الفهرس 
Breaking dormancy of some plantsOnly 14 pages are availabe for public view
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Abstract
This work was carried out from 2014 to 2018 in the Plant Biotechnology Department, National Research Center, in cooperation with Horticulture Department, Faculty of Agriculture, Ain Shams University. Jerusalem artichoke Balady, Fuza and Alba cultivars were used and this study was performed throughout five experiments as follows:
First: Experiments
1. Breaking dormancy in Jerusalem artichoke tubers for the three cultivars Balady, Fuza and Alba by gibberellic acid.
2. Establishment of a simple protocol for in vitro propagation of J. artichoke plant for the three cultivars Balady, Fuza and Alba.
3. Establishment of an applicable protocol for callus induction and production from different explants of J. artichoke plant for the three cultivars Balady, Fuza and Alba.
4. Establishment of an efficient protocol for indirect plantlets regeneration via callus cultures of J. artichoke for the three cultivars Balady, Fuza and Alba
5. Establishment of hairy roots culture of Balady cultivar by Agrobacterium rhizogenes.
1. The first experiment: Breaking dormancy in Jerusalem artichoke tubers
In the middle of February, after one month from harvest, The tubers of the three cultivars Balady, Fuza and Alba have been soaked separately after well washing for 12 hours in gibberellic acid solutions at concentrations (0, 400 and 800 mg L-1) by dissolving GA3 tablet (perlix) contains 90% gibberellic acid in tap water. The treated tubers were kept at room temperature and watered regularly.
After two weeks, number of sprouts/tuber and sprout length were estimated.
The obtained results can be summarized as follow:
1- The tubers of the three cultivars were successfully released of dormancy at the beginning of March; two months before the normal sprouting.
2- The best treatment was 400 mg L-1 gibberellic acid which achieves not only the maximum number of sprouts/tuber but also the highest value of sprout length for all cultivars.
3- The tubers of the three cultivars failed to sprout using the first treatment (zero mg L-1 gibberellic acid, tap water).
4- Alba cultivar was the superior for number of sprouts/tuber followed by Fuza then Balady while, Balady recorded the longest sprout followed by Alba then Fuza.
2. The second experiment: in vitro propagation of Jerusalem artichoke
This experiment included four stages as follow:
2.1. Establishment of Jerusalem artichoke aseptic cultures
This stage aimed to initiate aseptic cultures resulting from sprouts and/or stem node explants of the three cultivars in order to undergo the three following stages of in vitro propagation.
In this stage, stem nodes and sprouts of Balady, Fuza and Alba cultivars of Jerusalem artichoke were surface sterilized then cultured on half strength MS medium supplied with different concentrations of kanamycin or cefotaxime.
After two weeks of culture, contamination (%) and survival (%) were estimated.
The obtained results can be summarized as follow:
1- The lowest contamination that correlated with the highest survival percent were recorded with ½ MS + 62.5 or 125 mg L-1 cefotaxime for stem nodes and sprouts, respectively.
2- Due to the very limited number of sterilized cultures of sprouts compared to that of stem nodes. The following stages of in vitro propagation starting from multiplication stage were performed by using stem nodes cultures.
2.2. Shoot Multiplication
This stage aimed to know the best medium which gave the highest shootlets multiplication from stem node explants.
In this stage, stem nodes from in vitro plantlets were cultured on MS + BA and NAA with/without nano-selenium.
Number of shootlets, shootlets length (cm) and number of leaves were measured after five weeks from culture.
The obtained results can be summarized as follow:
1- Maximum number of shootlets per explant and optimum number of leaves per shootlet as well were observed on MS + 1 mg L-1 BA + 0.1 mg L-1 NAA + 50 mg L-1 nano-selenium for all cultivars.
2- The longest shootlets were noticed on MS basal medium which produced the minimum number of shootlets for all cultivars.
3- Nano-Se supplemented medium enhanced the multiplication rate of shootlets compared to that free of selenium.
2.3. Rooting in vitro
This stage aimed to know the best medium which gave the highest rooting percentage, maximum number of roots and roots length.
In this stage, shootlets produced from previous stage were separated and transferred onto MS medium fortified with different combinations of IBA and NAA for roots formation.
After five weeks of culture on rooting media, rooting (%), number of roots and roots length (cm) were estimated.
The obtained results can be summarized as follow:
1- The maximum rooting formation (100 %) was recorded on ½ MS + 0.1 mg L-1 NAA + 2 mg L-1 IBA + 0.5 mg L-1 kin for all cultivars.
2- While, the minimum rate of rooting (40 %) was obtained with ½ MS basal medium (free growth regulators).
3- Moreover, the longest root lengths and number of roots as well were noticed on ½ MS + 0.1 mg L-1 NAA + 2 mg L-1 IBA + 0.5 mg L-1 kin for Alba, Fuza and Balady, respectively.
4- However, the minimum root lengths and the lowest number of roots were obtained on ½ MS basal medium (free growth regulators) for Balady, Fuza and Alba, respectively.
5- It is concluded that ½ MS + 0.1 mg L-1 NAA + 2 mg L-1 IBA + 0.5 mg L-1 kin achieved the highest values for rooting percentage, roots length and roots number, respectively for all cultivars and the Alba was the best.
4.2. Acclimatization
This stage aimed to know the best acclimatization medium which gave the highest survival percentage for in vitro derived plantlets.
In this stage, the in vitro derived plantlets resulting from rooting were subjected to acclimatization process in which three different composition of nutrient media were used as follow:
1. Peat moss.
2. Peat moss: perlite (1:1)
3. Peat moss: perlite (2:1)
Survival (%) and plant length (cm) after one month of acclimatization were measured.
The obtained results can be summarized as follow:
1- The in vitro derived plantlets were successfully acclimatized on the mixture of peat moss and perlite (1:1) for all cultivars with highly survival percentage reach to 100 % for Alba.
2- The maximum plant height was recorded with the same mixture for all cultivars and the Alba was the longest (10.6 cm).
3. The third experiment: Callus induction and growth development
This experiment aimed to study the effect of MS medium fortified with different combinations of BA and NAA on callus induction and production from leaf, stem and root explants of in vitro derived plantlets of Jerusalem artichoke Balady, Fuza and Alba cultivars.
After four weeks, frequency of callus formation (%) and callus fresh weights (g/jar) were recorded.
The obtained results can be summarized as follow:
1- Callus cultures were successfully initiated on all tested media except MS free growth regulators medium for all explants and cultivars.
2- The stem explant was the superior explant followed by root and leaf explants for callus induction for all cultivars.
3- The best medium that achieved maximum values of callus fresh weight for all cultivars was MS supplemented with 1 mg L-1 BA + 2 mg L-1 NAA.
4- The best results for callus fresh weights were noticed when stem explant of Balady cultured on MS + 1 mg L-1 BA + 2 mg L-1 NAA followed by stem explant of Fuza and Alba on the same medium, respectively.
5- It could be concluded that MS medium augmented with 1 mg L-1 BA + 2 mg L-1 NAA was the best medium for callus induction and production as well.
6- The preference for callus induction and growth recorded for Balady as the best cultivar and stem as the superior explant.
7- About callus growth curve, there was a gradual increase in callus fresh weight week by week till it reached the maximum value at the eighth week for all explant of Balady cultivar also for all explants of Fuza and Alba.
8- The optimum value of callus fresh weight (5.18 g) was recorded for callus-derived stem at the 8th week from culturing on MS + 1 mg L-1 BA + 2 mg L-1 NAA While, it recorded (3.98 and 2.30 g for callus-derived root and leaf, respectively).
4. The fourth experiment: Plantlets regeneration via callus cultures
This experiment aimed to study the effect of MS medium supplemented with different concentrations of picloram (0, 0.5, 1.5, 3, 6 and 12 mg L-1) + 0.1 mg L-1 NAA on plantlets regeneration via stem-derived callus of Jerusalem artichoke Balady, Fuza and Alba cultivars.
After four weeks, regeneration percentage (%) and number of regenerated shootlets were recorded.
The obtained results can be summarized as follow:
1- The callus cultures were hardly regenerated into a few numbers of shootlets on picloram supplemented media.
2- The highest regeneration percent (73, 69 and 67%) and the maximum number of regenerated shootlets (2.2, 2.0 and 2.1) for Balady, Fuza and Alba, respectively were recorded with MS + 6.0 mg L-1 picloram + 0.1 mg L-1 NAA
3- Callus failed to regenerate into shootlets on free picloram medium (MS + 0 mg L-1 picloram + 0.1 mg L -1 NAA) for all cultivars.
4- By increasing the concentration of picloram in the tested media up to 6 mg L-1, either regeneration percent or number of regenerated shootlets was increased after that concentration they began to decrease.
5- It could be summarized that picloram is more efficient and superior for plantlets regeneration compared to NAA.
5. The fifth experiment: Establishment of hairy roots culture by Agrobacterium rhizogenes
1- A hairy root culture for Balady cultivar by Agrobacterium rhizogenes was established.
2- Hairy root culture could be used to enhance the accumulation of inulin in these cultures using bioreactors.
3- It very important to increase the awareness and interest for large scale production of inulin in the near future for medicinal and industrial applications.
Second: Chemical analyses
1- Inulin content in tubers and callus cultures
- The inulin content in tubers of the three cultivars Balady, Fuza and Alba represents about 4-5 fold their callus cultures of inulin content.
- Further studies should be done to enhance the accumulation of inulin in callus cultures of these cultivars of Jerusalem artichoke under investigation using either precursors or elicitors.
- In addition, hairy root cultures for optimizing inulin production could be initiated from callus cultures or in vitro plantlets of Jerusalem artichoke
2- Molecular characterization of DNA using RAPD, ISSR and SCoT based markers
- The total polymorphism percent after applying the RAPD technique was 61.53 % between the in vivo shoots and in vitro cultures (regenerates and callus) of the three cultivars.
- While, the total polymorphism after applying the RAPD method among the in vivo cultivars under study recorded 38.09%.
- The dendrogram of RAPD analysis showed highly similarity between Balady and Fuza (100%), while low similarity was recorded between Alba and the other cultivars (0.35 %).
- It could be concluded that primers OP-B9 and OP-K3 can be used to distinguish among in vitro derived cultures (regenerates and callus) from the three cultivars of Jerusalem artichoke. However, OP-K1 can be used for in vivo cultivars of Jerusalem artichoke.
- Further, the total polymorphic percentage after applying the ISSR technique was 67.8% between the in vivo shoots and in vitro derived cultures (regenerates and callus) of the three cultivars of Jerusalem artichoke.
- The total polymorphic percentage 42.3% after applying of ISSR technique among the in vivo cultivars under study.
- Dendrogram of ISSR analysis showed highly similarity between Balady and Fuza (100%), while low similarity was recorded between Alba and the other cultivars (0.33%).
- Dendrogram of combination RAPD and ISSR analysis showed highly similarity between Balady and Fuza (100 %), while similarity between Alba and the other cultivars was 0.71 (%).
- It could be concluded that the primer HP-12 could be used for distinguishing among in vitro derived cultures of Jerusalem artichoke. However, 44 –B primer can be used for distinguishing between in vivo cultivars of Jerusalem artichoke.
- The total polymorphism after applying the SCoT technique was 50 % between the in vivo shoots and in vitro cultures (regenerates and callus) of the three cultivars.
- While, the total polymorphism after applying the SCoT method among the in vivo cultivars under study recorded 34.61%.
- SCoT achieved highly similarity between Balady and Fuza following by Alba.
Conclusion
1- Establishment simplified protocol for in vitro propagation of the three cultivars of J. artichoke in order to intensive multiplication and expansion its cultivation in Egypt for different purposes and the production of inulin.
2- Propagate the Hungarian cultivar (Alba) in vitro to introduce it into the Egyptian agriculture as a new cultivar.
3- Achivement of friable callus cultures from stem explants of the three cultivars of J. artichoke aiming to maximize and scale up of inulin production.
4- Enhancing of indirect plantlets regeneration via callus culture for the three cultivars of J. artichoke which could be applied for genetic improvement
5- Obtaining of hairy roots cultures using Agrobacterium rhizogenes (A4) which could be useful for inulin production at the level of industry.