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Effects of medium composition on the morphological and physiological changes of Oncidium Sharry Baby plantlets.

Introduction

Orchids are among the most coveted of all the flowering plant families. Orchids have become increasingly popular due to the new and improved cultivation and propagation techniques. Such advances allowed the mass propagation of orchids at reasonable costs to customers, in association with a profitable product for growers. However, growers reported that the production does not meet the existing demand (Vendrame 2004). On of the orchids that faced the above mentioned problems was Oncidium Sharry Baby. Although the growth of O. Sharry Baby has been successfully commercialized, the establishment of aseptic cultures is often difficult due to contamination and production of phenolic compounds by the explants (Smith 2000). In addition, often published protocols might not fulfill the needs of certain conditions or provide a clear rationale for particular modifications. Furthermore, the appropriate medium compositions required for improved cultivation and at the same time, increasing the yield of O. Sharry Baby, is still unknown. These unsolved problems led to the biochemical testing of in vitro plantlets as a successful means to increasing production depends on an improved understanding of physiological and biochemical processes underlying environmental stresses and subsequent tolerance to these stresses (Yu and Rengel 1999). Therefore, studies were conducted to test the total chlorophyll content, total soluble protein, and specific activity of peroxidase of O. Sharry Baby plantlets subjected to different medium compositions and concentrations of a plant growth regulator, 6-benzylaminopurine (BAP).

Materials and methods

Plant materials

O. Sharry Baby (Fig. 1A) stocks that were maintained on half-strength Murashige and Skoog (1962) medium were separated into single plantlets on Petri dishes and the roots were removed. Selection of plantlets was done carefully by selecting single plantlets that had similar height and average weight (Fig. 1B). Plantlet length, number of leaves, and fresh weight were measured before being transfer into each culture vial with various treatments. All cultures were incubated in a culture room at 25 [+ or -] 2[degrees] C in a 16-hr photoperiod with light intensity of 1000 lux.

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Culture medium

Half-strength MS medium, which contained macroelements, microelements, vitamins and 87 mM sucrose (except for the experiment on effects of sucrose concentration) were used as the basal medium in this study. All media were adjusted to pH 5.8 [+ or -] 0.1 with 0.1 M NaOH or 0.1 M HCl. A total of 0.8% (w/v) plain agar was then added and dissolved. Approximately 10 mL of the medium was poured into each flat bottom culture vial (25 x 100 mm), covered with aluminum foil and autoclaved at 121[degrees] C and 15 psi for 15 min.

Effects of medium composition

To study the effects of magnesium, plantlets were grown on medium containing magnesium sulphate (MgS[O.sub.4]) (Sigma Aldrich, USA) at the concentrations of 0, 0.75, 1.50 and 3.00 mM. As for the nitrate treatment, the plantlets were grown in medium containing potassium nitrate (KN[O.sub.3]) (R&M Chemicals, UK) and ammonium sulphate (N[H.sub.4] N[O.sub.3]) (R&M Chemicals, UK) at a concentration of 0 mM KN[O.sub.3] and 0 mM N[H.sub.4] N[O.sub.3] , 19.7 mM KN[O.sub.3] and 10.3 mM N[H.sub.4] N[O.sub.3] , 39.4 mM KN[O.sub.3] and 20.6 mM N[H.sub.4] N[O.sub.3] , as well as 78.8 mM KN[O.sub.3] and 41.2 mM N[H.sub.4] N[O.sub.3] . The concentrations of magnesium and nitrate selected were 0, V2, 1 or 2-times higher than the MS basal medium.

Effects of sucrose concentrations

O. Sharry Baby plantlets were cultured on half-strength MS medium containing sucrose (Sigma, USA) at 0, 87, 146 or 204 mM.

Effects of BAP concentration

BAP has been widely reported to enhance the growth of Oncidium sp. as compared to other plant growth regulators (PGRs), thus, only BAP was selected for this part of the study. In order to study the effects of BAP on growth and biochemical properties of plantlets, different concentrations of BAP (0, 0.004, 0.013 and 0.022 mM) were added to the basal culture medium.

Morphological studies

To investigate the effects of medium compositions (magnesium, nitrate ion and sucrose concentrations) as well as the BAP on the growth changes of O. Sharry Baby, quantitative measurements were carried out after four weeks of culture. General measurements of increase in plantlet length, number of leaves and fresh weight were recorded on the basis of three replicates for two repeated experiments. Increment in plant length, leaf numbers and fresh weight were calculated by subtracting the initial measurement prior to four weeks of treatment.

Total chlorophyll content

Total chlorophyll content was determined using the Lichtenthaler (1987) method. Extraction was carried out by adding 2 g of CaC[O.sub.3] (Spectrum, USA) and 10 mL of 80% (v/v) acetone (Spectrum) to 1 of 4-week old plantlets. Sample were ground by mortar and pestle on ice at 4[degrees] C. The samples were then filtered with a Buchner Funnel through Whatman No. 1 filter paper followed by a wash with 80% (v/v) acetone. The extraction volume was adjusted to 50 mL with 80% (v/v) acetone using a measuring cylinder.

Chlorophyll extracts were monitored with a spectrophotometer (Bio-Rad smartspec plus, USA) at 646 and 663 nm. Total chlorophyll content in mg/l was determined using the formula below and further converted to mg/g fresh weight (FW) of plant material:

Total chlorophyll content, [C.sub.a+b] = 7.15 ([OD.sub.663]) + 1 8 . 71 ([OD.sub.646])

Total soluble protein content

A total of 1 g of sample was extracted with 3 mL of protein extraction buffer in an ice bath using a mortar and pestle. The enzyme extract was transferred into an Eppendorf tube and centrifuged (Heraeus, UK) at 12,000 rpm, 4[degrees] C for 20 min. The supernatant was used to determine the protein content and specific activity of peroxidase.

The total soluble protein content was determined by Coomasie Blue Dye (Fisher, USA) binding described by Bradford (1976) and bovine serum albumin (BSA) (Sigma, USA) prepared in distilled water was used as the standard. Protein standard was prepared by adding 10-100 [micro] g BSA in a volume up to 0.1 mL in test tubes. The volume in the test tubes was adjusted with protein extraction buffer to 0.1 mL and 5 mL of protein reagent was added and the mixture was mixed.

Absorbance at 595 nm was determined using a spectrophotometer (Bio-Rad Smartspec Plus, USA). A mixture of 0.1 mL protein extraction buffer and 5 mL protein reagent was prepared as blank. A standard curve was plotted using the corresponding absorbance against the concentration of BSA. The total soluble protein content was compared to the standard of BSA and expressed in mg/g FW of plant material.

Specific activity of peroxidase

2.6 mL of 0.1 M sodium phosphate buffer (pH 6.1), 0.3 mL of 30% [H.sub.2] [O.sub.2] (Fisher) and 0.3 mL of 1% guaiacol (Fisher) was added to 0.5 mL enzyme extract. After mixing, the absorbance was monitored at 420 nm in a spectrophotometer (Bio-Rad Smartspec Plus) for 3 min. Units (U) of peroxidase were calculated as U/mg of soluble protein. One unit of activity is defined as the amount of enzyme that caused a change of absorbance of 0.01/min. The specific activity of peroxidase was calculated using the formula below:

Total activities

Specific activity of peroxidase (U/mg) = Total activities/protein content of the sample in mg

Statistical analysis

The growth and physiological changes of O. Sharry Baby plantlets were assessed by analysis of variance (ANOVA) by using SPSS software (version 11.5) (SPSS Inc. USA). One-way ANOVA was used to test the significance of values obtained for both morphological and physiological studies. Differences between treatment means were analyzed by Duncan's Multiple Range Test (DMRT) to determine the significant difference at a 95% confidence level (P < 0.05).

Results and discussion

Effects of magnesium concentration

As magnesium (Mg) is the key metal in chlorophyll synthesis, the study of [Mg.sup.2+] nutrition is of importance in determining the growth and productivity of green tissues. In this study, the morphology of O. Sharry Baby was affected by the concentrations of the [Mg.sup.2+] ion supplied. The increment in plantlet length was highest at 0.75 mM MgS[O.sub.4] whereas leaf number increased at 1.50 mM MgS[O.sub.4] (Table 1). In agreement with this study, Madhok and Walker (1969) discovered that leaf number and plant height of in vitro sunflower also increased with an increase in Mg concentration in nutrient solution. In this study, plant length decreased significantly when Mg concentration was increased to 1.50 mM. This could be due to excessive Mg concentration which may become toxic to the plant cells. In accordance to the studies by Therios and Sakellariadis (1981), even though the number of leaves continued to increase, when Mg in nutrient solutions was further increased, the relative growth declined, indicating Mg toxicity. Although Mg has been reported to help to activate many plant enzymes needed for growth, the fresh weight of in vitro O. Sharry Baby was not affected by Mg. In contrast, a significant fresh weight increment was successfully observed when the Mg concentration in the nutrient solution was increased to four times in olive plantlets (Therios and Sakellariadis 1981). In another orchid, hybrid Cymbidium, Teixeira da Silva et al. (2005) found that the level of Mg in different media formulations affected the development of callus and protocorm-like bodies.

The total chlorophyll content of plantlets was slightly affected by the Mg concentrations: values increased gradually up to a [Mg.sup.2+] ion concentration of 3.00 mM (Fig. 2). This could be explained by the fact that Mg is part of chlorophyll in all green plants and essential for photosynthesis (Therios and Sakellariadis 1981). However, in agreement with Leonardi and Giuffrida (2006) in grafted tomatoes and eggplant, the lack of significant differences indicated that Mg was not the sole cation in the total chlorophyll content of the plantlets. The data subjected to statistical analysis strongly suggested that various concentrations of Mg did not affect the total soluble protein in the plantlets of O. Sharry Baby (Fig. 3). To understand this result, Mg is an integral component and the only mineral constituent of the chlorophyll molecule (Stamps and Chandler 2003). Thus, it was not surprising that the result obtained on the total soluble protein content of O. Sharry Baby plantlets cultured in various magnesium concentrations did not demonstrate a great differences.

[FIGURE 2 OMITTED]

The specific activity of peroxidase in O. Sharry Baby plantlets was greatly affected by the amount of Mg (Fig. 4). The lack of availability of [Mg.sup.2+] ions causes general physiological stress, with the plant attempting to redistribute any of the still available endogenous ion (Henry et al. 1987). However, high concentration (3.00 mM) of Mg in culture medium may have caused the damage to O. Sharry Baby cells due to the release of toxic towards the plant cells, resulting in increased peroxidase level released by the cells. High peroxidase activity observed in plants submitted to stress indicated the ability of plant cells to degrade toxic substances that were released (Campa 1991). The high [Mg.sup.2+] ion concentrations may have imposed additional stress on the plant cells. As evidence, many higher plants exhibited an increased in peroxidase activity after treatment with Mg.

Effects of nitrate concentrations

In this study, the nitrogen source is supplied as a mixture of nitrate ions (KN[O.sub.3] and N[H.sub.4] N[O.sub.3]). Potassium is associated with movement of water termed osmotic regulation, nutrients, and carbohydrates in plant tissue; while ammonium nitrate acts as nitrogen source for plants. If the nitrates are deficient or not supplied in adequate amounts, growth of plant will be stunted and yields are reduced. Malavolta (1954) first reported a favorable effect of nitrate on the growth of rice. Since then, several reports have indicated that rice growth, yield, and nitrogen acquisition are significantly enhanced when both N[H.sub.4] and N[O.sub.3] sources are provided simultaneously in solution culture, compared with growth on NH4 alone (Cox and Reiseenauer 1973; Ta and Koji 1982; Youngdahl et al. 1982; Raman et al. 1995).

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Results revealed that O. Sharry Baby plantlets cultured in adequate concentrations of nitrate contributed to the increment of plant length, leaf number and fresh weight (Table 2). The concentration of 39.4mM KN[O.sub.3] and 20.6mM N[H.sub.4] N[O.sub.3] showed significant increment in plant length and leaf number. This could mean that a particular concentration of nitrate is sufficient for the growth of plantlet. Many studies have shown that a mixed nitrogen source could contributed to the up-regulation of nitrogen uptake and metabolism by nitrate, further help in the growth and yield response of plant (Kronzucker et al. 1999). Studies on soybeans proved that nitrate is an important regulator in the processes that include leaf number increment (McDonald and Davies 1996; Walch et al. 2000), root branching and the allocation of resources on plant growth (Scheible et al. 1997). Similarly, nitrate stimulated the absorption of nitrogen by rice, thus resulting in increased growth and yield of rice (Duan et al. 2005). Further increment of nitrate concentration (78.8mM KN[O.sub.3] and 41.2mM N[H.sub.4] N[O.sub.3]) resulted in stunted growth, reduction in leaf numbers and also the fresh weight of the O. Sharry baby plantlets. This statement could be explained by the fact that plantlet growth is sensitive to stress conditions within the environment. It has been proven that salt-induced inhibition on plant growth could be attributed to specific ion toxicity (Huang and Redmann 1995). Excessive [K.sup.+] was harmful to the growth of O. Sharry Baby plantlets. It is possible that in the presence of high salt concentrations, the amount of naturally occurring [K.sup.+] may suppress plant growth (Chen et al. 2005).The ammonium ions, NH4+, in the nitrate solution can also be toxic to plant cells at high concentrations, causing acidification of the medium if excessive ammonium ions are consumed by the plantlets (Chapin et al. 1993).

The O. Sharry Baby plantlet cultured in the highest concentration of nitrate (78.8mM KN[O.sub.3] and 41.2mM N[H.sub.4] N[O.sub.3]) displayed significantly higher total chlorophyll content (Fig. 5). This indicated that the chlorophyll content of plantlets can be maximized using high concentration of nitrate but perform less well when the conditions are poor, particularly if nitrogen is in short supply or none, since they must commit significant resources to plantlet development which would otherwise have been available for chlorophyll production. This could be due to the fact that changes in the photosynthetic metabolism occurred rapidly in response to variations in the external nitrogen supply. Chapin et al. (1993) concluded that the assimilation of nitrogen was provided by photosynthesis because over 50% of the tissue's nitrogen is allocated in chloroplast (Chapin et al. 1993).

Various concentrations of nitrate did not affect the total soluble protein in the plantlets of O. Sharry Baby (Fig. 6). These results implied that the effect of nitrate supplementation on the biochemical events is not a general effect of total soluble protein content in the plantlets. Furthermore, the investigations of protein patterns were carried out with soybean leaf tissues, and there was no qualitative difference in the total soluble protein spectra of nitrate-fed and nitrogen-fixing soybean leaves neither with the progress of development nor under drought conditions (Kirova et al. 2005).

Cation [K.sup.+] is known to be the major inorganic ion playing a role in osmotic potential adjustment of plant cells. Accumulation of inorganic solutes can also play a role, singly or in combination with other mechanisms in maintaining the osmotic imbalance caused by salt stress (Asch et al. 1999). The higher specific activity of peroxidase observed in plants submitted to stress can indicate the ability of certain genotypes to degrade toxic substances, such as free radicals (peroxides) released under these conditions (Campa 1991). It has been shown that high salinity in the external medium leads to a higher rate of lipid peroxidation and oxidative stress in the tissues of sunflower (Helianthus tuberosus) (Mittal and Dubey 1991). However, the results in this study were in contrary to the above findings, where the O. Sharry Baby plantlets performed the highest peroxidase activity at the nitrate concentration of 19.7mM KN[O.sub.3] and 10.3mM N[H.sub.4] N[O.sub.3] , indicating that the specific activity of peroxidase were not affected by high concentrations of nitrate, and that the plantlet were not subjected to high salt stress (Fig. 7).

Effects of sucrose concentrations

Carbohydrate accumulation in plant has been well known for osmotic

adjustment (Cheeseman 1988). Significant differences among treatments of various sucrose concentrations in the plantlets indicated that the sucrose concentration affected the morphology and physiology of O. Sharry Baby plantlets. As stated by few researches, the availability of the sugar in the culture medium has been found to affect both plant growth and photosynthetic functionality in many plant species (Gray et al. 1993; Lou and Kako 1995; Luo et al. 1996). Besides their role as substrates for plant growth and development, soluble sugars are involved in either the up-or down-regulation of several photosynthetic enzymes (Kovtun and Daie 1995; Koch 1996). In addition, organic compounds that accumulate in response to stress, such as soluble sugars, apparently play a role in the development of tolerance against salt stress (Hu and Schmidhalter 1998).

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Media with high sucrose concentrations supported the best growth and shoot differentiation in the green plants (Percival and Gerritsen 1998). However, a sucrose concentration of 87mM was observed to provide the highest increment of plant length and fresh weight of O. Sharry Baby plantlets (Table 3). Therefore, it could be inferred that the plantlets preferred an average amount of sucrose because of the significant increment of plant length and yield achieved. It could also come to a consensus that the addition of excessive sugar to the culture medium was shown to negatively affect the growth. These results supported the hypothesis that upregulation of these carbohydrate-metabolizing enzymes might be an optimization process to facilitate the balance between carbon gain and its use (Percival and Gerritsen 1998). Thus, it is clear that an appropriate concentration of sucrose can improve the morphology of O. Sharry Baby plantlet, thus, increase the fresh weight of the plantlets simultaneously.

Changes in the total chlorophyll content of the epiphytic orchid hybrid O. 'Goldiana' were brought about by a variety of physiological stresses, leaf development and senescence, and such pigment variations related directly to the rate of primary production (Li et al. 2001). The demonstration of sugars acted as regulators of gene expression in plants has led to the characterization of a growing number of sugar-regulated genes (Koch 1996; Roland et al. 2006). Thus, glucose or sucrose treatment in the absence of abiotic stress usually represses photosynthesis-related genes in plants (Pego et al. 2000). Low total chlorophyll content would indicate reduced photosynthetic rates in O. Sharry Baby plantlets at 146mM sucrose. The results indicated that the chlorophyll production was favored by media with low sucrose content which was 87mM (Fig. 8). This is in accordance to the previous reports which showed that excessive uptake of exogenous sugars by intact plants resulted in growth inhibition and/or photosynthetic down-regulation (Grob et al. 1993; Hdider and Desjardins 1994; Kozai et al. 1995; Jones et al. 1996; Kilb et al. 1996; Ehness et al. 1997). Therefore, it can be inferred that incubation of plantlets in high concentration of sucrose solutions leads to the repression of photosynthetic genes, decreased rates of net photosynthesis, hence decreasing the total chlorophyll content of O. Sharry Baby plantlets.

[FIGURE 8 OMITTED]

Irrespective of various concentrations, the sucrose has no significant effects on the total soluble protein content in the plantlets of O. Sharry Baby (Fig. 9). This indicated that the plant cells did not depend on the sucrose concentrations to produce soluble protein. In accordance to the result, Ortiz and Donelly (2001) discovered that the total soluble protein content of potato tubers were not affected after 6 months of tuber storage in various sucrose compositions.

Soluble sugars have been involved in a number of stress-related processes, as metabolites that accumulate in response to stress conditions (Roitsch 1999) as well as act as substrates of carbon metabolism (Loreti et al. 2005). The specific activity of peroxidase was at the highest at 204mM sucrose (Fig. 10). The higher specific activity of peroxidase observed in O. Sharry Baby plantlets submitted to stress could indicate the ability of plant cells to degrade toxic substances released under high sugar conditions. Moreover, sucrose treatment had also been proven to be more efficient than glucose treatment in providing protection mechanisms against the stress condition (Ramel et al. 2007).

Effects of BAP

The most important development in culture media was the incorporation of growth regulators like cytokinins. The discovery of various plant hormones led to their utilization in attempts to promote photosynthesis and plant growth in orchids (Zarhloul et al. 1999). It has been well documented that extremely low concentrations of PGRs have the ability to regulate many aspects of plant growth and development from seed germination through senescence and death of the plant (Sengbusch 2007). However, growth hormones inhibit as well as promote orchid seed germination in orchids, depending on the type. Several researchers have tried various PGRs and various concentrations of growth hormones in order to promote seedling growth of various types of orchids (Arditti 1979).

[FIGURE 9 OMITTED]

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Certain plants react well with low concentration of BAP that reached below 0.09 [micro]M (Koroch et al. 2002), 0.004mM and above (Liu and Bao 2003; Dayal et al. 2003) or both high and low BAP concentrations (Hiregoudar et al. 2003). The present study demonstrated that the highest length increment of O. Sharry Baby plantlets was achieved at the highest BAP concentration (0.022mM) (Table 4). In Canola cultivar 'Drakkar', a length increment of 12% was achieved through the increase of the BAP concentrations and at 0.013mM, providing the best growth rate (Zarhloul et al. 1999). These experiments strongly indicated that cytokinins play a central role in the suppression of plantlet development in their length and fresh weight.

On the other hand, increment in leaf number was observed at the BAP concentration of 0.013mM, followed by reduction of leaf number as the concentration of BAP increased to 0.022mM (Table 4). This finding revealed that higher concentrations of BAP inhibited the leaf growth in O. Sharry Baby plantlet. Stimulation of plant growth by BAP was also reported in cymbidium seedlings, in which leaf induction worked best on the MS medium supplemented with only 0.0009mM of BAP (Ozel et al. 2007). There was no significant variation observed in the fresh weight of the plantlets cultured in various BAP concentrations. In contrary, Jheng et al. (2006) showed that a higher level of cytokinin (0.044mM) stimulated the increase of cell masses in O. 'Gower Ramsey'.

Specific cytokinins are required for stimulating the synthesis of chlorophylls in different tissues; but its influence on chlorophyll production in these systems differ (Kalimuthu et al. 2006). In the green tissues, chlorophyll synthesis was inhibited by cytokinin. In the tobacco tissue used by Stetler and Laetsch (1965), the cytokinin synthesizing system is partially functional and can synthesize enough cytokinin to keep the tissue growing, and not for photosynthetic purposes. The differences of chlorophyll content among the plantlets in different BAP concentrations were too small and there was no significant among each other (Fig. 11). In other words, the concentrations of BAP used did not affect the total chlorophyll content of O. Sharry Baby. Hence, the cytokinin synthesizing system in the plantlet seemed to be completely non-functional in this study.

Cytokinins usually have no beneficial effect on orchid seed germination (Kalimuthu et al. 2006). In agreement with this, Narain and Laloraya (1970) reported that the addition of BAP neither had significant effect on chlorophyll production of plant in L. rubellum, nor affected the total chlorophyll content in L. ledebourii. Thus, it can be assumed that cytokinins did not inhibit as well as promote the seed germination in orchids. This could be due to the reason that the amount of cytokinin produced was not sufficient to support chlorophyll synthesis. However, the possibility that in the green tissue, cytokinin inhibits chlorophyll synthesis by acting as a competitive inhibitor against endogenous cytokinins cannot be ruled out (Schaffert et al. 1996). Besides increasing the rate of DNA replication, cytokinin increases the general rate of protein synthesis (van Staden and Smith 1978). Apparently, it was learnt that cytokinins can be glycosylated or bound to a protein, thereby being temporarily inactive (Sengbusch 2007). In this study, various concentrations of BAP did not affect the total soluble protein in the plantlets of O. Sharry Baby (Fig. 12). Similarly, a study conducted by Ljudmila et al. (2000) on in vitro barley with maximal BAP concentration found that not much difference in the total soluble protein content was observed. The occurrence of the result might be due to the biosynthesis site is different from the site of action of a PGR. For example, the PGR was utilized for morphological growth as the site of action may be in specialized cells, located physically within a variety of other cells and tissues. Thus, it may be difficult to correlate levels of PGRs with particular physiological responses (Sengbusch 2007). Similarly, there was no significant difference in the specific activity of peroxidase among O. Sharry Baby plantlets cultured in various BAP concentrations (Fig. 13). This indicated that the presence of BAP did not affect the peroxidase activity of plantlets. This finding implied that the stress condition could not be directly linked to the BAP induction. A reason that the specific activity of peroxidase was not observed in BAP concentrations may be due to the little amount of BAP that was incorporated into the medium, and this little amount was insufficient to create a stress condition to the plantlets. This statement was proven by Vianello et al. (1997) who reported that the plasma membrane isolated from soybean (Glycine max) roots would only exhibit higher peroxidase activity in the presence of kinetin at 10.0mg/L (Mika and Luthje 2003).

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Conclusions

The results obtained from this study strongly suggested that plants are equipped with mechanisms to accumulate more and more inorganic ions with increasing concentrations of major ions such as [Mg.sup.2+] , [K.sup.+] and N[H.sub.4.sup.+] ions in the medium as the peroxidase in the plantlets was proven to decrease in its specific enzyme activities. However, it was unclear whether this increased uptake of inorganic ions was active uptake or as a mass flow due to loss of membrane permeability at high stress levels. Further research is needed to determine the mechanisms involved in the accumulation of inorganic ions under higher stresses, especially salt stresses in the plantlets.

A PGR may be applied but may not penetrate or be transported like the endogenous PGRs, so it might not get to the site of action. However, high concentrations of PGRs may cause non-physiological effects. The type of morphogenesis that occurs in a plant tissue culture largely depends upon the ratio and concentrations of auxin and cytokinins present in the medium. Thus, instead of only using BAP, manipulation of auxin and cytokinin levels could be done in defining a growth regulator balance necessary for the required behavior in culture.

Acknowledgements

This research was supported by Tunku Abdul Rahman University.

Abbreviations:

BAP, 6-benzylamino purine; MS, Murashige and Skoog medium; PGR, plant growth regulator References

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(1) Anna Pick Kiong Ling, (1) Hui Zhen Teoh, (1) Chong Siang Tee, (2) Sobri Hussein

(1) Department of Science, Faculty of Engineering and Science, Tunku Abdul Rahman University (UTAR), 53300 Setapak, Kuala Lumpur, Malaysia

(2) Agrotechnology and Bioscience Division, Malaysian Nuclear Agency, Bangi, 43000 Kajang, Selangor, Malaysia

Corresponding Author: Dr. Anna Pick Kiong Ling, Faculty of Engineering and Science, Tunku Abdul Rahman University (UTAR), 53300 Setapak, Kuala Lumpur, Malaysia

Tel: 603-41079802 ext 132 Fax: 603-41079803

E-mail: lingpk@utar.edu.my
Table 1: Increase in plant length (cm), leaf number, and fresh weight
(g) in plantlets cultured in different concentrations of MgS[O.sub.4].

Concentrations   Increase in Plant       Increase in Number
(mM)             Length (cm)             of Leaves

0.00             0.3 [+ or -] 0.4 ab     0  [+ or -] 0 ab
0.75             0.7 [+ or -] 0.7 ab     0  [+ or -] 0 ab
1.50             0.1 [+ or -] 0.5 ab     1  [+ or -] 2 b
3.00             -0.8 [+ or -] 0.5 a     1  [+ or -] 2 b

Concentrations   Increase in Fresh
(mM)             Weight (g)

0.00             0.043  [+ or -] 0.000 a
0.75             0.041  [+ or -] 0.000 a
1.50             0.038  [+ or -] 0.000 a
3.00             0.038  [+ or -] 0.000 a

Differences in letters represent statistical differences at P<0.05
based on Duncan Multiple Range Test. Error bars show the standard
deviation of mean.

Table 2: Increase in plant length (cm), leaf number, and fresh weight
(g) in plantlets cultured in different concentrations of nitrates.

Concentrations (mM)             Increase in Plant
                                Length (cm)

KN[O.sub.3]     NH4N[O.sub.3]

0.0             0.0             0.4 [+ or -] 0.7 ab
19.7            10.3            0.4 [+ or -] 0.7 ab
39.4            20.6            0.8 [+ or -] 0.6 b
78.8            41.2            0.3 [+ or -] 0.3 ab

Concentrations  Increase in Number    Increase in Fresh
(mM)            of Leaves             Weight (g)

KN[O.sub.3]

0.0             0 [+ or -] 0 ab       0.047 [+ or -] 0.000 a
19.7            0 [+ or -] 0 a        0.056 [+ or -] 0.000 a
39.4            0 [+ or -] 0 a        0.078 [+ or -] 0.000 ab
78.8            0 [+ or -] 1 ab       0.038 [+ or -] 0.000 a

Differences in letters represent statistical differences at P<0.05
based on Duncan Multiple Range Test. Error bars show the standard
deviation of mean.

Table 3: Increase in plant length (cm), leaf number, and fresh weight
(g) in plantlets cultured in different concentrations of sucrose.

Concentrations    Increase in Plant       Increase in Number
(mM)              Length (cm)             of Leaves

0                 0.0 [+ or -] 0.6 a      1 [+ or -] 1 ab
87                0.8 [+ or -] 0.6 b      1 [+ or -] 1 ab
146               -0.1 [+ or -] 0.7 a     1 [+ or -] 1 ab
204               0.1 [+ or -] 0.4 ab     1 [+ or -] 1 ab

Concentrations    Increase in Fresh
(mM)              Weight (g)

0                 0.050 [+ or -] 0.000 a
87                0.111 [+ or -] 0.000 b
146               0.046 [+ or -] 0.000 a
204               0.041 [+ or -] 0.000 a

Differences in letters represent statistical differences at P<0.05
based on Duncan Multiple Range Test. Error bars show the standard
deviation of mean.

Table 4: Increase in plant length (cm), leaf number, and fresh weight
(g) in plantlets cultured in different concentrations of BAP.

Concentrations   Increase in Plant        Increase in Number
(mM)             Length (cm)              of Leaves

0.000            0.1 [+ or -] 0.6 ab      1 [+ or -] 1 ab
0.004            0.1 [+ or -] 0.8 ab      1 [+ or -] 1 ab
0.013            0.3 [+ or -] 0.5 ab      2 [+ or -] 1 b
0.022            0.3 [+ or -] 0.6 ab      1 [+ or -] 1 ab

Concentrations   Increase in Fresh
(mM)             Weight (g)

0.000            0.050 [+ or -] 0.000 a
0.004            0.069 [+ or -] 0.000 a
0.013            0.046 [+ or -] 0.000 a
0.022            0.056 [+ or -] 0.000 a

Differences in letters represent statistical differences at P<0.05
based on Duncan Multiple Range Test. Error bars show the standard
deviation of mean.
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Title Annotation:Original Articles
Author:Ling, Anna Pick Kiong; Teoh, Hui Zhen; Tee, Chong Siang; Hussein, Sobri
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Geographic Code:9MALA
Date:Sep 1, 2009
Words:7182
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