Plant Growth Regulation 26: 57–61, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.
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Micropropagation of tea (Camellia sinensis (L.) O. Kuntze) using Thidiazuron Tapan Kumar Mondal, Amita Bhattacharya, Anil Sood & Paramvir Singh Ahuja Division of Biotechnology, Institute of Himalayan Bioresource Technology, Palampur-176061, H.P., India Fax: +91-1894-30433; Email:
[email protected] Received 10 March 1998; accepted in revised form 2 June 1998
Key words: axillary shoot multiplication, Camellia sinensis (L.) O. Kuntze, nodal segments, Thidiazuron, woody plant
Abstract The effect of thidiazuron (TDZ) on the micropropagation of Camellia sinensis (China hybrid) was compared with that of benzylaminopurine (BAP) using nodal segments from in vitro raised seedlings. Extremely low concentrations of TDZ (1pM–100nM) alone were effective in inducing shoot bud proliferation and maintaining high rates of shoot multiplication on hormone-free media. On the other hand, higher concentrations of BAP (1–10µM) and its continued presence were required to initiate and sustain shoot proliferation. While wider ranges of BAP combined favourably with auxins like NAA or IBA, only specific combinations of TDZ and NAA were effective for shoot proliferation. TDZ treated explants yielded healthy shoots, with sturdy leaves, even during the initial stages of growth, whereas, the effect of BAP was cumulative over subcultures in attaining a high proliferative rate. Abbreviations: BAP = 6-Benzyl aminopurine; 2,4-D = 2,4-dichlorophenoxy acetic acid; IBA = Indole-3-butyric acid; MS = Murashige and Skoog Medium (1962); NAA = Naphthaleneacetic acid; TDZ = Thidiazuron (N-phenylN0 -1,2,3-thiadiazol-5 yl urea); WPM = Woody Plant Medium (Lloyd and McCown, 1980) 1. Introduction Thidiazuron is a herbicide with intrinsic cytokinin-like activity [16] and is known to stimulate high rates of regeneration and axillary shoot proliferation in many woody plant species [14] such as in Malus, [22] Acer, [12]; Populus, [15]; Acanthopanax, [24]; Quercus [8] etc. Since thidiazuron has been considered to be more potent than most of the commonly used cytokinins currently available for tissue culture of many woody species [10], it is important that the effect of thidiazuron be tested on an important plantation crop like tea. Though there are a number of excellent reports on the micropropagation of tea [1, 2, 5, 11, 19, 21], there is still no report on the effect of thidiazuron on the micropropagation of tea [9]. The present paper reports the effect of thidiazuron on multiple shoot proliferation in Camellia sinensis var. China hybrid using nodal segments as explants from in vitro raised
seedlings. Specifically, the impact of TDZ or BAP on the multiplication rates of responsive explants after their subculture to hormone-free medium has been compared and evaluated.
2. Materials and methods Seeds (variety China hybrid) collected in the month of November from the Institute’s Tea Experimental Farm were surface sterilized with 4% (w/v) sodium hypochlorite solution for 10 minutes and washed five times with sterile deionized water. The surface sterilized seeds were germinated on 1/2 strength MS medium supplemented with 30 gl−1 sucrose and 8 gl−1 agar (Qualigens, Bombay). Plantlets which were obtained from the germinated seeds were then selected as the source material when they had attained a height of 3–4 cms after a period of 60 days. From
58 Table 1. Effect of TDZ/BAP in MS/WPM media on the explant response Explant Response (%) MS
WPM
Concentration
TDZ
BAP
TDZ
BAP
0
—
—
—
—
100µM 10µM 1µM
94.0 ± 1.4CF 95.8 ± 1.2CF 96.7 ± 0.8CF
— 33.8 ± 1.0SP 12.8 ± 0.5SP
94.7 ± 1.1CF 95.5 ± 1.1CF 96.0 ± 1.0CF
— 1.5 ± 0.2SP 1.3 ± 0.2SP
100nM 10nM 1nM
54.5 ± 1.2SP 49.2 ± 0.7SP 49.0 ± 0.9SP
— — —
43.5 ± 0.9SP 38.5 ± 0.7SP 39.0 ± 0.7SP
— — —
100pM 10pM 1pM
29.0 ± 0.9SP 27.5 ± 0.6SP 28.2 ± 0.8SP
— — —
28.7 ± 0.8SP 28.7 ± 0.8SP 28.3 ± 0.7SP
— — —
SP = Shoot proliferation; CF = Callus formation; — = No response
such in vitro grown plantlets, nodal segments of about 1.0 cm were taken as explants and inoculated horizontally on two different basal media i.e. Woody Plant Medium or WPM [13] or MS medium [17] supplemented with 30 gl−1 sucrose and 0.8% agar. The pH of the media was maintained at 5.8 prior to autoclaving. All cultures were maintained at 25 ± 2 ◦ C under a photoperiod of 16 hrs with cool fluorescent lights of 52 µmol m−2 s−1 each. Experiments were repeated four times. While the effect of either TDZ or BAP (0–100µM) alone was tested on both WPM and MS medium, the effect of different factorial combinations of auxins like 0, 5, 10 and 15µM 2,4-D, NAA and IBA were tested with 0, 5 and 10µM of either TDZ or BAP for shoot and bud proliferation on MS medium only. After 4 weeks, the number of responsive explants were recorded and they were then transferred either to the same media or to hormone-free media and their multiplication rates evaluated. Subculturing was done at regular intervals of 4 weeks up to 24 weeks. Observations on multiplication rates after each subculture were noted with respect to the number of shoots per explant, shoot length, shoot diameter and the number of internodes and their lengths. For rooting, shoots (above 3.0 cm high) were treated with 500 mgl−1 IBA solution for 30 minutes and transferred directly to potting mix comprising of 9:1:1 :: garden soil:river bed sand : farm yard manure (pH 5.4) in Hikkotrays (Sharma M, Sood A, Prakash Om, Ahuja P. S. unpub-
lished) and the number of established rooted shoots recorded after 60 days.
3. Results and discussion 3.1 Effect of either Thidiazuron or BAP alone At µM concentrations of TDZ, callus response was obtained in both MS and WPM basal media (Table 1). Shoot bud proliferation was observed only after 4 weeks when TDZ at concentrations ranging from 1pM–100nM or when BAP at 1–10µM were used. Although the optimal response of shoot proliferation was observed at 100nM TDZ and 10µM BAP when maximum numbers of the explants responded in both MS and WPM medium, yet TDZ was found to be more effective than BAP (Table 1). No response was, however, observed when BAP alone was used below micromolar concentrations. This is in accordance with an earlier report [10] that high rates of axillary shoot proliferation in many woody species is stimulated at very low concentrations of TDZ (i.e. 1nM to 10µM) when most of the aminopurines like BAP are not effective. Since combination of BAP with auxins like IBA, IAA or NAA is necessary for tea shoot multiplication [9], this may be the reason for lower number of responsive explants in media supplemented with BAP alone as compared to TDZ.
59 3.2 Effect of TDZ or BAP in combination with IBA, 2,4-D and NAA Callus (90–99%) was induced in all the explants when TDZ (5, 10 and 15µM) was used in combination with different concentrations (5, 10, 15µM) of 2, 4D or IBA. Multiple shoot bud primordia were formed when 5–10µM TDZ was used in combination with 5– 15µM NAA. Optimal response (98%) was observed at a combination of 5µM TDZ and 10µM NAA and this declined up to 8 to 9% with further increase or decrease in the concentrations of NAA or TDZ. While increased shoot elongation and proliferation has been reported in species like Sorbus and Tilia [7] due to combined effects of auxin(s) and TDZ, the inductive potential of TDZ alone in shoot formation only at specific concentrations is also known [18]. Shoot bud primordia induction and shoot proliferation were observed at all combinations of BAP (5, 10, 15µM) with IBA (5, 10, 15µM) and also at BAP (5, 10, 15µM) with only 5µM NAA. Optimal concentrations were 5µM NAA with 10µM BAP (77.9%) and 5µM IBA with 5µM BAP (84.8%) respectively. While all other combinations of BAP and IBA resulted in a decline in the response, an increase in the concentrations of NAA (10 and 15µM) resulted in callusing and finally necrosis with time. All combinations of 2,4-D and BAP resulted in compact nodular callus with profuse anthocyanin pigmentation. Similar observations were also made by Vieitez [23] when 2,4-D alone or in combination was used. 3.3 Effect of absence or continued presence of PGRs on shoot multiplication at different subculture periods When each of the responsive explants (with 3–5 shoots per explant) initiated on a medium containing BAP were transferred to a hormone-free basal MS medium, the shoots elongated but did not multiply despite regular subcultures. However, shoot multiplication increased considerably when they were transferred to media supplemented with optimal concentrations of BAP (Figure 1A). On the contrary, the shoot multiplication rates increased with each subculture to hormone-free medium when TDZ was used in the initiation medium. However, when the responsive explants were continuously grown on medium containing TDZ, an overall callus overgrowth and later necrosis were observed (Figure 1B). While the continued presence of BAP in the shoot multiplication media of tea is important [1, 2, 3, 11, 21], the deleterious effect of the continued presence of TDZ on the
growth and multiplication of chickpea and pea have been reported [4, 18]. This further supports our finding that the multiplication of shoots initiated on TDZ medium was considerably enhanced on hormone-free medium. This is probably due to the capacity of TDZ in stimulating endogenous cytokinin biosynthesis or in altering cytokinin metabolism [20, 25]. Moreover, calli initiated on media containing TDZ are known to become habituated with cytokinin autonomy [6, 10]. From our study it is evident that TDZ is effective at very low i.e. pM to nM concentrations and this may be the reason for its continued response to shoot proliferation. 3.4 Comparative performance of explants initiated on TDZ or BAP containing media after different subculture periods Although the number of shoots formed was higher in explants initiated on medium containing TDZ than that on medium supplemented with BAP, the multiplication rates were however, more or less similar (i.e. 2–3 times) after each subculture (Table 2). The general growth of the shoots during the earlier stages with respect to sturdy leaves, shoot length and shoot diameter were also better in explants initiated on TDZ medium in comparison to BAP but this became almost similar in the later stages. Persistent presence of BAP at optimal concentrations was necessary at different subcultures and there seemed to be a cumulative gain over subcultures in both the number as well as the sturdiness of the shoots. In contrast however, it was necessary to remove TDZ in subsequent subcultures in order to increase the proliferation rates or the sturdiness of the shoots. 3.5 Rooting of the microshoots Adventitious roots were formed in the microshoots after 60 days. Hardened young plants with healthy roots were then shifted to polysleeves in polytunnels after 60 days prior to field transfer.
4. Conclusions The present study accurately establishes a protocol for the method of use of TDZ in tea micropropagation. For high rates of shoot proliferation, it is necessary to subculture explants (initiated on medium containing TDZ) on to a hormone free medium. In contrast however, when initiation of shoot buds occur on medium
60
Figure 1. Shoot proliferation of explants which were initially cultured for 4 weeks on (A) MS with BAP and (B) MS with TDZ. After 4 weeks subsequent subculturing were done either on hormone-free MS medium or on MS medium with the respective PGRs. (The bars in the figure represent standard errors of means).
Table 2. Comparative performance of explants initiated on TDZ or BAP containing media Sub-culture No. of shoots/explant periods MS∗ MS+BAP 8 weeks 12 weeks 24 weeks
Shoot length (mm) MS∗ MS+BAP
Shoot diameter (mm) No. of internodes Internode length (mm) MS∗ MS+BAP MS∗ MS+BAP MS∗ MS+BAP
13.00±1.74 11.33±0.42 30.83±6.67 22.33±2.73 2.25±0.27 1.20±0.24 7.33±0.42 5.20±0.58 0.36±0.07 0.42±0.13 37.17±3.77 23.33±0.8 60.00±3.87 40.00±1.18 2.25±0.11 2.25±0.11 10.50±0.5 7.33±0.42 0.56±0.09 0.98±0.07 44.17±1.08 30.33±1.20 66.17±2.17 44.20±1.74 2.30±0.11 2.70±0.12 11.00±0.60 9.70±0.21 0.50±0.03 1.15±0.07
∗ Explants initiated on media containing TDZ were sub-cultured on to hormone-free media whereas explants initiated on media containing BAP
were maintained in the same medium.
61 containing BAP in combination with auxins, continued presence of plant growth regulators is required to evince sustained proliferation rates. During the initial stages of growth, the number of healthy shoots produced on medium containing TDZ are relatively more and this increases further with every subculture on to hormone free medium for up to over 24 subcultures. Since very low concentrations of TDZ are used only at the initial phase, the overall higher cost of TDZ is overcome. TDZ thus, appears to be a potent cytokinin for tea micropropagation with high proliferation rates.
8.
9.
10.
11. 12.
13.
Acknowledgements The authors thank Prof. Akshey K. Gupta, Director, Institute of Himalayan Bioresource Technology, Palampur for providing the necessary facilities. Tapan K. Mondal gratefully acknowledges the financial assistance from University Grants Commission, New Delhi.
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