In Vitro Cell.Dev.Biol.Plant (2007) 43:5964 DOI 10.

1007/s11627-006-9011-8

MICROPROPAGATION

In vitro propagation of Indian Kino (Pterocarpus marsupium Roxb.) using Thidiazuron

M. K. Husain & M. Anis & A. Shahzad

Received: 7 November 2006 / Accepted: 15 November 2006 / Published online: 14 February 2007 / Editor: S.S. Korban # The Society for In Vitro Biology 2007

Abstract An efficient protocol was developed for micropropagation of an economically important timber-yielding multipurpose tree, Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes (CNs) derived from 18-d-old axenic seedlings on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.110 M). The highest shoot regeneration frequency (90%) and maximum number (15.20.20) of shoots per explant was recorded on MS medium amended with 0.4 M TDZ. Continuous presence of TDZ inhibited shoot elongation. In the primary medium, TDZ-initiated cultures were transferred to the secondary medium supplemented with another cytokinin, 6-benzyladenine (BA), for shoot growth and elongation. Maximum (90%) shoot elongation with an average shoot length of 5.40.06 cm was observed at 5 M BA. To further enhance the number of shoots per explant, mother tissue was repeatedly subcultured on fresh shoot induction medium after each harvest of newly formed shoots. Thus, by adopting this strategy, an average of 44 shoots per explant could be obtained. About 65% of in vitro regenerated shoots produced a maximum number (4.40.2) of roots per shoot by a two-step culture procedure employing pulse treatment and subsequent transfer of treated shoots to a low concentration of 0.2 M indole-3-butyric acid along with phloroglucinol (3.96 M). The in vitroraised plantlets were successfully acclimatized first under culture room conditions, then to greenhouse with 70% survival rate.

Keywords Cotyledonary nodes . Leguminous tree . Multiple shoots . Plant regeneration . Pterocarpus marsupium . Thidiazuron

Introduction Pterocarpus marsupium. Roxb. (Fabaceae), commonly known as Bijasal, is one of the most important multipurpose forest tree legumes of India, valued greatly for its excellent timber. In addition, the trees are valued for its pharmaceutical properties as the gumkino obtained from the tree is a powerful astringent used for treatments of dysentery, diarrhea, fever, and toothache. An aqueous infusion of the wood is said to be of use in diabetes, and water stored in the vessel made of the wood is reputed to have antidiabetic properties (Anonymous, 2003). Two important phenolic constituents, marsupsin and pterostilbene, isolated from the heartwood of P. marsupium are reported to possess antihyperglycemic activity (Manickam et al., 1997). The aqueous extract of stem bark was found to reduce the blood glucose level in alloxan-induced diabetic rats (Vats et al., 2002). Natural propagation of P. marsupium through seeds is not overly successful because of poor seed germination (Kalimuthu and Lakhsmanan, 1995). It is also known that the regeneration rate of leguminous trees in natural habitats is quite low (Dewan et al., 1992). The indiscriminate exploitation of the species, together with low percentage of germination, has resulted in a decrease in the size of its natural stand, which ultimately has led to its inclusion in the list of depleted plant species (Chaudhuri and Sarkar, 2002). In view of its inherent qualities and restricted distribution, its propagation and multiplication through tissue culture is urgently required (Anis et al., 2005). Plant tissue

M. K. Husain : M. Anis (*) : A. Shahzad Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh 202 002, India e-mail: anism1@rediffmail.com

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culture offer advantages over conventional methods for multiplication and large-scale production of woody plants (Thorpe et al., 1991). Many woody plant species (McCown, 2000) and particularly tree legumes are known for their recalcitrant nature of regeneration (Jha et al., 2004; Anis et al., 2005). Thidiazuron, a substituted phenylurea (N-phenyl-1,2,3thidiazol-5-ylurea) with intrinsic cytokinin like activity (Mok et al., 1982) was shown to induce high rates of regeneration and shoot proliferation of several recalcitrant woody species including Quercus robur (Chalupa, 1988) and Pinus strobus (Pijut et al., 1991). Its effect as a plant growth regulator (PGR) facilitated efficient micropropagation of many woody plant species such as Malus (van Nieuwkerk et al., 1986), Rubus (Fiola et al., 1990), and Populus (McCown et al., 1991) is well known. Because TDZ is among the most active cytokinin-like substances for woody plant tissue culture (Huetteman and Preece, 1993), inevitably, therefore, it becomes imperative that the effect of TDZ be tested on an important multipurpose treelegume like P. marsupium. Although there are few preliminary reports (Das and Chatterjee, 1993; Kalimuthu and Lakhsmanan, 1994) or detailed reports (Chand and Singh, 2004; Anis et al., 2005) on in vitro plant regeneration available, the effect of TDZ on micropropagation of the species has not yet been investigated. In the present communication, an attempt was made to study the effect of TDZ on multiple shoot induction and plant regeneration from in vitro-grown seedling-derived cotyledonary node (CN) explants of P. marsupium.

(bacteriological grade, Hi-media, India). Cotyledonary nodes explants were excised from 6-, 12-, 18-, and 24-dold axenic seedlings. Culture media and conditions. Murashige and Skoog medium containing 3% (w/v) sucrose and 0.7% agar was used in all the experiments unless otherwise specified. Plant growth regulators at different concentrations were incorporated into the basal media as stated below. The pH of the medium was adjusted to 5.8 by 1 N NaOH or 1 N HCl before autoclaving at 1.06 kg cm2 (121C) pressure for 20 min. The cultures were incubated at 252C in a culture room with 50 mol m2 s1 irradiance provided by cool fluorescent tubes (40 W; Philips, India) and were exposed to a photoperiod of 16 h and 555% of relative humidity (RH). Multiple shoot induction. After removal of a radicle and primary shoots from the seedlings, the CNs (1 cm) were placed vertically onto MS medium supplemented with different concentrations (0.1, 0.2, 0.4, 0.8, 1.0, 2.5, 5.0, 7.5, and 10 M) of TDZ. Shoot induction medium was designated as primary medium. After 6 wk of culture, the frequency of explants producing shoot and the average number of shoots per explants were recorded. All cultures were transferred to fresh medium every 21 d. Growth regulator-free MS medium was used as control. Shoot growth and elongation. To facilitate shoot growth and elongation, a modified two-step culture procedure was employed. The first step involved the induction of shoots from CN explants on primary medium augmented with TDZ for 6 wk followed by step two in which TDZ-induced (optimum TDZ concentration) shoots were transferred to the secondary medium consisting of either growth regulatorfree MS medium or another cytokinin, 6-benzyladenine (BA) at different concentrations (2.5, 5.0 and 10 M). After 6 wk, percentage of elongated shoots and shoot length were recorded. In vitro root induction. The elongated individual microshoots (34 cm) with more than two fully expanded leaves were excised, and in vitro root induction was attempted either on half-strength MS medium without growth regulators or by a two-step culture procedure. In the first step, the induced microshoots were pulse-treated with indole-3butyric acid (IBA) (200 M) for 4 d on filter paper bridge on half-strength MS-liquid nutrient media with reduced concentration of sucrose (2%), followed by step two involving subsequent transfer of such pulse-treated shoots onto 0.6% agar-gelled half-strength MS salts augmented with IBA (0.2 M) alone or with phenolic acids. Phenolic compounds (Sigma, St. Louis, MO, USA) included

Materials and Methods Plant material and explant source. The mature winged fruits of P. marsupium were obtained from the Tropical Forest Research Institute, Jabalpur, India. The healthy seeds were excised from the fruits and washed thoroughly in running tap water for 30 min, rinsed with 5% (v/v) Teepol (a liquid detergent; Glaxo, India) for 10 min and kept in 1% (w/v) Bavistin (carnbandazim powder, BASF, India), a broad spectrum fungicide, for 15 min. The treated seeds were agitated in distilled water for 24 h to remove the chemical inhibitors to germination. The leachates were replaced with sterile distilled water. The seeds were surfacedisinfected with 70% (v/v) ethanol for 30 s, followed by with an aqueous solution of 0.1% (w/v) freshly prepared HgCl2 for 5 min, and finally rinsed five times with sterile distilled water. The surface-disinfected seeds were germinated aseptically in culture flask (100 ml, Borosil, India) containing growth regulator-free half-strength Murashige and Skoog (MS) (Murashige and Skoog, 1962) medium with 3% (w/v) sucrose (Qualigens, India) and 0.7% agar

IN VITRO PROPAGATION OF INDIAN KINO USING THIDIAZURON Table 1. Effect of TDZ on multiple shoot induction from CN explants of P. marsupium after 6 wk of culture TDZ (M) Percent of explants producing shoots 0.0 50 75 90 80 70 65 55 50 40 Mean number of shoots/explant 0.00.0g 3.80.37e 6.60.24c 15.20.20a 8.20.44b 5.20.35d 4.00.31de 3.20.20ef 2.40.24f 2.20.36f

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0.0 0.1 0.2 0.4 0.8 1.0 2.5 5.0 7.5 10

Values represent meansSE. Means followed by the same letter within columns are not significantly different by Tukeys test at the 5% probability level.

regulator-free half-strength MS medium. Cotyledonary nodes excised from axenic seedlings were used as explants in this study. The induction of multiple shoots varied with the age of CNs; a significantly greater percentage response was recorded from 18-d-old explant compared to 6-, 12-, and 24-d-old explants (data not shown). Explant age plays a significant role to induce multiple shoots in a number of plants, including Cercis canadensis (Distabanjong and Geneve, 1997) and Morus alba (Thomas, 2003). Cotyledonary nodes failed to respond morphogenically to a growth regulator-free MS medium. The addition of cytokinin to the medium was essential to induce multiple shoots. The CN explants swelled and started differentiating multiple shoot buds directly within 3 wk of inoculation at all concentrations of TDZ. The range of percentage of shoot regenerating explants was 4090%, and the average number of shoots formed per explant varied considerably and showed significant differences at different concentra-

phloroglucinol (PG) (1.98, 3.96, and 7.92 M), chlorogenic acid (0.70, 1.41, and 2.82 M), and salicylic acid (1.81, 3.62, and 7.24 M). After 4 wk of culture, percentage of rooted shoots, mean number of roots per shoot, and root length were recorded. Hardening and acclimatization. In vitro regenerated plantlets were washed carefully in running tap water to remove the traces of agar, followed by the transfer to culture tubes containing quarter-strength liquid MS nutrients without sucrose for 2448 h. Hardened plantlets were transferred to pots containing autoclaved soil and soilrite (1:1, w/w) and were covered with polybags for 4 wk to maintain high RH. The plantlets were initially irrigated with quarter-strength inorganic salts of MS medium for 2 wk followed by tap water. Potted plantlets were grown in culture room conditions (252C, 555% RH, under 16 h of photoperiod with a light intensity of 40 mol m2 s1) for 2 mo. Polybags were removed gradually upon emergence of new leaves and acclimatized plantlets were transferred to the greenhouse. Statistical analysis. All experiments were repeated three times with 10 replicates per treatment. A single explant was used per culture tube. The data on shoot formation and rooting were analyzed using SPSS version 10 (SPSS Inc., Chicago, USA), and significant differences between means were assessed by the Tukeys test at 5% level of significance.

Results and Discussion Multiple shoot induction. Eighty percent of P. marsupium seeds germinated within 45 d of inoculation on a growth

Figure 1. In vitro propagation of P. marsupium. (a) Multiple shoot induction from CN explant on MS medium amended with TDZ (0.4 M). (b) Shoot growth and elongation on MS medium with BA (5 M). (c) Rooting of in vitro regenerated shoot by a two-step culture procedure on half-strength MS medium with IBA (0.2 M) and PG (3.96 M). (d) Plant acclimatized to greenhouse conditions.

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Table 2. Effect of BA-containing secondary medium on shoot elongation from CNs of P. marsupium after 6 wk of culture Precontinued primary medium (optimum concentration) TDZ (0.4 M) BA-supplemented secondary medium (M) Percent of culture showing response 75 90 60 Mean shoot length (cm)

2.5 5.0 10.0

3.9.03b 5.4.06a 3.1.05c

grown continuously on TDZ-supplemented medium resulted in the formation of suppressed/fasciated shoots. The fasciation of shoots on TDZ-augmented medium was reported from time to time for several plant species including Rhododendron (Preece and Imel, 1991) and D. sissoo (Pradhan et al., 1998). Inhibition of shoot elongation may be because of the high cytokinin activity of TDZ, whereas the presence of a phenyl group in TDZ may be a possible cause of shoot bud fasciation (Huetteman and Preece, 1993). Shoot growth and elongation. A maximum number of shoots was initiated in the presence of MS medium supplemented with 0.4 M TDZ. A two-step culture procedure was initiated to address the problem of shoot elongation. This was done by transferring the shoot cultures (initiated on primary medium) to a secondary medium augmented with BA. Among the different concentrations of BA tested, the best elongation of shoots (90%) was recorded at 5 M BA with an average shoot length of 5.40.06 cm with four internodes after 6 wk (Table 2; Fig. 1b). The results obtained here are in accordance with an earlier report of Huetteman and Preece (1993) that the problem of shoot elongation can be solved by a two-stage culture procedure consisting of a TDZ-containing primary medium to induce shoot proliferation followed by a secondary medium with another PGR that promotes shoot development. This type of culture procedure of using primary (shoot induction) and secondary (shoot elongation) medium was successfully applied to a number of plant species, including Malus domestica (Fasolo et al., 1989), Cajanus cajan (Eapen et al., 1998), Nothapodites foetida (Satheeshkumar and Seeni, 2000), Rosa damascena (Kumar et al., 2001), M. alba (Thomas, 2003), and A. sinuata (Vengadesan et al., 2002). When TDZ-initiated cultures were transferred to growth regulator-free MS medium, shoots failed to elongate and

Values represent meansSE. Means followed by the same letter within columns are not significantly different by Tukeys test at the 5% probability level.

tions (Table 1). Axillary shoot proliferation from CNs of seedlings was well documented for several tree species such as Acacia nelotica (Dewan et al., 1992), Sterculia urens (Purohit and Dave, 1996), C. canadensis (Distabanjong et al., 1997), Dalbergia sissoo (Pradhan et al., 1998), Acacia sinuata (Vengadesan et al., 2002), and Sesbania rostrata (Jha et al., 2004). The frequency of axillary shoot proliferation and the number of shoots per explant increased with increasing concentration of TDZ up to the optimum level. Among various concentrations of TDZ used, the highest shoot regeneration frequency (90%) and maximum mean number of shoots (15.20.2) was recorded at 0.4 M TDZ after 6 wk of culture (Table 1, Fig. 1a). The finding is in accordance with an earlier report by Huetteman and Preece (1993) that higher rates of axillary shoot induction in many woody tree species, including recalcitrant ones, is stimulated at 1 nM to 10 M TDZ. The frequency of shoot regeneration ability declined markedly at higher concentrations of TDZ and was invariably associated with basal callusing along with thick and stunted shoots. Although a high multiple shoots rate was induced on TDZ-containing medium, the shoots failed to elongate. Moreover, cultures

Table 3. Effect of different phenolic compounds in combination with 0.2 M IBA supplemented to half-strength MS medium on in vitro root induction of pulse-treated (IBA 200 M; 4 d) microshoots of P. marsupium after 4 wk of culture Phenol compound Phloroglucinol Concentration (M) 1.98 3.96 7.92 0.70 1.41 2.82 1.81 3.62 7.24 Percent of rooted shoots 61 65 60 55 62 55 50 48 40 Mean number of roots per shoot 2.60.24b 4.40.25a 2.40.23bc 1.80.20bcd 2.20.21bcd 2.00.32bcd 1.60.24bcd 1.40.30cd 1.20.20d Mean root length (cm) 3.6.03b 4.0.04a 3.1.04cd 3.0.05d 3.2.02c 2.7.03e 2.4.02f 1.9.04g 1.7.05h

Chlorogenic acid

Salicylic acid

Values represent meansSE. Means followed by the same letter within columns are not significantly different by Tukeys test at the 5% probability level.

IN VITRO PROPAGATION OF INDIAN KINO USING THIDIAZURON

63

shed all their leaves within 2 wk. This indicates that the presence of cytokinin is essential for survival and elongation of shoots. The cultured mother explants exhibited the development of new shoots up to two culture passages only and retained the same number of shoots. During a third passage, shoot induction was poor, and mother explants turned brown thereafter. Adopting this procedure, an average of 44 shoots could be induced per explant. A similar approach was adopted earlier in Syzygium cumini (Jain and Babbar, 2000). In vitro root induction. Half-strength MS medium without any growth regulator failed to induce root formation even after 4 wk. Only 25% of pulse-treated (200 M, 4-d) microshoots rooted on half-strength MS medium with IBA (0.2 M) within 3 wk had about 12 thin and delicate roots per shoot. Two-step rooting procedure was used to advantage in many leguminous species including Prosopis cineraria (Shekhawat et al., 1993). Root development was accompanied by a little callus formation at the junction of root and shoot. Such type of rooting was found to be undesirable for successful transplantation of in vitroregenerated plantlets. The percentage of rooting was markedly enhanced by augmenting the medium with phenolic compounds along with IBA (Table 3). All three phenolic compounds stimulated in vitro rooting in combination with IBA but failed to produce roots when used alone. This may be because of the auxinphenol synergism resulting in suppression of the peroxidase activity in the culture, thereby protecting the endogenous auxin from peroxidase-catalyzed oxidation (De Klerk et al., 1999). A maximum frequency of root formation (65%) and the highest number of roots (4.40.25) with maximum root length (4.00.04 cm) was achieved on half-strength MS medium with IBA (0.2 M) and PG (3.96 M) after 4 wk (Fig. 1c). The promotive effect of PG on rooting was identified in several plant species including Malus pumila (James, 1983; Zanol et al., 1998) and Prunus avium (Hammatt and Grant, 1996). In all these reports, PG enhanced rooting in the presence of auxin, which is in line of our findings. Hardening and acclimatization. Hardening is a crucial step before transfer of plantlets to soil. The rooted plantlets were first hardened under in vitro conditions by transferring to culture tubes containing quarter-strength liquid MS nutrients lacking sucrose for 2448 h. Hardened plants were finally transferred to clay pots. After 2 mo., acclimatized plantlets were transferred to greenhouse. The survival rate of these plants after transfer was 70% (Fig. 1d). All the established plants showed a high degree of uniformity without any detectable phenotypic variation. Using this

protocol, it is estimated that a single CN explant could produce approximately 4345 plants within 4 mo. The present finding accurately establishes a protocol for the use of TDZ as a potent cytokinin for micropropagation of P. marsupium. Our findings suggested that TDZaugmented primary medium is required for high rate of shoot bud induction. Shoot buds must then be transferred to a secondary medium supplemented with another cytokinin, BA, for better shoot growth and elongation. In conclusion, the in vitro protocol described in this study for regenerating plantlets of P. marsupium using CNs of axenic seedlings is an improved method compared to earlier reports. This protocol could thus be useful for improvement, conservation, and large-scale planting of this economically and medicinally important timber-yielding tree legume.

Acknowledgment The authors are thankful to the Department of Biotechnology, Government of India, New Delhi, for the research grant.

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