Enhanced production of doubled haploid lines from parthenogenetic Iranian melon plants obtained of irradiated pollen (Cucumis melo L.

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1 International Research Journal of Applied and Basic Sciences. Vol., (8), , 2012 Available online at www. irjabs.com ISSN X 2012 Enhanced production of doubled haploid lines from parthenogenetic Iranian melon plants obtained of irradiated pollen (Cucumis melo L.) Mostafa Nasertorabi 1, Esmaeil Madadkhah 1, Hamid Moghbeli 2, Ali Roain, Ecehagh Moghbeli 1- Department of Horticulture, College of Aboureihan, University of Tehran, Tehran, Iran. 2- Department of Plant Science, Faculty of Agriculture, University of Jiroft, Jiroft, Iran. - Department of Horticulture. University of Islamic Azad, Jiroft Branch Jiroft, Iran. - Department of Horticulture, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. *Corresponding Author nasertorabi@ut.ac.ir Abstract Haploid and doubled haploid plants are valuable materials for plant breeding purposes such as resistance to many fungal and viral diseases that affect this crop. We produced 8 parthenogenetic melon plantlets via pollination with -irradiated pollen witch 550 Gray of dose cloned them by nodal cuttings, and tested the effects of in vitro and in vivo doubling treatment on survival, ploidy and fruit recovery. Chromosome number was counted in root tip cells from in vitro haploid plants.the most effective procedure for explants recovery was in vitro treatment of shoot tip explants with 500 mg/l colchicine for 12 h in standard medium and plant regeneration from nodal explants treated with 500 mg/l colchicine for 12 h was increased from to 76% by removing apical dominant one week before colchicine treatments.! " # $ regeneration. The most doubling occured in 2h treatment from soot tip explant. This treatment gave 9% survived of explants and 20% conservation to diploidy. In in vivo treatments, 2000 mg/l colchicine for 2h treatment was the effectiveness than other dose with 18% doubling rate. Keywords: Cucumis melo, Colchicine, Doubled haploid, Growth regulators, Parthenogenesis Abbreviations: ABA Abscisic acid, BA Benzyl adenine, DH Doubled haploid, DMSO Dimethyl sulfoxide, IAA Indole-acetic acid. Introduction Recently pure lines can be produced in a short time using in vitro techniques and therefore put away several years of conventional plant breeding program (Bajaj 1990). Doubled haploids (DH) can aid plant breeding programs by reducing the time required to obtain homozygous inbred lines. In melon, produce haploids of melon by anther or ovule culture were not successful (Dryanovska and Ilieva 198) but haploids have been recovered both by culture of unpollinated ovaries (Ficcadenti et al.1999) and from!"!, 1999; Lotfi et al. 200; Lim and Earle 2008). Such parthenogenetic haploids rarely show spontaneous doubling so chromosome Doubling using colchicines or other antimitotic agentsis required to obtain fruit set and DH seeds. Various strategies for colchicines treatment of parthenogenetic melon plants have been described (Caglarand Abak Koksal et #$$# Yashiro et al. #$$# Lotfi et al. 200, Lim and Earle 2008 and 2009) with widely disparate rates of conversion to diploidy (from 0 to 100%, of ten with small sample sizes). Lime and Erle 2008, reported in vitro treatment of cm shoot tips with 500 mg/l colchicine for h was selected for the highest duplication rate and nodes had poor survival and low doubling rates. They reported pollen counts are easy and efficient way for ploidy levels determination. Lim and Earle 2009 reported that transfer of nodes treated with colchicine for 12 h to E20A medium containing a combination of growth regulators (5 µm IAA, 5 µm BA, 1 µm ABA and 0 µm AgNO) increased their regeneration rate from 0 to 88% at weeks. In this study we described the production of parthenogenic haploid Iranian melon plants and improved procedure for recovery of

2 Intl. Res. J. Appl. Basic. Sci. Vol., (8), , 2012 chromosome doubling techniques to parthenogenetic melon obtained through gynogenesis after pollination with irradiated pollen and to compare the effects of these treatments on survival, ploidy, pollen production, and recovery of fruit and viable seed from the treated materials. Materials and Methods Plant materials and general methods Six cultivars of Iranian melon khatooni, Zard ivanake, sabz ivanake, garmak, saveh and samsoori (Cucumis melo L.) and one alien cultivar used as genotypes materials. Haploid/mixoploid parthenogenetic melon (Cucumis melo L.) plantlets were obtained from seeds produced after pollination with irradiated pollen and were maintained in Magenta boxes of E20A medium (Sauton and Dumas de Vaulx 1987) solidified with 0.8% Phytoblend (Caisson Laboratories, Inc., North Logan, UT).The procedure for recovery of plants from partheno genetic embryos was based on Lim and Earle (2008) with some modifications. In vitro colchicines treatments Colchicine (Sigma) was dissolved in dimethyl sulfoxide (DMSO), diluted in distilled water to 10 mg/ml, filter sterilized. The stock was further diluted in sterile distilled water to final concentrations of 500 mg/l (Lim and Earle 2008, 2009). Water containing 0.2% DMSO only was used as a control for effects of DMSO on regeneration. Rapidly growing in vitro plantlets were stripped of leaves. types of explants: shoot tips, nodes and nodal growthed (the nodal explants that their apical dominant or shoot tips one week before colchicine treatments had removed) (~1.5cm), were placed in baby food jars containing 25 ml of colchicines solution for treatments of time:12, 18 and 2 h on a rotary shaker at 100 RPM. Treated explants were washed three times with sterile water and transferred to 2 kind colchicine-free medium: standard solid E20A medium and solid E20A supple mented with a combination of growth regulators [5 µm %&&$ '()!µm *& # '()µ+&*&$ #, '()-$µm AgNO (5 mg/l)] (Yadav et al. 1996). Finaly after one week, whole of explants transfered to standard E20A medium. Transfer of plants out of culture Plantlets with roots were transferred to 8 cm pots with pit and perlite. Plants in pots were covered with plastics. Corners of which were gradually cut open to allow ventilation. The plastic was removed after about one week. About 108 healthy plants were taken to a greenhouse, eventually transferred to 25 cm pots, and grown to maturity. Greenhouse conditions were 25groth C day and 20 C night, with a 16 h photoperiod. Plants were repeatedly self-pollinated between 8 and 10 a.m. Pollen from one to three male flowers was used for each female flower. In vivo colchicines treatments After transferring plantlets to greenhouse, the plants who that self-pollinating were not successful on them the tips and the first leaf from the top were dipped into 250 ml jars containing a solution either 500 or 1000 mg/l colchicines for 12 or 2 h (Fig 1 c). After treatments, the dipped parts were washed times with running tap water. Plants were repeatedly self-pollinated until they either set fruit or were unhealthy/dead. Determination of ploidy level After colchicines treatments the ploidy levels of plants were determined by tow way: 1- Chromosome counting in root tips and pollen counting from male flower. For observation of the chromosomes in the regenerated plantlets, root tips were treated with 0.05% colchicines solutionfor 2 h, then, fixed in ethanol: glacial acetic acid (:1) and finally stained in 1% aceto-carmine stain as described by Darlington and Lacour (1976). 2- Pollen counts: All of the anthers Pollen from plants that flowered in the greenhouse was examined as described in Lim and Earle (2008, 2009). All the anthers were removed from a male flower were teased apart in a 0 µl drop 0 µl drop of Alexander s stain (Alexander 1969). Pollen grains were covered with an 18 mm cover slip and observed with an Olympus CX21 inverted microscope ( 0, 100). Stained large round pollen grains were considered fertile and viable; unstained shriveled pollen was considered sterile. Stained pollen grains in microscope fields of each sample was counted and averaged. This average value is the pollen number presented in the text and tables. For a sample of flowers (~15), the pollen number was compared with the total number of viable pollen grains in a whole male flower. All the anthers from a male flower were teased apart in 100 µl of Alexander s stain. The stained pollen grains in aliquots of the pollen suspension were counted on a Fuchs-Rosenthal hemacytometer, and appropriate calculations were done to determine the approximate number of stained pollen grains in the whole flower. Pollen number in the same samples was determined by the methods described above except that the average microscope

3 Intl. Res. J. Appl. Basic. Sci. Vol., (8), , 2012 slide count was multiplied by 2.5 to compensate for the fact that the anthers were crushed in 100 µl of stain rather than in 0 µl. Pollinations of culture-derived plants Greenhouse-grown plants were self pollinated between 8 and 10 a.m. Pollen from one to three male flowers was used for each female flower. Pollinations were continued until each plant either had fruit or was dead. Statistical analysis The statistical analysis for the categorical data was modified from Alan et al. (200). A logistic regression model was used since the dependent variable was dichotomous. The analyses were performed using the genmod procedure (SAS Institute 200). The mean separation was performed by least square means. Results In vitro flowering In vitro flowering in melon reported by Lotfi et al 200, Lim and Earle 2008, In this research this event, occured rarely, in some genotype before and after colchicine treatment. They were male flower and in most time flower buds died before opening. Therefore unlike Lim and Earle 2009 we don t have enough flowers for in vitro pollen counting. Effects of treatment with colchicine in vitro The survival rate of colchicine-treated explants varied from 2 81% depending on the methods of application. Effects of kind of explants in vitro colchicine treatments Shoot tips, nodes and nodal growthed explants from haploid plants were treated with colchicine to induce chromosome doubling. The shoot tips treated with 500 mg/l colchicine for 12 h in standard medium showed a significantly greater survival rate (82%) than other treatments. Melon plantlets grown in vitro usually have only one shoot tip but can have 5 10 nodes. The plantlets are easily propagated in vitro by nodal cuttings, but like Lim and Earle s report (2008), after colchicines treatment plant regeneration from nodes is much lower than from shoot tips. By cutting terminal bud and removing apical dominant one week before colchicine treatment we stimulated lateral buds to growthing and it caused plant regeneration from 6% in lateral buds increased to 78% nodal growthed in 12 h colchicines treatments. Effects of during the time in in vitro colchicine treatments By increasing the time rate of survival had decreased but rate of doubling had increased. Most rate of survival had occured in 12 h treatment by 50% average and most rate of doubling occurred in 2 h treatment by 0% average. Effects growth regulators in culture medium in vitro colchicine treatments In all treatments, using growth regulators (5 µm IAA, 5 µm BA, 1 µm ABA and 0 µm AgNO), in E20Amedium nether decreased rate of explants regeneration, also callus production was caused as a result of delayed, decreased or stopped regeneration. After one week, explants transfered to standard medium by removing the callus but their regeneration was slow yet (Fig.1). Therefore this research did not agree with Lim and Earle 2009 claimed plant regeneration from nodal explants treated with 500 mg/l colchicine for 12 h was increased from 0 to 88% by transferring the treated explants to medium supplemented with a combination of growth regulators. Effects of treatment with colchicine in vivo A total of 52 plants were treated with colchicine in vivo (Table 2). Of these, 26 (67%) survived. Flowers on these plants were checked for pollen number. Flowers from seed-grown plants in the greenhouse and haploid plants in vitro were used as positive and negative controls.

4 Intl. Res. J. Appl. Basic. Sci. Vol., (8), , 2012 Table 1. The survival, pollen number, and fruit set of parthenogenic melon plants derived from nodal explants treated with for different times of in vitro treatment with 500 mg/l colchicine Kind of Treatment Duration(h) Explants Explants that Plants in Pollen number (%) explant treated formed plants (%) greenhouse < >20 tip A (8) 2 (67) 0 (0) 1 () (80) 1 (25) 2 (50) 1 (25) (75) 2 (50) 1 (25) 1 (25) B (5) 2 (60) 1 (20) 0 (0) 18 8 (50) 2 (50) 1 (25) 1 (25) (5) 6 2 () 2 () 2 () total 52 (67) a 2 11 (5) 7 (29) 6 (21) node A (6) (75) 1 (25) 0 (0) () 2 (75) 1 (25) 0 (0) (0) 6 (50) 2 () 1 (16) B (25) 1 1 (100) 0 (0) 0 (0) (2) 1 () 1 () 1 () 2 15 (20) 1 () 1 () 1 () total (29) c (56) 5 (28) (16) Nodal grwothed A (78) (75) 0 (0) 1 (25) (75) 2 (67) 1 () 0 (0) (70) 5 2 (8) 2 (7) 1 (25) B 12 6 (50) 1 1 (100) 0 (0) 0 (0) 18 7 (2) 1 () 1 () 1 () 2 10 (0) 0 (0) 2 (66) 1 () total 50 0(60) b 19 9 (7) 6 (2) (21) Total/mean (5) 6 0 (8) 1 (67) 1 (22) Values followed by the same letter are not significantly different at a = 0.05 using least square means. Treatments: A: explants transfer to E20 medium after treated with colchicine B: explants transfer to E20 medium after treated with colchicine contain growth regulators Table 2. The survival, pollen number, and fruit set of parthenogenic melon plants derived from no plants treated with 500 and 1000 mg/l colchicine for different times Colchicine (mg/l) Duration( plants Plants Pollen number (%) h) treated survived (%) < > (70) a 5 (71) 2 (29) 0 (0) d (67) a (50) 2 (25) 1 (16) c (50) c 1 (25) 2 (50) 1 (25) b (50) c 2 (22) () () a total 26 (67) 11 (2) 10 (8) 6 (20) Controls Haploid adapted to green (100) 0 (0) 0 (0) hose from in vitro Diploid (seed grown) 12 0 (0) 0 (0) 12 (100) Values followed by the same letter are not significantly different at a = 0.05 using least square means Determination of ploidy levels by chromosome counting Generally the plants that gain from partenogenic plants athwart seeds grown, tended to grow more slowly and with less vigor than normal diploid melons; flowers were produced late; flower buds died before opening, resulting in fewer open flowers; male flowers did not produce pollen. Results of chromosome counting approved haploidy of plantlets recovered (x=12) a by comparison with chromosome number of seeds grown pelants (2x= 2). Pollen counting Pollen released from anthers of greenhouse-grown plants, after adaptation growthing and flowering, was examined microscopically and compared with controls. Figure 1 shows stained and unstained pollen from a seed grown and parthenogenic plants flower. Almost all untreated haploid plants who had 12 chromosomes, shed no visible pollen and self pollinating were not successful on them whereas all seed grown plants visibly shedding pollen had pollen numbers >20 (table 2) and self pollinating caused grwothig the ovary. flowers with pollen numbers >20 contained approximately 700 pollen grains (Fig 1 b). After colchicine treatments three type plants of male flowers detected: 1- Male flower with pollen numbers ranging from 0 2 (Fig 1 c) and 12 chromosome considered haploid who that doubling were not successful on them

5 Intl. Res. J. Appl. Basic. Sci. Vol., (8), , 2012 and self pollinating were not successful on them too. 2- Male flower by pollen numbers between 5 and 20, - Male flower with pollen numbers >20. In total, by increasing the time of treatments, percentage of plants with a high pollen number (>20) was increased and it was significantly greater in the 2 h treatment by % in in vitro and in vivo treatment. Survival of plants transferred to the greenhouse after treatments was 69%. A total of 250 pollinations (average of /plant) was done on the 8 plants in Table, regardless of ploidy or pollen number. Fruits were obtained from 1 (15%) of self pollinated plants (Table ) and there were not significant different between genotypes in fruit set. Pollen from 8 plants treated with colchicine in vitro and in vivo was examined to assess the effects of the treatments and fruit set on them with different pollen numbers was compared (Table ). Pollen number in relation to fruit set of plants recovered and Fruit set was significantly higher on the plants with pollen numbers <20, as measured either in vitro or in vivo and no one fruit had set in plant with <5 pollen. Figure 1. (a) 2 callus production in plantlets cultured in medium contain growth regulators after in vitro colchicine treatment (b) Stained and pollen from a diploid melon plant (100 ) (c) unstained pollen from partenogenic plant. Table. Fruit set after pollination of plants derived from in vitro and in vivocolchicine treatment classified by pollen number Genotype Pollen Total (%) Fruit set number (%) (%) Melons <5 21 (25) 0 (0) b (15) 1 (8) b >20 10 (12) 6 (60) a Contalapine <5 20 (2) 0 (0) (1) 0 (0) b >20 9 (11) 6 (66) Total 8 1 (15) Values followed by the same letter are not significantly different at a = 0.05 using least square means Discussion We detected no spontaneous doubling, even after repeated checks of ploidy by chromosome and pollen counting. Chromosome doubling was therefore required to produce fertile plants. We did not observe genotypic variation for chromosome doubling. This result agrees with Lotfi et al. (200). Yashiro et al. (2002) reported that five melon cultivars showed different chromosome duplication rates after being sprayed with 1000 mg/l colchicine in vivo. We used two approaches to evaluate the efficacy of chromosome doubling procedures: chromosome counting and pollen counts. Chromosome counting provides a direct measurement for deteminig poloidy levels. However, because of melons had very little choromosome, this way was not efficient. Counts of pollen number are way that Lime and Erle reported 2008, are simple and inexpensive. Plants with pollen numbers>20 gave fruit recovery rates of 60% (Table ). On the other hand, pollen number cannot be determined until the treated plants have male flowers, so considerable greenhouse space is required for several months unless the plants flower in vitro, as sometimes happens (Lotfi et al. 200). Thus the best approach depends on the resources and space available. Our comparison of in vivo and in vitro colchicines treatments was based on pollen numbers. As expected, haploid plants had little or no stained pollen. In vitro treatment of shoot tips with 500 mg/l colchicine for 2 h was selected for comparison the highest duplication rate. For the in vivo treatment increasing the level of colchicine to 1000 mg/l was beneficial (Table 2) and 2 h treatments with 1000 mg/l colchicine had highest duplication rate. A melon plant in vitro has 5 10 nodes and only one shoot tip. It

6 Intl. Res. J. Appl. Basic. Sci. Vol., (8), , 2012 would therefore be desirable to have an efficient doubling procedure for nodal explants.we found that nodes had poor survival and low doubling rates after h exposure to 500 or 1000 mg/l colchicine. Since the doubling rate at 1000 mg/l was lower than at 500 mg/l, increasing the time of treatment is unlikely to be helpful. We used growth regulators (5 µm IAA, 5 µm BA, 1 µm ABA and 0 µm AgNO) in other to increasing their regeneration rate since Erle reported (2009) reports they caused increasing regeneration rate from 0 to 88%. But in our study they decreased rate of explants regeneration, also callus production was caused as a result of delayed, decreased or stopped regeneration. We recommend colchicine treatment of nodal as well asshoot tip explants for enhanced recovery of DH lines from parthenogenetic melon plants. By cutting terminal bud and removing apical dominant one week before colchicine treatment we stimulated lateral buds to growthing and it caused plant regeneration from 6% in lateral buds increased to 78% nodal growthed in 12 h colchicines treatments. Thus using nodal explants as described above, in addition toshoot tip explants, promises the greatest success in obtaining the large numbers of DH progeny needed for usein breeding programs. References Anagnostou K, Jahn M, Perl-Treves R, Inheritance and linkage analysis of resistance to zucchini yellow mosaic virus, watermelon mosaic virus, papaya ringspot virus and powdery mildew in melon. Euphytica 116: Bajaj YPS, In vitro production of haploid and their use in cell genetic and plant breeding. In: Bajaj YPS (ed.), Biotechnology in Agriculture and Forestry 12: Haploid in Crop Improvement I. Springer 126:1. Darlington CD, Lacour LE, The Handling of Chromosomes, 6th ed. Allen and Unwin, London. Dore C, Boulidard L, Sauton A, Rode J-C, Cuny F, Niemirowiecz- Szczytt K Interest of irradiated pollen for obtaining haploid vegetables. Acta Hortic 92: Dryanovska OA, IlievaI N, 198. Invitro anther and ovule cultures in muskmelon (Cucumis melo L.). C R Acad Bulg Sci 6: Ficcadenti N, Sestili S, Annibali S, Di Marco M, Schiavi M, In vitro gynogenesis to induce haploid plants in melon Cucumis melo L. J Genet Breed 5: Ficcadenti N, Veronese P, Sestili S, Crino P, Lucretti S, Schiavi M, Influence of genotype on the induction of haploidy in Cucumis melo L. by using irradiated pollen. J Genet Breed 9:59 6. Frantz JD, Jahn MM, 200. Five independent loci each control monogenic resistance to gummy stem blight in melon (Cucumis melo L.). Theor Appl Genet 108: Koksal N, Yetisir H, Sari N, Abak K, Comparison of different invivo methods for chromosome duplicationin muskmelon (Cucumis melo). ActaHortic588: Lim W, Earle ED, Effect of in vitro and in vivo colchicines treatments on pollen production and fruit recovery on melon plants obtained after pollination with irradiated pollen. Plant Cell Tiss Organ Cult 95: Lim W, Earle ED, Enhanced recovery of doubled haploid lines from parthenogenetic plants of melon (Cucumis melo L.). Plant Cell Tiss Organ Cult 98: Lotfi M, Alan AR, Hennings MJ, Jahn MM, Earle ED, 200. Production of haploid and doubled haploid plants of melon (Cucumis melo L.) for use in breeding for multiple virus resistance. Plant Cell Rep 21: Sari N, Abak K, 199. Induction of parthenogenetic haploid embryos after pollination by irradiated pollen in watermelon. HortScience 29: SAS Institute, 200. SAS/STAT 9.1. User s guide. SAS Institute Inc., Cary, USA Sauton A, Dumas de Vaulx R, Induction of gynogenetic haploid plants in muskmelon (Cucumis melo L.) by use of irradiated pollen. Agronomie 7: doi: / agro: Sauton A, Olivier C, Chavagnat A, Use of soft X-ray technique to detect haploid embryos in immature seeds of melon. Acta Hortic 25: Sauton A, Effect of season and genotype on gynogenetic haploid production in muskmelon, Cucumis melo L. Sci Hortic (Amsterdam) 5: doi: /00-28 (88) Yashiro K, Hosoya K, Kuzuya M, Tomita K, Ezura E, Efficient production of doubled haploid melon plants by modified colchicines treatment of parthenogenetic haploids. Acta Hort 588:5 8.