IN VITRO MORPHOGENETIC RESPONSE OF APPLE (Malus domestica Borkh.) AND PEAR (Pyrus communis L.) TO THE ELEVATED LEVELS OF COPPER AND MYO-INOSITOL

The elevated levels of copper and myo-inositol in the MS medium were demonstrated to enhance culture growth and morphogenetic response of apple and pear explants. The results revealed that the highest number of branches per explant (2.80) for apple was obtained from the levels of 0.0+ 100 and 0.050+ 400 mg/l of both copper and myo-inositol, respectively (C1M2 and C4M4)), while for pear 3.40 branches per explant were achieved from the same treatment. The mean length of branches was significantly lower in the case of the control treatment (the absence of copper and inositol). The highest number of leaves per explant (29.73 and 29.80) for both apple and pear, respectively, was recorded for treatment C4M4 (0.050+ 400 mg/l of both copper and myo-inositol, respectively. At the rooting stage, the elevated levels of copper and myo-inositol were very effective in stimulating root formation in both apple and pear shoots. The highest number of roots in apple (2.00 roots/ explant) was achieved while using 0.100+ 800 (C5M5) of both copper and myo-inositol, whereas the highest number of roots for pear (3.17 roots/ explant) was recorded for C6M6 (0.200+ 1600). The highest mean length of roots for apple reached 1.23 cm in treatment C3M3 and 1.10 cm for pear in treatment C6M6. These data suggest that the higher levels of copper and myo-inositol enabled shoot and root formation in the explants, and it might be necessary to use higher levels of these two medium components in order to enhance morphogenetic potential of explants.


INTRODUCTION
Inositol is one of the four most frequently used vitamins in plant tissue culture media and is classified as a member of the vitamin B complex. Inositol is ne-cessary for the growth of plants, yeasts and fungi, and in humans it is especially abundant as part of a phospholipid found in the brain (F a r l e x , 2010). The most ubiquitous and biologically important inositol is myo-inositol, a water-soluble crystalline compound. Many biological functions require myo-inositol, and thus it is identified as an essential metabolite ( Answers. com, 2010). Myo-inositol (also sometimes described as meso-inositol or i-inositol) is the only one of the nine theoretical stereoisomers of inositol which has significant biological importance (G e o r g e et al. 2008).
Myo-inositol plays many important roles in plants. It is involved in signal transduction, the transport of indole-3-aceticacid (IAA), the control of IAA--induced growth and cell elongation (L o e w u s , 1974) as well as the transport of cytokinins (G u r et al. 1988). It is commonly used in culture media and is considered to be a promoting or even an essential medium component in many types of tissue cultures (L o e w u s and D i c k i n s o n , 1980). Its presence in the culture medium is especially important when cytokinins are added to the culture medium (L e t h a m , 1966).
Copper is a necessary micronutrient element in the human diet and essential to plant growth. It is known to be a component or activator of many enzymes involved in electron transport, protein and carbohydrate biosynthesis (P u r n h a u s e r and G y u l a i , 1993). Studies dealing with the optimum concentrations of Cu in tissue culture media are limited. M u r as h i g e and S k o o g (1962) reported that the effect of Cu on culture growth was insignificant at concentrations starting from 0.03 to 30 μM, while S c h e n c k and H i l d e b r a n d t (1972) reported stimulatory effects of higher Cu concentrations on callus growth; hence, they used 0.8 μM Cu in the culture medium. Starting in the 1990s, studies on tissue culture of different species have shown that high concentrations of Cu could enhance morphogenetic responses of cultures (G a r c i a -S o g o et al. 1991; P u r n h a u s e r and G y u l a i , 1993; Y a n g et al. 1999; S a h r a w a t et al. 1999).
The current study aims to test the effects of elevated levels of myo-inositol and copper in the culture medium on the growth and morphogenetic response of apple and pear explants. The culture medium was supplemented with 1 mg × L -1 BA for the multiplication stage and 0.5 mg × L -1 NAA for the rooting stage. Furthermore, the medium was supplemented with 1 mg.L -1 GA 3 , 10 mg × L -1 thiamine-HCl, and 30 g × L -1 sucrose. After pH was adjusted to 5.7 using NaOH or HCl, 7 g × L -1 , agar (7 g/l) was added to the medium. The medium was autoclaved at 121 o C and 1.05 kg × m -2 for 20 minutes.

This investigation was carried out in the Plant
Three single axillary buds of apple and pear were cultured in each culture jar.
Five replications were assigned for each level of treatment and the experiment was set up as a Completely Randomized Design (CRD). The comparison between means was carried out according to Duncan's multiple range test (P < 0.05) using a computerized program of SAS (SAS, 2001). Observations for both the multiplication and rooting stage were recorded after 6 weeks of culture period.

RESULTS AND DISCUSSION
At the shoot multiplication stage, the elevated levels of copper and myo-inositol affected the ability of explants to produce shoots (caulogenesis) in both apple and pear. Table 1 shows that the highest number of branches per explant (2.80) for apple was obtained from the levels of 0.0 + 100 (C1M2) and 0.050 + 400 mg × L -1 (C4M4)) of both copper and myo-inositol, respectively. This number of branches was significantly higher than the control, C2M2, C3M3, C5M5 and C6M6 for apple. On the other hand, the highest number of branches per explant for pear (3.40) was achieved from treatment C1M2 and this number was significantly higher than the rest of treatments, except C6M6. The mean length of branches was significantly lower in the case of the control treatment (the absence of copper and inositol). But in the case of pear, the control did not show significant differences as compared with C2M1 and C1M2. The highest number of leaves per explant (29.73 and 29.80) for both apple and pear, respectively, was recorded for treatment C4M4. The promoted caulogenic response of explants was due to the elevated levels of both copper and myo-inositol in combination or individually ( Fig. 1 and 2).  At the rooting stage, the elevated levels of copper and myo-inositol were very effective in stimulating root formation in both apple and pear shoots. Table 2 shows that the highest number of roots in apple (2.00 roots/ explant) was achieved while using 0.100+ 800 (C5M5) of both copper and myo--inositol, which was significantly different from the rest of treatments except C4M4. On the other hand, the highest number of roots for pear (3.17 roots/ explant) was recorded for C6M6 and this number was significantly higher than the rest of treatments. The longest roots were also significantly affected by the elevated copper and myo-inositol levels. The highest mean length of roots for apple reached 1.23 cm in treatment C3M3 and 1.10 cm for pear in treatment C6M6 (Fig. 3).
In plants, rapid cell division and differentiation require adequate amounts of precursors of cell wall biosynthesis. Both copper and myo-inositol play important roles in cell wall biosynthesis, signal transduction (L o t t et al. 1995), and cell wall lignification (M a r s c h n e r , 1998). The enhancing effect of high concentrations of copper on morphogenetic responses of different species has been reported (G a r c i a -S og o et al. (1991) for Cucumis melo; P u r n h a u s e r and G y u l a i , (1993) for wheat ; C h o et al. (1998) for barley; Y a n g et al. (1999) for rice). The results of the current study are in agreement with those published by G a r c i a -S o g o et al. (1991) who reported that the addition of CuSO 4 × 5H 2 O at high concentrations (0.1 to 5.0 mg × l -1 ) to the culture medium increased the organogenic response of explants in Cucumis melo.

CONCLUSIONS
The positive effects of elevated copper and myo-inositol in the culture medium and the lack of any apparent negative effect with any genotype tested at higher levels of treatment indicate that it will likely be safe, if not beneficial, to use higher levels of these two medium components in order to enhance the morphogenetic potential of explants. These findings are highly supported by Nas's hypothesis that a composition of mineral and organic substances in proportions similar to those found in the seed could provide an optimum culture medium for the growth of plant tissues (N a s and R e a d , 2004).
Finally, it can be said that higher levels of these two components in the culture medium could enhance morphogenetic response of explants and the success of many micropropagation programs.