OPTIMUM MICRONUTRIENT LEVEL FOR PHALAENOPSIS DELICIOSA ORCHID SEEDLING IN VITRO GROWTH

There were many researches about the influence of m icronutrient concentration toward growth of plants. However, there was no clear statement about the mic ronutrient level for growth of Phalaenopsis seedlings. Hence it was worth to investigate the optimum level of mi cronutrient for Phalaenopsis, an endangered orchid species. Using Phalaenopsis deliciosa as the subject, germinated seedlings were grown on defi ed culture media containing different MS micronutrient level. After 90 days with a subculture at day 45, fresh and dry weights of shoots and roots of the seedlings were measured. It was found that the optimum micronutrient level for P. deliciosa seedling growth was observed between 0.50× and 1.0 0× of MS micronutrient level. Higher micronutrient level caused roots and seedlings to d e erioration except for a minority of seedling vari ants that grew exceptionally well, suggesting that high micronutri ent level was selective for a small number of varia nts. The study demonstrated the importance of appropriate mi cronutrient level for supporting growth and develop ment for wide range of genotypes in P. deliciosa. This micronutrient level may as well be optimum f or other species under the genus Phalaenopsis and should be considered for maintaining genotype d iversity in vitro.


INTRODUCTION
Micronutrients are important for plant growth and morphogenesis (George and De Klerk, 2008).Micronutrients are essential elements required at minute amount and constitute less than 0.01% of plant tissue dry mass while beneficial elements are those that may enhance plant growth or required by certain plant species only (Barker and Pilbeam, 2007).Murashige and Skoog (1962) micronutrients consist of Iron (Fe), Manganese (Mn), Zinc (Zn), Boron (B), Iodine (I), Molybdenum (Mo), Copper (Cu) and Cobalt (Co), all of which are essential micronutrients except for Co and I, which are beneficial elements.The functions of these elements are described in detail elsewhere (George and De Klerk, 2008).
Non-optimum concentration of micronutrient may adversely affect plant growth.High concentration of B and Mn caused poor root development due to the enhancement of auxin destruction according to a research by Galston and Hillman (1957); the presence of Mn (II) decreased auxin level through degradation by Indole Acetic Acid (IAA)-oxidase (George and De Klerk, 2008).High B concentration resulted in decrease of number of roots formed, because high B concentration inhibited growth of roots through degradation of auxin (Jarvis, 1986).Excessive Zn concentration is inhibitory for root growth (George and De Klerk, 2008).Besides, some microelements like Cu are toxic to plant cells at high concentration (Kopsell and Kopsell, 2007).
Insufficient concentration of micronutrients also affects plants growth.Mo is essential and occurs in numerous oxido-reductase enzymes in plants (Hamlin, 2006).Zn deficient plants underwent low enzymatic activities and subsequently low production of protein,

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nucleic acid and chlorophyll (George and De Klerk, 2008).Insufficient B led to the cease of cell division and root elongation in plants via its effect on the metabolism of RNA (Ali and Jarvis, 1988).
Phalaenopsis species seedlings were found to grow well on (Choong et al., 2013) medium which was shown to be non-genotype selective.However, optimum level of MS micronutrient in this medium is unknown; therefore the optimum MS micronutrient level for P. deliciosa seedling yield on CCT medium was investigated.P. deliciosa is an endangered orchid species listed in the Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES, 2013) and therefore deserves to be propagated.

MATERIALS AND METHODS
Seedpods of Phalaenopsis deliciosa Rchb.f. were harvested 110 days after pollination.The seedpods were washed briefly under tap water before immersed into 5.25% sodium hypochlorite for 5 min and subsequently 70% ethanol for 1 min.This was for the sterilization of the seedpods.The seedpods were then flamed briefly until traces of ethanol burnt off, excised with a scalpel and seeds within the seedpods were inoculated onto CCT medium (Choong et al., 2013).Seedlings were maintained on the same medium with a 60-day subculturing regime.
After that, roots and shoots of the seedlings were separated and respectively weighed to obtain the Shoot Fresh Weight (SFW) and Root Fresh Weight (RFW), subsequently dried at 70°C for 2 days before measuring the Shoot Dry Weight (SDW) and Root Dry Weight (RDW).Total Fresh Weight (TFW) and Total Dry Weight (TDW) of the seedlings were obtained by the addition of shoot and root fresh weight and dry weight respectively.Root to Shoot Ratio (R/S) was calculated by dividing RDW with SDW.Water content was calculated using on the formula (TFW-TDW)/TDW.
Data obtained from the measurements were tested for normality based on z-score of kurtosis and skewness at α = 0.05.Treatment means of the parameters mentioned above were analyzed by single-factor Analysis of Variance (ANOVA) and two-way pairwise comparisons between those treatment means were performed with Fisher's Least Significant Difference (LSD) test at α = 0.05.

RESULTS
Most of the data from the experiment were approved by normality tests in terms of kurtosis and skewness.Statistical analysis was only run with normal data, to prevent inaccuracy due to not normal data.
Analysis Of Variance (ANOVA) on various parameters of seedlings grown on different micronutrient levels showed that RFW, RDW, TFW, TDW, R/S ratio and WC were significantly different (ρ>0.995),while SFW and SDW not significantly different (Table 1).
The significance in ANOVA of RFW and RDW indicated that the micronutrient level affected the growth of roots.The significance in TFW and TDW indicated that micronutrient level also affected the total yield of seedlings.R/S ratio showed significant difference, which demonstrated that root development was also affected by micronutrient level.From ANOVA, there was no significant difference in SFW and SDW, which meant that shoot growth was not significantly influenced by micronutrient level tested in this experiment.
However, the ANOVA only indicated whether there were significant differences between the treatments but did not indicate differences among individual treatment means.Therefore multiple pairwise comparisons Fisher's LSD analysis was run after ANOVA (Table 2).
In terms of Fisher's LSD analysis, RFW, RDW, TFW, TDW and R/S ratio had a same distribution, which were resolved into two distinct groups where micronutrient level 0.25, 0.50 and 1.00× in group a, while 2.00× was in group b.For WC, there was three groups assigned, 0.25× and 0.50× in group a, 1.00× in group b and 2.00× in group c.SFW and SDW had no significance; all treatments were within one group.
Graph (Fig. 1) was used to represent the response of various parameters according to micronutrient level.OJBS RFW (Fig. 1c) and TFW (Fig. 1e) had similar response pattern, which increased from 0.25× to 0.50× micronutrient level and steadily decreased from 0.50× to 2.00× micronutrient level; peaked when micronutrient level was at 0.50×.As it can be seen, root weight contributed mainly to total weight due to the fact that roots were at least 3.5 times heavier than shoots.R/S ratio (Fig. 1g) presented a similar trend as TFW.Apart from that, SDW (Fig. 1b), RDW (Fig. 1d) and TDW (Fig. 1f) had a peak between 0.50× and 1.0× micronutrient level, while SFW (Fig. 1a) had a maximum point between 0.5× to 0.75× micronutrient level.Graph for WC (Fig. 1h) had a same trend as RFW and R/S, which reflected that larger amount of fresh root and root development may contribute to the higher WC in seedlings.Thus, when RFW decreased, WC was observed to decrease as well.Notes: SFW-shoot fresh weight, SDW-shoot dry weight, RFW-root fresh weight, RDW-root dry weight, TFW-total (seedling) fresh weight, TDW-total (seedling) dry weight, R/S-root to shoot ratio, WC-water content 0.328 c Notes: SFW-shoot fresh weight, SDW-shoot dry weight, RFW-root fresh weight, RDW-root dry weight, TFW-total (seedling) fresh weight, TDW-total (seedling) dry weight, R/S-root to shoot ratio, WC-water content

DISCUSSION
Micronutrients are nutrients required in minute amount, which are components of many plant cell proteins involved in metabolic and physiological processes (George and De Klerk, 2008).In a previous study by (Murashige and Skoog, 1962), they introduced a medium called the MS medium that contained micronutrients (µM) Fe (100), B (100), Mn (100), Zn (30), I (5), Mo (1), Cu (0.2) and Co (0.2).Our experiment was based on the CCT medium containing different levels of MS micronutrients (2.00, 1.00, 0.50 and 0.25×).According to our observation, MS micronutrient level between 0.50× and 1.00× gave a better growth of the seedlings (Fig. 2a).Medium used for Phalaenopsis germination and growth in vitro often used 0.5× MS micronutrient level, such as those used for Phalaenopsis bellina (Khoddamzadeh et al., 2010) and Phalaenopsis amabilis (Chen and Chang, 2006).
In our observation, we found several replicates containing seedlings variants with very vigorous growth at 2.00× MS micronutrient level.The size of their leaves was larger than other treatments and the leaf color appeared as dark green; their roots also appeared unusually thick.These seedlings were variants with a phenotype that was tolerant to high concentration of micronutrients which was toxic to other seedlings.The appearance of these seedlings caused a second distribution which peaked at the higher end the first distribution.These data were removed, reducing the number of replicates from 23 to 19 for statistical analysis.This observation indicated that high concentration of micronutrient could select for several vigorous P. deliciosa seedlings and is undesirable for conservation purpose.

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Loci responsible for tolerance to copper were identified in wheat (Bálint et al., 2007), supporting the existence of micronutrient tolerance trait in plant.

Micronutrient Excess
Micronutrient level higher than 1.00× affected the growth and development of roots and led to the decrease of yield.When micronutrient level was at 2.00×, the root tips became yellow and dark brown eventually (Fig. 2b).This revealed that micronutrient could be toxic to seedlings at high concentration.In a previous study done by (Sarkar et al., 2004) they found that excess of Mn supply was toxic for potato microplant and it caused stem streak necrosis as well as significant inhibition in seedling rooting and growth (Sarkar et al., 2004).
Mn 2+ was one of the cofactors IAA oxidases in plant cell (Galston and Hillman, 1957) as cited by (George and De Klerk, 2008).Therefore presence of Mn in culture medium could activate the IAA oxidases and promoted the degradation of auxin; thus it reduced the development of roots.In addition, high concentration of B could cause excessive IAA degradation as well, reducing root growth (Jarvis, 1986).B excess could cause yellowing of leaf margin and tip (Gupta, 2006), which occurred in some of the seedlings treated with 2.00× MS micronutrient.
Besides, high level of Zn was found to be inhibitory and prevent root growth, such as the result obtained by (Malik et al., 2011) in their experiment concerning effect of different levels of Zn on growth and yield of red amaranth and rice.Sedberry et al. (1988) found that Zn application resulted in a reduction of P concentration in rice plant tissue at the first joint and this may also be another reason for yield reduction on medium containing 2.00× MS micronutrient.
In XER medium (Ernst, 1994) and NDM medium (Tokuhara and Mii, 1993) used for culturing Phalaenopsis, the Zn concentration was even lower (0.11 mg L  1 ).Thus, Zn concentration higher than 1.00× may be excessive and inhibitory to P. deliciosa seedling growth and development and this was probably due to Zn-induced P deficiency (Storey, 2007).

Comparisons Between Different Culture Media
When comparing the components of 0.50× MS micronutrient with other media, it was found that the B concentration was similar to the B5 medium (Gamborg et al., 1968) but much higher than NDM and XER media (Tokuhara and Mii, 1993;Ernst, 1994) (Table 3).The concentration of Cu and Co in 0.50× MS medium was half of the other 3 media.The amount of Cu and Co in the CCT medium could be doubled to achieve better seedling growth.In B5 medium, the Zn concentration was approximately half of 0.50× MS micronutrient and in NDM medium the concentration of Zn was even much lower than 0.50× MS micronutrient Science Publications

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(near one ninth).Hence, CCT medium could be better for seedling growth if the amount of Zn in the medium was reduced.Experiments involving different levels of individual micronutrient could be done to identify the optimum level of each micronutrient for P. deliciosa.

CONCLUSION
Based on our observation and data analysis, we found out that the optimum micronutrient level for P. deliciosa was between 0.50× to 1.00× MS micronutrient level in CCT medium.Micronutrient level between 0.50× to 1.00× gave the maximum development and growth of roots.High micronutrient level inhibited growth and development of roots probably due to excessive Mnassociated and B-associated IAA degradation and Zninduced P deficiency.However some of variants had the capability of tolerating the high micronutrient level and grew vigorously.Low micronutrient level could cause deficiency of micronutrients and non-optimum growth.Besides, WC of the seedlings varied when grown on different micronutrient level; the roots of P. deliciosa contained most of the water, which predominantly contributed to total water content of whole seedlings.Micronutrient level did not have a significant influence on the growth of shoots.MS micronutrient of 0.50× or slightly higher is recommended for in vitro seedling growth and root development of P. deliciosa.Alternatively, a new micronutrient mixture can be designed based on literature or empirically determined by experimenting with individual micronutrient one at a time.

ACKNOWLEDGEMENT
The research was carried out at and financially supported by INTI International University, Nilai, Malaysia, part of the Laureate International Universities network under the research grant ORD/12/FAS/(57).

Table 1 .
Analysis Of Variance (ANOVA) on various parameters of seedlings grown on different micronutrient levels ns-not significant.

Table 2 .
Treatment means of parameters of seedlings grown on different micronutrient level and resolved with Fisher's LSD analysis at α = 0.05 into groups denoted by small caps superscript alphabets

Table 3 .
Comparison of the micronutrient content of 4 different culture media Medium