An effective in-vitro acclimatization using uniconazole

Scientia Horticulturae 121 (2009) 468–473

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An effective in-vitro acclimatization using uniconazole treatments and ex-vitro adaptation of Phalaenopsis orchid Suriyan Cha-um a,*, Ouk Puthea b, Chalermpol Kirdmanee a a National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Paholyothin Road, Klong 1, Klong Luang, Pathumthani, 12120, Thailand b Faculty of Forestry, Royal University of Agriculture, P.O. Box 2696, Chamkar Daung, Dangkor District, Phnom Penh, Cambodia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 December 2008 Received in revised form 11 February 2009 Accepted 27 February 2009

The in-vitro acclimatization of Phalaenopsis plantlets under photoautotrophic conditions, with 0 (control), 3.43, 6.86 and 13.72 mM uniconazole (UCZ) treatments for 30 days was investigated before the plantlets were transferred to ex-vitro environments for 14 days. The physiological and growth characters of in-vitro acclimatized, and ex-vitro adapted plantlets were measured. Chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll (TC) and total carotenoid (Cx+c) content in plantlets treated with 6.86 mM UCZ were maintained at higher levels than those in plantlets of the control, by 1.82, 1.85, 1.83 and 1.93 times, respectively, leading to enrichment of the pigments in ex-vitro conditions. The maximum quantum yield of PSII (Fv/Fm), photon yield of PSII (FPSII), photochemical quenching (qP) and non-photochemical quenching (NPQ) in UCZ treated plantlets and in ex-vitro adaptation were not significantly different. Proline was accumulated in the control plantlets in both in-vitro acclimatization and ex-vitro conditions, while proline in those plantlets with UCZ treatments was maintained at a low level, which was defined by unstressed conditions. Net photosynthetic rate (Pn) in 6.86 mM UNZ treated plantlets peaked at a higher level than that of the control plantlets, both in-vitro and ex-vitro, by 3.27 and 2.93 times, respectively. In addition, proline content and Pn were inversely related in both in-vitro acclimatization and ex-vitro adaptation. The Pn in UCZ acclimatized plantlets was negatively correlated with plant dryweight. In-vitro photoautotrophic Phalaenopsis plantlets were successfully acclimatized using a 6.86 mM UCZ treatment which caused them to adapt quickly to ex-vitro environments. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Chlorophyll a fluorescence Net photosynthetic rate Photosynthetic pigment Proline Orchid

1. Introduction Phalaenopsis, or the moth orchid, is one of the most important ornamental plant species in the world. 75% of the market share of orchid sales in the year 2000 consisted of potted Phalaenopsis orchids, with a market value of 75 million US dollars. Large scale production of Phalaenopsis is carried out in The Netherlands, Germany, China, Taiwan, The United States of America and Japan (Griesbach, 2002). Phalaenopsis has been reported as being sensitive to high temperatures, especially hybrid types (Kano, 2001). In the floral transition stage, low temperatures are required for endogenous cytokinin and gibberellin accumulation, as well as photosynthetic enhancement, leading to sucrose accumulation for flower bud initiation and stalk elongation (Chou et al., 2000; Su et al., 2001; Kataoka et al., 2004; Blanchard and Runkle, 2006; Lee et al., 2007; Chen et al., 2008; Penfield, 2008). On a commercial scale, Phalaenopsis plantlets are produced using micropropaga-

* Corresponding author. Tel.: +66 2 564 6700; fax: +66 2 564 6707. E-mail address: [email protected] (S. Cha-um). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.02.027

tion through plant tissue culture, which is widely used in many countries, i.e., Japan, Taiwan and China (Griesbach, 2002). There are many reports on developing effective protocols for Phalaenopsis micropropagation via protocorm-like-bodies (Islam et al., 1998; Chen et al., 2000; Park et al., 2000; Tokuhara and Mii, 2001; Park et al., 2002; Tokuhara and Mii, 2003; Liu et al., 2006; Shrestha et al., 2007). Environmental conditions for ex-vitro growth are quite different from those used for in-vitro cultivation (Kozai et al., 1997; Hazarika, 2006). In-vitro acclimatization is one of the key factors in producing healthy plantlets before they are transplanted to exvitro conditions (Pospı´sˇilova et al., 1999; Hazarika, 2003). Plant growth retardants, i.e., uniconazole (UCZ), paclobutrazol (PBZ), triapenthenol (TPN), triadimefon (TDM) and hexaconazole (HCZ) have been reported as effective agents in reducing the size of plants, but retaining dark-green leaves and thick roots, which define them as healthy plantlets, and aiding anti-wilting, leading to better survival and growth in ex-vitro conditions (Smith et al., 1990; Smith et al., 1991; Murali and Duncan, 1995; Hazarika, 2006; Kozak, 2006; Thakur et al., 2006). Uniconazole [(E)-1-(4chlorophynyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol]

S. Cha-um et al. / Scientia Horticulturae 121 (2009) 468–473

is a member of the triazole group, which is well known as plant growth retardant (Izumi et al., 1984). The function of UCZ is as an inhibitor of gibberellic acid (GA) biosynthesis in higher plants (Izumi et al., 1984; Nishijima et al., 1992; Nishijima et al., 1997; Noguchi et al., 1999), leading to reduced seed germination (Fellner et al., 2001; Li et al., 2005) and plant growth (Oda, 1994; Sankhla et al., 1994; Ogawa et al., 1996; Nishijima et al., 1997; Pasian, 1999; Inada and Shimmen, 2000; Cavins et al., 2002; Hays et al., 2002; Blanchard and Runkle, 2007; Hwang et al., 2008; Zhao and Wang, 2008). Moreover, there are many purposes for which UCZ can be applied in higher plants, i.e., in-vitro acclimatization (Murali and Duncan, 1995), water deficit tolerance (Li and van Staden, 1998; Li et al., 1998; Zhang et al., 2007; Duan et al., 2008), salt tolerance (AlRumaih and Al-Rumaih, 2007; Kandil and Eleiwa, 2008), waterlogging tolerance (Leul and Zhou, 1998; Qiu et al., 2005) and extreme temperature tolerance (Zhou and Leul, 1998; Zhou and Leul, 1999; Fukuta et al., 2001; Kaneko and Suzuki, 2006). Exogenous application of UCZ is an effective way to produce healthy plantlets in-vitro, causing them to adapt quickly to ex-vitro environments. The aim of this investigation was to apply UCZ for in-vitro acclimatization, prior to ex-vitro adaptation of Phalaenopsis orchids. 2. Materials and methods 2.1. Plant materials and in-vitro acclimatization Phalaenopsis orchid plantlets provided by Prayoon Orchid Lab (Prayoon Orchid Co. Ltd., Pathumthani Thailand) were transferred to sugar-free MS medium (Murashige and Skoog, 1962) using vermiculate as supporting material and cultured for 7 days at 65 5% relative humidity (RH), 25 2 8C ambient temperature and 70 5 mmol m2 s1 photosynthetic photon flux (PPF) using fluorescent lamps with a 16 h d1 photoperiod. Then, the culture medium was adjusted to 0 (control), 3.43, 6.86 and 13.72 mM UCZ. The opentopped glass vessels containing the orchid plantlets were placed in an aseptic culture chamber (Carry Box Model P-850, size 26 cm 36 cm 19 cm) in which the RH conditions were maintained at 60 5% by 1500 ml saturated NaCl solution. The air exchange rate in the culture chamber was increased to 5.13 0.3 h1 by punching the side of the plastic chambers with 32 holes and placing a gas permeable microporous polypropylene film (0.22 mm pore size) over each hole. After 30 days, proline content, photosynthetic pigments, Chlorophyll a (Chla) fluorescence and net photosynthetic rate (Pn) were measured. Fresh-weight (FW), dry-weight (DW), shoot height (SH) and leaf area (LA) measurements were collected as growth characters. 2.2. Ex-vitro adaptation Plantlets acclimatized for 30 days were directly transplanted to a glasshouse, at 30 2 8C ambient temperature, 75 5% RH and 300–400 mmol m2 s1 PPF at plant level with 10 h d1 photoperiod and grown on for 14 days. Proline content, photosynthetic pigments, Chlorophyll a fluorescence and net photosynthetic rate were measured. FW, DW, SH and LA measurements were collected as growth parameters. 2.3. Measurement of physiological and morphological characteristics Proline from the leaves of the plantlets was extracted according to the method of Bates et al. (1973). One hundred milligrams of leaf tissue was ground in liquid nitrogen. The homogenate powder was mixed with 1 mL aqueous sulfosalicylic acid (3%, w/v) and filtered through filter paper (Whatman #1). The extracted solution was reacted with an equal volume of glacial acetic acid and ninhydrin reagent (1.25 mg ninhydrin in 30 mL of glacial acetic acid and

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20 mL of 6 M H3PO4) and incubated at 95 8C for 1 h. The reaction was terminated by placing the container in an ice bath. The reaction mixture was vigorously mixed with 2 mL toluene. After warming to 25 8C, the chromophore was measured with a spectrophotometer at 520 nm. L-proline was used as a standard. Chlorophyll a, chlorophyll b (Chlb), total chlorophyll (TC), and total carotenoids (Cx+c) concentrations were analyzed following the methods of Shabala et al. (1998) and Lichtenthaler (1987), respectively. One hundred milligrams of leaf material was collected. The leaf samples were placed in a 25 mL glass vial, along with 10 mL of 95.5% acetone, and blended using a homogenizer. The glass vials were sealed with parafilm to prevent evaporation and then stored at 4 8C for 48 h. The Chla and Chlb concentrations were measured using a UV–visible spectrophotometer at 662 and 644 nm wavelengths. The Cx+c concentration was also measured by spectrophotometer at 470 nm. A solution of 95.5% acetone was used as a blank. Chlorophyll a fluorescence emission from the adaxial surface of the leaf was monitored using a fluorescence monitoring system in the pulse amplitude modulation mode, as previously described by Loggini et al. (1999). A leaf grown in dark conditions was initially exposed to the modulated measuring beam of far-red light. Original (F0) and maximum (Fm) fluorescence yields were measured under weak modulated red light (6.8 mmol m2 s1 PAR) and calculated using FMS software for Windows1. The variable fluorescence yield (Fv) was calculated by the equation of Fm F0. 0. The ratio of variable to maximum fluorescence (Fv/Fm) was calculated as maximum quantum yield of PSII photochemistry. The photon yield of PSII (FPSII) in the light was calculated by FPSII ¼ ðF0m FÞ=F0m after 45 s of illumination, when steady state was achieved. In addition, photochemical quenching (qP) and nonphotochemical quenching (NPQ) were calculated as described by Maxwell and Johnson (2000). The Pn) of Phalaenopsis plantlets was measured under dark conditions using an infra-red gas analyser. The flow-rate of air in the sample line was adjusted to 500 mmol s1. The micro-chamber temperature was set at 25 8C (Cha-um et al., 2007). Phalaenopsis plantlets were dried at 110 8C in a hot-air oven for 4 days, and then incubated in desiccators before measurement of dry-weight. The leaf area of plantlets was measured using a leaf area meter DT-scan. 2.4. Experiment design and data analysis The experiment was arranged as a completely randomized design (CRD) with four replicates and four plantlets per replicate. The mean values were compared by Duncan’s new Multiple Range Test (DMRT) and analyzed using SPSS software. The correlations between proline content and Pn as well as Pn and plant dry-weights were evaluated. 3. Results 3.1. In-vitro acclimatization In-vitro acclimatized plantlets of Phalaenopsis orchids using different uniconazole treatments were established. Photosynthetic pigments including chlorophyll a, chlorophyll b, total chlorophyll and total carotenoids in UCZ acclimatized plantlets were exhibited at high levels when compared to untreated plantlets (0 mM UCZ) (Table 1). The photosynthetic pigments in acclimatized plantlets were significantly increased, related to UCZ concentration in the culture medium, except for the 13.72 mM UCZ treatment. Under the 6.86 mM UCZ treatment, the photosynthetic pigments of the plantlets peaked at a higher level than those of plantlets grown

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Table 1 Chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll (TC) and total carotenoids (Cx+c) of Phalaenopsis acclimatized in-vitro using uniconazole treatments for 30 days and subsequently transferred to ex-vitro for 14 days. Acclimatization In-vitro

Uniconazole (mM)

Chla (mg g1 FW)

Chlb (mg g1 FW)

TC (mg g1 FW)

Cx+c (mg g1 FW)

0 3.43 6.86 13.72

23.69b 25.69b 43.13a 32.79ab

14.55b 15.97b 26.95a 22.87a

38.21b 41.66b 70.08a 55.66ab

6.67b 9.00ab 12.90a 9.39ab

** 30.36b

* 15.24b

* 45.60b

** 10.00b

33.94b 48.16a 39.41ab

16.77ab 22.87a 21.74a

50.71b 71.03a 61.15ab

10.63b 16.04a 12.53ab

ANOVA 0 Ex-vitro

3.43 6.86 13.72

ANOVA

*

*

*

*

Different letters in each column show significant difference at **p 0.01 and *p 0.05 by Duncan’s New Multiple Range Test (DMRT).

under control conditions by 1.82, 1.85, 1.83 and 1.93 times, respectively (Table 1). On the other hand, the chlorophyll a fluorescence parameters, maximum quantum yield of PSII (Fv/Fm), photon yield of PSII (FPSII), photochemical quenching (qP) and non-photochemical quenching (NPQ), were not significantly different in the statistical analysis (Table 2). The proline content of 6.86 and 13.72 mM UCZ acclimatized plantlets was significantly lower when compared to that of the control and the 3.43 mM UCZ treatment (Fig. 1). Proline content in the acclimatized plantlets was an effective biochemical indicator of plant adaptation to in-vitro acclimatization. In contrast, net photosynthetic rate in UCZ acclimatized plantlets was maintained better than in that of the control plantlets (Fig. 2). There was a negative correlation between proline content and Pn (Fig. 3A; r2 = 0.76). In addition, Pn was inversely related to plant dry-weight (Fig. 4A; r2 = 0.95). Growth performance, fresh-weight, dry-weight, shoot height and leaf area in UCZ acclimatized plantlets were significantly retarded, related to the UCZ concentration in the culture medium, especially in the cases of the 6.86 and 13.72 mM UCZ treatments (Table 3). Morphological characters of UCZ acclimatized plantlets include; green leaves, and healthy shoots and roots, prior to transplantation to ex-vitro conditions (Fig. 5A).

a greater degree when compared to those under control conditions (0 mM UCZ) (Table 1). Chla, Chlb, TC and Cx+c in UCZ acclimatized plantlets were significantly increased, related to UCZ treatments, except in the case of 13.72 mM UCZ. In the 6.86 mM UCZ acclimatization, Chla, Chlb, TC and Cx+c were enriched to a greater

3.2. Ex-vitro adaptation Orchid plantlets, acclimatized using UCZ treatments, were subsequently transplanted to an ex-vitro environment for 14 days. Photosynthetic pigments, Chla, Chlb, TC and Cx+c of UCZ acclimatized plantlets grown in ex-vitro conditions were exhibited to

Fig. 1. Proline content of Phalaenopsis acclimatized in-vitro using uniconazole treatments for 30 days (white bars) and subsequently transferred to ex-vitro for 14 days (black bars). Different letters in each bar show significant difference at **p 0.01 by DMRT. Error bars represent SE.

Table 2 Maximum quantum yield of PSII (Fv/Fm), photon yield of PSII (FPSII), photochemical quenching (qP) and non-photochemical quenching (NPQ) of Phalaenopsis acclimatized in-vitro using uniconazole treatments for 30 days and subsequently transferred to ex-vitro for 14 days. Acclimatization In-vitro

Uniconazole (mM)

Fv/Fm

FPSII

qP

NPQ

0 3.43 6.86 13.72

0.763 0.763 0.782 0.778

0.594 0.608 0.638 0.623

0.805 0.845 0.863 0.807

0.176 0.111 0.107 0.141

NS 0.756

NS 0.590

NS 0.789

NS 0.246

0.760 0.781 0.767

0.591 0.620 0.619

0.793 0.846 0.815

0.097 0.062 0.083

NS

NS

NS

NS

ANOVA 0 Ex-vitro

ANOVA

3.43 6.86 13.72

Different letters in each column show significant difference at **p 0.01 and *p 0.05 by Duncan’s new multiple range test (DMRT). NS: represents nonsignificant in statistical analysis.

Fig. 2. Net photosynthetic rate (Pn) of Phalaenopsis acclimatized in-vitro using uniconazole treatments for 30 days (white bars) and subsequently transferred to ex-vitro for 14 days (black bars). Different letters in each bar show significant difference at **p 0.01 by DMRT. Error bars represent SE.


471

Fig. 5. Morphological characters of Phalaenopsis acclimatized in-vitro using uniconazole treatments for 30 days (A) and subsequently transferred to ex-vitro for 14 days (B). Table 3 Fresh-weight (FW), dry-weight (DW), shoot height (RL) and leaf area (LA) of Phalaenopsis acclimatized in-vitro using uniconazole treatments for 30 days and subsequently transferred to ex-vitro for 14 days. Acclimatization Fig. 3. Relationship between proline and net photosynthetic rate (Pn) of Phalaenopsis acclimatized in-vitro using uniconazole treatments for 30 days (A) and subsequently transferred to ex-vitro for 14 days (B). Error bars represent SE.

In-vitro

Uniconazole (mM)

FW (g)

DW (mg)

SH (cm)

LA (cm2)

0 3.43 6.86 13.72

3.20a 2.92a 2.30ab 1.74b

0.37a 0.20b 0.13c 0.11c

17.91a 11.94b 11.24b 9.60b

33.91a 22.94b 22.38b 20.66b

0

* 3.30a

* 0.46a

* 16.52a

* 38.02a

3.43 6.86 13.72

2.65b 2.51b 2.52b

0.27b 0.22b 0.21b

14.73ab 12.43bc 10.58c

28.35b 28.32b 26.39b

*

*

*

*

ANOVA

Ex-vitro

ANOVA

Different letters in each column show significant difference at *p 0.05 by Duncan’s new multiple range test (DMRT).

degree than those in control conditions by 1.59, 1.50, 1.56 and 1.60 times, respectively (Table 1). On the other hand, the chlorophyll a fluorescence parameters, Fv/Fm, FPSII, qP and NPQ, in all treatments showed no difference (Table 2). Proline content of 6.86 and 13.72 mM UCZ acclimatized plantlets in ex-vitro conditions was significantly lower in comparison to the control and the 3.43 mM UCZ treated plantlets (Fig. 1). In contrast, Pn of UCZ acclimatized plantlets grown in ex-vitro environments was maintained better than in the control plantlets (Fig. 2). There was a negative correlation between proline content and Pn (Fig. 3A; r2 = 0.81). In addition, Pn was inversely related to plant dry-weight (Fig. 4A; r2 = 0.94). Fresh-weight, dry-weight, shoot height and leaf area, of UCZ acclimatized plantlets in ex-vitro conditions were significantly reduced, especially in plantlets with the 6.86 and 13.72 mM UCZ treatments (Table 3). In addition, the morphological characters in UCZ acclimatized plantlets showed rapid adaptation to ex-vitro environments (Fig. 5B). 4. Discussion Fig. 4. Relationship between net photosynthetic rate (Pn) and plant dry-weight (DW) of Phalaenopsis acclimatized in-vitro using uniconazole treatments for 30 days (A) and subsequently transferred to ex-vitro for 14 days (B). Error bars represent SE.

Growth performance indicators, including shoot length, shoot dry-weight, number of roots, root length and root dry-weight, in banana plantlets acclimatized using UCZ treatments, decrease depending on UCZ concentrations in the culture medium (Murali

472


and Duncan, 1995). In the present study, the fresh-weight, dryweight, shoot height and leaf area of acclimatized Phalaenopsis plantlets were retarded, relating to UCZ concentrations. In the acclimatization process, environmental factors such as sugar-free media (photoautotrophic conditions), low relative humidity (RH), high light intensity and CO2 enrichment have been modified to produce healthy Phalaenopsis plantlets before they are transplanted to ex-vitro environments (Lin and Hsu, 2004; Ali et al., 2005; Jeon et al., 2005; Ali et al., 2006; Yoon et al., 2009). Proline content in acclimatized plantlets is a good indicator of biochemical responses to acute environments in higher plants (Ashraf and Foolad, 2007; Jaleel et al., 2007; Manivannan et al., 2008). Proline in orchid plantlets acclimatized under 0 mM UCZ reached the highest level, whereas that of plantlets under 6.86 mM UCZ was lowest. In contrast, proline in 34.27 mM UCZ treated beans (Phaseolus vulgaris) is accumulated at a greater level than that in control plants (Mackay et al., 1990). Photosynthetic pigments in the UCZ acclimatized plantlets were stabilized, leading to high Pn. Photosynthetic pigments in UCZ treated maize (Li and van Staden, 1998; Zhang et al., 2007) and soybean crops are maintained, causing high Pn, water use efficiency, transpiration rate and stomatal conductance, when exposed to water deficit stress (Duan et al., 2008). In addition, antioxidation systems in UCZ treated maize and soybeans, prior to being subjected to water stress, are expressed better than those in control plants (Li and van Staden, 1998; Li et al., 1998; Zhang et al., 2007). Whereas, chlorophyll a fluorescence parameters, Fv/Fm, FPSII and qP, in UCZ treated plantlets are not significantly different in statistical analysis. The results are similar to previous reports into Viola wittrockiana treated with PBZ (Gliozˇeris et al., 2007). The UCZ acclimatized plantlets quickly adapted to ex-vitro conditions, defined by fresh-weight, dry-weight, shoot height and leaf area. There are many reports on the successful acclimatization of in-vitro cultivated plantlets using triazoles, namely paclobutrazole (PBZ), and subsequent transplantation to ex-vitro environments with high survival percentage rates and rapid adaptation (Kozak, 2006; Thakur et al., 2006; Gliozˇeris et al., 2007). In contrast, UCZ acclimatized banana plantlets do not survive in ex-vitro conditions (Murali and Duncan, 1995). Water stress, or water loss from plantlets in ex-vitro conditions is a major problem that can be controlled using UCZ applications, which are successfully applied in the case of many crop species, such as wheat (Duan et al., 2008), soybeans (Zhang et al., 2007), maize (Li and van Staden, 1998; Li et al., 1998) and beans (Mackay et al., 1990). The growth rate of UCZ acclimatized plantlets grown ex-vitro is still delayed, depending on the long term effects of plant growth retardant (Smith et al., 1990; Kozak, 2006). Chla, Chlb, TC and Cx+c content in UCZ acclimatized plantlets grown in a glasshouse stabilized and functioned better, defined by chlorophyll a fluorescence. The results are quite similar to a previous study into photosynthetic pigment stabilization in UCZ treated plantlets (Li and van Staden, 1998; Zhang et al., 2007) as well as in PBZ acclimatized plantlets after transferal to ex-vitro environments (Smith et al., 1990; Kozak, 2006; Thakur et al., 2006; Gliozˇeris et al., 2007; Gopi et al., 2007), leading to enhanced Pn and better survival percentages. In Phalaenopsis orchids, UCZ in-vitro acclimatization is an effective protocol to produce healthy plantlets leading to rapid adaptation and continuing growth in ex-vitro conditions. In conclusion, plantlets of Phalaenopsis orchid, acclimatized in-vitro using a 6.86 mM UCZ treatment, increased levels of photosynthetic pigments and higher net photosynthetic rates, while the growth characters were retarded leading to compact plantlets. Moreover, the in-vitro acclimatized plantlets quickly adapted to ex-vitro environments, identified by low proline contents, high photosynthetic pigments and high Pn. The basic knowledge of this investigation can be further applied for the rapid acclimatization of plantlets of transplanted Phalaenopsis orchids in large scale production.

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