Morphological, Cytological, and Molecular Characterization of Interspecific Cotton Hybrids Derived from Gossypium hirsutum and G. arboreum Crosses

Morphological, Cytological, and Molecular Characterization of Interspecific Cotton Hybrids Derived from Gossypium hirsutum and G. arboreum Crosses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Morphological, Cytological, and Molecular Characterization of Interspecific Cotton Hybrids Derived from Gossypium hirsutum and G. arboreum Crosses Saptarshi Mondal, Dharminder Pathak, Salil Jindal, Neha Agrawal, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5015023/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Oct, 2024 Read the published version in Genetic Resources and Crop Evolution → Version 1 posted 9 You are reading this latest preprint version Abstract Diploid cotton Gossypium arboreum (2n = 2x = 26, A d A d ) is a valuable genetic resource to improve widely cultivated American cotton G . hirsutum (2n = 4x = 52, A t A t D t D t ). In this study, successful generation of four unique interspecific hybrids ( G. hirsutum × G. arboreum ) was confirmed through morphological, cytological, and molecular characterization. The morphological evaluation included different stem and leaf characters, trichome density, floral characters, and cotton leaf curl disease reaction. Interspecific hybrids were recorded with either complete dominance of some characters or an intermediate expression. However, variation among the F 1 s was observed for some traits, especially for pollen size and fertility. Two of the four F 1 s were found to possess relatively bigger pollen sizes with partial fertility which enabled their utilization as male parents in backcrosses. Mitotic analysis of already established F 1 s was made possible through the induction of fresh roots by air layering. Of the four hybrid plants, two were triploid (2n = 39) and the other two were tetraploids (2n = 52), the latter case probably was a result of the fusion of normal n (A t D t ) gametes from female parent ( G. hirsutum ) and unreduced gametes 2n (A d A d ) from male parent ( G. arboreum ). Meiotic irregularities in interspecific hybrids were also observed. Successful amplification of polymorphic parental bands in all four F 1 s using simple sequence repeat markers added another line of evidence supporting their hybridity. Results of this experiment provided useful insight and base material to introgress leaf-hopper tolerance from G . arboreum to G . hirsutum . Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights G. arboreum is highly cross incompatible with G. hirsutum . Here, we report the development and characterization of four unique interspecific G. hirsutum × G. arboreum hybrids Out of four hybrid plants, two were triploid (2n = 39) and the other two were tetraploids (2n = 52) The latter case indicates the fusion of normal n gamete from female parent and unreduced gamete 2n from male parent. Tetraploid hybrids showed partial pollen fertility which enabled their utilization as pollen donors in backcrosses Introduction The importance of cotton may be judged from the fact that it is cultivated in more than 80 countries across the world. Economy of several countries is directly or indirectly dependent on cotton. Cotton is grown on an area of 30–36 million ha worldwide. Presently, ten countries viz ., Australia, Brazil, Burkina Faso, China, India, Pakistan, Türkiye, Turkmenistan, Uzbekistan and the USA produce greater than 90% of the total cotton globally (Kranthi, 2019 ). Australia has the highest global cotton productivity of more than 2000 kg lint per ha which is at least 2.5 times higher than the global average productivity of cotton. Besides lint (long fibers borne on the seed coat), which is the major product and the chief source of natural fiber for textile industry, cotton is also an important source of edible oil. Factually, cotton is the fifth major source of vegetable oil worldwide (Chen et al., 2021 ). The genus Gossypium , to which cotton belongs, includes about 50 species distributed in the arid to semi-arid regions of the tropics and subtropics (Wendel et al., 2003 ). Four cotton species namely, G. hirsutum , G . barbadense , G. arboreum , and G. herbaceum are cultivated, of which cotton acreage is dominated by G. hirsutum (~ 98%), aka Upland cotton/American cotton across the world. A longer crop duration in cotton provides a large number of insect pests and pathogens more opportunities to affect cotton at all the crop stages. The nuisance of bollworms has largely been managed through the commercialization of transgenic Bt cotton cultivars. However, this has resulted in shifting of pest profile to sap sucking insect pests such as whitefly, leafhopper, mealy bug, mirid bug, aphid etc. The whitefly epidemic in north Indian cotton growing states destroyed cotton crop on nearly 1.5 million ha during 2015 (Kumar et al., 2020 ). Similarly, cotton leaf curl disease (CLCuD)- caused by Geminiviruses and vectored by whitefly [ Bemisia tabaci (Gennadius)] led to huge financial losses of ~ USD 5 billion between 1992–1997 to Pakistan economy (Briddon & Markham, 2000 ). Related species of cotton are invaluable sources of genes of economic importance. For example, Gossypium armourianum , a wild non-progenitor D-genome species has been reported to possess resistance to several biotic stresses such as pink bollworm (Brazzel & Martin, 1956 ), jassid (Pushpam & Raveendran, 2006 ), whitefly and cotton leaf curl disease (CLCuD) (Suthar et al., 2022 ). Similarly, G. arboreum (A d A d ) is resistant to CLCuD (Naqvi et al., 2017 ; Singh et al., 1997 ), jassid (Jindal et al., 2022 ; Nibouche et al., 2008 ; Sidhu & Dhawan, 1980 ) and whitefly (Miyazaki et al., 2013 ). Availability of genetic variation is vital to crop improvement. Domestication and extensive use of limited number of genotypes in the breeding programs gradually narrowed down the genetic base of crop plants, which made them vulnerable to various biotic and abiotic stresses. Limited genetic variation among hirsutum cotton cultivars and their vulnerability to pathogen/insect epidemics has been documented (Bowman et al., 1996 ; Brubaker & Wendel, 1994 ). Related species of crop plants are invaluable sources of genes of economic importance and can play a pivotal role in enhancing genetic diversity through the introgression of useful novel alleles in the adapted germplasm. At Punjab Agricultural University, Ludhiana, India, related Gossypium species, especially belonging to secondary gene pool, are being used in the pre-breeding programs. We crossed widely grown Upland cotton ( G . hirsutum ) with desi cotton ( G. arboreum ) with the objective to introgress favorable alleles such as for resistance to CLCuD, leafhopper, and whitefly which are serious threats to Upland cotton cultivation in various parts of India and the world. Identification and characterization of true hybrids are of utmost importance to carry forward the introgression program and give an insight into the dominance/recessive relationships of alleles, genomic constitution of the hybrids etc. In this article, we report the development of interspecific hybrids between G. hirsutum and G. arboreum and their hybridity confirmation based on morphology, chromosome number, and molecular markers. Materials and Methods Plant materials: A number of G . hirsutum and G . arboreum lines were hybridized in different combinations to develop wide-hybrids. G . hirsutum was used as the seed parent, whereas G . arboreum served as the pollen parent in all the crosses. G . arboreum (A d A d ) belongs to the secondary gene pool of cotton and is not easily crossable with G . hirsutum . Substantial efforts are required to generate interspecific hybrids between these species. A total of 12,392 flowers of hirsutum parents were pollinated to obtain the interspecific hybrids (Jindal et al., 2022 ). Hormones (a mixture of GA3 @ 50ppm + NAA @ 100 ppm) were applied to the base of pedicel of the cross pollinated ( G. hirsutum × G. arboreum ) buds for three consecutive days to enhance crossed bud retention and four true interspecific hybrids were obtained through the direct crosses. The pedigree of cotton interspecific hybrids is presented in Table 1 . F 846 and LH 2108 are commercial cultivars, whereas LH 2107 is an advanced line of hirsutum cotton. Similarly, LD 491 and LD 949 are commercial cultivars of arboreum ( desi ) cotton. All these cotton cultivars/lines have been developed at Punjab Agricultural University, Ludhiana (Punjab), India. The resulting four unique interspecific putative cotton hybrids were characterized at morphological, cytological, and molecular levels. Table 1 Pedigree of the cotton interspecific F 1 hybrids S. No. G. hirsutum (seed parent) G. arboreum (pollen parent) Designation of F 1 plant 1 F 846 LD 949 F 1 -1 2 LH 2108 LD 949 F 1 -2 3 LH 2107 LD 949 F 1 -3 4 LH 2107 LD 491 F 1 -4 Morphological observations: The morphological investigation comprised the following characters recorded from the parental lines and their respective interspecific F 1 hybrids: plant stem coloration, plant height, leaf shape, leaf lobe number, leaf nectary, leaf size, leaf trichome density, petiole length, number of serrations on epicalyx, sepal number, petal number, petal color, petal spotting, anther color, anther number, pollen viability, pollen size, and reaction to cotton leaf curl disease (CLCuD). Fully matured leaves and fresh flowers at anthesis were taken for morphological characterization. To analyze pollen fertility, flower buds at the candle stage were identified and tied with a thread at the top to prevent stray pollen admixtures. Flowers were collected in the morning on the day of anthesis between 9 am to 10 am. Freshly collected pollens were subjected to acetocarmine test, a widely followed colorimetric method of pollen viability estimation that depends on the reactions of dye with cytoplasm proteins, nuclei, and nucleic acids, to discriminate fertile pollens from sterile ones (Shekari et al., 2016 ; Skrzypkowski et al., 2023 ). Average pollen size was determined using micrometry method. In order to facilitate natural CLCuD inoculation under the field conditions, F 846 (a highly CLCuD susceptible Upland cotton variety) was planted in the experimental plot to provide inoculum of the CLCuV throughout the season. Further, no management of whitefly (vector of CLCuV) through the insecticides was undertaken. Adaxial surface of fully expanded leaves of parents and hybrids was examined for the occurrence of CLCuD. Cytological observations: For mitotic analyses, root tips of parental lines ( G . hirsutum and G . arboreum ) were collected from germinated seeds. However, collection of the same through seed germination was not possible for the already established interspecific hybrids under investigation. Thus, air layering (Fig. 1 ) was followed for root induction as an alternative. Fresh milky white root tips were harvested between 8 a.m. to 10 a.m. avoiding any damage to the tip portion. Three to four root tips were collected in each micro-centrifuge tube containing 2 mM of 8-hydroxyquinoline (a metaphase arresting chemical) and then transferred to freshly prepared fixative (3 Ethanol: 1 Glacial acetic acid). The fixed root tips were enzymatically digested and on-slide maceration technique (Ma et al., 1996 ) was followed under a stereo microscope to squash the root meristem. This preparation was examined under a phase contrast microscope. The slides with the highest mitotic index with promising chromosome spread were selected for staining with 4,6- diamino-2-phenylindole (DAPI) and visualized and photographed under a fluorescent microscope. For meiotic analysis, young flower buds were collected in the early morning (just after sunrise) and fixed in Carnoy’s solution (70% ethanol, chloroform, and glacial acetic acid in 6:3:1 ratio). Taking anthers from the fixed buds, squash technique was followed to prepare slides with 2% acetocarmine as a staining agent. Cover slip was placed carefully on it and a shy heat treatment was provided to facilitate chromosomal spreading. Molecular analyses: Moisture free tender leaves were collected from the parental lines and F 1 hybrids for genomic DNA extraction by Cetyltrimethylammonium bromide (CTAB)-method following Saghai-Maroof et al. (1984). Extracted DNA was purified from impurities like phenolics, proteins etc. DNA quality and quantity were assessed using Nano-Drop Spectrophotometer (Thermo Scientific NanoDrop™ 8000 Spectrophotometer). Eight polymorphic cotton specific SSR markers were employed for hybridity confirmation of the putative interspecific hybrids (Supplementary Tables: ST1, ST3, and ST4). Seven of these eight SSR primers were specific to A-sub genome of cotton. Virus-specific universal primer A and primer B (Deng et al., 1994 ) were used to detect the presence/absence of the begomovirus component of CLCuD complex (ST2). Amplified PCR products for hybridity confirmation were resolved on 3% agarose gel and the presence of virus specific bands was detected on 2% agarose gel. PCR reaction was followed as described by (Deng et al., 1994 ) with an annealing temperature of 52°C. Statistical analysis: Graphical representations and mean separation using Fisher’s protected LSD at p-value < 0.05 for the quantitative parameters were carried out in SAS JMP Pro 17.0.0 software. Results Morphological characterization: Comparison of morphological characters of G . hirsutum parents viz. , F 846, LH 2108, and LH 2107; G. arboreum parents namely LD 949 and LD 491 with their respective hybrids are presented in Table 2 . Variable phenotypic expression was observed among the F 1 hybrids. They manifested either dominance or intermediate expression for various morphological traits. All the female parents (LH 2107, LH 2108, and F 846) and one of the male parents LD 491 have green stems, whereas LD 949 possesses deep red stem coloration. Stems of F 1 -1 (F 846 × LD 949) and F 1 -2 (LH 2108 × LD 949) were observed to be red, which is an indication of a successful cross as the character was inherited from male parent. Interestingly, F 1 -3 (LH 2107 × LD 949) even having the same parent as those of F 1 -1 and F 1 -2 was found to possess a green stem like its hirsutum parent. This hybrid is actually tetraploid with 2n = 52 (explained later). F 1 -1 and F 1 -2 recorded intermediate leaf shape, whereas, other two F 1 s (F 1 -3 and F 1 -4) were more similar to their maternal parent i.e. G. hirsutum cv. LH 2107. All three hirsutum parents and LD 949 recorded similar leaf lengths, but LD 491 registered a lower leaf length. Both the tetraploid hybrid plants (F 1 -3 and F 1 -4) possessed relatively smaller leaf lengths than that of their parents as well as F 1 -1 and F 1 -2 (F 1 -4 < F 1 -3 < F 1 -2 ≤ F 1 -1). Average petiole lengths of all the G. hirsutum parents were higher than that of G. arboreum parents. Significant differences were observed between both the F 1 -1 and F 1 -2 with their respective maternal ( G. hirsutum ) parents; however, F 1 -2 was at par with its G. arboreum parent (LD 949) for petiole length. On the other hand, F 1 -3 and F 1 -4 possessed significantly smaller petioles as compared to their respective parents and other two hybrids (F 1 -4 = F 1 -3 < F 1 -2 = F 1 -1). All the parental lines, except LD 949 possessed nectary on the mid-rib of the abaxial side of leaves, whereas all the F 1 hybrids were found to possess nectary. In the present study, tetraploid Upland cotton genotypes recorded significantly lesser TD as compared to desi cotton parents. Young leaves of all the test entries were found to possess higher TD than that of mature leaves. A significant (at p < 0.05 ) positive correlation (r = 0.73*) was found between TD of mature and young leaves. All the interspecific cotton hybrids recorded a higher TD than Upland cotton parents, however, lesser TD in the younger leaves than that of the arboreum cotton parents. F 1 -1 and F 1 -2 possessed a significantly higher TD than F 1 -3 and F 1 -4 in young leaves. Nonetheless, the per cent reduction of TD at later growth stages was much lesser in F 1 -3 and F 1 -4, due to which TD of all four hybrids did not show significant differences in mature leaves (Supplementary Fig: SF1). Female parents, F 846, LH 2108, and LH 2107 possessed cream-colored petals, whereas, male parents LD 949 and LD 491 had bicolored and white (colorless) petals, respectively. F 1 hybrids showed variable expressions for petal color. In the present investigation, male parents as well as all four interspecific F 1 hybrids manifested petal spot phenotype. Pollens in female parents were cream-colored, whereas, male parents LD 491 and LD 949 possessed yellow and dark yellow pollens, respectively (Fig. 2 ). F 1 -1 and F 1 -2 had yellow pollens, whereas F 1 -3 and F 1 -4 possessed cream-colored anthers. Pollen viability test revealed high pollen fertility of parental lines, but a little fertility (F 1 -3 and F 1 -4) to complete sterility (F 1 -1 and F 1 -2) in the interspecific hybrids. Tetraploid hybrids (F 1 -3 and F 1 -4) recorded an average pollen fertility of 40.5% and 17.8%, respectively Table 2 . All three hirsutum parents possessed larger pollens (119.4- 122.3 µm) than desi cotton genotypes and interspecific hybrids. Due to the pollen sterility, F 1 -1 and F 1 -2 recorded the lowest pollen size, whereas a higher pollen fertility of F 1 -3 and F 1 -4 was also reflected through their larger pollen size Table 2 . Pollen size of F 1 -3 was even significantly higher than its male parent (Fig. 3 ; SF2). Table 2 Comparison of morphological characters among parents and interspecific hybrids Character F 846 LH 2108 LH 2107 LD 949 LD 491 F 1 -1 (F846 × LD949) F 1 -2 (LH 2108 × LD949) F 1 -3 (LH 2107 × LD949) F 1 -4 (LH 2107 × LD 491) Stem coloration Green Green Green Red Green Red Red Green Green Plant height (cm) 159 171 166 186 172.6 195 183 156 144 Leaf shape Palmate Palmate Palmate Digitate Digitate Intermediate Intermediate Palmate Palmate Nectaries on mid-rib Present Present Present Absent Absent Present Present Present Present Average leaf length (cm) 8.9 ab 8.6 bc 8.8 ab 9.1 a 8.2 c 8.6 bc 8.8 ab 7.4 d 6.6 e Trichome density (ML) 44.33 b 44.22 b 43.67 b 82.78 a 83.89 a 83.89 a 80.33 a 85.67 a 85.11 a Trichome density (YL) 87.67 d 85.67 d 86.33 d 178.78 a 177.78 a 136.89 b 130.11 b 113.56 c 108.0 c Petiole length (cm) 7.3 ab 7.6 a 7.1 b 6.1 c 6.2 c 5.6 d 5.9 cd 3.9 e 4.3 e Flower petal color Cream Cream Cream Bicolor White Bicolor Bicolor Cream with pink margins Cream with pink margins Flower petal spotting Absent Absent Absent Present Present Present Present Present Present Anther color Cream Cream Cream Dark Yellow Yellow Yellow Yellow Cream Cream Pollen fertility (%) 96.7 93.7 94.5 80.1 87.6 0 0 40.5 17.8 Average pollen size (µm) 121.1 a 119.4 a 122.3 a 91.5 c 89.6 c 79.4 d 82.2 d 103.1 b 91.4 c CLCuD symptoms Present Present Present Absent Absent Absent Absent Present Present Cytological observations: Cytological characterization of F 1 hybrids and their parents was carried out by both mitotic and meiotic analyses. Mitosis: Air-layering of the F 1 plants with rooting hormone helped to induce fresh roots, which were collected for mitotic slide preparation. Slide preparation from freshly collected roots from Upland cotton and desi cotton parental lines along with their respective F 1 hybrids revealed the ploidy level of the plants. As expected, G. hirsutum (LH 2108 and LH 2107) and G. arboreum (LD 949 and LD 491) genotypes were found to have 2n = 52 and 2n = 26 chromosomes, respectively (Fig. 3 ). In the present study, accordance, as well as deviation from the expected ploidy status of interspecific Gossypium species hybrids, were observed. F 1 -2 was found to possess 2n = 3x = 39 chromosomes. F 1 -1 is expected to have the same chromosome number as that of F 1 -2 based on the observation that F 1 -1 and F 1 -2 have the same morphological features such as pollen sterility, leaf shape, stem color, petal color, intensity of petal spot, pollen color etc. (Table 2 ). Interestingly, F 1 -3 and F 1 -4 both registered somatic chromosome numbers of 2n = 4x = 52 (Fig. 3 ). Meiosis: Meiotic analysis on the pollen mother cells of interspecific hybrids revealed several interesting observations. Meiotic irregularities were observed in both F 1 -1 and F 1 -2 at the different phases of the meiosis viz. early metaphase I, metaphase I and metaphase II, and late anaphase in hybrids (SF3). The presence of univalent chromosomes (as indicated with arrows in SF3) which were lagging to reach the equatorial position was observed at early metaphase-I in both triploid F 1 plants. Normal metaphase-II and abnormal metaphase-II were observed in F 1 -3 and F 1 -2, respectively. Cytokinesis after the first meiotic division and the formation of dyads in F 1 -2 were observed. Interestingly, the occurrence of both radial (tetragonal) and tetrahedral tetrad was observed in this interspecific hybrid (SF3). Molecular characterization: In the present study, besides phenotypic and cytological characterization, hybridity confirmation was also carried out using cotton specific SSR markers to provide another line of evidence supporting the hybrid status of G. hrsutum × G. arboreum F 1 s. Eight SSR primers viz ., BNL 0946, BNL 1679, BNL 2652, BNL 2921, BNL 3888, NAU 0922, NAU 1222, and CIR 0183 were employed for hybridity confirmation of the interspecific cotton F 1 s (Fig. 4 ). Reaction to cotton leaf curl disease (CLCuD): Phenotypic reaction to CLCuD in parents and their respective F 1 s was observed. Female hirsutum parents were found to possess clear symptoms, whereas male parents ( G. arboreum ) were symptomless (Fig. 5 ). All the four interspecific hybrids were initially found to be CLCuD susceptible and exhibited typical symptoms of the disease during 2017 (Pathak et al., 2017 ). However, in the next season (2018), F 1 -1 and F 1 -2 became symptomless, whereas F 1 -3 and F 1 -4 maintained symptoms of CLCuD such as interveinal thickening, leaf curling, and the presence of enation on the underside of the leaves (Table 2 ; Fig. 5 ). CLCuD reaction of the test genotypes was also confirmed at the molecular level through the amplification of viral DNA using virus specific primers. Virus specific bands were amplified in all the G. hirsutum parents namely, F 846, LH 2108, and LH 2107; whereas no virus specific band was observed in G. arboreum parental lines (LD 949 and LD 491). Two of the interspecific hybrids namely, F 1 -1 and F 1 -2 did not show the amplification of virus specific bands, whereas amplification of the same was found in the other two hybrids (F 1 -3 and F 1 -4) (Fig. 5 ). Amplified viral DNA of the susceptible genotypes was approximately 500 bp in size. Discussion Upland cotton genotypes owing to their narrow genetic base are susceptible to many pathogens and insect pests and demand further improvements. On the other hand, desi cotton species such as G. arboreum serve as valuable genetic resources for the breeders to perform interspecific crosses for broadening the narrow genetic base of Upland cotton. However, reports on successful interspecific hybrids are limited due to fertilization barriers between different species. Therefore, identification of true interspecific hybrids, a clear understanding of their ploidy level, and genetic constitution help formulate appropriate breeding strategies, facilitating their further utilization in enhancing trait value of the recipient species. In the present study, we discuss the successful generation and characterization of four unique interspecific hybrids at the morphological, cytological, and molecular levels in comparison to their respective parents. Morphological characterization: The phenotypic expression of F 1 s was variable. Some of the characters showed dominance, whereas others displayed intermediate expression in the interspecific hybrids. Occurrence of red stems in triploid hybrids (F 1 -1 and F 1 -2) indicated the manifestation of a dominant character from the male parent (LD 949), showing consistency with the findings of Tahir et al. ( 2011 ) and Chen et al. ( 2015 ). Interestingly, showing contradictions to the above-mentioned reports, the tetraploid F 1 -3 (male parent LD 949) possessed green stems.Both F 1 -3 and F 1 -4 possessed significantly smaller leaf and petiole lengths than their respective parents and the other two hybrids, which counters the finding of Ahmad et al. ( 2011 ) in 2( G . arboreum ) × G . hirsutum where the average leaf size was observed to be larger in interspecific hybrid as compared to the arboreum parent. Cotton plants possess minute hair like structures known as trichomes on most of the aerial parts. Trichome density (TD) on leaves is correlated with the varying degree of resistance/ susceptibility to sucking insect pests, as well as drought resistance (Karkkäinen et al., 2004 ; Meagher et al., 1997 ). In this study, the presence of higher TD in desi cotton entries than American cotton genotypes for both types of leaves (mature and young) was observed (SF1). A higher tolerance to sucking insect pests including leaf hoppers due to the presence of denser trichomes in G. arboreum than G. hirsutum has been reported (Aherkar et al., 2023 ; Jindal et al., 2022 ). Also, the occurrence of denser trichomes on the young leaves of all the test entries than that of the mature leaves supports the findings of Wright et al. ( 1999 ), Turley and Vaughn ( 2012 ), and Grover et al. ( 2016 ). Here we found a strong positive correlation between TD of mature and young leaves (r 2 = 0.73*) which was consistent with the findings of (Grover et al., 2016 ). Although triploid F 1 s were found to possess a significantly higher TD than the tetraploid F 1 s in young leaves, the latter two showed a much lesser reduction of TD at later growth stages (SF1). Consequently, TD of all four hybrids was the same in mature leaves. Presence of petal spots in all four interspecific cotton hybrids served as a major morphological marker to confirm their hybridity supporting paternal inheritance (Fig. 2 ). The observation is consistent with the findings of Tahir et al. ( 2011 ) and Chen et al. ( 2015 ) in the G . hirsutum × G . arboreum hybrids, Saravanan et al. ( 2007 ) in G . hirsutum × G . raimondii hybrids, and Pushpam and Raveendran ( 2006 ) in G . hirsutum × G . armourianum hybrids. Pollen fertility test confirmed complete sterility of the triploid hybrids, which is expected from a cross between tetraploid and diploid parents and also corresponds to Ahmad et al. ( 2011 ). However, it was interesting to find F 1 -3 and F 1 -4 to have an average pollen fertility of 40.5% and 17.8%, respectively, which does not align with the report of Ahmad et al. ( 2011 ) where only 1.90% of pollen fertility was observed in tetraploid interspecific hybrids. The higher male fertility of two tetraploid hybrids allowed their utilization as pollen parents in backcrossing with Upland cotton. Development of backcross derivatives using these fertile hybrids followed by their screening for leafhopper tolerance has been reported by Jindal et al. ( 2022 ). It is worth mentioning that while the commercialization of Bt cotton could effectively manage the menace of bollworms, it has shifted the pest profile to sap sucking insect-pests such as leafhopper, whitefly, mealy bug, cotton aphid, mirid bug etc. The Upland cotton Bt hybrids cultivated in India are susceptible to sap-feeding insects (Kranthi & Stone, 2020 ). Leafhopper is one of the most economically important sap feeding insects of cotton in the country and can cause 25–45% reduction in seed cotton yield (Kalyan et al., 2017 ). We found that two genes introgressed from arboreum (located on chromosomes A5 and A11) either individually or in combination conferred tolerance to leafhopper in the interspecific backcross derivatives (Jindal et al., 2022 ). This also underscores the importance of 2n gametes formation in nature as well as doubling the chromosome number of parent with lower ploidy as a breeding strategy in the wide-hybridization programs so as to increase the probability of developing an interspecific hybrid. Cytological observations: Cytological observations on interspecific hybrids provide insights into their genomic constitution, ploidy status, and meiotic behavior. It is quite important to devise a future strategy and ensure the proper utilization of the interspecific hybrids. In this current investigation, mitotic analysis confirmed the ploidy status of interspecific hybrids. It was made possible by inducing roots through air-layering (Fig. 1 ). The somatic chromosome number of G . hirsutum genotypes namely, LH 2108 and LH 2107 (2n = 52) and G . arboreum cv. LD 949 and LD 491(2n = 26) was found to be consistent with the findings of Beasley ( 1940 ), Mehetre et al. ( 2004 ) Kale et al. ( 2007 ), Ahmad et al. ( 2011 ), Tahir et al. ( 2011 ), Newaskar et al. ( 2013 ), and Montes et al. ( 2017 ). In this present study, 39 somatic chromosomes of F 1 -2 showed accordance with the findings of Ahmad et al. ( 2011 ), Tahir et al. ( 2011 ) and Chen et al. ( 2015 ) who obtained putative triploid hybrids from crosses between G . hirsutum and G . arboreum . Based on several morphological similarities such as leaf shape, stem color, petal color, anther color etc., F 1 -1 is expected to have the same chromosome number as that of F 1 -2 (Table 2 ). Deviation from the expected chromosome count (2n = 3x = 39) in F 1 -3 and F 1 -4, where both registered a somatic chromosome number of 2n = 4x = 52, may question their true hybridity drawing attention towards the probability of self-fertilization of maternal parents (Fig. 3 ). However, the morphological (for example, the presence of prominent petal spot in these F 1 hybrids) as well as molecular characterization of these F 1 plants present unequivocal evidence against that perplexity (Fig. 2 ; Table 2 ). Triploidy of two interspecific hybrids points out the formation of normal haploid gametes from both parental species, whereas tertaploidy of two F 1 s seeks an explanation for this deviation. One probable reason can be the formation of unreduced (2n = 26) gametes from the diploid male parent G . arboreum and fertilization with normal female gamete (n = 26) of G . hirsutum resulting into F 1 plants with 52 somatic chromosome number. In-situ hybridization of these interspecific hybrids may provide more insight to this question. Formation of unreduced gametes in low frequency is common in plants. Diverse mechanisms for unreduced gamete formation including premeiotic doubling of chromosome number, complete loss of the first or second meiotic division, and defects in meiotic cell plate formation, spindle orientation, or cytokinesis have been observed in flowering plants (Mason & Pires, 2015 ). In cotton, Sheidai ( 2008 ) reported the occurrence of larger unreduced pollen grain in G . hirsutum cv. C-200 (R). Similarly, Noormohammadi et al. ( 2012 ) published a report indicating the occurrence of relatively larger unreduced pollens (87–136 µm) as compared to normal (n) pollens (63-101.7 µm). More recently, Montes et al. ( 2017 ) obtained an interspecific hybrid of G . herbaceum × G . hirsutum , having somatic chromosome number of 52 (A d A d A t D t ) resulted from a fertilization of unreduced female gamete (2n) of herbaceum with normal pollen of hirsutum . Meiotic irregularities have been reported in several interspecific cotton hybrids (Konan et al., 2007 ; Newaskar et al., 2013 ; Skovsted, 1934 ) and this present investigation was not an exception. The observation of abnormal metaphase II in this experiment corresponds to the finding in Salix alba by Khalili et al. ( 2012 ) who described this phenomenon as the fusion of non-sister chromatids in a single pole due to the partial separation of spindle. The presence of univalent chromosomes at the early metaphase of both F 1 -1 and F 1 -2 indicates the transmission of D t genomes from their maternal parents (A t A t D t D t ) only (SF3). Probable fate of such univalents is eventual degeneration which might be the prime reason for the sterility of those plants. Whereas a normal metaphase-II in tetraploid F 1 -3 (A t D t A d A d ) might be the reason for their pollen fertility. Although cotton is a eudicot, the formation of dyads and separation pattern of chromatids in anaphase II (that suggests tetragonal tetrad formation) of F 1 -2 are the key characteristic features of a monocot (SF3). However, interestingly, the occurrence of both radial (tetragonal) and tetrahedral tetrad was observed in this interspecific hybrid. A similar observation for the dyad formation was also reported by Wu et al. ( 2017 ) in interspecific hybrid between G . herbaceum and G . raimondii . Molecular confirmation of hybridity and CLCuV: SSR markers have been extensively used for fingerprinting of cultivars and hybridity confirmation. Several reports on the use of SSR markers for hybridity confirmation in cotton are available (Mehetre et al., 2004 ; Tahir et al., 2011 ; Wu et al., 2017 ; Zhang et al., 2014 ). In the present investigation, eight SSR primer pairs (BNL 0946, BNL 1679, BNL 2652, BNL 2921, NAU 0922, BNL 3888, NAU 1222, and CIR 0183) unambiguously confirmed the hybrid status of the interspecific G . hrsutum × G. arboreum hybrids (Fig. 4 ). Chen et al. ( 2015 ), Tahir et al. ( 2011 ), and Virk ( 2014 ) also employed SSR primer pairs to confirm the hybrid status of putative interspecific F 1 s obtained from G . hirsutum and G . arboreum crosses. Cotton leaf curl disease is reported to significantly reduce the seed cotton yield and emerged as one of the most serious threats to Upland cotton cultivation in north-western India and Pakistan (Monga et al., 2008 ; Nazeer et al., 2014 ; Sattar et al., 2013 ). Clear symptoms in hirsutum parents and symptomless male parents ( G . arboreum ) showed similarity to the previous reports (Iqbal et al., 2015 ; Tahir et al., 2011 ). The interspecific hybrids between G . hirsutum × G . arboreum have been reported to be resistant to CLCuD (Ahmad et al., 2011 ; Tahir et al., 2011 ). In our study, although susceptibility of all four hybrids toward CLCuD was recorded during 2017 (Pathak et al., 2017 ), F 1 -1 and F 1 -2 did not show any symptoms during 2018 and later on, whereas F 1 -3 and F 1 -4 maintained symptoms (Fig. 5 ). Molecular confirmation for the presence/absence of CLCuD in the present study aligned with the phenotypic observations. Amplification of viral DNA of the susceptible genotypes (female parents, F 1 -3, and F 1 -4,) produced bands of approximately 500 bp each. These results corroborate the findings of Deng et al. ( 1994 ), Maruthi et al. ( 2006 ), and Mahesh et al. ( 2010 ). Conclusion G . arboreum , one of the Asiatic cotton species is an important source of genes/charactersfor trait enhancement of G . hirsutum ,the predominantly cultivated cotton species worldwide. The study underscores the natural occurrence of 2n gametes in plants and highlights its relevance during wide-hybridization programs. Generation of roots in already established cotton interspecific hybrids through air layering provides another means of mitotic analyses and may be replicated in other woody plant species. Partial fertility of tetraploid hybrids allowed their utilization as pollen parent to introduce leafhopper tolerance from G . arboreum to G . hirsutum . Abbreviations CLCuD Cotton leaf curl disease CLCuV Cotton leaf curl virus CTAB Cetyltrimethylammonium bromide DAPI 4,6- diamino-2-phenylindole SSR Simple sequence Repeat TD trichome density. Declarations Conflict of interest: The authors have declared that no competing interests exist. Author Contribution Saptarshi Mondal: Conducted major field and laboratory works, data analysis, and prepared the draft manuscript; Dharminder Pathak: Conceptualization of the project, generation of the interspecific hybrids, principal investigator, funding acquisition, review and editing of the manuscript; Salil Jindal: Helped in molecular characterization of hybrids; Neha Agrawal: Helped in mitotic analysis; Mehak Gupta: Guidance in cytological analysis; Pankaj Rathore: Conceptualization of the project. Acknowledgement The interspecific hybrids used in the present study were developed under the Program Support on ‘Enhancing Durability of Resistance to Biotic Stresses in Selected Cereal and Fiber Crops through Biotechnological Approaches (BT/01/CE1B/121/01)’ funded by the Department of Biotechnology, Government of India. Ministry of Science and Technology (Grant No. 102/IFD/SAN/1307/2014-15). Thanks are due to Dr S. S. 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University","correspondingAuthor":false,"prefix":"","firstName":"Mehak","middleName":"","lastName":"Gupta","suffix":""},{"id":353431518,"identity":"22fec2d8-f809-4f1d-bf1c-e1964550c047","order_by":5,"name":"Pankaj Rathore","email":"","orcid":"","institution":"Punjab Agricultural University Regional Research Station","correspondingAuthor":false,"prefix":"","firstName":"Pankaj","middleName":"","lastName":"Rathore","suffix":""}],"badges":[],"createdAt":"2024-09-02 03:06:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5015023/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5015023/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10722-024-02220-y","type":"published","date":"2024-10-30T16:05:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66942880,"identity":"0b5e6b81-2ebc-47da-b6e5-b08a8a7ab137","added_by":"auto","created_at":"2024-10-18 09:12:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":56806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInduction of fresh roots in interspecific hybrids through air-layering\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5015023/v1/aba3f9e0b782f33b65c73774.jpg"},{"id":66942876,"identity":"99515375-5c79-4912-8ff4-1fb1c09133ef","added_by":"auto","created_at":"2024-10-18 09:12:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1446188,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of morphological traits among parents and their respective hybrids\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5015023/v1/b80991a289b8eb73bd62feab.jpg"},{"id":66942877,"identity":"ad2123f9-73cb-4403-baed-022cc107fc6d","added_by":"auto","created_at":"2024-10-18 09:12:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1040874,"visible":true,"origin":"","legend":"\u003cp\u003eChromosome number and Pollen Fertility of parents and their respective hybrids\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5015023/v1/b1ee9304a8faebe316a0d397.jpg"},{"id":66943104,"identity":"78fce3be-9444-4b2e-84c2-70da23baa284","added_by":"auto","created_at":"2024-10-18 09:20:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105040,"visible":true,"origin":"","legend":"\u003cp\u003eHybridity confirmation of the interspecific hybrids through SSR markers (M: Marker, H\u003csub\u003e1\u003c/sub\u003e: F 846, H\u003csub\u003e2\u003c/sub\u003e: LH 2108, H\u003csub\u003e3 \u003c/sub\u003eand H\u003csub\u003e4\u003c/sub\u003e: LH 2107, A\u003csub\u003e1\u003c/sub\u003e, A\u003csub\u003e2\u003c/sub\u003e and A\u003csub\u003e3\u003c/sub\u003e: LD 949, A\u003csub\u003e4\u003c/sub\u003e: LD 491, and F\u003csub\u003e1\u003c/sub\u003es of their respective parents)\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5015023/v1/f26030e857a8e1520cd73e65.jpg"},{"id":66944028,"identity":"5f6595b2-c7c5-4849-9fc0-8624ebc0d424","added_by":"auto","created_at":"2024-10-18 09:28:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":60572,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular confirmation (top) of viral DNA presence/absence in parents and their hybrids and phenotypic response (bottom) of CLCuD in parents and hybrids in 2018\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5015023/v1/81ff68298a3bc73eed3abcfe.jpg"},{"id":68206622,"identity":"41400321-7486-4ba3-afc8-1e29f7ce810b","added_by":"auto","created_at":"2024-11-04 16:33:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3580794,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5015023/v1/be2e9c31-becc-4a0a-a706-1153b5b3025b.pdf"},{"id":66942882,"identity":"476cecad-0c98-4653-8d90-8082700f5155","added_by":"auto","created_at":"2024-10-18 09:12:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1174637,"visible":true,"origin":"","legend":"","description":"","filename":"PBrevsupplementaryfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-5015023/v1/1ba07ef35cc5be046e45bf7b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Morphological, Cytological, and Molecular Characterization of Interspecific Cotton Hybrids Derived from Gossypium hirsutum and G. arboreum Crosses","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003e\u003cem\u003eG. arboreum\u003c/em\u003e is highly cross incompatible with \u003cem\u003eG. hirsutum\u003c/em\u003e.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHere, we report the development and characterization of four unique interspecific \u003cem\u003eG. hirsutum \u0026times; G. arboreum\u003c/em\u003e hybrids\u003c/li\u003e\n \u003cli\u003eOut of four hybrid plants, two were triploid (2n = 39) and the other two were tetraploids (2n = 52)\u003c/li\u003e\n \u003cli\u003eThe latter case indicates the fusion of normal n gamete from female parent and unreduced gamete 2n from male parent.\u003c/li\u003e\n \u003cli\u003eTetraploid hybrids showed partial pollen fertility which enabled their utilization as pollen donors in backcrosses\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe importance of cotton may be judged from the fact that it is cultivated in more than 80 countries across the world. Economy of several countries is directly or indirectly dependent on cotton. Cotton is grown on an area of 30\u0026ndash;36\u0026nbsp;million ha worldwide. Presently, ten countries \u003cem\u003eviz\u003c/em\u003e., Australia, Brazil, Burkina Faso, China, India, Pakistan, T\u0026uuml;rkiye, Turkmenistan, Uzbekistan and the USA produce greater than 90% of the total cotton globally (Kranthi, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Australia has the highest global cotton productivity of more than 2000 kg lint per ha which is at least 2.5 times higher than the global average productivity of cotton. Besides lint (long fibers borne on the seed coat), which is the major product and the chief source of natural fiber for textile industry, cotton is also an important source of edible oil. Factually, cotton is the fifth major source of vegetable oil worldwide (Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eGossypium\u003c/em\u003e, to which cotton belongs, includes about 50 species distributed in the arid to semi-arid regions of the tropics and subtropics (Wendel et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Four cotton species namely, \u003cem\u003eG. hirsutum\u003c/em\u003e, \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ebarbadense\u003c/em\u003e, \u003cem\u003eG. arboreum\u003c/em\u003e, and \u003cem\u003eG. herbaceum\u003c/em\u003e are cultivated, of which cotton acreage is dominated by \u003cem\u003eG. hirsutum\u003c/em\u003e (~\u0026thinsp;98%), aka Upland cotton/American cotton across the world. A longer crop duration in cotton provides a large number of insect pests and pathogens more opportunities to affect cotton at all the crop stages. The nuisance of bollworms has largely been managed through the commercialization of transgenic Bt cotton cultivars. However, this has resulted in shifting of pest profile to sap sucking insect pests such as whitefly, leafhopper, mealy bug, mirid bug, aphid etc. The whitefly epidemic in north Indian cotton growing states destroyed cotton crop on nearly 1.5\u0026nbsp;million ha during 2015 (Kumar et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, cotton leaf curl disease (CLCuD)- caused by \u003cem\u003eGeminiviruses\u003c/em\u003e and vectored by whitefly [\u003cem\u003eBemisia tabaci\u003c/em\u003e (Gennadius)] led to huge financial losses of ~\u0026thinsp;USD 5\u0026nbsp;billion between 1992\u0026ndash;1997 to Pakistan economy (Briddon \u0026amp; Markham, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Related species of cotton are invaluable sources of genes of economic importance. For example, \u003cem\u003eGossypium armourianum\u003c/em\u003e, a wild non-progenitor D-genome species has been reported to possess resistance to several biotic stresses such as pink bollworm (Brazzel \u0026amp; Martin, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1956\u003c/span\u003e), jassid (Pushpam \u0026amp; Raveendran, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), whitefly and cotton leaf curl disease (CLCuD) (Suthar et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, \u003cem\u003eG. arboreum\u003c/em\u003e (A\u003csup\u003ed\u003c/sup\u003eA\u003csup\u003ed\u003c/sup\u003e) is resistant to CLCuD (Naqvi et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), jassid (Jindal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nibouche et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Sidhu \u0026amp; Dhawan, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) and whitefly (Miyazaki et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAvailability of genetic variation is vital to crop improvement. Domestication and extensive use of limited number of genotypes in the breeding programs gradually narrowed down the genetic base of crop plants, which made them vulnerable to various biotic and abiotic stresses. Limited genetic variation among \u003cem\u003ehirsutum\u003c/em\u003e cotton cultivars and their vulnerability to pathogen/insect epidemics has been documented (Bowman et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Brubaker \u0026amp; Wendel, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Related species of crop plants are invaluable sources of genes of economic importance and can play a pivotal role in enhancing genetic diversity through the introgression of useful novel alleles in the adapted germplasm. At Punjab Agricultural University, Ludhiana, India, related \u003cem\u003eGossypium\u003c/em\u003e species, especially belonging to secondary gene pool, are being used in the pre-breeding programs. We crossed widely grown Upland cotton (\u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e) with \u003cem\u003edesi\u003c/em\u003e cotton (\u003cem\u003eG. arboreum\u003c/em\u003e) with the objective to introgress favorable alleles such as for resistance to CLCuD, leafhopper, and whitefly which are serious threats to Upland cotton cultivation in various parts of India and the world. Identification and characterization of true hybrids are of utmost importance to carry forward the introgression program and give an insight into the dominance/recessive relationships of alleles, genomic constitution of the hybrids etc. In this article, we report the development of interspecific hybrids between \u003cem\u003eG. hirsutum\u003c/em\u003e and \u003cem\u003eG. arboreum\u003c/em\u003e and their hybridity confirmation based on morphology, chromosome number, and molecular markers.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePlant materials:\u003c/h2\u003e\n \u003cp\u003eA number of \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e and \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e lines were hybridized in different combinations to develop wide-hybrids. \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e was used as the seed parent, whereas \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e served as the pollen parent in all the crosses. \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e (A\u003csup\u003ed\u003c/sup\u003eA\u003csup\u003ed\u003c/sup\u003e) belongs to the secondary gene pool of cotton and is not easily crossable with \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e. Substantial efforts are required to generate interspecific hybrids between these species. A total of 12,392 flowers of \u003cem\u003ehirsutum\u003c/em\u003e parents were pollinated to obtain the interspecific hybrids (Jindal et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Hormones (a mixture of GA3 @ 50ppm\u0026thinsp;+\u0026thinsp;NAA @ 100 ppm) were applied to the base of pedicel of the cross pollinated (\u003cem\u003eG. hirsutum\u003c/em\u003e \u0026times; \u003cem\u003eG. arboreum\u003c/em\u003e) buds for three consecutive days to enhance crossed bud retention and four true interspecific hybrids were obtained through the direct crosses. The pedigree of cotton interspecific hybrids is presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. F 846 and LH 2108 are commercial cultivars, whereas LH 2107 is an advanced line of \u003cem\u003ehirsutum\u003c/em\u003e cotton. Similarly, LD 491 and LD 949 are commercial cultivars of \u003cem\u003earboreum\u003c/em\u003e (\u003cem\u003edesi\u003c/em\u003e) cotton. All these cotton cultivars/lines have been developed at Punjab Agricultural University, Ludhiana (Punjab), India. The resulting four unique interspecific putative cotton hybrids were characterized at morphological, cytological, and molecular levels.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePedigree of the cotton interspecific F\u003csub\u003e1\u003c/sub\u003e hybrids\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS. No.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eG. hirsutum\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e(seed parent)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eG. arboreum\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e(pollen parent)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDesignation of F\u003csub\u003e1\u003c/sub\u003e plant\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF 846\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLD 949\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e-1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLH 2108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLD 949\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e-2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLH 2107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLD 949\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e-3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLH 2107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLD 491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e-4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eMorphological observations:\u003c/h2\u003e\n \u003cp\u003eThe morphological investigation comprised the following characters recorded from the parental lines and their respective interspecific F\u003csub\u003e1\u003c/sub\u003e hybrids: plant stem coloration, plant height, leaf shape, leaf lobe number, leaf nectary, leaf size, leaf trichome density, petiole length, number of serrations on epicalyx, sepal number, petal number, petal color, petal spotting, anther color, anther number, pollen viability, pollen size, and reaction to cotton leaf curl disease (CLCuD). Fully matured leaves and fresh flowers at anthesis were taken for morphological characterization. To analyze pollen fertility, flower buds at the candle stage were identified and tied with a thread at the top to prevent stray pollen admixtures. Flowers were collected in the morning on the day of anthesis between 9 am to 10 am. Freshly collected pollens were subjected to acetocarmine test, a widely followed colorimetric method of pollen viability estimation that depends on the reactions of dye with cytoplasm proteins, nuclei, and nucleic acids, to discriminate fertile pollens from sterile ones (Shekari et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Skrzypkowski et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Average pollen size was determined using micrometry method. In order to facilitate natural CLCuD inoculation under the field conditions, F 846 (a highly CLCuD susceptible Upland cotton variety) was planted in the experimental plot to provide inoculum of the CLCuV throughout the season. Further, no management of whitefly (vector of CLCuV) through the insecticides was undertaken. Adaxial surface of fully expanded leaves of parents and hybrids was examined for the occurrence of CLCuD.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eCytological observations:\u003c/h2\u003e\n \u003cp\u003eFor mitotic analyses, root tips of parental lines (\u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e and \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e) were collected from germinated seeds. However, collection of the same through seed germination was not possible for the already established interspecific hybrids under investigation. Thus, air layering (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) was followed for root induction as an alternative. Fresh milky white root tips were harvested between 8 a.m. to 10 a.m. avoiding any damage to the tip portion. Three to four root tips were collected in each micro-centrifuge tube containing 2 mM of 8-hydroxyquinoline (a metaphase arresting chemical) and then transferred to freshly prepared fixative (3 Ethanol: 1 Glacial acetic acid). The fixed root tips were enzymatically digested and \u003cem\u003eon-slide maceration\u003c/em\u003e technique (Ma et al., \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e) was followed under a stereo microscope to squash the root meristem. This preparation was examined under a phase contrast microscope. The slides with the highest mitotic index with promising chromosome spread were selected for staining with 4,6- diamino-2-phenylindole (DAPI) and visualized and photographed under a fluorescent microscope.\u003c/p\u003e\n \u003cp\u003eFor meiotic analysis, young flower buds were collected in the early morning (just after sunrise) and fixed in Carnoy\u0026rsquo;s solution (70% ethanol, chloroform, and glacial acetic acid in 6:3:1 ratio). Taking anthers from the fixed buds, squash technique was followed to prepare slides with 2% acetocarmine as a staining agent. Cover slip was placed carefully on it and a shy heat treatment was provided to facilitate chromosomal spreading.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eMolecular analyses:\u003c/h2\u003e\n \u003cp\u003eMoisture free tender leaves were collected from the parental lines and F\u003csub\u003e1\u003c/sub\u003e hybrids for genomic DNA extraction by Cetyltrimethylammonium bromide (CTAB)-method following Saghai-Maroof et al. (1984). Extracted DNA was purified from impurities like phenolics, proteins etc. DNA quality and quantity were assessed using Nano-Drop Spectrophotometer (Thermo Scientific NanoDrop\u0026trade; 8000 Spectrophotometer). Eight polymorphic cotton specific SSR markers were employed for hybridity confirmation of the putative interspecific hybrids (Supplementary Tables: ST1, ST3, and ST4). Seven of these eight SSR primers were specific to A-sub genome of cotton.\u003c/p\u003e\n \u003cp\u003eVirus-specific universal primer A and primer B (Deng et al., \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e) were used to detect the presence/absence of the begomovirus component of CLCuD complex (ST2). Amplified PCR products for hybridity confirmation were resolved on 3% agarose gel and the presence of virus specific bands was detected on 2% agarose gel. PCR reaction was followed as described by (Deng et al., \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e) with an annealing temperature of 52\u0026deg;C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis:\u003c/h2\u003e\n \u003cp\u003eGraphical representations and mean separation using Fisher\u0026rsquo;s protected LSD at \u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for the quantitative parameters were carried out in SAS JMP Pro 17.0.0 software.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMorphological characterization:\u003c/h2\u003e \u003cp\u003eComparison of morphological characters of \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e parents \u003cem\u003eviz.\u003c/em\u003e, F 846, LH 2108, and LH 2107; \u003cem\u003eG. arboreum\u003c/em\u003e parents namely LD 949 and LD 491 with their respective hybrids are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Variable phenotypic expression was observed among the F\u003csub\u003e1\u003c/sub\u003e hybrids. They manifested either dominance or intermediate expression for various morphological traits. All the female parents (LH 2107, LH 2108, and F 846) and one of the male parents LD 491 have green stems, whereas LD 949 possesses deep red stem coloration. Stems of F\u003csub\u003e1\u003c/sub\u003e-1 (F 846 \u0026times; LD 949) and F\u003csub\u003e1\u003c/sub\u003e-2 (LH 2108 \u0026times; LD 949) were observed to be red, which is an indication of a successful cross as the character was inherited from male parent. Interestingly, F\u003csub\u003e1\u003c/sub\u003e-3 (LH 2107 \u0026times; LD 949) even having the same parent as those of F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 was found to possess a green stem like its \u003cem\u003ehirsutum\u003c/em\u003e parent. This hybrid is actually tetraploid with 2n\u0026thinsp;=\u0026thinsp;52 (explained later).\u003c/p\u003e \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 recorded intermediate leaf shape, whereas, other two F\u003csub\u003e1\u003c/sub\u003es (F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4) were more similar to their maternal parent i.e. \u003cem\u003eG. hirsutum\u003c/em\u003e cv. LH 2107. All three \u003cem\u003ehirsutum\u003c/em\u003e parents and LD 949 recorded similar leaf lengths, but LD 491 registered a lower leaf length. Both the tetraploid hybrid plants (F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4) possessed relatively smaller leaf lengths than that of their parents as well as F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 (F\u003csub\u003e1\u003c/sub\u003e-4\u0026thinsp;\u0026lt;\u0026thinsp;F\u003csub\u003e1\u003c/sub\u003e-3\u0026thinsp;\u0026lt;\u0026thinsp;F\u003csub\u003e1\u003c/sub\u003e-2\u0026thinsp;\u0026le;\u0026thinsp;F\u003csub\u003e1\u003c/sub\u003e-1). Average petiole lengths of all the \u003cem\u003eG. hirsutum\u003c/em\u003e parents were higher than that of \u003cem\u003eG. arboreum\u003c/em\u003e parents. Significant differences were observed between both the F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 with their respective maternal (\u003cem\u003eG. hirsutum\u003c/em\u003e) parents; however, F\u003csub\u003e1\u003c/sub\u003e-2 was at par with its \u003cem\u003eG. arboreum\u003c/em\u003e parent (LD 949) for petiole length. On the other hand, F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4 possessed significantly smaller petioles as compared to their respective parents and other two hybrids (F\u003csub\u003e1\u003c/sub\u003e-4\u0026thinsp;=\u0026thinsp;F\u003csub\u003e1\u003c/sub\u003e-3\u0026thinsp;\u0026lt;\u0026thinsp;F\u003csub\u003e1\u003c/sub\u003e-2\u0026thinsp;=\u0026thinsp;F\u003csub\u003e1\u003c/sub\u003e-1). All the parental lines, except LD 949 possessed nectary on the mid-rib of the abaxial side of leaves, whereas all the F\u003csub\u003e1\u003c/sub\u003e hybrids were found to possess nectary. In the present study, tetraploid Upland cotton genotypes recorded significantly lesser TD as compared to \u003cem\u003edesi\u003c/em\u003e cotton parents. Young leaves of all the test entries were found to possess higher TD than that of mature leaves. A significant (at \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) positive correlation (r\u0026thinsp;=\u0026thinsp;0.73*) was found between TD of mature and young leaves. All the interspecific cotton hybrids recorded a higher TD than Upland cotton parents, however, lesser TD in the younger leaves than that of the \u003cem\u003earboreum\u003c/em\u003e cotton parents. F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 possessed a significantly higher TD than F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4 in young leaves. Nonetheless, the per cent reduction of TD at later growth stages was much lesser in F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4, due to which TD of all four hybrids did not show significant differences in mature leaves (Supplementary Fig: SF1).\u003c/p\u003e \u003cp\u003eFemale parents, F 846, LH 2108, and LH 2107 possessed cream-colored petals, whereas, male parents LD 949 and LD 491 had bicolored and white (colorless) petals, respectively. F\u003csub\u003e1\u003c/sub\u003e hybrids showed variable expressions for petal color. In the present investigation, male parents as well as all four interspecific F\u003csub\u003e1\u003c/sub\u003e hybrids manifested petal spot phenotype. Pollens in female parents were cream-colored, whereas, male parents LD 491 and LD 949 possessed yellow and dark yellow pollens, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 had yellow pollens, whereas F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4 possessed cream-colored anthers. Pollen viability test revealed high pollen fertility of parental lines, but a little fertility (F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4) to complete sterility (F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2) in the interspecific hybrids. Tetraploid hybrids (F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4) recorded an average pollen fertility of 40.5% and 17.8%, respectively Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. All three \u003cem\u003ehirsutum\u003c/em\u003e parents possessed larger pollens (119.4- 122.3 \u0026micro;m) than \u003cem\u003edesi\u003c/em\u003e cotton genotypes and interspecific hybrids. Due to the pollen sterility, F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 recorded the lowest pollen size, whereas a higher pollen fertility of F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4 was also reflected through their larger pollen size Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Pollen size of F\u003csub\u003e1\u003c/sub\u003e-3 was even significantly higher than its male parent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; SF2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of morphological characters among parents and interspecific hybrids\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF 846\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLH 2108\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLH 2107\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLD 949\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLD 491\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e-1\u003c/p\u003e \u003cp\u003e(F846 \u0026times; LD949)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e-2\u003c/p\u003e \u003cp\u003e(LH 2108 \u0026times; LD949)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e-3\u003c/p\u003e \u003cp\u003e(LH 2107 \u0026times; LD949)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e-4\u003c/p\u003e \u003cp\u003e(LH 2107 \u0026times; LD 491)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStem coloration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGreen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGreen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGreen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eGreen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGreen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant height (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e166\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e172.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e195\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaf shape\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePalmate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePalmate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePalmate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDigitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDigitate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIntermediate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIntermediate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePalmate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ePalmate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNectaries on mid-rib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage leaf length (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.9\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.6\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.8\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.6\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.8\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.4\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6.6\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrichome density (ML)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e82.78\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e83.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e83.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e80.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e85.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e85.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrichome density (YL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e87.67\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.67\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86.33\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e178.78\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e177.78\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e136.89\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e130.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e113.56\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e108.0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePetiole length (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.3\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.6\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5.9\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.9\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4.3\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlower petal color\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCream\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCream\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCream\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBicolor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWhite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBicolor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eBicolor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCream with pink margins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCream with pink margins\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlower petal\u003c/p\u003e \u003cp\u003espotting\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnther color\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCream\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCream\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCream\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDark Yellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCream\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCream\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePollen fertility (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e87.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e40.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e17.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage pollen size (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e121.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e119.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e122.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e91.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e89.6\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e79.4\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e82.2\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e103.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e91.4\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLCuD symptoms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCytological observations:\u003c/h2\u003e \u003cp\u003eCytological characterization of F\u003csub\u003e1\u003c/sub\u003e hybrids and their parents was carried out by both mitotic and meiotic analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMitosis:\u003c/h2\u003e \u003cp\u003eAir-layering of the F\u003csub\u003e1\u003c/sub\u003e plants with rooting hormone helped to induce fresh roots, which were collected for mitotic slide preparation. Slide preparation from freshly collected roots from Upland cotton and \u003cem\u003edesi\u003c/em\u003e cotton parental lines along with their respective F\u003csub\u003e1\u003c/sub\u003e hybrids revealed the ploidy level of the plants. As expected, \u003cem\u003eG. hirsutum\u003c/em\u003e (LH 2108 and LH 2107) and \u003cem\u003eG. arboreum\u003c/em\u003e (LD 949 and LD 491) genotypes were found to have 2n\u0026thinsp;=\u0026thinsp;52 and 2n\u0026thinsp;=\u0026thinsp;26 chromosomes, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the present study, accordance, as well as deviation from the expected ploidy status of interspecific \u003cem\u003eGossypium\u003c/em\u003e species hybrids, were observed. F\u003csub\u003e1\u003c/sub\u003e-2 was found to possess 2n\u0026thinsp;=\u0026thinsp;3x\u0026thinsp;=\u0026thinsp;39 chromosomes. F\u003csub\u003e1\u003c/sub\u003e-1 is expected to have the same chromosome number as that of F\u003csub\u003e1\u003c/sub\u003e-2 based on the observation that F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 have the same morphological features such as pollen sterility, leaf shape, stem color, petal color, intensity of petal spot, pollen color etc. (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Interestingly, F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4 both registered somatic chromosome numbers of 2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;52 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMeiosis:\u003c/h2\u003e \u003cp\u003eMeiotic analysis on the pollen mother cells of interspecific hybrids revealed several interesting observations. Meiotic irregularities were observed in both F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 at the different phases of the meiosis \u003cem\u003eviz.\u003c/em\u003e early metaphase I, metaphase I and metaphase II, and late anaphase in hybrids (SF3). The presence of univalent chromosomes (as indicated with arrows in SF3) which were lagging to reach the equatorial position was observed at early metaphase-I in both triploid F\u003csub\u003e1\u003c/sub\u003e plants. Normal metaphase-II and abnormal metaphase-II were observed in F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-2, respectively. Cytokinesis after the first meiotic division and the formation of dyads in F\u003csub\u003e1\u003c/sub\u003e-2 were observed. Interestingly, the occurrence of both radial (tetragonal) and tetrahedral tetrad was observed in this interspecific hybrid (SF3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMolecular characterization:\u003c/h2\u003e \u003cp\u003eIn the present study, besides phenotypic and cytological characterization, hybridity confirmation was also carried out using cotton specific SSR markers to provide another line of evidence supporting the hybrid status of \u003cem\u003eG. hrsutum \u0026times; G. arboreum\u003c/em\u003e F\u003csub\u003e1\u003c/sub\u003es. Eight SSR primers \u003cem\u003eviz\u003c/em\u003e., BNL 0946, BNL 1679, BNL 2652, BNL 2921, BNL 3888, NAU 0922, NAU 1222, and CIR 0183 were employed for hybridity confirmation of the interspecific cotton F\u003csub\u003e1\u003c/sub\u003es (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eReaction to cotton leaf curl disease (CLCuD):\u003c/h2\u003e \u003cp\u003ePhenotypic reaction to CLCuD in parents and their respective F\u003csub\u003e1\u003c/sub\u003es was observed. Female \u003cem\u003ehirsutum\u003c/em\u003e parents were found to possess clear symptoms, whereas male parents (\u003cem\u003eG. arboreum\u003c/em\u003e) were symptomless (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). All the four interspecific hybrids were initially found to be CLCuD susceptible and exhibited typical symptoms of the disease during 2017 (Pathak et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, in the next season (2018), F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 became symptomless, whereas F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4 maintained symptoms of CLCuD such as interveinal thickening, leaf curling, and the presence of enation on the underside of the leaves (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCLCuD reaction of the test genotypes was also confirmed at the molecular level through the amplification of viral DNA using virus specific primers. Virus specific bands were amplified in all the \u003cem\u003eG. hirsutum\u003c/em\u003e parents namely, F 846, LH 2108, and LH 2107; whereas no virus specific band was observed in \u003cem\u003eG. arboreum\u003c/em\u003e parental lines (LD 949 and LD 491). Two of the interspecific hybrids namely, F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 did not show the amplification of virus specific bands, whereas amplification of the same was found in the other two hybrids (F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Amplified viral DNA of the susceptible genotypes was approximately 500 bp in size.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eUpland cotton genotypes owing to their narrow genetic base are susceptible to many pathogens and insect pests and demand further improvements. On the other hand, \u003cem\u003edesi\u003c/em\u003e cotton species such as \u003cem\u003eG. arboreum\u003c/em\u003e serve as valuable genetic resources for the breeders to perform interspecific crosses for broadening the narrow genetic base of Upland cotton. However, reports on successful interspecific hybrids are limited due to fertilization barriers between different species. Therefore, identification of true interspecific hybrids, a clear understanding of their ploidy level, and genetic constitution help formulate appropriate breeding strategies, facilitating their further utilization in enhancing trait value of the recipient species. In the present study, we discuss the successful generation and characterization of four unique interspecific hybrids at the morphological, cytological, and molecular levels in comparison to their respective parents.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMorphological characterization:\u003c/h2\u003e \u003cp\u003eThe phenotypic expression of F\u003csub\u003e1\u003c/sub\u003es was variable. Some of the characters showed dominance, whereas others displayed intermediate expression in the interspecific hybrids. Occurrence of red stems in triploid hybrids (F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2) indicated the manifestation of a dominant character from the male parent (LD 949), showing consistency with the findings of Tahir et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and Chen et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Interestingly, showing contradictions to the above-mentioned reports, the tetraploid F\u003csub\u003e1\u003c/sub\u003e-3 (male parent LD 949) possessed green stems.Both F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4 possessed significantly smaller leaf and petiole lengths than their respective parents and the other two hybrids, which counters the finding of Ahmad et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) in 2(\u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e) \u0026times; \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e where the average leaf size was observed to be larger in interspecific hybrid as compared to the \u003cem\u003earboreum\u003c/em\u003e parent.\u003c/p\u003e \u003cp\u003eCotton plants possess minute hair like structures known as trichomes on most of the aerial parts. Trichome density (TD) on leaves is correlated with the varying degree of resistance/ susceptibility to sucking insect pests, as well as drought resistance (Karkk\u0026auml;inen et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Meagher et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). In this study, the presence of higher TD in \u003cem\u003edesi\u003c/em\u003e cotton entries than American cotton genotypes for both types of leaves (mature and young) was observed (SF1). A higher tolerance to sucking insect pests including leaf hoppers due to the presence of denser trichomes in \u003cem\u003eG. arboreum\u003c/em\u003e than \u003cem\u003eG. hirsutum\u003c/em\u003e has been reported (Aherkar et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jindal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Also, the occurrence of denser trichomes on the young leaves of all the test entries than that of the mature leaves supports the findings of Wright et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), Turley and Vaughn (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and Grover et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Here we found a strong positive correlation between TD of mature and young leaves (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.73*) which was consistent with the findings of (Grover et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Although triploid F\u003csub\u003e1\u003c/sub\u003es were found to possess a significantly higher TD than the tetraploid F\u003csub\u003e1\u003c/sub\u003es in young leaves, the latter two showed a much lesser reduction of TD at later growth stages (SF1). Consequently, TD of all four hybrids was the same in mature leaves.\u003c/p\u003e \u003cp\u003ePresence of petal spots in all four interspecific cotton hybrids served as a major morphological marker to confirm their hybridity supporting paternal inheritance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The observation is consistent with the findings of Tahir et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and Chen et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) in the \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum \u0026times; G\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e hybrids, Saravanan et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) in \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum \u0026times; G\u003c/em\u003e. \u003cem\u003eraimondii\u003c/em\u003e hybrids, and Pushpam and Raveendran (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) in \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum \u0026times; G\u003c/em\u003e. \u003cem\u003earmourianum\u003c/em\u003e hybrids. Pollen fertility test confirmed complete sterility of the triploid hybrids, which is expected from a cross between tetraploid and diploid parents and also corresponds to Ahmad et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, it was interesting to find F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4 to have an average pollen fertility of 40.5% and 17.8%, respectively, which does not align with the report of Ahmad et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) where only 1.90% of pollen fertility was observed in tetraploid interspecific hybrids. The higher male fertility of two tetraploid hybrids allowed their utilization as pollen parents in backcrossing with Upland cotton. Development of backcross derivatives using these fertile hybrids followed by their screening for leafhopper tolerance has been reported by Jindal et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is worth mentioning that while the commercialization of Bt cotton could effectively manage the menace of bollworms, it has shifted the pest profile to sap sucking insect-pests such as leafhopper, whitefly, mealy bug, cotton aphid, mirid bug etc. The Upland cotton Bt hybrids cultivated in India are susceptible to sap-feeding insects (Kranthi \u0026amp; Stone, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Leafhopper is one of the most economically important sap feeding insects of cotton in the country and can cause 25\u0026ndash;45% reduction in seed cotton yield (Kalyan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We found that two genes introgressed from \u003cem\u003earboreum\u003c/em\u003e (located on chromosomes A5 and A11) either individually or in combination conferred tolerance to leafhopper in the interspecific backcross derivatives (Jindal et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This also underscores the importance of 2n gametes formation in nature as well as doubling the chromosome number of parent with lower ploidy as a breeding strategy in the wide-hybridization programs so as to increase the probability of developing an interspecific hybrid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCytological observations:\u003c/h2\u003e \u003cp\u003eCytological observations on interspecific hybrids provide insights into their genomic constitution, ploidy status, and meiotic behavior. It is quite important to devise a future strategy and ensure the proper utilization of the interspecific hybrids. In this current investigation, mitotic analysis confirmed the ploidy status of interspecific hybrids. It was made possible by inducing roots through air-layering (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The somatic chromosome number of \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e genotypes namely, LH 2108 and LH 2107 (2n\u0026thinsp;=\u0026thinsp;52) and \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e cv. LD 949 and LD 491(2n\u0026thinsp;=\u0026thinsp;26) was found to be consistent with the findings of Beasley (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1940\u003c/span\u003e), Mehetre et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) Kale et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), Ahmad et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), Tahir et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), Newaskar et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and Montes et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this present study, 39 somatic chromosomes of F\u003csub\u003e1\u003c/sub\u003e-2 showed accordance with the findings of Ahmad et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), Tahir et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and Chen et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) who obtained putative triploid hybrids from crosses between \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e and \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e. Based on several morphological similarities such as leaf shape, stem color, petal color, anther color etc., F\u003csub\u003e1\u003c/sub\u003e-1 is expected to have the same chromosome number as that of F\u003csub\u003e1\u003c/sub\u003e-2 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Deviation from the expected chromosome count (2n\u0026thinsp;=\u0026thinsp;3x\u0026thinsp;=\u0026thinsp;39) in F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4, where both registered a somatic chromosome number of 2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;52, may question their true hybridity drawing attention towards the probability of self-fertilization of maternal parents (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, the morphological (for example, the presence of prominent petal spot in these F\u003csub\u003e1\u003c/sub\u003e hybrids) as well as molecular characterization of these F\u003csub\u003e1\u003c/sub\u003e plants present unequivocal evidence against that perplexity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Triploidy of two interspecific hybrids points out the formation of normal haploid gametes from both parental species, whereas tertaploidy of two F\u003csub\u003e1\u003c/sub\u003es seeks an explanation for this deviation. One probable reason can be the formation of unreduced (2n\u0026thinsp;=\u0026thinsp;26) gametes from the diploid male parent \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e and fertilization with normal female gamete (n\u0026thinsp;=\u0026thinsp;26) of \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e resulting into F\u003csub\u003e1\u003c/sub\u003e plants with 52 somatic chromosome number. \u003cem\u003eIn-situ\u003c/em\u003e hybridization of these interspecific hybrids may provide more insight to this question. Formation of unreduced gametes in low frequency is common in plants. Diverse mechanisms for unreduced gamete formation including premeiotic doubling of chromosome number, complete loss of the first or second meiotic division, and defects in meiotic cell plate formation, spindle orientation, or cytokinesis have been observed in flowering plants (Mason \u0026amp; Pires, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In cotton, Sheidai (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) reported the occurrence of larger unreduced pollen grain in \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e cv. C-200 (R). Similarly, Noormohammadi et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) published a report indicating the occurrence of relatively larger unreduced pollens (87\u0026ndash;136 \u0026micro;m) as compared to normal (n) pollens (63-101.7 \u0026micro;m). More recently, Montes et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) obtained an interspecific hybrid of \u003cem\u003eG\u003c/em\u003e. \u003cem\u003eherbaceum\u003c/em\u003e \u0026times; \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e, having somatic chromosome number of 52 (A\u003csup\u003ed\u003c/sup\u003eA\u003csup\u003ed\u003c/sup\u003eA\u003csup\u003et\u003c/sup\u003eD\u003csup\u003et\u003c/sup\u003e) resulted from a fertilization of unreduced female gamete (2n) of \u003cem\u003eherbaceum\u003c/em\u003e with normal pollen of \u003cem\u003ehirsutum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eMeiotic irregularities have been reported in several interspecific cotton hybrids (Konan et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Newaskar et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Skovsted, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1934\u003c/span\u003e) and this present investigation was not an exception. The observation of abnormal metaphase II in this experiment corresponds to the finding in \u003cem\u003eSalix alba\u003c/em\u003e by Khalili et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) who described this phenomenon as the fusion of non-sister chromatids in a single pole due to the partial separation of spindle. The presence of univalent chromosomes at the early metaphase of both F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 indicates the transmission of D\u003csup\u003et\u003c/sup\u003e genomes from their maternal parents (A\u003csup\u003et\u003c/sup\u003eA\u003csup\u003et\u003c/sup\u003eD\u003csup\u003et\u003c/sup\u003eD\u003csup\u003et\u003c/sup\u003e) only (SF3). Probable fate of such univalents is eventual degeneration which might be the prime reason for the sterility of those plants. Whereas a normal metaphase-II in tetraploid F\u003csub\u003e1\u003c/sub\u003e-3 (A\u003csup\u003et\u003c/sup\u003eD\u003csup\u003et\u003c/sup\u003eA\u003csup\u003ed\u003c/sup\u003eA\u003csup\u003ed\u003c/sup\u003e) might be the reason for their pollen fertility. Although cotton is a eudicot, the formation of dyads and separation pattern of chromatids in anaphase II (that suggests tetragonal tetrad formation) of F\u003csub\u003e1\u003c/sub\u003e-2 are the key characteristic features of a monocot (SF3). However, interestingly, the occurrence of both radial (tetragonal) and tetrahedral tetrad was observed in this interspecific hybrid. A similar observation for the dyad formation was also reported by Wu et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) in interspecific hybrid between \u003cem\u003eG\u003c/em\u003e. \u003cem\u003eherbaceum\u003c/em\u003e and \u003cem\u003eG\u003c/em\u003e. \u003cem\u003eraimondii\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMolecular confirmation of hybridity and CLCuV:\u003c/h2\u003e \u003cp\u003eSSR markers have been extensively used for fingerprinting of cultivars and hybridity confirmation. Several reports on the use of SSR markers for hybridity confirmation in cotton are available (Mehetre et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Tahir et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the present investigation, eight SSR primer pairs (BNL 0946, BNL 1679, BNL 2652, BNL 2921, NAU 0922, BNL 3888, NAU 1222, and CIR 0183) unambiguously confirmed the hybrid status of the interspecific \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehrsutum\u003c/em\u003e \u0026times; \u003cem\u003eG. arboreum\u003c/em\u003e hybrids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Chen et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), Tahir et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and Virk (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) also employed SSR primer pairs to confirm the hybrid status of putative interspecific F\u003csub\u003e1\u003c/sub\u003es obtained from \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e and \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e crosses.\u003c/p\u003e \u003cp\u003eCotton leaf curl disease is reported to significantly reduce the seed cotton yield and emerged as one of the most serious threats to Upland cotton cultivation in north-western India and Pakistan (Monga et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Nazeer et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sattar et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Clear symptoms in \u003cem\u003ehirsutum\u003c/em\u003e parents and symptomless male parents (\u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e) showed similarity to the previous reports (Iqbal et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tahir et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The interspecific hybrids between \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e \u0026times; \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e have been reported to be resistant to CLCuD (Ahmad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Tahir et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In our study, although susceptibility of all four hybrids toward CLCuD was recorded during 2017 (Pathak et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), F\u003csub\u003e1\u003c/sub\u003e-1 and F\u003csub\u003e1\u003c/sub\u003e-2 did not show any symptoms during 2018 and later on, whereas F\u003csub\u003e1\u003c/sub\u003e-3 and F\u003csub\u003e1\u003c/sub\u003e-4 maintained symptoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Molecular confirmation for the presence/absence of CLCuD in the present study aligned with the phenotypic observations. Amplification of viral DNA of the susceptible genotypes (female parents, F\u003csub\u003e1\u003c/sub\u003e-3, and F\u003csub\u003e1\u003c/sub\u003e-4,) produced bands of approximately 500 bp each. These results corroborate the findings of Deng et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), Maruthi et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and Mahesh et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e, one of the Asiatic cotton species is an important source of genes/charactersfor trait enhancement of \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e,the predominantly cultivated cotton species worldwide. The study underscores the natural occurrence of 2n gametes in plants and highlights its relevance during wide-hybridization programs. Generation of roots in already established cotton interspecific hybrids through air layering provides another means of mitotic analyses and may be replicated in other woody plant species. Partial fertility of tetraploid hybrids allowed their utilization as pollen parent to introduce leafhopper tolerance from \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e to \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLCuD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCotton leaf curl disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLCuV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCotton leaf curl virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCTAB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCetyltrimethylammonium bromide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e4,6- diamino-2-phenylindole\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSSR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSimple sequence Repeat\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etrichome density.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest:\u003c/h2\u003e \u003cp\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSaptarshi Mondal: Conducted major field and laboratory works, data analysis, and prepared the draft manuscript; Dharminder Pathak: Conceptualization of the project, generation of the interspecific hybrids, principal investigator, funding acquisition, review and editing of the manuscript; Salil Jindal: Helped in molecular characterization of hybrids; Neha Agrawal: Helped in mitotic analysis; Mehak Gupta: Guidance in cytological analysis; Pankaj Rathore: Conceptualization of the project.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe interspecific hybrids used in the present study were developed under the Program Support on \u0026lsquo;Enhancing Durability of Resistance to Biotic Stresses in Selected Cereal and Fiber Crops through Biotechnological Approaches (BT/01/CE1B/121/01)\u0026rsquo; funded by the Department of Biotechnology, Government of India. Ministry of Science and Technology (Grant No. 102/IFD/SAN/1307/2014-15). Thanks are due to Dr S. S. Banga, ICAR- National Professor, Department of Plant Breeding and Genetics, Punjab Agricultural University (PAU), Ludhiana, India for providing us facility to conduct cytological experiments in the DBT Centre of Excellence on Brassicas, Punjab Agricultural University, Ludhiana, India.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAherkar, S. S., Deshmukh, S. B., Konde, N. M., Paslawar, A. N., Joshi, T., Messmer, M. M., \u0026amp; Riar, A. (2023). 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The Crop Journal, \u003cem\u003e2\u003c/em\u003e(5), 272\u0026ndash;277. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cj.2014.06.009\u003c/span\u003e\u003cspan address=\"10.1016/j.cj.2014.06.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genetic-resources-and-crop-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gres","sideBox":"Learn more about [Genetic Resources and Crop Evolution](https://www.springer.com/journal/10722)","snPcode":"10722","submissionUrl":"https://submission.nature.com/new-submission/10722/3","title":"Genetic Resources and Crop Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5015023/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5015023/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiploid cotton \u003cem\u003eGossypium arboreum\u003c/em\u003e (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;26, A\u003csup\u003ed\u003c/sup\u003eA\u003csup\u003ed\u003c/sup\u003e) is a valuable genetic resource to improve widely cultivated American cotton \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;52, A\u003csup\u003et\u003c/sup\u003eA\u003csup\u003et\u003c/sup\u003eD\u003csup\u003et\u003c/sup\u003eD\u003csup\u003et\u003c/sup\u003e). In this study, successful generation of four unique interspecific hybrids (\u003cem\u003eG. hirsutum\u003c/em\u003e \u0026times; \u003cem\u003eG. arboreum\u003c/em\u003e) was confirmed through morphological, cytological, and molecular characterization. The morphological evaluation included different stem and leaf characters, trichome density, floral characters, and cotton leaf curl disease reaction. Interspecific hybrids were recorded with either complete dominance of some characters or an intermediate expression. However, variation among the F\u003csub\u003e1\u003c/sub\u003es was observed for some traits, especially for pollen size and fertility. Two of the four F\u003csub\u003e1\u003c/sub\u003es were found to possess relatively bigger pollen sizes with partial fertility which enabled their utilization as male parents in backcrosses. Mitotic analysis of already established F\u003csub\u003e1\u003c/sub\u003es was made possible through the induction of fresh roots by air layering. Of the four hybrid plants, two were triploid (2n\u0026thinsp;=\u0026thinsp;39) and the other two were tetraploids (2n\u0026thinsp;=\u0026thinsp;52), the latter case probably was a result of the fusion of normal n (A\u003csup\u003et\u003c/sup\u003eD\u003csup\u003et\u003c/sup\u003e) gametes from female parent (\u003cem\u003eG. hirsutum\u003c/em\u003e) and unreduced gametes 2n (A\u003csup\u003ed\u003c/sup\u003eA\u003csup\u003ed\u003c/sup\u003e) from male parent (\u003cem\u003eG. arboreum\u003c/em\u003e). Meiotic irregularities in interspecific hybrids were also observed. Successful amplification of polymorphic parental bands in all four F\u003csub\u003e1\u003c/sub\u003es using simple sequence repeat markers added another line of evidence supporting their hybridity. Results of this experiment provided useful insight and base material to introgress leaf-hopper tolerance from \u003cem\u003eG\u003c/em\u003e. \u003cem\u003earboreum\u003c/em\u003e to \u003cem\u003eG\u003c/em\u003e. \u003cem\u003ehirsutum\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Morphological, Cytological, and Molecular Characterization of Interspecific Cotton Hybrids Derived from Gossypium hirsutum and G. arboreum Crosses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-18 09:12:39","doi":"10.21203/rs.3.rs-5015023/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-13T06:04:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-13T05:58:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-12T01:39:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280474759368717224695669846644532662121","date":"2024-09-02T09:31:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337241077606248669929865823789101131701","date":"2024-09-02T08:54:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-02T08:48:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-02T07:27:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-02T07:27:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genetic Resources and Crop Evolution","date":"2024-09-02T03:03:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genetic-resources-and-crop-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gres","sideBox":"Learn more about [Genetic Resources and Crop Evolution](https://www.springer.com/journal/10722)","snPcode":"10722","submissionUrl":"https://submission.nature.com/new-submission/10722/3","title":"Genetic Resources and Crop Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"257f993c-a4b7-4fd8-abcd-6112a5e08922","owner":[],"postedDate":"October 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-04T16:22:56+00:00","versionOfRecord":{"articleIdentity":"rs-5015023","link":"https://doi.org/10.1007/s10722-024-02220-y","journal":{"identity":"genetic-resources-and-crop-evolution","isVorOnly":false,"title":"Genetic Resources and Crop Evolution"},"publishedOn":"2024-10-30 16:05:04","publishedOnDateReadable":"October 30th, 2024"},"versionCreatedAt":"2024-10-18 09:12:39","video":"","vorDoi":"10.1007/s10722-024-02220-y","vorDoiUrl":"https://doi.org/10.1007/s10722-024-02220-y","workflowStages":[]},"version":"v1","identity":"rs-5015023","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5015023","identity":"rs-5015023","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}