الفهرس 
ACKNOWLEDGEMENTOnly 14 pages are availabe for public view
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Abstract
This study was conducted during three consecutive seasons 2018, 2019 and 2020 on some pomegranate cultivars at the farm of the Faculty of Agriculture, Assiut University, to evaluate six pomegranate cultivars: Manfaluty, Hegazy, Wonderful, Nab El-Gamal, Al-Muntakhab and Al-Wardy, as well as studying the effect of nanoscale fertilizers on Manfaluty, Hegazi, and Wonderful trees. These cultivars were evaluated as well as the effect of fertilizers on them was studied by studying some vegetative and fruiting traits, as well as SDS protein electrolysis, which provides valuable evidence for the taxonomic and evolutionary relationships of plant species. It should be noted that leaf protein profiles are often species-specific, highly stable, and unlikely to be affected by environmental conditions and seasonal fluctuations. In addition to the molecular genetics assessment of pomegranate cultivars based on ISSR and SCoT, a wide range of molecular markers were used to assess the genetic diversity of pomegranate cultivars as well as wild genotypes from different parts of the world. Inter simple sequence repeat (ISSR) analysis is an effective molecular marker.
All horticulture practices were carried out as usual. Five trees from each cultivar were chosen. The experiment was arranged in a randomized complete block design with five replications, one tree each. The following parameters were measured during the three studied seasons.
A- Vegetative growth:
Data of the vegetative characteristics included
1– Shoot length (cm). 2– Leaves number/m of shoot.
3– Leaf area (cm2). 4– Leaf chlorophyll content.
B – Yield component:
1- Fruit set percentage. 2- Number of fruit/tree.
3- Yield/tree. 4- Fruit cracking percentage.
C- Fruit physical properties:
1- Fruit weight (g). 2- Weight of 50 grains (g)
3- Arils percentage 4- Juice %
D- Fruit chemical properties:
1- Total soluble solids percentage (TSS%).
2- Total acidity.
3- Percentage of reducing sugars in juice.
4- Vitamin C content (mg ascorbic acid/100 mL juice).
5- Total anthocyanin content is peel and juice.
6- Total tannin.
E- General evaluation:
General evaluation of the tested of pomegranate cultivars was calculated on the basis of 100 units which were divided among the vegetative growth and fruiting.
F- Molecular analysis:
DNA isolation procedure
Fresh pomegranate leaf tissues were collected separately from different cultivars. The bulked DNA extraction from each cultivar was performed using DNeasy plant Mini Kit (QIAGEN).
Polymerase for ISSR and SCoT
The DNA amplifications were performed in an automated thermal cycle (model Techno 512) programmed for one cycle at 94º C for 4 min followed by 45 cycles of 1 min at 94º C, 1 min at 57º C, and 2 min at 72º C. the reaction was finally stored at 72º C for 10 min.
Molecular data analysis
The similarity matrices were done using Gel works ID advanced software UVP-England Program. The relationships among genotypes as revealed by dendrograms were done using SPSS windows (Version 10) program. DICE computer package was used to calculate the pairwise difference matrix and plot the phenogram among cultivars
The obtained results during the three studied seasons could be summarized under follow main items:
A - Vegetative characteristics:
1- Tree growth:
Data declared that great variability in vegetative growth (shoot length and leaves number/shoot) of different studied pomegranate cultivars during three studied seasons.
Data indicated that Wonderful and Hegazy cultivar gave the highest values of shoot length followed Manfalouty and Nab El-Gamal and smallest values of shoot length were found on Montkhab and Wardy cultivar. The intermediate values of leaf number/shoot were found on Wonderful and Montkhab cultivars. Then, these data indicated that Wonderful cultivar was biggest trees, whereas the Montkhab tree was the smallest ones in comparison to other studied pomegranate cultivars.
2- Leaf traits:
Hegazy cultivar had the highest leaf area followed by Wonderful in the three studied seasons, respectively. Whereas, Montkhab cultivar had the highest, leaf chlorophyll content during the three studied seasons, respectively. On other hand, the lowest values of leaf area, chlorophyll content were4 Montkhab and Hegazy, respectively.
The intermediate values of leaf area and total chlorophyll content were recorded on Nab El-Gamal trees. from the aforementioned data that there is a positive relationship between leaf area and total leaf area. On other hand, there is a negative relationship between the leaf area and chlorophyll content.
B- Yield components:
The data indicated that significantly varied in fruit set and number of fruit/tree as well as yield/tree and fruit cracking percentage.
The highest fruit set percentage, number fruit, fruit/tree and heaviest yield were recorded on Manfalouty, Wardy and Wonderful pomegranate cultivars, respectively. On other hand, the lowest values of these traits, fruit set, fruit/tree and yield/tree were recorded on Wonderful, Hegazy and Wardy pomegranate cultivars, respectively.
So, it could be arranged these cultivars for their yield in descending order as follow, Wonderful, Hegazy, Manfalouty, Nab El-Gamal, Montakhab and Wardy pomegranate cultivars, respectively.
Fruit cracking percentage ranged from 8.41 to 20.45% as an av. of the three studied seasons, Nab El-Gamal cultivar, gave the highest values of fruit cracking percentage followed in descending order by Wonderful, Hegazy, Manfalouty, Wardy and Montakhab.
from the economic view, the fruit cracking is one of the most serious problems facing the pomegranate production. Therefore, the varieties that are characterized by the lack of this phenomenon are considered required items. Thus, the Montakhab, Wardy and Manfalouty cultivars are the best in this respect.
C- Fruit physical properties:
Data indicated that there is a significantly differences in physical fruit properties.
The recorded heaviest weight for Wonderful and Hegazy, whereas, the lightest one was Wardy cultivars. Then, it can be arranged the fruit weight in descending order as follow, Wonderful, Hegazy, Manfalouty, Nab El-Gamal, Montakhab and Wardy, respectively.
Wardy had the highest juice volume followed Wonderful and Manfalouty fruits. On other hand, the Nab El-Gamal was heaviest of 50 grain followed Hegazy and Manfalouty fruits. There were insignificant differences among Manfalouty, Wonderful and Nab El-Gamal fruits of their juice content. Wardy fruits gave the highest peel anthocyanin content followed Montakhab and Manfalouty whereas, Nab El-Gamal fruits recorded the least peel anthocyanin.
Also, data declared that Hegazy fruit had the highest peel tannins followed by Wonderful fruits, whereas, Manfalouty had the lowest values. Hence, there are a decrement percent of tannin content for Manfalouty, Wonderful, Nab El-Gamal, Montakhab and Wardy compared to Hegazy fruits, respectively.
D- Fruit chemical properties:
Manfalouty and Montkhab fruits had the highest values of total soluble solids, reducing sugar and V.C. content compared to other studied cultivars during the seasons, respectively. On other hand, Wardy, Montakhab and Manfalouty had the highest the anthocyanin content. Nab El-Gamal fruits was the lowest values of most studied fruit chemical characteristics compared to other studied cultivars. The intermediate values of all studied chemical constituents were recorded of other studied pomegranate cultivars. No significantly difference in reducing sugars of Hegazy, Wonderful and Nab El-Gamal and anthocyanin contents of Manfalouty and Montakhab fruits. Such results indicated that Manfalouty, Wonderful and Montkhab pomegranate cultivars had the best chemical constituents compared to other studied.
E- General evaluation of the studied pomegranate cultivars:
The numerical evaluation of pomegranate cultivars grown under Assiut condition, declared that Manfalouty and Hegazy cultivars recorded the highest units according to the general evaluation, as it attained the uppermost score units followed by Wonderful cultivar which occupied the third ranked. On other hand, Nab El-Gamal cultivar recorded the least score compared to other studied pomegranate cultivar. Moreover, data showed that no significant difference obtained between Manfalouty, Hegazy and Wonderful cultivar where that recorded un almost similar score.
F- Molecular analysis
1- ISSR molecular analysis
Total number of 31 bands with molecular weight from 130 to 10000 bp, 14 of total amplified bands were polymorphic (45.16%) and the highest polymorphic percentage was80 % produced with primer 44B and the lowest polymorphic percentage was 16.66 % produced with primer HB-15. On the other hand, total of 17 monomorphic bands were appeared and five specific marker bands in all five primers. Finally, Primer HB-14 was the highest amplified bands (9bands) whereas, primers (14A and 44B) were the lowest amplified bands (5 bands).
The similarity between the six pomegranate cultivars ranged from 0.800 to 0.947 and the highest similarity was between Manfalouty and Wonderful cultivars at 0.947 and the lowest similarity was between Montakhab and Nab El-Gamal cultivars. UPGMA cluster and analysis based on genetic similarity values for ISSR markers from all cultivars from pomegranate were used to construct classify six pomegranate cultivars. Results from this analysis showed two main groups. The first main group was Nab El-Gamal cultivar alone and the second main group was divided into two sub main groups: the first sub main group included Wardy cultivar only and the second sub main group was divided into two sub sub group: the first sub sub group was included Hegazy cultivar and the second sub sub group was included other cultivars Montakhab, Manfalouty and Wonderful.
2- SCOT molecular analysis
Total number of 30 bands were visualized across the six investigated cultivars of pomegranate, the results obtained 30 total bands with molecular weight from 145 to 1640 bp, 9 of total amplified bands were polymorphic (30%) and the highest polymorphic percentage was 42.85% produced with primer SCoT 3 and the lowest polymorphic percentage was 25% produced with primers (SCoT 2 and SCoT 6) . While Primer SCoT 1 was appeared no polmorism. On the other hand, total of 21 monomorphic bands were appeared and three specific marker bands in the six primers. Finally, primer SCoT 3 was the highest amplified bands (7 bands) whereas, each of primer ScoT 2 and SCoT 6 were lowest amplified bands (4bands).
The similarity between the six pomegranate cultivars ranged from 0.844 to 0.979 and the highest similarity was between Montakhab and Hegazy cultivars at 0.979 and the lowest similarity was between Wardy and Manfalouty cultivars. UPGMA cluster and analysis based on genetic similarity values for SCoT markers from all cultivars from pomegranate were used to construct classify six pomegranate cultivars. Results from this analysis showed two main groups. The first main group was Wardy cultivar alone and the second main group was divided into two sub main groups: the first sub main group included Wonderful cultivar only and the second sub main group was divided into two sub sub group: the first sub sub group was included Montakhab and hegazy cultivars and the second sub sub group was included other cultivars Manfalouty and Wonderful.
3- Combining ISSR and SCoT analysis
Six cultivars of pomegranate across five primers of ISSR and six primers of SCoT, the results obtained 61 total bands with molecular weight from 130 to 10000 bp, 23 of total amplified bands were polymorphic (37.70%) and the highest polymorphic percentage was 45.16% produced with ISSR primers and the lowest polymorphic percentage was 30%% produced with SCoT primers. On the other hand, total of 38 monomorphic bands were appeared and eight specific marker bands in eleven primers.
Combining ISSR and SCoT, the similarity between the six cultivars of pomegranate ranged from 0.853 to 0.934 and the highest similarity was between Hegazy and Manfalouty cultivars at 0.934 and the lowest similarity was between Wardy and Hegazy cultivars at 0.853. UPGMA cluster and analysis based on genetic similarity values for combining ISSR and SCoT markers from all the pomegranate cultivars was used to construct classify six pomegranate cultivars. Results from this analysis showed two main groups. The first main group was only Wardy cultivar and the second main group was divided into two sub main groups: the first sub main group included Nab El-Gamal cultivar alone and the second sub main group was divided into two sub sub group: the first sub sub group was concluded Wonderful cultivar alone and the second sub sub group was included other cultivars Montakhab, Hegazy and Manfalouty.
In general, the pomegranate cultivars under the present study were widely differed in their growth, yield and physiochemical properties. Variations among these cultivars could be mainly due to their genetically and adaptability differences. Hence these results are important for economic and horticultural point of view. The best one must be selected for vegetative propagation and planting.
Second experiment
The effect of nano-nutrients fertilization on some pomegranate cultivars was concerned. The effect included vegetative growth yield and fruit quality.
1- Effect of different nano-nutrients fertilizers on vegetative growth:
1.1- Shoot length:
Foliar spraying nano-fertilizer of boron or calcium singly or in combination significantly increased the shoot length and leaves number/shoot compared to water foliar spraying (check treatment). The highest shoot length and leaves number/shoot were recorded for foliar spraying with 0.1% nano-boron plus 1% nano-calcium compared other studied treatments during the three studied seasons. No significant differences were observed on shoot length and leaves number/shoot due to spray nano-boron or nano-calcium whatever singly or in combination.
The highest length shoot and leaves number/shoot were recorded on Wonderful pomegranates cultivars compared to other two studied cultivars during three studied seasons, respectively. No significant differences were observed on shoot length of Manfalouty and Hegazy pomegranate cultivars during three studied seasons.
All combinations of nano-boron plus nano-calcium spraying gave the highest values of shoot length and leaves number/shoot. In other hand, all combination of water spraying gave the least values of length shoot and leaves number/shoot, compared to other studied combinations during the three studied seasons. At the same time, all combination of Wonderful pomegranate cultivar significantly increased the shoot length and leaves number/shoot compared to other combinations of Manfalouty or Hegazy pomegranate cultivars during the three studied seasons. The maximum values of shoot length were found in Wonderful trees sprayed 0.1 nano-boron plus 1% nano-calcium during the three studied seasons.
Thus it can be concluded that spraying nano-boron plus nano-calcium were effective in improving the growth of Manfalouty, Hegazy and Wonderful pomegranate trees. Moreover, such treatment more effective in Wonderful pomegranate cultivar. In other words, Wonderful cultivar is more desirable for these nano-nutrients foliar spraying, followed by Manfalouty and then Hegazy trees.
1.2- Leaf area:
Spraying nano-boron or nano-calcium singly or in combination increased significantly the leaf area compared to water spraying (check treatment). The highest area of leaf was recorded on the tree that spraying with 0.1% nano-boron plus 1.0% nano-calcium compared to other treatments, respectively. No significant differences on leaf area due to spray nano-boron singly or compared with nano-calcium.
The highest leaf area was recorded for Hegazy pomegranate cultivar compared to other studied cultivars during the three studied seasons.
All combinations of nano-boron plus nano-calcium or 0.1% nano-boron gave the highest values of leaf area. In other hand, all combination of water spraying recorded the lowest values compared to other studied combinations during the three studied seasons. The maximum values of leaf area were found in Hegazy trees sprayed with combined nano-boron and nano-calcium during the three studied seasons.
Moreover, all combinations of Hegazy significantly increased the leaf area compared to combine with Manfalouty or Wonderful pomegranate cultivars during the three studied seasons.
As, a general view, it could be seen that the increment of the leaf surface expansion of trees of Wonderful cultivar due to the combination between either nano-nutrients spraying and studied cultivar was better than increment for Manfalouty or Hegazy pomegranate cultivars during the three studied seasons.
Thus, it could be concluded that spraying nano-boron singly or combined with nano-calcium were more effective in improving the leaf surface expansion of Manfalouty, Hegazy and Wonderful pomegranate trees.
2- Effect of different nano-nutrients fertilizers on yield/tree and fruit cracking percentage:
Spray the trees with nano-boron or nano-calcium singly or in combination significantly increased the yield/tree and significantly decreased the fruit cracking percentage compared with water spray (check treatment).
The highest values of yield/tree was recorded on trees that sprayed 0.1% nano-boron plus 1% nano-calcium compared to other treatments and check treatment. Whereas, the lowest values of fruit splitting percentages were found in the trees sprayed with mixture of nano-boron and nano-calcium compared to other spraying treatments.
No significant differences were found on yield/tree and fruit cracking percentage due to spray nano-boron, or nano-calcium whatever, singly or in combination application during the three studied seasons.
from these results, it can be arranged the yield/tree is descending order as follow Wonderful, Hegazy and Manfalouty pomegranate cultivars during the three studied seasons. On other hand, fruit cracking percentage in ascending order were Manfalouty, Hegazy and Wonderful pomegranate cultivar during the three studied seasons, respectively.
The results also declared that the nano-nutrients application are more effect on Wonderful, then Hegazy and Manfalouty pomegranate cultivars during the three studied seasons, respectively.
In addition, the interaction between the two studied factors on yield/ tree and fruit cracking percentage, declared that nano-nutrients spraying induce a significant increase in yield/tree and significantly decreased the the fruit splitting percentage. All combinations of 0.1% nano-boron plus 1% nano-calcium spraying gave the heaviest yield/tree and the least values of fruit splitting, hence, it gave the highest values of commercial fruit percentage compared to other studied combinations during the three studied seasons. Whereas, all combinations of water spraying gave the least values of yield/tree and the maximum values of fruit and splitting percentage of fruits during the three studied seasons.
The maximum values of yield kg/tree were recorded on Wonderful tree that sprayed with combined nano-boron plus nano-calcium. On other hand, the minimum values of fruit splitting percentage was recorded on Hegazy trees that spraying with mixture of nano-boron and nano-calcium during the three studied seasons.
As a general view, the results cleared that using combined of sprayed Wonderful and Hegazy trees with nano-nutrients significantly increased the yield and significantly decreased the fruit splitting percentage than increasing or decreasing effects occurred of Manfalouty pomegranate trees.
3- Effect of different nano-nutrients application on fruit properties:
3.1- Physical fruit properties:
Foliar spraying fertilize the Manfalouty, Hegazy and Wonderful pomegranate trees with 0.1 nano-boron, 0.2 nano-boron, 1% nano-calcium, 2% nano-calcium and 0.1% nano-boron plus 1% nano-calcium significantly increased the fruit weight and arils % as well as peel anthocyanin grain and extracted juice percentage relative to whole fruit weight compared with foliar spraying with water (check treatment). The highest fruit weight and arils percentage as well as juice and peel anthocyanin were found on the trees that sprayed nano-boron plus nano-calcium compared to other treatments and control. No significant differences were found due to spray nano-boron, nano-calcium in singly or in combination. Whereas, the lowest values of such fruit traits was found in the trees that sprayed with water (check treatment).
Studied cultivars significantly varied for fruit weight and arils percentage as well as peel anthocyanin contents.
The highest values of weight, arils percentage and peel anthocyanin contents were detected on the Wonderful, Manfalouty and Manfalouty pomegranate fruits compared other studied cultivars, respectively.
On other hand, the lowest values of fruit weight, arils percentage and peel anthocyanin contents were recorded on Manfalouty, Hegazy and Wonderful pomegranate fruits during the three studied seasons, respectively.
All nano-nutrients spraying induced a significant increase in these fruit traits. All combinations of nano-boron plus nano-calcium spraying gave the highest values of fruit weight, arils %, juice and peel anthocyanin contents compared to other studied combinations during the three studied seasons.
The maximum fruit weight, arils percentage and peel anthocyanin contents were recorded on fruit of Wonderful, Manfalouty and Hegazy trees that sprayed by 0.1% nano-boron plus 1% nano-calcium. On other hand, the least values were recorded in these fruit traits found of fruits on the trees that water spraying (check treatments) during the three studied seasons.
As, an overview, the results showed that combination nano-boron plus nano-calcium significantly improved the physical fruit properties than increment due to nano-boron or nano-calcium singly. So, it can be concluded that it is necessary of used a combined of nano-boron plus nano-calcium spraying together.
3.2- Chemical constituents:
Spraying the Manfalouty, Hegazy and Wonderful pomegranate trees with 0.1 nano-boron, 0.2% nano-boron, 1% nano-calcium, 2% nano-calcium and 0.1% nano-boron plus 1% nano-calcium was preferable in improving the chemical fruit quality than spraying with water check treatment. Thus improvement of chemical quality parameters of fruits was occurred in terms of increasing total solids, reducing sugars, juice anthocyanin and V.C. contents and decreasing the total acidity during the three studied seasons.
The highest values of total soluble solids, reducing sugars, vitamin C contents and juice anthocyanin and least values of total titratable acid were observed due to spray the mixed nano-boron and nano-calcium compared to other fertilization treatments. Whereas, the least values of these traits and highest total acidity were detected in fruits of trees sprayed with water.
These chemical qualities show a significant difference between the studied cultivars. The highest values of total soluble solids, reducing sugar and juice anthocyanin were recorded on the Manfalouty pomegranates. All combination of nano-nutrients spraying significantly improved these chemical properties compared to combine with water spraying. Moreover, the improvement occur due to spray nano-nutrients was more effective on Hegazy pomegranate fruits than on Manfalouty or Wonderful pomegranates.
Conclusion
In general, the pomegranate cultivars under the present study were widely different in their vegetative growth, yield and physiological properties. Variation among the studied cultivars could be mainly due to their genetically and adaptability differences. These results revealed that Manfalouty, Hegazy and Wonderful are considered promising cultivars to obtain a high yield with good fruit quality compared to other studied cultivars. selection assisted by these molecular markers may also be helpful to produce new cultivars with improved productivity, abiotic tolerance and pest resistance. Furthermore, marker-assisted selection is important in the assessment of genotypes used in local pomegranate cultivars improvement programs in Egypt. Hence these results are important for economic and horticultural point of view. The best one must be selected for vegetative propagation and planting.
Also, it could be concluded that foliar spraying the pomegranate trees with nano-boron or nano-calcium thrice at middle of May, June and August significantly improved the vegetative growth and fruiting. The foliar application nano-boron at 10 ppm plus nano-calcium at 0.1% was found most suitable for increase vegetative growth and get the maximum yield with good fruit quality and net return.