الفهرس 
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Abstract
5. SUMMARY
This study was carried out in plant Tissue Culture Laboratory in Faculty of Environmental Agricultural Sciences El-Arish, North Sinai, Suez Canal University during the period from 2006 to 2008 .The molecular studies were carried out in the Plant Biotechnology Laboratory at El-Sheik Zuwyed Research Station, Desert Research Center (DRC), North Sinai Governorate, Egypt. The work was done to display in details the whole protocol for Stevia rebaudiana propagation through tissue culture technique to produce and introduce Stevia rebaudiana plants as anew sweetener crop to Egyptian agriculture as it is a potential source of low caloric –sweeteners. In order to maximize efficiency of plant propagation via direct organogenesis, it is important to study the effect of temperature, light intensity and growth regulators as well as plant material on the growth and development of Stevia rebaudiana grown in vitro via micropropagation and consequently the production of identical true to type plantlets.
The results of this study are shown in the following topics:
The first experiments: In vitro culture experiments:
1. Establishment stage:
1.1. Shoot tip explant gave the highest number of shoots compared with nodal segments, while the maximum number of shoots induced without adding benzyl adenine (BA). The highest number of shoots obtained from the interaction between shoot tip explant and zero BA concentration.
1.2. The tallest shoot was achieved by the interaction between nodal segment and 0.5 mgl-1 BA. In other words, shoot tip explant improved shoot length by 4.21% compared with nodal segment.
1.3. Shoot tip explant gave the highest number of nodes compared with nodal segments, while the maximum number of nodes induced at 0.5 mgl-1 BA. The highest number of nodes obtained from the interaction of 0.5 mgl-1 BA for the both studied explant types.
1.4. Shoot tip explant gave the highest number of leaves compared with nodal segments, while the maximum number of leaves gained by adding 0.5 mgl-1 BA. The highest number of leaves was achieved by the interaction between shoot tip and 0.5 mgl-1 BA.
2. Multiplcation stage:
2.1. Shoots number.
1- The concentration of 0.5 mgl-1 BA gave the highest number of shoots compared with the control treatment. Concerning to light intensity, 3000 lux was the best treatment which recorded the highest number of shoots. While, using 25oC gave the highest number of shoots.
2- The highest number of shoots was induced with 0.5 mgl-1 BA under light intensity 3000 lux.
3- The highest number of shoots was encouraged with 0.5 mgl-1 BA at 25oC.
4- The highest number of shoots was induced at 25oC under light intensity 2000 or 3000 lux.
5- The highest number of shoots was induced with 0.5 mgl-1 BA at 25oC with light intensity of 3000 lux.
2.2. Shoot length.
1- The highest shoot length value was resulted from free hormone treatment compared with 0.5 mgl-1 BA. Also, results given allowed that 2000 lux was the best light intensity which recorded the tallest shoot.while the best treatment is at temperature 20oC (4.81).
2-The tallest shoot was achieved by control treatment (0.0 mgl-1 BA) under light intensity of 1000 lux.
3- The maximum shoot length was induced with 0.0 mgl-1 BA with 20oC treatment.
4- The tallest shoots were induced at 20oC under light intensity 1000 lux.
5- The interaction of 0.5 mgl-1 BA, 20oC and 2000 lux gave the highest shoot length.
2.3. Nodes number.
1-The control treatment (0.0 mgl-1 BA) gave the highest number of nodes compared with 0.5 mgl-1 of BA. Concerning to light intensity, the given results allowed that 2000 lux was the best light intensity which recorded the highest number of nodes, meanwhile, using 30 oC gave the lowest ones.
2- The highest number of nodes was achieved by 0.0 mgl-1 BA under light intensity of 1000 lux.
3- The maximum number of nodes was induced with 0.0 mgl-1 BA with 20oC treament.
4- The maximum number of nodes was induced at 30oC under light intensity 3000 lux.
5- The highest number of nodes was induced with 0.0 mgl-1 BA under 20oC with light intensity of 1000 lux.
2.4. Leaves number.
1- The concentration of 0.0 mgl-1 BA gave the highest number of leaves compared with 0.5 mgl-1 BA. While, 2000 lux was the best treatment Also, using 20oC gave the maximum number of leaves.
2- The highest number of leaves was induced with 0.0mgl-1 BA under light intensity 1000 lux.
3- The Control treatment (0.0 mgl-1 BA ) with 20oC gave the maximum number of leaves.
4- The highest number of leaves was induced at 20oC under 1000 lux.
5- The concentration of 0.0 mgl-1 BA at 20oC under light intensity of 1000 lux.
3. Rooting stage:
3.1. Roots number.
According to hormone type, auxin (IBA) gave the highest number of roots compared with NAA. Over the hormone type, 1 mgl-1 was the best concentration. The interaction between IBA and 1 mgl-1 concentration gave the maximum root number.
3.2. Roots length.
The tallest root was achieved by IBA when compared with NAA. In other words, using IBA improved number of roots by 37.31% comparing with NAA. Results given allowed that 1 mgl-1 was the best concentration. The maximum number of roots was achieved by the interaction between IBA and 1 mgl-1 concentration.
3.3.Shoots number.
The NAA hormone gave the highest number of shoots compared with IBA, while the maximum number of shoots induced with 2 mgl-1 of auxin. The highest number of shoots obtained from the interaction between IBA and 2 mgl-1.
3.4. Shoot length.
The maximum shoot length was obtained with NAA compared with IBA. Concerning to hormone concentration, adding 2 mgl-1 gave the tallest shoot. The interaction of NAA at 3 mgl-1 concentration was the best treatment for shoot length.
3.2. Effect of MS medium salt strength and IBA concentrations.
3.2.1. Root number:
The full MS medium gave the highest number of roots compared with half treatment. The concentration of 1.5 mgl-1 was the best concentration which recorded the highest number of roots. The maximum number of roots was achieved by the interaction between full MS and 1.5 mgl-1 .
3.2.2. Roots length.
The full MS medium gave the tallest roots compared with half treatment. The given results given allowed that 1.5 mgl-1 concentration was the best concentration which recorded the highest roots length. The maximum number of roots was achieved by the interaction between IBA 1 mgl-1 concentration and full MS medium.
3.2.3. Shoots number.
Full MS medium gave the highest number of shoots compared with half MS, while the maximum number of shoots induced without adding IBA. The highest number of shoots obtained from the interaction between full MS medium and zero IBA.
3.2.4. Shoot length.
The maximum shoot length induced with full MS medium. Also, the tallest shoots were achieved by 0.5 mgl-1 IBA. The maximum shoot length obtained from the interaction between full MS medium and 1.5 mgl-1 IBA concentration.
3.2.5. Nodes number.
Full MS gave the highest number of nodes compared with half MS, while the maximum number of nodes induced at 0.5 mgl-1 of IBA concentration. The highest number of nodes obtained from the interaction of 1.5 mgl-1 of IBA and full MS medium.
3.2.6. Leaves number.
Full MS gave the highest number of leaves compared with half MS, while the maximum number of leaves gained by adding 1.5 mgl-1 IBA. The interaction between full MS and 0.5 and 1.5 mgl-1 BA was the best treatment.
3.3. Effect of explant and IBA concentrations.
3.3.1. Effect of explant.
The highest relative water content (RWC) was gained when plantlet was used at rooting stage.
3.3.2. Effect of IBA concentration.
The highest RWC was used at 1.5 mgl-1 of IBA.
3.3.3. Effect of interaction.
The highest value of RWC was the interaction between plantlet and 1.5 mgl-1 of IBA.
4. Acclimazation stage.
Hydroaeropoinc system gave the highest number of shoots compared with mixture soil, while the tallest shoot induced using Hydroaeropoinc system. The highest number of nodes obtained from Hydroaeropoinc system were used. The highest number of leaves induced using Hydroaeropoinc system. The maximum number of roots obtained from Hydroaeropoinc system were used. The tallest roots induced using Hydroaeropoinc system compared with mixture soil. Hydroaeropoinc system gave the highest survival rate compared with mixture soil.
5. Molecular studies:
The present work aimed to study the genetic stability for Stevia rebaudiana as produced from in vitro technique to compare Stevia plant through studying the genetic stability of regenerated explants and their parental line using Inter simple sequence repeat (ISSR).
Molecular ISSR –PCR of Stevia rebaudiana:
Molecular level of polymorphic bands.
The ISSR analysis was performed on the twenty seven DNA samples representing the Stevia rebaudiana using nine primers composed of short random repeat sequences with or without anchor A total of 81 amplicons – amplified fragments – (ranged from 141 to 3037 Pb ). The total number of Polymorphic bands was 13 with an average of 1.44 polymorphic amplicons per primer. This represents a level of polymorphism about of 14.4%. The total number of monomorphic band was 68 with an average of 7.56 monomorphic amplicons per primer. This represents a level of monomorphism about of 75.6 %. Primer 844A and 17899A generated 8 polymorphic bands 50 and 40 %, respectively. Primer 844A exhibited high polymorphic differences and were useful in Stevia rebaudiana identification.
Molecular level of monomorphic bands.
The number of monomorphic level marker varied among the different primers. Primer HB1, 17899B, 17898B and HB10 exhibited high monomorphic bands 100%. However, primer 17898B generated 10 monomorphic bands with 14.71% monomorphism, while primers 17899A showed low level of monomorphism of 4.41 %.
Conclusion
The study concluded that we can produce seedlings from Stevia rebaudiana after in 14 weeks the fllowing establishment stage by using tissue culture technique and the genetic stability will be 96%.