الفهرس 
SterilizationOnly 14 pages are availabe for public view
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Abstract
This study was carried out at the Tissue Culture Laboratory of Horticulture Department, faculty of Agriculture Menoufia University, Egypt, and Laboratory of Plant Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Sadat City University during the period 2010 – 2014.
1- Optimize tissue culture technique for propagation of the Nemaguard rootstock:
a- In vitro control of bacterial and fungal infection of Nemaguard, the most famous peach rootstock in Egypt
Experiment (1): Effect of exposure period of sodium hypochlorite (NaOCl) concentrations on success of sterilization and survival of Nemaguard peach
Explants were sterilized using different concentrations of sodium hypochlorite (NaOCl) (0.37, 0.5, 0.75, 1.5, 2.25 and 3 %), for (2, 4 and 6 minutes).
The explants which were sterilized with 3% NaOCl over all exposure period recorded the highest number and percentage of uncontaminated explants (9.7). The same result was obtained when explants were sterilized with 2.25 % NaOCl for 6 min as exposure period.
The high concentrations of NaOCl (2.25) with all exposure period 2, 4 and 6 min resulted in the lowest number and percentage of survived shoots (2.0 and 1.3 respectively). On the other hand, the lowest concentration of NaOCl (0.37%) at all exposure periods possessed the highest number of survival shoots (9.7).Experiment (2): Effect of exposure period of mercuric chloride (HgCl2) concentrations on success of sterilization and survival percentage of Nemaguard peach
Explants were sterilized by using different concentrations of mercuric chloride (HgCl2) (0.1% and 0.2%), for (1, 2, 3 and 4 minutes).
Mercuric chloride (HgCl2) at concentration 0.2% cleared an efficient sterilization compare with 0.1% HgCl2 in all exposure period, while, low HgCl2 concentration (0.1%) gave the higher number of survived shoots (9.7)
Experiment (3): Effect of exposure period of ethanol on success of sterilization and survival of Nemaguard peach
Explants were sterilized by using 70% ethanol for different exposure periods (3, 5, 10, 20, 30, 35, 40, 45, 50, 55 and 60 seconds).
Shoots which were sterilized with ethanol 70% ethanol for a period of 15 second maximized number of both uncontaminated shoot and survived shoots number (7.0)
Experiment (4): Effect of different concentrations of both mercuric chloride (HgCl2) and sodium hypochlorite (NaOCl) on success of sterilization and survival of Nemaguard peach
Explants were sterilized by using different concentrations of mercuric chloride (HgCl2) (0.1% and 0.2%) for (3 min) and different concentrations of sodium hypochlorite (NaOCl) (0.37, 0.5 and 0.75 %) for 8 minutes. Results cleared that use 0.50% and 0.75% NaOCl in combination with 0.2% HgCl2 increased number of uncontaminated explants (7.0).
The lowest concentration of NaOCl (0.37%) in combination with both concentrations of HgCl2 (0.1% and 0.2%) possessed the highest survival shoots number.
b- Establish a protocol for in vitro proliferation of Nemaguard
The effect of P.G.Rs as Benzyl-Amino-Purine (BAP), Kinitine (KIN), Adenine Sulphate (ADSO4) and indole acetic acid (IAA) on in vitro growth parameters during multiplication stage.
Experiment (5): Effect of both Benzyl-Amino-Purine (BAP) and indole acetic acid (IAA) concentrations on multiplication parameters of Nemaguard peach
Effect of different concentrations of BAP (0; 2; 3; 4 and 5 mg/L) in combination with various concentrations of IAA (0.0; 0.25 and 0.5 mg/L) on microshoots proliferation were examined .
Interaction between BAP and IAA concentrations cleared that 2.0 mg/L BAP and 0.00 mg/L IAA gave the highest shoot number/explant (9.0 shoots/explant), while, data showed that MS medium supplemented with 0.0 mg/L BAP in combination with 0.5 mg/L IAA gave the highest shoot length (3.5 cm) followed by 3.0 and 2.0mg/L BAP in absence of IAA (2.0 and 1.5 cm, respectively). The highest leaves number were observed on MS medium supplemented with 2 mg/L BAP without IAA (12.0 leaves/explant) followed by 0.25 and 0.50 mg/L IAA without BAP (11.0 leaves/explant for the both concentrations). Experiment (6): Effect of Kinetin (KIN) and indole acetic acid (IAA) concentrations on multiplication parameters of Nemaguard peach
Effect of MS medium supplemented with different concentrations of KIN (0; 2; 3; 4 and 5 mg/L) in combination with different concentrations of IAA (0.0; 0.25 and 0.5 mg/L) on Nemaguard peach proliferation was examined.
- MS medium supplemented with 4 mg/L KIN in combination with 0.25 or 0.5 mg/L IAA resulted in the highest shoot number (2.25 shoots/explants). The highest shoot length (3.50cm) was obtained from MS medium supplemented with 0.0 mg/L KIN and 0.5 mg/L IAA . Leaves number was maximized (22 leaves/explants) when MS medium supplemented with 5 mg/L KIN in combination with 0.25 mg/L IAA or 0.0 mg/L KIN with 0.5 mg/L IAA.
Experiment (7): Effect of adenine sulphate (ADSO4) and indole acetic acid (IAA) concentrations on growth parameters of Nemaguard peach
Effect of MS medium supplemented with different concentrations of (ADSO4) (0; 2; 3; 4 and 5 mg/L) individual or in combination with different concentrations of IAA (0.0; 0.25 and 0.5 mg/L) on Nemaguard peach proliferation was examined.
Adding different concentrations of ADSO4 and IAA to MS medium did not induce multiplication of Nemaguard peach. While adding 5 mg/L ADSO4 in combination with 0.5 mg/L IAA to MS medium gave the highest shoot length (3.7 cm). Also, MS medium supplemented with 3 or 5 mg/L ADSO4 in combination with 0.5 mg/L IAA cleared significantly high leaves number (22 leaves/explants). 2- Establish a protocol for photoautotrophic and photomixotrophic micropropagation as a new micropropagation system for Nemaguard peach rootstock
The effect of rates of gas exchange of culture vessels (0.85, 2, 3, 4 and 5 h-1) and sucrose concentrations (0.0, 10, 15 and 30 g/l) on growth parameters under phototrophic and photomixotrophic conditions was examined. A comparison between regular tissue culture condition and CO2 enriched condition was observed through the following experiments:
Experiment (8): Effect of both gas exchange rates of culture vessel and sucrose concentrations on shoot multiplication parameters of Nemaguard peach
When rate of gas exchange was 2 or 3 h-1 in combination with 30 g/l sucrose the highest shoot number of Nemaguard peach (9 shoots/ explant) was observed. Concerning shoot length, rate of gas exchange 3 (h-1) in combination with 15 g/l sucrose resulted in the highest shoot length (3 cm). The same result was observed for leaves number (20 leaves/ microshoot).
Experiment (9): Effect of both gas exchange rates of culture vessel and sucrose concentrations on shoot multiplication of Nemaguard peach under CO2 enriched condition
Nemaguard peach plantlets possessed the best growing and development when incubated under CO2 enriched conditions with 3 h-1 rate of gas exchange in combination with 15 g/l sucrose compare with all treatments. Shoot multiplication reached to 11 shoot /explant, shoot length possessed 3cm and leaves number 25 leaves /shoot.*Photosynthetic pigments analysis:
Effect of gas exchange rates of culture vessel and sucrose concentrations on Chlorophyll a+b content of Nemaguard peach leaves under regular condition and CO2 enriched condition
Result cleared that 3 h-1 gas exchange rate and 15 g/l sucrose gave the highest chlorophyll a+b content (4.2),while The lowest value was obtained when gas exchange rate was 0.85 h-1 and 0 in combination with 10 g/l sucrose (1.8 and 2.1, respectively) under tissue culture regular condition. On the other side, chlorophyll a+b content under CO2 enriched condition results cleared that 2 and 3 h-1 gas exchange rate in combination with 15 g/l sucrose gave the highest chlorophyll a+b content, the same result was observed when gas exchange rate was 4 h-1 in combination with 30 g/l sucrose.
Effect of gas exchange rates of culture vessel and sucrose concentrations on Carotenoids content of Nemaguard peach under tissue culture regular condition and CO2 enriched condition
Result cleared that under tissue culture regular condition gas exchange rates negatively affected carotenoid contents value. While, under CO2 enriched condition the relationship was positive. Regarding the effect of sucrose, under tissue culture regular condition, sucrose concentrations did not affected carotienoid content value, while, under CO2 enriched condition there were a positive relationship between sucrose concentration and carotenoid value. , Anyway, under tissue culture regular condition, gas exchange rate 0.85 h-1 in combination with 0 g/l sucrose gave the highest carotenoids content (1.7). On the other sid, under CO2 enriched condition, 2, 3, 4 and 5 h-1 gas exchange rate in combination with 10, 15 and 30 g/l sucrosegave the highest carotenoids content. These result may indicate that high gas exchange rate of culture vissel may be allowed to increase CO2 concentration which may enhance photosynthesis pigments.
◘ Leaf anatomy of ventilated and non-ventilated conditions on Growth of in vitro Nemaguard peach shoots
a- Stomata density study of Nemaguard peach leaves
Effect of gas exchange rates of culture vessel and sucrose concentrations and combination on stomatal density of Nemaguard peach leaves under CO2 enriched and non-enriched condition
Microscopic observations of leaf abaxial surface showed that plantlets grown in ventilated vessels showed the lowest stomatal density, whereas the highest value was observed when plantlets were grown in non-ventilated vessels.
Interaction between gas exchange rate and sucrose concentrations cleared the same effect under tissue culture regular condition and CO2 enriched condition, data cleared that 0.85 h-1 gas exchange rate with all sucrose concentrations gave the highest stomatal density with no significant difference, while 5 h-1 gas exchange rate with 30 g/l sucrose gave the lowest stomatal density.
b-Histological study of ventilated, non-ventilated conditions of in vitro Nemaguard peach
Leaves of plantlets in non-ventilated vessels failed to build up a distinct upper epidermis layer (UE), palisade parenchyma layer (PP) and spongy mesophyll (SPand had greater air space (IS) within the PP and SP. The cells of these leaves were regular shape and in a loose structure. In contrast, the anatomy of leaves under ventilated condition displayed comparatively well organized UE and layers of palisade cells. The mesophyll of those leaves had one PP layer and two to three layers of SP and smaller air spaces within SP. The vascular system of leaves under ventilated condition displayed very well developed xylem (XY) which was arranged in a line of protozylem to metazylem from the abaxial to the adaxial. The vascular system for leaves under non-ventilated condition was not well organized.
◘ rooting and Acclimatization sage
In vitro rooting rate are 64% by using 0.5 mg/L of IAA. The rooted plants showed 85% survival rate in acclimatization stage.