration 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 cultivars
number of culture cycles resulting from the residual effect (Figs 1-5). This finding suggests that TDZ had a high carry
of TDZ treatment in the basal cycle. The results of over effect which enabled the shoots to continue
proliferation rates of cultivar Sukalindizi are presented in proliferating on the hormone free medium. Similar
Figure 2. Just like Kibuzi, shoot proliferation of Sukalindizi observations were made by Neuman et al.(1993), on eastern
was lower on 22.2 mM BAP than on TDZ irrespective of the black walnut (Juglans nigra ) cotyledon cultures in which
light incubation conditions and subculture cycles. Shoot they found that when cultured on a primary medium

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 concentrations
Figure 1: Proliferation rates of Kibuzi on Hormone free MS medium at different culture cycles and light incubation
conditions (A-dark, B- 8 hours and C-16 hours light/d) after a basal cycle with various TDZ concentrations and 22.2 µM
BAP (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 concentrations
Figure 2: Proliferation rates of Sukalindizi on Hormone free MS medium at different culture cycles and light incubation
conditions (A-dark, B- 8 hours and C-16 hours light/d) after a basal cycle with various TDZ concentrations and 22.2 µM
BAP (control)

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 concentrations
Figure 3. Proliferation rates of Gros Michel on Hormone free MS medium at different culture cycles and light incubation
conditions (A-dark, B- 8 hours and C-16 hours light/d) after a basal cycle with various TDZ concentrations and 22.2 µM
BAP (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 concentrations
Figure 4 Proliferation rate of Bwara on Hormone free MS medium at different culture cycles and light incubation
conditions (A-dark, B-8 hours light/d) after a basal cycle with various TDZ concentrations and 22.2µMBAP (control)

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 concentrations
Figure 5. Proliferation rates of Kifuba on Hormone free MS medium at different culture cycles and light incubation
conditions (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 cultivars
cultured on secondary medium lacking growth regulators. therefore account for the expression of variations in cultivar
Similar 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 in
5 mM TDZ was sufficient to evoke higher embryogenic proliferation decline indicating that high TDZ levels
response 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 excessive
TDZ (manifested in shoot proliferation rates on hormone suppression of lateral buds, consequently resulting in
free 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 concentrations
Cultivar Bwara proliferated highest (7.6-9.9) at the lowest inhibit axillary shoot proliferation and stimulate formation
TDZ concentration of 5.14 µM used in the basal cycle (Fig. of undistinguishable bulbous structures in bananas while
4) suggesting that it is a highly prolific (highly non- Thomas and Katterman (1986) reported that high TDZ
recalcitrant) cultivar as reported by Talengera et al. (1994). concentrations resulted in a higher number of extremely
It has a high content of endogenous cytokinins hence stunted and undifferentiated shoots in tobacco cultures. A
requiring 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 rates
relatively high TDZ concentration of 9.14 µM (Fig. 5) with culture cycles on a hormone free medium after TDZ
suggesting that as a recalcitrant cultivar (Talengera et al., had been used in the basal cycle, the highest being in the
1994). 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 medium
proliferation. Cultivars Kibuzi, Sukalindizi and Gros Michel had a resultant reduction in the amount of TDZ carried over
proliferated maximally at 7.14 µM TDZ suggesting that they hence releasing the dormant buds that result in increased
have moderate endogenous cytokinin content (Fis 1-3). The shoot proliferation. Similar observations were made by

6. Christena et al . (1995) in somatic embryogenesis of Crouch, J.H., Vuylsteke, D.and Oritz, R. 1998. Perspectives of
Geranium 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 in
5ìM 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 potent
incubation conditions on mean proliferation rates that cytokinin forA
depends 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 of
In 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 and
by 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. Physiologia
while 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 of
Highland 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 adventitious
for 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.
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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 East
this 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.
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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 somaclonal
production 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 .
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