Essay Example on Plant material and Sterilization Shoot tips was selected and excised From Peach









Plant material and Sterilization Shoot tips was selected and excised from peach baladai CV plant grown in Taif governorate The explants were used of peach was used as a plant materials The explants were disinfested for 1 min in 70 ethanol followed by 20 or 30 of Clorox solution 5 25 active ingredient sodium hypochlorite containing also 1500 ppm citric acid and 1500 ppm ascorbic acid for 20 or 30 minutes plus 0 01 Tween 20 and then rinsed three times with sterile distilled water Pooler and Scorza 1995 Growth chamber conditions were set to 24 ºC and a photoperiod of 16 hours at a light intensity of 2000 lx provided by cool white fluorescent tubes Media preparation Murashige and Skoog MS m supplemented with 3 sucrose w v and different concentrations of BAP 0 0 0 5 1 0 1 5 and 2 mg l were used for shoot initiation shoot multiplication and shoot elongation stages while different concentrations of IBA 0 0 0 1 1 0 and 2 mg l and 0 1 mg l NAA were used for root formation The pH of all media was adjusted to 5 7 using 1 0 N potassium hydroxide KOH and 1 0 N hydrochloric acid HCl before adding 0 8 w v phytoagar Media were autoclaved at 121 C for 20 min and 1 5 k cm2 pressure In vitro culture Meristems tip of 1 0 5 mm in length were taken under the stereoscope and cultured individually within glass test tubes and Shoot tips explants were cultured for three to four weeks on MS medium with various concentrations of BAP 0 0 0 5 1 0 1 5 and 2 mg l and 3 w v sucrose for shoot initiation multiplication and elongation Induced shoots were subcultured on the same media for another three to four weeks Rooting stage elongated shoots 3 to 4 cm length were transferred on MS medium with different concentrations of IBA 0 0 0 1 1 0 and 2 mg l in combination with 0 1 mg l of NAA for a further four weeks MS medium without growth regulators was used as a control for all the in vitro cultures 

All the in vitro cultures were incubated at 26 2 C in a growth room on a 16 8 hour dark light and 3000 lux light intensity provided by coolwhite fluorescent light After successful rooting the rooted plantlets with 5 to 6 roots of length 3 to 5 cm were carefully washed with warm H2O to remove agar and traces of medium then they were transplanted to plastic pots containing sterile peat moss The top of the pots was covered with transparent plastic Thermotherapy incubated at 250umol m 2s 1 light condition with increasing temperature from 28 oC to 39 oC Two separate experiments were effectuated the first heat therapy experiment had a duration of 15 days setting a 39 oC temperature at the Imago 3000 alphanumeric keypad and the second experiment had a duration of 22 days at 37 oC In the preliminary thermotherapy phase the plants were acclimated step by step 1 oC day until the set temperature was achieved Humidification varied between 32 75 rH Detection of PPV using DAS ELISA

 For the detection of PPV with DAS ELISA Clark Adams 1977 commercial polyclonal antibodies were used IgG and IgG conjugated with alkaline phosphatase from the Loewe Company Germany Six leaves were collected from different parts of the tested tree and were combined to prepare a representative sample 1 g of leaves was homogenized in extraction buffer using manual homogenizer and 200 µl was pipetted into 2 wells of microtiter plate Results were evaluated on the spectrophotometer Dynatech MR 5000 at 405 nm ELISA tests were done before treatment 2 weeks after the treatment and 9 T1 eventually 7 T2 months after the treatment Detection of PPV by RT PCR RT PCR was used for testing of the trees for which negative DAS LISA results were obtained immediately after the thermotherapy and 7 9 months later

The total RNA was isolated from samples of 0 1 g of leaf tissue using an RNeasy Plant Mini Kit Qiagen according to the manufacturer's instructions Primers P1 P2 Wetzel et al 1991 and GO Taq polymerase Promega Corporation were used for the RT PCR amplification Aliquots of PCR products were run on 1 agarose gel DNA was stained with SYBR Green Invitrogen Experimental design For shoot initiation multiplication and elongation experiments every treatment contained of 40 shoot tips and internodes segments containing axillary bud explants For rooting experiments every treatment contained of 30 elongated shoots All experiments were carried out in three replicates The results were recorded from different experiments four weeks after culture For statistical analysis of data analysis of variance ANOVA and mean separation were carried out using Duncan's multiple range test and significance was determined at the p 0 01 level Data analysis was performed using ASSISTANT version 7 7 beta 2016 computer package

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