الفهرس 
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Abstract
The current study aimed to characterize ten plum cultivars grown in Egypt based on their physiological traits and organelle genetic markers. Also, this research aimed to enhance Mariana rootstock’ capacity to better adjustment to salty soils through foliar application of ZnSO4, CaSO4 and Validamycin A and investigate their effectiveness on some biochemical aspects and gene expression in the mitochondria and chloroplasts under salinity stress.
Samples of leaves and fruits of ten plum cultivars (Prunus salicina L.) were collected from El-Kanater El-Khairia, Agriculture Research Center, Egypt and from private farms located in El-Menofia governorate and Alexandria Desert Road, Egypt.
The amounts of carotenoid and chlorophyll a and b were measured in fresh leaves. Total sugar, total soluble solids, titratable acidity (TA), pH value and ascorbic acid were evaluated to test the fruit quality. Total genomic DNA and mitochondrial DNA were isolated and amplified using five chloroplast primers (C-D, E-F, trnH-psbA, matK and rbcl) and two mitochondrial primers (nad4 and nad9).
An experiment was carried out in the Department of Botany, Faculty of Women, Ain Shams University during (2019–2021) to investigate the response of ‘Mariana 2624’ rootstock; the only plum rootstock in Egypt; to salt stress and foliar treatment with ZnSO4, CaSO4 and Validamycin A at different growing periods (leaves falling, buds growing, leaves growing).
Photosynthetic pigments, free amino acids, proline, total soluble protein, total phenolics, antioxidant, total soluble sugars, trehalose contents, Malondialdehyde and antioxidant enzyme activity were determined. In addition, Reverse transcriptase polymerase chain reaction (RT-PCR) using gene specific primers to analyze the expression patterns of chloroplast (rbcL and matK) genes and mitochondrial (nad4 and nad9) genes.
- The results showed that the photosynthetic pigments were significantly different among ten plum cultivar leaves, where the higher content was found in Africanrose but the lowest was found in Celebration, Pioneer and Kelsey respectively.
- The sugar content of ten plum cultivars varied significantly, ranging from 13.67 % in Kelsey cultivar to 33.56 % in Golden Japan cultivar, respectively.
- The Celebration cultivar showed the greatest total soluble solids (TSS), while the lowest was in Climax cultivar.
- In addition, the total acid content (TA) varied significantly between plum cultivars that ranging from 0.83 % in Celebration to 1.94 % in Climax. With the low TA level, all cultivars were found to have slightly high pH values. Regardless of the cultivar, there was no significant difference between all cultivars which ranged from 3.42 to 3.95.
- Ascorbic acid content ranged from (10.63 mg/100 g FW) in Golden Japan to (12.50 mg/100 g FW) in Africanrose and Climax with no significant difference between plum cultivars.
- The (C-D) primer is a non-coding trnL (UAA) intron, whereas the (E-F) primer is a non-coding trnL(UAA) -trnF(GAA) intergenic region. The PCR amplification of (C-D) was successfully obtained for (Santa Rosa, Songold, Methley, Kelsey and Climax) cultivars with 607 bp length.
- The PCR amplification of the (E-F) primer was successfully obtained for (Golden Japan, Methley, Kelsey, Climax and Celebration) cultivars with a length ranging from 407 to 429 bp.
- In addition, the trnH- psbA intergenic region was successfully amplified by PCR for Africanrose, Hollywood, Kelsey, Climax and Celebration cultivars with a length ranging from 370 to 483 bp.
- Amplification of the matK coding region was successful in all plum cultivars except Pioneer, Methley, Kelsey and Climax at a length of 835 bp, whereas amplification of the rbcL coding region was successful in all cultivars with a length range of 551-662 bp, except Hollywood, Pioneer, Songold and Methley cultivars.
- About mitochondrial DNA, Amplification of the nad4 coding region was successful in all plum cultivars at length of 117 bp, whereas amplification of nad9 coding region was successful in Africanrose, Hollywood, Pioneer, Golden Jaban and Celebration plum cultivars at length of 140 bp.
- In the current study, we compared the genetic variations in the mtDNA, nad4 and nad9 genes as, the nad9 gene has a higher genetic variation than the nad4 gene, indicating that it is more variable and valuable for researching genetic diversity in the Prunus salicina L. species.
- Among the loci, the PCR success rate varied greatly; the locus nad4 in the mitochondria had the greatest success rate, at 100% followed by each chloroplast loci rbcL and matK at (60%) then the success amplified rate at (50%) with other loci (trn L, trn L-trnF, trnH- psbA and nad9).
- The PCR success rate for the cultivars against cpDNA and mtDNA primers ranged between 85.71% in the Celebration cultivar and 28.57% in Pioneer cultivar.
- The genetic distances between the cultivars ranged from 0.00 to 1.00; the Kelsey and Climax cultivars had the lowest distance, indicating that they have the most genetic similarities. A greater distance was observed between the Africanrose and the Methley cultivars, reflecting their significant differences.
- The chlorophyll content of Marianna rootstock decreased under salinity stress conditions. Zinc sulphate foliar treatment recorded the highest chlorophyll content, followed by Validamycin A, calcium sulphate, respectively without salinity stress. Validamycin A treatment gave the highest values of chlorophyll content at the two salinity levels.
- Total free amino acids increased as salinity levels increased in all treatments. The highest value was recorded in plants under salt stress (100 mM NaCl) followed by plants treated with calcium sulphate and Validamycin A under high levels of salt stress, respectively.
- Proline content increased with increasing salinity stress in all treatments recording the highest values in plants sprayed with validamycin A under salinity stress conditions but recording the lowest value in control plants irrigated by tap water.
- Total protein was slightly decreased with increasing salt stress level with and without foliar treatment. Control plants have higher amounts of total protein than plants treated with foliar zinc sulphate, calcium sulphate and Validamycin, respectively.
- Total phenolics increased with increasing salt stress levels. Plants treated with foliar application of zinc sulphate and Validamycin A recorded the highest values under high level of salt stress, respectively. While the lowest values were recorded in plants with foliar treatment of zinc sulphate and Validamycin A without salt stress, respectively.
- The total soluble sugars increased with increasing salinity stress levels in all treatments except zinc sulphate foliar treatment. Control plants recorded the highest value, while foliar application of validamycin A and calcium sulphate without salt stress recorded the lowest value, respectively.
- Trehalose content increased as salinity levels increased. Also, all foliar treatments increased trehalose compared to control plants.
- Malondialdehyde (MDA) recorded the greatest values under salinity levels. Application of all foliar treatments decreased the level of MDA as compared to control.
- The antioxidant enzymes activities (GR, PPO, APX and CAT) increased as salinity levels increased in all foliar treatments. The foliar applications of zinc sulphate and validamycin A were effective in reducing the negative impact of salinity stress followed by calcium sulphate foliar application.
- Gene expression patterns showed differences between all treatments. The expression pattern of the rbcL gene was strongly induced under stress conditions in both control and ZnSO4/ CaSO4 treated plants but reduced in control and Validamycin A treated plants without salt stress.
- The matK patterns showed an increase in expression in plants treated with validamycin A under salt stress conditions, while showing a reduction in plants treated with both ZnSO4 and CaSO4.
- Mitochondrial gene expression patterns for nad4 revealed differing patterns; no patterns were obtained in CaSO4 treated plants under normal conditions, whereas an increase in CaSO4 and Validamycin A treated plants under salt stress conditions was observed. Furthermore, under normal and stressful conditions, the nad9 transcripts showed a similar pattern of expression in ZnSO4 treated and untreated plants.
- The amplified chloroplast genes of rbcL and matK were sequenced to confirm gene identities, but only the rbcL gene was confirmed as rbcL, producing a 312 bp. long sequence with an average GC content of 42.72%. While low amplification of the matK gene prevented adequate sequencing. rbcL showed 98.72% identity with Prunus salicina, Prunus domestica, Prunus dulcis, Prunus cerasifera, Prunus triloba and Prunus mira, whereas matK didn’t match any sequence alignments in the gene bank.
- The sequences from the rbcL were used to estimate trees with another Prunus species in the genebank database using the maximum likelihood (ML) algorithm. The tree was constructed using the sequences obtained from rbcL loci grouped Mariana rootstock in one clade with Prunus dulcis and Prunus salicinia. Meanwhile, it separate another species in the genbank from other clades.
According to the phylogenetic tree and cytoplasmic genetic studies of plum cultivars, scientists should conduct more studies to use the more combatable cultivar as a new rootstock.
The study recommended applying foliar treatments with ZnSO4, Ca SO4 and Validamycin A to plum plants grown in the new reclaimed area to study the effectiveness of these materials in alleviating salinity stress in the field and their impact on the growth and productivity.
This is a research gap that must be filled in future research programs.