Thidiazuron in the Improvement of Banana Micropropagation

Transcription

1 Thidiazuron in the Improvement of Banana Micropropagation Sin-Wan Lee Taiwan Banana Research Institute, P.O. Box 18, Chiuju, Pingtung Taiwan 904, R.O.C. Keywords: Paclobutrazol Abstract In 1983 a commercial micropropagation system was established in Taiwan to produce disease free planting material to control the spread of Fusarium wilt (race 4). With the rapid increase in production costs, it was necessary to search for a more active cytokinin than 6-Benzylaminopurine (BA) to improve the efficiency of the micropropagation system. Thidiazuron (TDZ) was tested in the range of to 2.0 mg/l for its influence on the proliferation of adventitious buds. TDZ at 0.2 mg/l almost doubled the multiplication rate of Cavendish cultivars in medium containing BA at 4.0 mg/l. The effect of TDZ on the multiplication rate varied among the commercial cultivars 'Pei-Chiao', 'Tai-Chiao No. 1' (AAA) and Latundan (AAB). The multiplication rate of shoots decreased gradually with increasing numbers of subculture cycles. The addition of paclobutrazol (PP333) ( mg/l) to TDZ ( mg/l) resulted in a further increase of proliferation of buds when compared with medium containing only TDZ, while also suppressing shoot elongation. The dwarf bud clusters induced in the TDZ + PP333 medium returned to normal shoots after one to two subcultures in medium containing only BA. The sequence of application of Dropp (containing TDZ as active ingredient) in combination with both BA and PP333 in the micropropagation system is discussed. Tissue culture plantlets produced from adventitious buds treated with TDZ and PP333 grew vigorously during the nursery stage. Field trials of 1,800 plants treated by TDZ showed 2.4% off-types, while the control group consisting of 400 BA treated plants showed 3.0% off-types. INTRODUCTION The triploid Cavendish banana is one of the most important fruit crops for export and local consumption in Taiwan. The rapid spread of Fusarium wilt caused by Fusarium oxysporum f. sp cubense (race 4) made it necessary to produce large quantities of disease free planting material. Therefore, since 1983 (Hwang et al., 1984) the Taiwan Banana Research Institute (TBRI) has applied the banana meristem culture technique (Ma and Shii, 1972, 1974) to mass propagate disease free plantlets for commercial planting. From 1983 to 2005, a total of 45 million plantlets have been supplied to growers at a minimal fee through a joint program between TBRI and the Taiwan Fruit and Marketing Cooperative. In order to meet the demand for export, over 2 to 3 million plantlets are propagated annually for field establishment within a short planting season (from February to April). Continuous efforts have been made to increase the efficiency of the system through improvement in culture media and techniques (Lee, 1993). The multiplication rate of adventitious buds under the influence of Benzylaminopurine (BA) is one of the determining factors deciding on the efficiency of the micropropagation system. The most commonly used cytokinin is BA at a range of 2 to 5 mg/l, often in combination with an auxin, indole 3-acetic acid (IAA), at a concentration of 0.1 to 0.2 mg/l (Vuylsteke, 1998). The commercial cultivars propagated in Taiwan belong to the Cavendish subgroup (AAA). The optimal range of BA at 4 to 5 mg/l gives a 3 to 4 fold increase with each cycle of subculture. Further increase in the multiplication rate required the search for a more effective cytokinin. Thidiazuron (TDZ) has been considered one of the most active phenylureas having cytokinin-like activities. It is stable and more active at lower concentrations than the adenine-type cytokinins (Mok et al., 1987). Huettman and Preece (1993) described TDZ Proc. IInd IS on Biotech. of Trop & Subtrop. Species Eds: W.-C. Chang and R. Drew Acta Hort 692, ISHS

2 as the most potent currently available cytokinin-like substance in the tissue culture of woody plants. At low concentrations (less than 1μM), TDZ induces greater proliferation of axillary shoots than other cytokinins. At higher concentrations, TDZ stimulates the formation of callus, shoots or somatic embryos. Herman (1997) reviewed the results of a comparison of the effect of thidiazuron from different sources in promoting adventitious shoot regeneration from leaf discs of Limonium perigrinum. Dropp, Schering AG and Sigma Chemicals were used. Results confirmed that Dropp, a relatively inexpensive powder used as a cotton defoliant, is an effective source of thidiazuron. In a comparison of the effect of cytokinins on the proliferation rate of banana cultivars, Arinaitwe et al. (2000) reported that cultivars differed significantly in their shoot proliferation responses to different TDZ concentrations and that TDZ is more economical than BA. Paclobutrazol (PP333) is a potent plant growth regulator that inhibits gibberellin biosynthesis. The use of growth retardants (PP333) in liquid culture has been found to increase the number of buds, reduce leaf elongation and enhance cormlet production in Gladiolus as compared with untreated buds (Ziv, 1989). Also, in Philodendron, ancymidol and PP333 induced the proliferation of bud clusters in liquid shake or bioreactor cultures (Ziv and Ariel, 1991). El-Otmani et al. (1992) reported that PP333 used on greenhousegrown bananas in Morocco had no significant effect on fruit quality and composition. The objective of the present study was to evaluate the effect of TDZ on the proliferation of adventitious buds of bananas and its application in commercial micropropagation. The combination of TDZ (Dropp), BA and PP333 and their sequence of application in the micropropagation system are reported. MATERIALS AND METHODS Culture Initiation and Proliferation of Adventitious Buds Young suckers were selected from healthy and true-to-type mother plants. The outer layers of leaves and corm tissue of the sucker were removed to obtain a block measuring 10 cm long, containing the shoot apex. This block was surface sterilized by wiping with 75% alcohol. Under aseptic conditions, the sheaths and bases of the leaves were trimmed to expose the meristematic region. The shoot tip was decapitated and a block of tissue measuring 1.5 x 1.5 x 1.5 cm was excised and inoculated on to multiplication medium. The explant (designated as S0) was incubated at with 14 hours light/dark cycle (daylight fluorescent tubes). After 5 to 6 weeks, 15 to 25 buds were obtained from each explant. The shoots/buds induced from the explant were divided into smaller pieces (usually 2 to 4) and subcultured onto fresh multiplication medium. This stage of subculture was designated as the first cycle of subculture (S1). The shoot/bud clusters were subcultured every 5 to 6 weeks for up to 6 to 7 cycles. The multiplication rate is referred to as the increase in number of propagules (each consisting of 2 to 3 shoot/bud) obtained from one piece of shoot/bud cluster from the previous subculture. The percentage of elongated shoots referred to those shoots above 1 cm tall with small leaves. Globular buds were short and round with scale-like leaves. Multiplication medium consisted of MS macro and micro salts supplemented with thiamin HCl 0.4 mg/l, myo-inositol and L-tyrosine both at 100 mg/l, sugar 30 g/l, agar 5.5 g/l. Growth regulators included 6-benzylaminopurine (BA) 4 mg/l, indole-3-acetetic acid (IAA) 1.6 mg/l and adenine sulfate 80 mg/l. ph was adjusted to 5.8 before autoclaving. Glass cylindrical culture vessels were 7 cm. in diameter, 10 cm. in height and contained 50 ml of medium. Each glass vessel contained one explant (sucker), and two to three propagules in later cycles of subculture. Other plant growth regulators used were: Thidiazuron TDZ (Sigma P 6186, tissue culture tested), Dropp (cotton defoliant, 490 g/kg thidiazuron, wettable powder), and Paclobutrazol PP333 (Cultar, containing 250 g/l of Paclobutrazol as a flowable suspension concentrate, ICI). Dropp and Cultar stock solutions were prepared by adding 68

3 the appropriate amount of powder or suspension to distilled water to give 1.0 mg/l of TDZ or PP333 as active ingredient. Regeneration of Plantlets and Acclimatization in Screened Nursery Elongation and rooting of adventitious buds was accomplished by adding liquid regeneration medium to the established shoot/bud culture. Regeneration medium consisted of HypoNex no.1 (5.0 g/l), MS micro salts, thiamin HCl (0.4 mg/l), sugar (10 g/l) and activated charcoal (1.6 g/l). Culture flasks were immediately transferred to the screen house under 60% shade. After 30 to 35 days under natural light, elongated shoot clusters were removed from the flask, and separated manually into large and small plantlets. Large ones were transplanted directly into plastic pots (350 cc) and small ones into soil boxes at high density. All plantlets were kept under a clear plastic tunnel for 2 to 3 weeks to retain moisture. Plantlets were maintained in the screened nurseries for 1.5 to 2 months before planting in the field. RESULTS Effective Range of TDZ in the Proliferation of Adventitious Buds The effect of TDZ on the multiplication of adventitious buds in the cultivars 'Pei- Chiao' (AAA) and Latundan (AAB) was tested by adding TDZ (0, 0.002, and 2.0 mg/l) to MS medium without BA. Propagules consisted of one finely trimmed single bud from the 4th subculture in BA. The fresh weight and number of buds obtained from each propagule were measured after 48 days and recorded in Table 1. In both cultivars, TDZ at 0.2 mg/l induced the largest number of shoots which were 7.3 and 4.4 while the number of shoots induced in the BA medium (2.0 mg/l) were 5.8 and 3.7. At higher concentrations of TDZ (2.0 mg/l), elongation of shoots was greatly suppressed and clumps of small globular buds appeared at the base of the large shoots. Effect of TDZ in Combination with BA for 3 Commercial Cultivars The effect of TDZ on two Cavendish cultivars 'Pei Chiao', 'Tai Chiao No. 1' (AAA) and Latundan (AAB) was compared with TDZ (0, 0.05, 0.1, 0.2, 0.5 and 2.0 mg/l) added to the multiplication medium (4.0 mg/l BA). Each propagule consisted of one finely trimmed single bud from the second cycle of subculture in BA. Shoot clusters were cultured for 8 weeks and the effective multiplication rate was recorded in Table 2. In the above 3 commercial cultivars, the optimum multiplication rate in TDZ (0.2 mg/l) was 8.2, 6.4 and 2.6 respectively, and was 1.8, 2.4 and 2.0 times higher than the BA check (4.0 mg/l). At higher concentrations of TDZ (0.5, 1.0 and 2.0 mg/l), shoot clusters were stunted, and the proportion of elongated shoots decreased drastically. Comparison of the Effect of TDZ or Dropp with or without PP333 on the Multiplication Rate of Adventitious Buds Adventitious buds of the commercial Cavendish cultivar Pei-Chiao from three different cycles of subculture (S1, S2, S3) were cultured for 8 weeks in medium containing BA at 4.0 mg/l as the control. The four other treatments were TDZ or Dropp at 0.2 mg/l with or without PP333 at 1.0 mg/l. The average of the multiplication rate obtained from the three different subculture cycles of the four treatments and the control were listed in Table 3. The average multiplication rate of buds in medium containing TDZ or Dropp without the addition of PP333 was 7.0 and 6.4 respectively. With the addition of PP333, the average multiplication rate increased to 8.1 in TDZ and 9.0 in Dropp, while the BA control was 4.8. The above observation indicated that TDZ and Dropp had a similar effect in enhancing the multiplication of buds and the addition of PP333 could further increase the multiplication rate. The advantage of adding PP333 in TDZ or Dropp was the induction of compact bud clusters due to the inhibition of the growth retardant on the elongation of leaves. These compact bud clusters were easier to dissect and hence could save manual labor in the process of subculture in the commercial propagation 69

4 system. In the medium containing Dropp and PP333, the average multiplication rate was 9.0, which was significantly higher than the BA control at 4.8. Since both Dropp and PP333 are agrochemicals, this medium is the most economical and effective formulation for commercial production of adventitious buds. Effect of Dropp and PP333 on the Cumulative Multiplication Rate after three Consecutive Cycles of Subculture and Regeneration of Plantlets The multiplication rate of shoots on medium containing Dropp combined with PP333 after three consecutive cycles of subculture was studied for the cultivar 'Pei Chiao'. Propagules were derived from shoots propagated in BA. Material from the 2nd subculture in BA, each consisting of 2 to 3 buds, were transferred to Dropp and PP333 medium and subcultured 3 times (S3, S4 and S5) on the same culture medium. Media tested included D4 (Dropp 0.2 mg/l and PP mg/l) and D7 (Dropp 0.1 mg/l and PP mg/l) in combination with 4.0 mg/l of BA. Shoot/bud clusters were subsequently subcultured on BA at S6 for four weeks. Regeneration liquid medium (HypoNex #1 5mg/L) was added to the bud clusters and plantlets were then regenerated at S7. Results are shown in Table 4. The cumulative multiplication rate (S3, S4, S5) on BA, the D7 and the D4 media were 52, 453 and 296 respectively. At S6, propagules of all treatments were transferred to medium containing BA only. At S7, the regeneration liquid medium containing activated charcoal had the effect of inducing the elongation and rooting of the shoot/bud clusters resulting in small plantlets ranging from 3 to 5 cm in height. The average number of plantlets per culture flask from the BA control, the D7 and D4 media were 15, 13.5 and 11 respectively. Results of this experiment indicated that in the D7 treatment, the cumulative multiplication rate of 453 after three consecutive cycles of subculture was obviously much higher than the BA control, which was 52. Bud clusters in the D7 medium, upon return to BA at S6, also showed a higher multiplication rate than the BA control. The number of plantlets from the D7 medium was comparable to that of the BA control. In the overall performance, the optimal medium is D7 (Dropp 0.1 mg/l, PP mg/L.) Effect of Time Intervals between Two Subcultures on the Multiplication Rate in Dropp and PP333 Adventitious buds cultured in BA were transferred to D7 medium and subcultured for 3 cycles (S3, S4, S5) in the same medium. Three groups with different time intervals of subculture were compared. The subculture intervals were 50, 60 and 70 days. The cumulative multiplication rate of shoots on the BA medium for three time intervals (50, 60, 70 days) were 47, 62 and 53 respectively (Table 5). On the D7 medium, the cumulative multiplication rates for the three time intervals were 214, 358 and 453 respectively, showing an increase with an increase of subculture interval. Effect of Dropp and PP333 on the Mutliplication Rate of Four Cultivars The cumulative multiplication rate of four cultivars after three subculture cycles (S3, S4, S5) in D7 medium were compared (Table 6). The length of each subculture was 50 days with the exception of Formosana which was 40 days. 'Pei Chiao' and 'Tai Chiao No. 2' were quite similar in their response on D7 medium with multiplication rates of 214 and 211 respectively. The cultivar 'Tai Chiao No.3' showed a slightly lower rate of multiplication while Formosana was the lowest. The Effect of TDZ or Dropp and PP333 on Growth of Plants The effect of TDZ or Dropp and PP333 on growth and occurrence of somaclonal variation in the resulting banana plantlets was observed during the nursery stage, after field planting to harvest. Tissue culture plantlets propagated using TDZ and PP333 grew vigorously during the nursery stage. A field trial consisting of banana plantlets derived from adventitious buds treated with 0.2mg/L TDZ showed no abnormalities in both cultivars 'Pei Chiao' and Latundan compared to the untreated plants. A field trial of 1,800 70

5 plants treated with TDZ (0.2 mg/l) + PP333 (1.0 mg/l) showed 2.4% off-types while the control group consisting of 400 BA treated plants showed 3.0% off-types. DISCUSSION In preliminary studies on the effect of TDZ on the proliferation of adventitious buds, the optimal concentration of TDZ (tissue culture grade) was 0.2 mg/l for the cultivars 'Pei Chiao' (AAA) and Latundan (AAB). In later experiments, when BA (4.0 mg/l) was used in combination with TDZ, results again indicated that the optimal concentration of TDZ was 0.2 mg/l In Cavendish bananas, TDZ at 0.2 mg/l slightly suppressed the elongation of shoots. At 2.0 mg/l, shoots became stunted and clumps of globular buds occurred at the base of large shoots. In woody species, TDZ also suppressed the elongation of shoots. These could however return to normal sizes after transfer to an elongation medium containing a lower TDZ level or a different cytokinin (Huettman and Preece, 1993). In the present study, stunted buds returned to normal size after one to two subcultures on BA medium. When micropropagagating Cavendish banana cultivars, the general trend is a gradual decrease in the rate of multiplication with increasing subculture cycles. This phenomenon was also observed when Dropp and PP333 were used in the multiplication medium. The effect of TDZ was different for the cultivars 'Pei Chiao', 'Tai Chiao #1' and Latundan. It was also observed that within the same cultivar, propagules from different cycles of subculture showed a slight difference in the rate of shoot/bud proliferation. This observation was in agreement with past experience in commercial micropropagtion that multiplication rates decrease with the increase in number of subcultures in the BA medium. Response of propagules after the 1st, 2nd, 3rd or 4th subculture cycle on TDZ were slightly different for the cultivar 'Pei Chiao'. The optimal subculture cycle for TDZ treatment was the second or third cycle. Results from the present study showed that TDZ (tissue culture grade) can be replaced by Dropp (wettable powder used as cotton defoliant). Pure crystalline PP333 can be replaced by Cultar (flowable suspension). This could result in a substantial reduction in production costs in terms of growth regulators used. In the present system, the agar used in culture medium was manufactured locally. According to our experience (results not shown), the use of Gelrite in the multiplication medium containing BA or TDZ showed a lower rate of multiplication of adventitious buds. The addition of PP333 increased the multiplication rate when combined with TDZ or Dropp. Another advantage of PP333 in combination with Dropp was the stunting of shoots into compact bud clumps and suppression of roots so that propagules could be maintained for a longer time without subculturing. The proposed strategy for Cavendish banana micropropagation consists of using BA to induce adventitious shoots from the explant (meristematic region of the sucker) followed by one or two subcultures on BA to obtain more buds. Thereafter, two or three subcultures in Dropp (0.1 mg/l) supplemented with PP333 (0.5 mg/l) should be executed. Finally, bud clusters should be subcultured once or twice in BA to reduce the inhibitory effect of Dropp and PP333 on the elongation of shoots before the regeneration of plantlets from the shoot clusters can take place. ACKNOWLEDGEMENTS This research was supported by the Council of Agriculture, Executive Yuen, Republic of China (89BT-2.1-FAD-63 and FD-Z3). Technical assistance of Ms L.K. Chen is gratefully appreciated. Literature Cited Arinaitwe, G., Rubaihayo, P.R. and Magambo, M.J.S Proliferation rate effects of cytokinins on banana (Musa spp.) cultivars. Scientia Horticulturae 86:

6 El-Otmani, M., Cheikh, N. and Sedki, M Effects of paclobutrazol on greenhousegrown bananas in Morocco. Scientia Horticulturae 49: Herman, E.B Recent Advanaces in Plant Tissue Culture V. Agritech Consultants, Inc. NY U.S.A. 126pp. Huetteman, C.A. and Preece, J.E Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell, Tissue and Organ Culture 33: Hwang, S.C Variation in banana plants propagated through tissue culture. Journal of Chinese Society of Horticultural Science 32: (in Chinese with English summary). Hwang, S.C., Chen, C.L., Lin, J.C. and Lin, H.L Cultivation of banana using plantlets from meristem culture. HortScience 19: Lee, S.W Improvement of methods used in the regeneration of micropropagated banana plantlets. p In: R.V. Valmayor, S.C. Hwang, R. Ploetz, S.W. Lee and N.V. Roa (eds.), Proceedings of International Symposium on Recent Developments in Banana Cultivation Technology. Pingtung, Taiwan, December, INIBAP/ASPNET. Ma, S.S. and Shii, C.T In vitro formation of adventitious buds in banana shoot apex following decapitation. Journal of Chinese Society of Horticultural Science 18: (In Chinese with English summary). Ma, S.S. and Shii, C.T Growing banana plantlet from adventitious buds. Journal of Chinese Society of Horticultural Science 20:6-12. (In Chinese with English summery). Mok, M.C., Mok, D.W.S., Turner, J.E. and Mujer, C.V Biological and biochemical effect of cytokinin-active phenylurea derivatives in tissue culture systems. HortScience 22: Ziv, M Enhanced shoot and cormlet proliferation in liquid cultured Gladiolus buds by growth retardants. Plant Cell Tissue Organ Culture 17: Ziv. M. and Ariel, T Bud proliferation and plant regeneration in liquid-cultured Philodendron treated with ancymidol and paclobutrazol. Journal of Plant Growth Regulation 10:53-7. Vuylsteke, D.R Shoot-tip culture for the propagation, conservation and exchange of Musa germplasm. International Institute of Tropical Agriculture, Ibadan, Nigeria. 82 pp. Tables Table 1. Effect of TDZ in the proliferation of shoots. Concentration of growth regulators (mg/l) Pei Chiao (AAA) Fresh wt. (g) Number of shoots Cultivar Latundan (AAB) Fresh wt (g) Number of shoots 0 TDZ 1.9± ± ± ± TDZ 2.7± ± ± ± TDZ 3.2± ± ± ± TDZ 3.2± ± ± ± TDZ n r*** 3.7+gb** n r 1.5+gb 2.0 BA 3.3± ± ± ±2.2 * mean of 15 sample with standard deviation ** gb: clumps of globular buds, *** n r: not recorded 72

7 Table 2. Effect of TDZ in combination with BA in 3 banana cultivars. TDZ was added to multiplication medium containing 4 mg/l BA. Cultivar Concentration of TDZ (mg/l) Multiplication rate (A) Elongated Shoots (%) (B) Effective multiplication rate (AxB) 'Pei-Chiao' 0 4.9±1.0 A (100)* (AAA) ± (174) ± (165) ± (178) ± (170) ± (67) ± (85) 'Tai-Chiao 0 3.2± (100) No. 1' ± (186) (AAA) ± (224) ± (221) ± (110) ± (72) ± (45) Latundan 0 2.5± (100) (AAB) ± (126) ± (95) ± (137) ± (95) ± (89) ± (79) * multiplication rate relative to 0 mg/l TDZ (only BA) A Multiplication rate are mean ± s.d. of 12 samples Table 3. Multiplication rate of adventitious buds from different cycles of subculture treated with TDZ, Dropp and PP333. Values in brackets are multiplication rates relative to 0 mg/l TDZ (only BA). S1 to S3 represent number of subcultures. Concentration of growth Multiplication rate regulators (mg/l) TDZ Dropp PP333 BA S1 S2 S3 Average ±1.4 (100) 5.2±1.4 (100) 4.4±0.7 (100) 4.8 (100) ±1.6 (151) 8.5±1.6 (163) 6.9±0.9 (157) 7.6 (158) ±1.1 (127) 8.1±1.9 (156) 6.2±2.0 (141) 6.8 (142) ±0.8 (141) 8.9±1.4 (171) 8.7±1.3 (198) 8.1 (169) ±1.8 (161) 10.8±2.6 (207) 8.4±2.0 (191) 9.0 (205) Multiplication rate data are means ± s.d. of 18 samples of the cultivar 'Pei-Chiao' 73

8 Table 4. Effect of Dropp and PP333 on the mass propagation of 'Pei Chiao'. Multiplication rates are means ± s.d. of 35 propagules of the cv. 'Pei Chiao' (interval between subcultures = 9 to 10 weeks). Concentration of cytokinin (mg/l) Multiplication rate S3 S4 S5 Cumulative multiplication rate S6 (BA) Plantlet regeneration Plants/flask BA ±0.4 BA Dropp 0.1 ±0.7 PP BA 4.0 Dropp 0.2 PP ± ± ± ± ± ± ± (100)* 453 (861) 296 (562) * values in brackets are multiplication rates relative to BA medium. 3.1 ± ± ± ± ± ±1.3 Table 5. Effect of time intervals between two subcultures on the multiplication rate of 'Pei Chiao'. BA: BA 4.0 mg/l. D7: BA 4.0 mg/l; Dropp 0.1 mg/l; PP mg/l. Multiplication rates relative to BA medium. Multiplication rates are mean of 35 propagules of 'Pei Chiao'. Days in Medium Multiplication rate Cumulative subculture S3 S4 S5 multiplication rate 50 BA (100)* D (455) 60 BA (100)* D (577) 70 BA (100)* D (855) Table 6. Effect of Dropp and PP333 on the multiplication rate of 4 cultivars. BA: BA 4.0 mg/l; D7: BA 4.0 mg/l; Dropp 0.1 mg/l; PP mg/l. Cultivar Culture Multiplication rate Cumulative medium S3 S4 S5 multiplication rate 'Pei Chiao' BA (100)* D (455) 'Tai Chiao BA (100)* No. 2' D (330) 'Tai Chiao BA (100)* No. 3' D (426) Formosana BA (100)* D (194) * multiplication rates relative to BA medium 74