Comparative physiological and biochemical mechanisms in Di, Tri, and 1 Tetraploid Watermelon ( Citrullus lanatus L.) grafted by Branches 2

11 Polyploid seeds production is laborious, complicated, and costly work. Tetraploid and 12 triploid plants produce a fewer number of seeds/fruit, and triploid embryos are fairly 13 weak, covered with a more hardened seed coat as compared to diploid seeds. Here we 14 investigated the interactive effect of new grafting technique of polyploid watermelon 15 scion onto rootstock on plants' survival rate, biochemical, and hormones contents. In 16 this study, three different branches, apical meristem (AM), branch with 1 node (1N), 17 and 2 nodes (2N) from di, Tri, and tetraploid watermelon plants, were used as scion and 18 grafted onto squash rootstock. The results showed highly significant differences 19 between polypoid watermelon when 1N using as a scion, tetraploid showed maximum 20 survival rates, higher contents of hormones, and antioxidants (AOX) activities, 21 compared to diploid, these may be the possible reasons for high compatibility in 22 tetraploid and degrading the grafting zone in diploid. RTq-PCR results confirm that the 23 expression of genes linked to compatibility is consistent with the hormonal and AOX 24 activities. This study provides an alternative and economical approach to produce more 25 tetraploid and triploid plants for breeding or seeds production by using branches as 26 scions. 27 The results showed that, the starch in Tri and Tetra highly significant than di, where it at 0 DAG were and in Tri and Tetra higher than Di in the first season, while in the second season, no significant differences between polyploidy levels. Also, the contents of starch start at 15 DAG in all polyploids, but the increment rates at Tri and Tetra were more than Di 9.68, 14.09, and 16.58% at the first season in Di, Tri, and Tetra watermelon, respectively.


Introduction 31
Watermelon is the fifth most-consumed flesh fruit worldwide and occupies 7% of the 32 cultivated land for fruits and vegetables (FAOSTAT 2018). Seedless watermelons are 33 the most desirable watermelon cultivars presently available to consumers and command 34 a high price and more excellent quality than seeded watermelon [1]. Seedless 35 watermelons are produced by crossing a tetraploid seed parent with a diploid pollen 36 parent [2]. Tetraploid induction can be done by different methods like applying aqueous 37 chemicals (colchicine, Oryzalin, and Dinitroaniline) solution to the growing apex of 38 diploid seedlings or by soaking diploid seeds before germination [3]; these chemicals 39 are toxic to the plant material treated, so the percentage of success was low [4]. 40 However, seedless watermelon production has been hampered by high seed cost and 41 poor seed germination. High seed cost has generally been because of difficulties in 42 obtaining a sufficient number of tetraploid individuals and the low number of seeds 43 only 5-20 seeds/fruit as compared to diploid fruits )600 seeds/fruit). It is important to 44 find a more economical way to increase the number and quality of triploid seeds [5][6][7][8][9][10][11][12][13] 45 Grafting has a very long history, and evidence of its use has been found in ancient 46 civilizations, e.g., in 1560 BC in China, as discussed by Aristotle (384-322 BC) [14]. 47 vascular formation; auxin is important for the differentiation of vascular tissues and 64 wound healing [15,18,32,33,[39][40][41][42][43][44][45]. Reactive Oxygen Species (ROS) accumulation leads 65 to cell death, and oxidative damage depends on the balance between the production of 66 antioxidant AOX enzymes and ROS [46,47]. Increase the activity of defense enzymes 67 such as Peroxidase POD, and catalase CAT can scavenge ROS in plants, and improving 68 the resistance of plants to stress [48,49]. POX and CAT convert H2O2 to H2O [50]. So the 69 difference relation between AOX and ROS during the healing process could be used as 70 a rapid mechanism to verify incompatibility [51]. Lignin is abundant in woody plants 71 and primarily contributes strength to the cell wall; so, decreased lignin would be 72 antagonistic to a strong graft union [52]. 73 Up to date, the cause and underlying mechanism for grafting incompatibility 74 remain elusive, and there is no report on genes associated with grafting 75 incompatibility [53,54]. More research work needs to be done to fully understand the 76 mechanisms of graft compatibility and incompatibility [41,52]. 77 The purpose of this research was to study the effect of genome duplication on graft 78 compatibility, by comparing the factors and parameters which lead to graft 79 compatibility or incompatibility. This study provides information at the molecular and 80 physiological levels for the mechanism compatibility in polyploid watermelons, which 81 should explain the mechanism of compatibility in polyploid watermelon, also to try a 82 new technique for vegetable propagation by using branches as scions, three different 83 branches were taken from mother plants and graft on squash rootstock to study the 84 success of this method. This method will add an option for breeders and seed producers 85 to save time and money using an asexual method to increase plant numbers. 86

1. Plant materials 89
Seedless watermelons are triploids (3n=33) produced by crossing a tetraploid seed 90 parent with a diploid (2n=22) pollen parent. Tetraploid induction was done by applying 91 colchicine to the growing apex of diploid seedlings. Polyploid seeds for one variety 92 (mimei), which is homozygous and genetically stable, and passed the achievement 93 appraisal of the Chinese Department of Agriculture in 1990 and won the second prize 94 of science technology progress of the Department of Agriculture in 1991 [55], were 95 used as a mother plant and squash interspecific hybrid (xijiaqiangsheng) which widely 96 used in China as a rootstock, obtained from the polyploid watermelon group -97 Zhengzhou Fruit research institute (CAAS) China. Tetraploid, triploid, and diploid 98 watermelon and rootstock seeds were sown in seedling cell trays with 32 cells at the 99 intelligent greenhouse of Zhengzhou, Henan province, China. After 50-60 days from 100 the date of transplanting, good and healthy mother plants free of pests and diseases, 101 especially virus-free, were selected for obtaining different types of scions. From the 102 healthy mother plants, suitable branches were chosen for scions. Three types of 103 branches (Figure 1) were taken from mother plants, (a) apical meristem (AM), (b) the 104 branch having one node and one leaf (1N), and (c) the branch has two nodes and one 105 leaf (2N) as mentioned by [10]. The grafting process was performed after 15 and 20 106 days after rootstock seeds sowing. The splice grafting method was used [19] (Figure 107 S1). Rootstock seedlings were subjected to adaptation before and after grafting to 108 increase the survival rate, as mentioned by [56,57]. Database. MEGA 6 software was used to draw phylogenetic trees of selected genes. 155 The clustalW tool was first used for the alignment. Afterward, the neighbor-joining 156 method, with 1000 bootstrap replicates, was used [58,59]. The phylogenetic trees were 157 constructed using protein sequences of the compatibility mechanisms. Reverse Transcriptase (Promega, USA) and diluted 20 ng/ul. 178

RTq-PCR expression analysis of genes involved in compatibility mechanisms 179
Genes linked to the compatibility mechanisms were selected from the watermelon 180 genome database (http://cucurbitgenomics.org/organism/21). To check the expression 181 patterns of selected genes in grafted watermelon at 0, 3, and 15 DAG, quantitative 182 reverse transcription PCR (RTq-PCR) was performed. The entire data were analyzed 183 using the 2-ΔΔCt method [61]. Three independent biological replicates were used for 184 gene expression analysis [62]. Actin "cla016178" was used as a reference gene [58].    The content of IAA was higher in Tetra, followed by Tri and Di watermelon at 0, 3, and 230 15 DAG, respectively. 231 Whereas ZR showed no significant differences between Di and Tetra at 0 DAG, while 232 at 3 and 15 DAG, there were highly significant differences in both seasons (Figure 4). 233 The results at 3 and 15 DAG showed that the content of the ZR starts to increase 234 significantly in Tetra than Di and Tri. At 3 DAG, the content of ZR was increased by 235 59.43 and 42.9% during the first, and second season respectively, and by 18.69 and 236 37% at 15 DAG during both seasons.

264
SOD activity was observed higher in Tetra and Tri watermelon as compared to Di 265 watermelon ( Figure 5). At 0 DAG, SOD activities were 3.76 and 3.19-fold higher in Tri 266 than diploid in both seasons, respectively. At 3 DAG, SOD activities were 2.9 and 267 2.97-fold higher than Di in the first and second season, respectively. In comparison, at 268 15 DAG, the SOD activities in Tri were 4.32 and 2.7-fold higher than Di in both 269 seasons. In Tetra SOD activities at 0 DAG were 3.64 and 4.49-fold higher than Di in 270 the first and second season, respectively, while at 3 DAG, it was 2.53 and 3-fold higher 271 than Di in the first and second season, respectively. Moreover, at 15 DAG, the contents 272 of SOD in Tri were 4.47 and 4.19-fold higher than Di in the first and second season, 273 respectively. Generally, there are significant differences between Tetra and Di at 0, 3, 274 and 15 DAG. 275 Significantly higher CAT content was observed in Tetra than Di and Tri ( Figure 5). At 276 0 DAG it was 1.85 and 2.35-fold higher in Tetra than Di in the first and second season 277 respectively, and 1.31 and 1.7-fold higher than Tri in the first and second season, respectively. At 0 DAG, CAT activity was 1.4 and 1.38-fold higher than Di in the first 279 and second season, respectively. CAT activities in the graft union start to increase at 3 280 DAG in all polyploids, but the increase at Tri was more than Di and Tetra, with 281 increment rates of 9.19, 122.29, and 33.47% in the first season and 26. 35, 35.52, and 282 25.34% in the second season in Di, Tri, and Tetra watermelon respectively. 283 Reactive Oxygen Species (ROS) like hydrogen peroxide (H2O2) accumulation leads to 284 cell death, and oxidative damage depends on the balance between the production of 285 AOX and ROS [46,47]. H2O2 contents were higher in Triploid than Di and Tetra at 0 286 DAG stage (Figure 6). It was 1.49 and 1.65-fold higher than Di in the first and second 287 season, respectively, and 1.14 and 1.2-fold higher than Tetra in the first and second 288 season, respectively. But the increment rates of H2O2 activities in the graft union at 289 Tetra starts to increase at 3 DAG less than Di, and, Tri; the increment rates were 51.51,

Measurement of Lignin, phenols, and starch contents in the grafting union among 299
Di, Tri, and tetraploid watermelon at different days after grafting 300 Lignin content was significantly different between Di and Tetra watermelons in both 301 seasons (Figure 7). Lignin contents were higher in Tri, and Tetra than Di at 0 DAG, 302 where it at Tetra were 2.7 and 2.41-fold higher than Di in the first and second season 303 respectively, and at Tri were 2.67 and 2.41-fold higher than Di in the first and second 304 season, respectively. But because of callus induction (branchime cells) in the graft 305 union at 3 DAG stage [18], the lignin contents start to decrease; nevertheless, the 306 decrement rate in Tetra was less than Di. The increment rates of lignin contents in the 307 graft union at 3 DAG were 51.68, 21.5, and 6.33% in the first season, and 51.68, 16.98, 308 and, 12.32% in the second season at Di, Tri, and, Tetra watermelon respectively. At 15 309 DAG, the lignin contents start to increase by 164.32, 55, 08, and, 59.18% in the first 310 season, and 251.37, 264, and, 188.11% in the second season at Di, Tri, and, Tetra 311 watermelon respectively. 312 The results of phenols showed significant differences between Di and Tetra 313 watermelons in both seasons at 3 DAG (Figure 7). The contents of phenols in Tetra 314 were 1.4, 1.7, and 1.1-fold higher than Di at 0, 3, and 15 DAG, respectively, in the first 315 season, and 1.1, 1.3, and 1.2-fold higher than Di at 0, 3, and 15 DAG respectively, in 316 the second season. 317 The results of starch showed highly significant differences between ploidy watermelons 318 in the first season during the grafting process, while in the second season, no significant 319 differences between ploidy levels ( Figure 7). The results showed that, the starch 320 contents in Tri and Tetra highly significant than di, where it at 0 DAG were (1.57 and 321 1.48-fold in Tri and Tetra higher than Di in the first season, while in the second season, 322 no significant differences between polyploidy levels. Also, the contents of starch start 323 to increase at 15 DAG in all polyploids, but the increment rates at Tri and Tetra were 324 more than Di 9.68, 14.09, and 16.58% at the first season in Di, Tri, and Tetra 325 watermelon, respectively. Triploid watermelon seeds are produced by cross-pollination between Di and Tetra, but 386 Tetra seeds production is a very hard process [11]. So it is important to find a more 387 economical way to increase the number and quality of triploid seeds [13]. In this study, 388 we tested three different parts from mother plants as a scion, apical meristem (AM), 389 branch with 1, and 2 nodes (1N and 2N)  Plant hormones play important roles in plant growth, development, and response to 398 biotic and abiotic cues and vascular formation in the graft junction [15,32,33,[40][41][42][43][44][45]. 399 Auxin, and cytokinin, play an important role in regulating stock-scion interactions 400 [18,36]. Cell divisions happened within 2-3 DAG in the graft junction and have the 401 highest hormone levels during this period [18,39,40,64]. During the grafting process, the 402 peak of IAA is observed to occur within 3 DAG in the scion, as reported by [65]. IAA 403 and Zeatin Riboside (ZR) are required for vascular bundle regeneration in the graft 404 union [37,38]. The main cause of incompatibility is the occurrence of hormonal 405 imbalance [34]. A low indole-3-acetic acid (IAA) content in incompatible combinations 406 may then affect the differentiation of xylem and phloem, as well as lignification 407 [17,28,66]. We compared the IAA and ZR contents in the graft union between Di, Tri, 408 and Tetra watermelon (Figure 4). The IAA concentration in the Tetra combination was 409 significantly higher than that in the Di, and Tri combination (p < 0.05). Our results 410 showed high compatibility in Tetra which has high content and high increment rates of 411 hormones than Di especially at 3 DAG (critical period) and 15 DAG, this can explain 412 high survival rates in Tetra, this results were agreement with [67][68][69] 413 On the other side, incompatibility results from the stress-induced during the healing 414 response, as reported earlier by [41]. In this study in (Figure 5), POD, SOD, and CAT 415 activities showed a high increase during the healing process in Tetra and Tri. Also, the 416 content of H2O2 ( Figure 6) didn't increase during the healing process in Tri, because of the high activities of antioxidants, which leads to scavenging oxygen radicals. These 418 results were in accordance with [70][71][72][73][74], who found that genome duplication gave high 419 resistance for salt stress because of the high contents of hormones and highly activities 420 of antioxidants more than Di. Also, ploidy results in chromosome doubling leading to 421 gene doubling, which results in higher expression, thus causing an increase in protein 422 contents [75]. Polyploidy is more tolerant of abiotic stress than Di it was reported in 423 watermelon [76], rice [73,77] [81], and rangpur lime [82]. Peroxidase and catalase activities were 425 increased in the grafted plants [83]. 426 Generally, antioxidant enzymes were usually studied in the responses to abiotic and 427 biotic stresses, but not often for graft stress; in this study, we suggest grafting as stress 428 because of, cut or wound stress, complete dark, and high humidity stress, especially in 429 the first three days after grafting (healing response) [12,41]. The most important critical 430 period in the grafting healing process was at 2 and 3 days after grafting [18]. The results 431 in both seasons showed that the activities of SOD, POD, and CAT were significantly 432 different at the grafting healing process between polyploid watermelons ( Figure 6). It 433 has been reported already by [49,83] that high activities of POD and CAT, during the 434 healing process have a high ability to scavenge reactive oxygen species (ROS) and 435

Conclusion 451
Seedless watermelons seeds production faces many problems. The current study 452 concludes that vegetative propagation by branches grafting could be used in Tri and 453 Tetra watermelon seed production. The high contents of hormones and high activities 454 of antioxidants in Tetra more than Di because of the genome duplication, which gave 455 high compatibility and high survival rates of grafting. Grafting using branches can be a 456 good alternative to promote vegetative propagation in Tetra watermelon. These