MULTIPLICATION OF NEMAGUARD AND MARIANA ROOTSTOCKS: INFLUENCE OF CYTOKININS AND ADENINE SULFATE AND THEIR CONCENTRATIONS

Maha I. Salih 1 , Farqad M.K. Al Dabagh 2 and Ibrahim A. Al Shmarey 3 . 1. Genetic Engineering and Biotechnology institute for higher studies. 2. Ministry of Agriculture. 3. College of Agriculture, University of Baghdad. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Additional additives (such as AS) used in culture medium have proved their efficiency and beneficially at the plant species that have a difficult reproduction in vitro by stimulating the growth and development for number of plant tissues (Kulcarni et al., 2007).

Materials and methods:-
Micropropagation study:-Preparation of explants:-Five to eight years old stock plants of Nemaguard and Mariana rootstocks were taken from fruit cultivation station/ Iraqi ministry of agriculture (Al Hawija, at the north of Iraq). Actively growing shoots of the donor plants were cut off, then the leaves were removed and cut into nodal parts with approximate size of 4-5 cm to be disinfected and cultured. Explants were washed under running tap water 5 times, surface sterilization was carried out under complete aseptic conditions at the Laminar air flow cabinet. These explants were sterilized with 70% ethanolalcohol for a minute and rinsed them three times with sterile distilled water, followed by sterilization with (10% v/v) commercial Clorox solution with a drop of Tween-20 for 20 minutes with continues shaking and rinsed. Finally, the nodal parts were washed 3 times with sterile distilled water to remove the bleach off the explants and trimmed to 2 cm long (V-shape) to increase the surface area of absorption.
The basic nutrient medium:-Nodal explants were planted vertically on solid basal medium of MS supplemented with vitamins (100mgl -1 myoinositol and 30 gl -1 sucrose. BA, Kin, GA 3 , and AS were used independently or in combination at different concentrations. The value of pH was adjusted to 5.7±0.1 with few drops of 0.1N either HCl or NaOH prior to agar addition and autoclaving. The media were dispensed into 40 ml jars and autoclaved for 20 minutes at 121 C° and 1.2kg/cm 2 pressure, then left at room temperature (22±2C°) to cool.

Culture growth conditions:-
Tissue culture jars were placed in an incubation room at 25±1C° under 16 hours photoperiod of 1000 lux supplied with cool white fluorescent lamps.

Influence of different growth regulators on shoot induction:-
The shoot induction development from the stem node segments was attempted with MS medium supplemented with different concentrations of BA (0.0, 1.0, 2.0, 3.0 mgl -1 ) in combination with GA 3 (0.0, 0.1, 0.2, 0.3 mgl -1 ) in the presence of 0.3 mgl -1 NAA.
Percentage of explants forming axillary shoots% and mean length of these shoots (cm) were recorded after 6 weeks of culture.
Multiplication stage:-Shoots were placed in MS hormone-free medium for 4 weeks in order to improve shoot elongation. 0.7-0.8 cmlong shoots from previous culture were subjected to be multiplied on MS medium containing different concentration of Kin (0.0, 2.0, 4.0, 6.0 mgl -1 ) in combination with AS (0.0, 25.0, 50.0, 75 mgl -1 ) in the presence of 0.1 mgl -1 GA 3 . For further multiplication, the shoots were subcultured 2 times on the best medium. Mean number and length (cm) of axillary shoots/explant were recorded after 6 weeks of subculture.

Statistically analysis:-
This experiment was carried out based on Completely Randomized Design (CRD), each treatment was done in 10 replications. SPSS 16 software was used for statistically analysis of the data, and differences among means of treatments were compared by using (LSD) Least Significance Design (Steel et al., 1997).

Influence of growth regulators on shoot induction:-Influence of growth regulators on shoot induction(%):-
The basic goal of the establishment stage is to get a large percentage of free pathogens explants (Murashige, 1974). There are many factors that affect the success of establishment stage involved:  The choice of plant material  Elimination of contamination  Culture conditions (Hartman and Kester, 1983) In this experiment, various concentrations of BA as cytokinin and GA 3 in the presence of NAA as an auxin were added to MS medium for shoot induction of the studied rootstocks; Nemaguard and Mariana. Table 1 showed the effect of different concentrations of BA and GA 3 with 0.3mgl -1 NAA on the shoot induction (%) of both rootstocks stem node sections. The interaction between genotype, BA and GA 3 and concentration was highly significant for shoot induction. For Nemaguard, the highest percentage (100%) was observed on media supplemented with 2mgl -1 BA and 0.1mgl -1 GA 3 . For Marina, the optimum percentage of shoot induction (100%) was on media with 1mgl -1 BA and 0.3mgl -1 GA 3 .
It´s well known that BA promotes cell division and cell expansion in plant tissue culture, These results agreed with those gained by Magalhaes et al. (2012) who reported that addition of GA3 to the culture medium was superior in increasing proliferation of Japanese plum (Prunus salicina Lindl.) cv. América shoots.  Table 2 showed the effect of different concentrations of BA and GA 3 with 0.3mgl -1 NAA on the induced shoot length (cm)of both rootstocks stem node sections. It is important to note the interaction of BA, GA 3 and hormonal concentrations, highest average (1.95 cm) was obtained from the treatment (3mgl -1 BA+ 0.6 mgl -1 GA 3 ). Highly shoot length of Nemaguard rootstock was observed between the interaction of genotype, growth regulator type and hormonal concentrations producing maximum shoot length (2.22 cm) in treatment containing (3mgl -1 BA+ 0.6 mgl -1 GA 3 )which proved to be the best treatment.Peirik (1987) reported that the cytokinin promoted division of cell by activating the synthesis of DNA; promoting the growth of an axillary buds and inducing shoot formation.

Influence of growth regulators on induced shoot length (cm):-
These results were agreed to this obtained by Yepes and Aldwinckle (1994) who found that the balance between GA 3 and auxin affected shoot elongation positively.

multiplication stage: Influence of growth regulators and AS on Mean number and length of axillary shoots/explant:-
following the successful growth of plant tissue culture, the establishment stage is followed by multiplication, which is defined as a rapid increase of organs which is achieved by enhancing axillary shoot initiation, through repeated this process, hundreds or thousands of plants may be produced from a single explant sample (Smith and Murashige, 1970;Murashige, 1974).
In this experiment, lateral shoots produced from the previous stage were excised and cut into 2 bud segments. Tables  3 & 4   The effect of adenine sulfate in known in the tissue cultures in many plant species and types of vegetal tissues, affect that is superior in combination with a balanced dose of cytokinin and auxin (Yepes and Aldwinckle, 1994).