Influence of ventilation closure, gelling agent and explant type on ...

In Vitro Cell. Dev. Biol.—Plant 42:445–449, September–October 2006 q 2006 Society for In Vitro Biology 1054-5476/06 $18.00+0.00

DOI: 10.1079/IVP2006791

INFLUENCE OF VENTILATION CLOSURE, GELLING AGENT AND EXPLANT TYPE ON SHOOT BUD PROLIFERATION AND HYPERHYDRICITY IN SCROPHULARIA YOSHIMURAE – A MEDICINAL PLANT HSIN-SHENG TSAY*, CHEN-YU LEE, DINESH CHANDRA AGRAWAL,

AND

SHOKKANNAGOUNDER BASKER

Graduate Institute of Biotechnology, Chaoyang University of Technology, 168, Gifong E. Road, Wufong, Taichung 41349, Taiwan, Republic of China (Received 20 December 2005; accepted 20 April 2006; editor W. Y. Soh)

Summary We report an improved procedure of in vitro propagation of Scrophularia yoshimurae – a medicinally important plant species indigenous to Taiwan. Induction of maximum shoot buds (22.75 per explant) was obtained with shoot tip explant cultured on Murashige and Skoog medium supplemented with 1.0 mg l21 benzyladenine (BA) and 0.2 mg l21 a-naphthaleneacetic acid and gelrite using dispense paper (DP) for ventilation closure of culture vessels. The type of gelling agents (agar and Gelrite) affected both quantity and quality of the shoots induced. Using aluminum foil for ventilation closure resulted in a higher number of hyperhydric shoots. Hyperhydricity was reduced by culturing shoots on a medium devoid of plant growth regulators in conjunction with the use of DP. Plantlet growth in vessels using DP was healthier and all plantlets survived after being transplanted to soil. Key words: gelling agent; hyperhydricity; in vitro propagation; Scrophularia yoshimurae; ventilation closure of vessels. important for in vitro plant regeneration. To maintain the sterility of cultures, it is essential to cover culture vessels with sealing. Different types of sealing are commonly used. Some sealings cause restriction of gaseous exchange between the vessel atmosphere and the outside environment (Buddendorf-Joosten and Woltering, 1994), which can result in poor aeration and hyperhydric conditions of cultures. Hyperhydricity is a morphological abnormality that has often been observed in micropropagated plants (Kevers et al., 1984). Solving the problem of hyperhydricity would be of immense help for commercial production and conservation of S. yoshimurae. A method of de novo regeneration of S. yoshimurae has been established in our laboratory (Sagare et al., 2001), but low proliferation rate and high frequency of hyperhydric shoots were major concerns (Lai et al., 2005). In this study, we evaluated the influence of different factors such as ventilation closure of vessel, explant type, plant growth regulators (PGR) and gelling agent with the aim of enhancing the multiplication rate and reducing hyperhydricity in S. yoshimurae.

Introduction Different members of the genus Scrophularia have been used as crude drugs in traditional Chinese medicine. Scrophularia yoshimurae Yamazaki, belonging to the family Scrophulariaceae, is a herbaceous perennial plant of 40– 60 cm height. This plant is indigenous to Taiwan, growing in the mountainous areas at an elevation of 1000– 1300 m. It is very difficult to locate plants of S. yoshimurae in the wild. S. yoshimurae has been known as ‘Xuanshen,’ which is a substitute for Scrophularia ningpoensis used for treatment of inflammation, laryngitis, tonsillitis, abscesses of carbuncles, and constipation (Chiu and Chang, 1998). In Taiwan, the processed roots of S. ningpoensis are imported from China as crude drugs because S. yoshimurae is not cultivated on a commercial scale and roots collected from the natural habitat are insufficient to meet the local demand. In vitro culture techniques have been used successfully for the propagation of many medicinally important plant species (Tsay, 1999; Rout et al., 2000; Mulabagal et al., 2004; Sunandarkumari et al., 2005). Plants propagated by tissue culture showed less variation in the content of secondary metabolites than their cultivated or wild counterparts (Yamada et al., 1991). It has been reported that the growth rate and other physiological and morphological characteristics of plants developed under in vitro conditions can be influenced by the physical and chemical microenvironments of culture vessels (Walker et al., 1988). The choice of explant type (Pua, 1999) and gelling agent (Debergh, 1983) are also

Materials and Methods Plants of S. yoshimurae collected from Chitou, Nantou County, Taiwan (altitude of c. 1300 m) were replanted in 18 cm diameter pots containing a mixture of soil : peat moss : vermiculite (1:1:1, v/v/v). These plants were maintained in growth chambers (Hotech Instruments Corp., Model 624 HD, Taipei, Taiwan) under the light intensity of 100 mmol m22 s21 in a 16 h photoperiod at day and night temperatures of 20 and 168C, respectively. Aseptic shoot cultures of S. yoshimurae were established as described previously (Sagare et al., 2001). Shoot tips and nodal segments excised from in vitro grown shoots were used as explants. These explants were cultured in 500 ml conical flasks, each containing 100 ml of medium. The medium consisted of MS (Murashige and Skoog, 1962), salts and vitamins (hereinafter referred as MS basal medium) supplemented with 1.0 mg l21 benzyladenine (BA) and 0.2 mg l21 a-naphthaleneacetic acid (NAA)

*Author to whom correspondence should be addressed: Email hstsay@ cyut.edu.tw

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(hereinafter referred as PGR or multiple shoot induction medium) or without PGR. Sucrose (3%), Difco BactoTM agar (1%) (Difco Laboratories, Detroit, MI, USA) or 0.325% Gelrite (CP Kelco US Inc.) was added to each medium, and the pH of the medium was adjusted to 5.7 ^ 0.1 before autoclaving at 1218C, 105 kPa for 15 min. In order to optimize each factor, four experiments were carried out. In the first experiment, 20 nodal segments (in five replicates) were cultured in 500 ml flasks, each containing medium with appropriate PGR and agar as gelling agent for multiple shoot induction. The vessel head space was capped with (1) two layers of aluminum foil (AF) or (2) two, three, and four layers of pharmaceutical dispense paper [DP; 9.5 £ 9.5 cm, 0.046 mm thick, gas flow 0.5 ml s21, made from soft- and hard-wood fiber (50:50); Cheng Loong Corp., Taiwan]. In the second experiment, 20 nodal segments (in five replicates) were cultured as described in the first experiment and head space was capped with (1) two layers of AF or (2) four layers of DP and one additional layer of Parafilm Mw. Parafilm was removed after 7, 14, 21 and 28 d of culture. In the third experiment, nodal segments were cultivated in a 500 ml flask as described in the first experiment except that the number of explants in each flask differed; each flask contained 10, 15, 20 or 30 explants, with each treatment consisting of five replicates. The head space of the vessel was capped with four layers of DP and sealed with one layer of Parafilm, which was removed after 28 d of culture. In the fourth experiment, 20 nodal or shoot tip explants were cultured in each 500 ml flask and the vessel was capped as described in the third experiment. In all the experiments, the number of multiple shoot buds induced and their hyperhydric conditions were recorded after 60 d of culture. In the next set of experiments, we evaluated the combined effect of explant type, PGR and ventilation closure on multiple shoot induction and hyperhydricity. This was carried out by culturing 20 nodal segments or shoot tips in 500 ml flasks, each containing MS medium containing 1.0 mg l21 BA and 0.2 mg l21 NAA or free of PGR. The vessel’s head space was covered with (1) two layers of AF or (2) four layers of DP. In both cases, the head space was further sealed with one layer of Parafilm. All cultures were incubated at 25 ^ 18C in a 16 h photoperiod at the light intensity of 38 mmol m22 s21 provided by cool white fluorescent lamps (Philips, The Netherlands). After 4 wk, the culture vessels were ventilated by removal of the Parafilm layer. The number of shoot buds, shoot length, and root number per explant were recorded at 4 wk after removal of Parafilm (after a total of 8 wk of incubation). Well-rooted plantlets were transferred to pots containing a mixture of peat moss, perlite, and soil (1:1:1, v/v/v) and kept in a growth chamber at 258C for hardening, as described previously (Sagare et al., 2001). The survival rate was recorded at 5 wk after potting. Data were analyzed statistically by using Fisher’s protected least significant difference (LSD) test at the 5% probability level.

Results and Discussion In initial experiments on the two container closures, i.e. AF and DP, AF induced a higher number of multiple shoot buds with 11.2 shoots per explant compared to 3.1– 5.1 shoots per explant in DP. However, 87.1% shoots derived from the former were hyperhydric but all shoots from the latter were normal (non-hyperhydric) (Table 1). It was observed that the number of shoot buds increased with an increase in the DP layers, with the highest number of shoot buds (5.1 per explant) using four layers of DP. Hence, four layers of DP were selected for subsequent experiments. Our results showed that, under tight-sealing conditions by using AF, there is a direct correlation between the number of multiple shoot bud formed and hyperhydricity. Decreasing the number of multiple buds without hyperhydricity by increasing aeration using DP was also observed in another experiment, in which the Parafilm layer was removed from the vessel at weekly intervals (Table 2). Results of these experiments indicate that formation of shoot buds from explants of S. yoshimurae requires tight sealing of culture vessels. Furthermore, we observed the highest number of shoots per explant in a culture vessel containing 20 explants (data not shown).

TABLE 1 EFFECT OF VENTILATION CLOSURE ON MULTIPLE SHOOT BUD INDUCTION AND HYPERHYDRICITY IN S. YOSHIMURAE z Ventilation closure type AF DP

Number of layers

Number of multiple shoots/explant

2 2 3 4

11.2 ay 3.1 c 3.6 c 5.1 b

% of hyperhydric shoots 87.1 0.0 0.0 0.0

(84.2–90.0) (0.0–3.5) (0.0–3.1) (0.0–2.2)

z Nodal explants were cultured on MS basal medium with 1.0 mg l21 BA, 0.2 mg l21 NAA, 3% sucrose, and 1% agar after 8 wk. y Means followed by the same letter are not significantly different at 5% level by LSD test. Data in parentheses are 95% confidence limits of binomial distribution.

Hence, 20 explants per culture vessel were used for subsequent experiments. It has been reported that the type of ventilation closure affects gaseous exchange, availability of water, micronutrients, and balance of hormones in the vessel (Kataeva et al., 1991). Hyperhydricity is a consequence of the plant’s response to an unsuitable in vitro environment. It has been reported that a higher number of hyperhydric shoots in potato, observed under a completely sealed vessel, was associated with high concentrations of ethylene and CO2 accumulated in culture (Park et al., 2004). In carnations, proper ventilation in culture vessels has been shown to minimize hyperhydricity (Jo et al., 2002). With respect to the combined affect of ventilation closure, explant type, and growth regulators in this study, multiple shoot buds could be induced from both the shoot tips and nodal segments cultured on MS basal medium with or without PGR. However, the quantity and quality of shoot buds induced from these two media varied. In general, the medium with PGR induced a higher number of shoot buds when compared to PGR-free medium (Table 3). When the shoot tips and nodal segments were compared, the former gave rise to a higher number of multiple shoot buds than the latter. Among the two gelling agents used, higher number of shoot buds was obtained with Gelrite than with agar in both types of explants (Table 3). Pua (1999) reported that the capacity of de novo shoot regeneration from cultured explants could be influenced by the type of explant, its age, and its position on the medium. In this study, we found that the combinations of PGR, less ventilation closure, and the use of Gelrite as Gelling agent drastically improved the number of shoot buds induced in S. yoshimurae (Table 3). The occurrence of hyperhydric shoots was also affected by explant type, PGR in the medium, and type of ventilation closure used. In general, the number of hyperhydric shoots was higher on the medium with PGR and Gelrite, while shoot tip explant gave rise to more hyperhydric shoots than nodal segments (Table 3). It was observed that the number of hyperhydric shoots decreased markedly in ventilated culture vessels after the removal of the parafilm layer. Fewer or no hyperhydric shoots were also observed in explants grown on PGR-free MS medium using DP, whereas the medium with PGR was less effective in reducing hyperhydric shoots. The beneficial effect of DP in reducing shoot hyperhydricity may be due

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IN VITRO STUDY IN SCROPHULARIA YOSHIMURAE TABLE 2 EFFECT OF VENTILATION CLOSURE AND DAYS OF PARAFILM SEALING REMOVAL ON MULTIPLE SHOOT BUD INDUCTION AND HYPERHYDRICITY IN S. YOSHIMURAE z Ventilation closure type

Number of days after culture where parafilm layer was removed

Number of multiple shoots/explant

0 0 7 14 21 28

12.5 ax 6.0 d 5.5 d 7.5 c 9.6 b 10.7 ab

AFy DP (4 layers)y

% of hyperhydric shoots 85.4 0.0 0.0 0.0 5.9 12.2

(82.2–88.6)w (0.0–1.3) (0.0–1.5) (0.0–0.8) (3.0–8.9) (8.9–15.5)

z

Nodal explants were cultured on MS basal medium with 1.0 mg l21 BA, 0.2 mg l21 NAA, 3% sucrose, and 1% agar after 8 wk. Culture vessels were further sealed with a layer of Parafilm Mw. x Means followed by the same letter are not significantly different at 5% level by LSD test. w Data in parentheses are 95% confidence limits of binomial distribution. y

TABLE 3 INFLUENCE OF EXPLANT TYPE, GROWTH REGULATORS, GELLING AGENT AND VENTILATION CLOSURE OF CULTURE VESSELS ON MULTIPLE SHOOT BUD INDUCTION IN S. YOSHIMURAE z MS þ 1.0 mg l21 BA and 0.2 mg l21 NAA

PGR-free MS Ventilation closure type

Shoot tip Nodal segment Shoot tip Nodal segment

AF DP z

Explant

Agar

% of hyperhydric shoots

1.80 gy 2.05 g 1.80 g 1.70 g

0 cy 0c 0c 0c

Gelrite


3.80 fgy 4.55 de 1.45 g 2.01 g

0 cy 10 bc 0c 0c

Agar


Gelrite


18.50 aby 13.40 bcde 13.98 bcd 8.15 ef

65 ay 50 ab 57 a 6 bc

22.75 ay 11.10 cde 16.04 bc 12.90 cde

75 ay 40 ab 78 a 5 bc

Basal medium: MS basic salts supplemented with 1.0 mgl21 BA, 0.2 mgl21 NAA and 3% sucrose. Culture duration: 8 weeks. Means followed by the same letter are not significantly different at 5% level by LSD test.

y

to the interaction of multiple factors leading to reduced relative humidity (Maene and Debergh, 1987), increased gaseous exchange (Ziv, 1986), and decreased water and nutrient levels in the medium (Debergh et al., 1981). Several attempts have been made to reduce hyperhydricity and to improve the efficiency of in vitro propagation of plants. These include an increase in the carbohydrate level in the medium (Zimmerman and Cobb, 1989), change of light intensity (Sutter and Langhans, 1979), reduction of humidity (Maene and Debergh, 1987), and the use of bactopeptone and its subfractions (Sato et al., 1993). It has been reported that hyperhydricity can be controlled by using the gelling agent at a higher concentration or with higher gel strength (Debergh et al., 1981). In our laboratory, we have previously shown a positive correlation between high levels of ethylene and CO2 in culture vessels and hyperhydricity (Lai et al., 2005). Plants grown on Gelrite-solidified medium produced a significantly higher number of roots, with the root system forming a compact network (Fig. 1f), using DP as a ventilation closure, compared to those grown on agar medium with DP (Fig. 1e). In general, shoots derived from shoot tip explants using both ventilation closures produced more roots (Table 4). These results may facilitate the development of an efficient root culture of

S. yoshimurae as roots are the main source of drugs in this plant (Sagare et al., 2001). It was observed that the content of harpagoside (quantitatively predominant iridoid glycoside) in both the aboveground and underground parts of in vitro-grown S. yoshimurae plants was significantly higher than that in plants grown in the wild (Sagare et al., 2001). Plantlets developed under AF produced fewer and smaller leaves (Fig. 1a, b) than those grown under DP (Fig. 1c, d). Shoot tips exhibited a better growth response than nodal segments. On the other hand, the use of Gelrite resulted in a higher number of multiple shoots with improved growth compared to agar. Improved shoot growth on medium solidified with Gelrite has also been reported in other plant species, including Cercis canadensis var. mexicana (Zimmerman and Robacker, 1988) and Artemisia dracunculus (Mackay and Kitto, 1988). The rooted plantlets were transplanted to pots containing a mixture of peat moss, perlite, and soil. It was found that all plantlets obtained from DP as ventilation closure successfully acclimatized and survived (data not shown). Similar observations have been reported in some Australian plants (Rossetto et al., 1992). Majada et al. (1997) reported that diffused ventilation of culture vessels lowered the relative humidity, increased the evapotranspiration

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FIG . 1. Shoot tip derived in vitro plantlets and in vitro root system in S. yoshimurae influenced by gelling agent and ventilation closure of vessel. a, Agar, aluminum foil (AF) as ventilation closure; b, Gelrite, AF; c, agar, dispense paper (DP) as ventilation closure; d, Gelrite, DP; e, roots in agar medium, DP; f, roots in Gelrite medium, DP.

TABLE 4 EFFECT OF EXPLANT TYPE, GELLING AGENT AND VENTILATION CLOSURE OF VESSEL ON NUMBER AND LENGTH OF ROOTS IN S. YOSHIMURAE z Root number Ventilation closure type

Explant source Shoot tip Nodal segment Shoot tip Nodal segment

AF DP

Agar

Root length (cm) Gelrite

y

7.85 cd 6.94 d 13.20 ab 8.10 cd

Agar y

13.81 ab 10.50 bc 15.15 a 14.46 a

y

3.35 c 3.34 c 5.36 b 4.02 bc

Gelrite 5.19 by 5.48 b 9.20 a 8.06 a

z

Basal medium: MS basic salts supplemented with 1.0 mgl21 BA, 0.2 mgl21 NAA and 3% sucrose. Culture duration:8 weeks. Means followed by the same letter are not significantly different at 5% level by LSD test.

y

rate, and, in turn, diminished the water potential of the culture medium. This favored a micro-environment suitable for epicuticular wax formation and higher survival rate of plantlets after transplanting. This has been supported by a scanning electron microscope study on leaf surfaces of in vitro- and ex vitro-derived plants of Bupleurum kaoi in our laboratory (unpublished results). The leaves derived from air-tight containers lacked epiculticular wax and possessed higher stomatal density, larger stomata, and fewer functional stomata when compared to those grown in diffusive containers, thus lowering the survival percentage (unpublished results).

In conclusion, results of this study demonstrate that induction of multiple shoots and culture hyperhydricity of S. yoshimurae are affected by several factors, including explant type, PGR, gelling agent, and ventilation closure of vessels. The use of PGRfree medium and DP reduced hyperhydricity significantly. Our results indicate that AF can be used for an initial shoot induction and bud proliferation of S. yoshimurae, while DP is recommended for subsequent stages of in vitro propagation. In addition, shoot tips with Gelrite are the preferred explants and gelling agents to be used for micropropagation of S. yoshimurae.

IN VITRO STUDY IN SCROPHULARIA YOSHIMURAE

Acknowledgment Financial support from the National Science Council of Taiwan (grant NSC 94-2313-B324-001) is gratefully acknowledged.

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