Balestrazzi, A.

Buttafava, A.

Carbonera, D.

Caser, Matteo

Ghione, G. G.

Giovannini, A.

Mansuino, A.

Sabatini, M. E.

Savona, M.

Scariot, Valentina

English

Institutional Research Information System University of Turin

    This is an author version of the contribution published on: Giovannini A, Scariot V, Caser M, Buttafava A, Mansuino A,  Ghione GG, Savona M, Sabatini ME, Carbonera D, Balestrazzi A Mutation breeding using gamma rays to increase seed germination in Rosa Hybrida ACTA HORTICULTURAE (2015) 1087, 373-378 DOI: 10.17660/ActaHortic.2015.1087.49  The definitive version is available at: http://www.actahort.org/books/1087/1087_49.htm   1 Mutation breeding using gamma-rays to increase seed germination in Rosa hybrida  A. Giovannini1, V. Scariot2, M. Caser2, A. Buttafava3, A. Mansuino4, G.G. Ghione4,  M. Savona4, M.E. Sabatini5, D. Carbonera5 and A. Balestrazzi5  1CRA-FSO, Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, Unità di ricerca per la floricoltura e le specie ornamentali, Corso Inglesi 508, 18038 Sanremo (IM), Italy 2Department of Agricultural, Forest and Food Sciences, University of Torino, Largo Paolo Braccini 2, 10095, Grugliasco (TO), Italy 3Department of Chemistry, University of Pavia, via Taramelli 12, 27100, Pavia, Italy 4NIRP International, Azienda Agricola di Ghione L. & Figli, Via San Rocco, 1 - Fraz. Bevera, 18039, Ventimiglia (IM), Italy 5Department of Biology and Biotechnology ‘L. Spallanzani, University of Pavia, Via Ferrata 9, 27100, Pavia, Italy  e-mail presenting author: annalisa.giovannini@entecra.it  Keywords: dose rate, Hybrid tea rose, ionizing radiation, mutagenic agents, seed quality  Abstract Breeders are always looking for new and novel varieties to be competitive in the flower market. Low seed germination rate is still a major problem encountered in traditional rose breeding programs. Mutagenic agents, such as ionizing radiation, may be used to enhance seed vigor and increase the productivity, improving sprouting and emergence of buds that are carried out through seed coating. The effects of ionizing radiation on seed vigor are in most cases genotype-dependent, thus irradiation treatments need to be optimised for each different cultivar. In the present study, gamma-rays (0, 50, 100 and 200 Gy) were applied on hybrid tea rose seeds in order to set up a radiation protocol for increasing seed germination. Seeds coming from six different crossings of Rosa hybrida commercial cultivars were used in order to gain information on radiotolerance and germination ability.   INTRODUCTION  Hybrid tea roses (Rosa hybrida) are the most important ornamental cut-flowers worldwide. Breeders are always looking for new and novel varieties to be competitive in the flower market. Rose breeding programs center mainly on the introduction of new flower colors, thornless stems, higher production, and good post-harvest performance (Pipino 2011). Rose propagation by seed is used in breeding new cultivars as well as in the production of rootstock plants. Poor seed germination is one of the major problems encountered in rose breeding programs (Anderson and Byrne 2007). This is mainly due to endogenous and exogenous dormancy (Pipino et al. 2012, Bosco et al. 2014), which requires prolonged and expensive treatments (Zlesak 2006, Pipino et al., 2011) or techniques (Caser et al., 2012, Caser et al. 2014) to be overcome.  Mutation breeding is the process of exposing seeds to chemicals or radiation in order to generate mutants with desirable traits to be bred with other cultivars. Mutagenic  2 agents are commonly used to generate genetic variation and thus select the desired characters with a global impact of mutation-derived varieties on food production and quality enhancement (Ahloowalia et al., 2004). Recent reports have underlined the potential of gamma rays as tools for seed priming, a process used in seed industry to increase seed vigor and to enhance plant tolerance to biotic/abiotic stresses (Macovei et al., 2014). Exposing plants to radiation is sometimes called radiation breeding, the biological effects of ionizing radiation (IR) are correlated with the energy absorbed per unit mass (dose) and, for a given dose, to the rate of energy deposition (dose rate) (Vilenchik and Knudson, 2000). The optimal dose is based on radiosensitivity, which is dependent both on genotype and physiological conditions of plant material (Esnault et al., 2010). Gamma-rays have been successfully exploited to enhance seed vigor in okra and hard wheat (Hegazi and Hamideldin, 2010; Melki and Marouani, 2010), however the irradiation dose should be carefully assessed to avoid the occurrence of undesirable effects. In Rosa hybrida, few studies on mutagenesis with ionizing radiation have been carried out. Ibrahim et al. (1998) applied X-irradiation at 25, 50 and 100 Gy on leaf explants for adventitious bud regeneration. Authors observed a decreasing rate of leaf explants regenerating buds with increasing dose (from 47% to 0%). Recently, in vitro radio-sensitivity of leaf explants of R. hybrida was tested with different doses of gamma-irradiation (0-50 Gy) (Kahrizi et al., 2013). Authors found that the LD50 for leaf explants is 20-30 Gy. No studies on seed mutagenesis are currently present in literature.   In the present work, gamma-rays (0, 50, 100 and 200 Gy) were applied to hybrid tea rose seeds in order to investigate the potential use of ionizing radiation as a tool to improve seed quality and increase seed germination.   MATERIALS AND METHODS Seed collection and irradiation Six crosses of R. hybrida were made by manual pollinations in the greenhouses of NIRP International (New International Rosebushes Protected, Bevera, Ventimiglia, IM, Italy) during June 2012 (Table 1). A total of 200 seeds were extracted from mature hips of each cross and dry seeds were separated in four groups (50 seeds each) and exposed to 50, 100, and 200 Gy doses of gamma rays from Co60 gamma source (high dose rate = 5.15 Gy min-1) available at the University of Pavia. Untreated controls (NT, fifty seeds) were used.     Seed germination  Irradiated and control seeds were sowed in perlite seed beds for germination in the greenhouses of the NIRP International in February 2013. On the basis of the International Rules for Seed Testing Association (ISTA 2014), seed germination was weekly recorded for a total of four months. The final germination percentage (FGP), half-time of germination (T50), and germination period (GPD) were calculated using Czabator’s index (1962) where FGP = total number of seeds germinated/total number of seeds in all replicates × 100; T50 = days from seeding to the 50% of the total germinated seeds, and GPD = days from seeding to when maximum number of seeds germinated. Moreover, the day of the first emergence (Em) and the germination energy (Ge) were counted. Germination energy is defined as the germination percentages when the mean daily germination (cumulative germination percent divided by the time elapsed since sowing date) reached its peak. Seeds were considered to have germinated when hypocotyl with cotyledons emerged.   3 Seed quality For each tested irradiation dose, changes in final germination percentage (±%) were estimated in Rosa hybrida crosses in comparison with the untreated sample (NT). After 16 weeks from sowing, the following growth criteria were recorded: flowering time and the percentage of flowering seedlings on total germinated seeds (Fl).   Statistical analysis  Arcsine transformation was performed on all percent incidence data before statistical analysis in order to improve homogeneity of variance. Effect of radiation treatments on FGP, Em, T50, GPD, and Ge were evaluated by the analysis of variance (one-way ANOVA) using Ryan-Einot-Gabriel-Welsch’s multiple stepdown F (REGW-F) test (p ≤ 0.05). All analyses were performed with SPSS 21.0 Inc. software (Chicago, Illinois, USA).  RESULTS In Table 2 are reported the effects of radiation treatments on seed germination of the studied crosses. The final germination percentage (FGP) was not significantly affected by the treatments. Few differences were noted for the T50 and the GPD while the day of the first emergence (Em) varied in all the progenies, even if with different radiation-dose effect. The 50 Gy-dose shortened Em in the progenies of four out of six crosses: 8, 187, 325 and 331. The seed progeny coming from the cross 187 (3940 orange code x 1225 orange code) showed a positive trend in germination percentage at 50, 100 and 200 Gy, while seed germination of cross 331 (2466 red code x 3741 red code) was negatively affected by high dose radiation (Table 3). All the obtained seedlings were then monitored at flowering time. The percentage of seedlings which started flowernig in June 2013 is listed in Table 2. Although no significant differences were counted also for this parameter, seedlings obtained from seeds treated with 50 Gy showed a flowering percentage trend superior than the others in crosses 8 (Fl = 68%), 325 (Fl = 67%) and 331 (Fl = 75%). Furthermore in the cross 51, the 100% of seedlings obtained from 50 and 100 Gy treated seeds produced flowers.  DISCUSSION  High ionizing radiation doses (> 10 kGy) are routinely used for the sterilization of food products, while low doses, ranging from 60 to 700 Gy, are used to improve characters and productivity in many seed propagated crops, such as rice, wheat, maize, beans and rape seed (Ahloowalia and Maluszynski, 2001). In the present study we evaluated the effects of gamma-rays at 50, 100 and 200 Gy on seed quality and germination, in order to assess the response of six crosses of Rosa hybrida and acquire useful information for developing novel seed vigorization protocols.   The range of gamma rays doses tested did not significantly affect final germination percentage and germination energy of the seeds regardless to the cross. These results are in contrast with different studies performed on seeds of several species such as Vigna unguiculata L. (Bind and Dwivedi, 2014), Citrus jambhiri Lush. (Sharma et al., 2013) and Withania somnifera L. (Bhosale and More, 2014) where a gradual decrease in seed germination and seedling vigor from lower doses to higher doses in given treatments  4 of gamma rays were detected. This is a controversial issue since other reports are available which demonstrate the beneficial effects of ionizing radiation on seed vigor. Few differences among rose radiated seeds were observed for the other evaluated parameters (T50, GPD and Em). In particular, the 50 Gy dose fasten the emergency period. Only one cross showed a positive trend in the germination percentage related to the radiation dose applied, in agreement with Bottino et al. (1975) who highligted that relatively low-doses ionizing irradiation on plant materials accelerated seed germination rate. With the aim to better explain the potential of gamma ray-based biostimulation in hybrid tea rose, the range of total dose will be expanded and the effects of different dose rate conditions (low dose rate versus high dose rate) will be also conducted. Seeds from a wider number of R. hybrida crosses will be tested in order to acquire more detailed information on a large sample population. This will allow a better understanding of the cultivar-related ability to withstand genotoxic-stress as well as the seed profile in terms of stress tolerance and vigor. ACKNOWLEDGEMENTS This research was supported by the ‘MUTROS-OIGA’ project (D.M. 18829) of the Italian Ministry of Agriculture - MIPAAF.   Literature Cited Ahloowalia, B.S. and Maluszynski, M. 2001. Induced mutations - A new paradigm in plant breeding. Euphytica, 118: 167-173. Ahloowalia, B.S., Maluszynski, M. and Nichterlein K. 2004. Global impact of mutation-derived varieties. Euphytica, 135: 187-204. Anderson, N. and Byrne, D.H. 2007. Methods for Rosa germination. Acta Hortic, 751: 503-507. Bhosale, R.S. and More, A.D. 2014. Effect of gamma radiation on seed germination, seedling height and seedling injury in Withania somnifera (L.) Dunal. Int. J. of Life Sciences, 2 (3): 226-228. Bottino, P.J., Sparrow, A.H., Susan, S.S. and Thompson K.H. 1975. Interrelation of exposure and exposure rate in germinating seeds of barley and its concurrence with dose-rate theory. Radiation Botany, 15 (1): 17-27. Bind, D. and Dwivedi, V.K. 2014. Effect of mutagenesis on germination, plant survival and pollen sterility in M1 generation of in cowpea (Vigna unguiculata (L.) Walp). Indian Journal of Agricultural Research, 48 (5): 398-401. Bosco, R., Caser, M., Ghione, G.G., Mansuino, A., Giovannini, A. and Scariot, V. 2014. Dynamics of abscisic acid and indole-3-acetic acid during the early-middle stage of seed development in Rosa hybrida. Plant Growth Regulation, 75: 265-270. Caser, M., Pipino, L., Van Labeke, M.C., Mansuino, A., Giovannini, A. and Scariot, V. 2012. Immature seed rescue and abscisic acid quantification in Rosa hybrida L. suggest early and transient endodormancy. Acta Horticolturae, 961: 593-598. Caser, M., Dente, F., Ghione, G.G., Mansuino, A., Giovannini, A. and Scariot, V. 2014. Shortening of selection time of Rosa hybrida by in vitro culture of isolated embryos and immature seeds. Propagation of Ornamental Plants, 14(3): 139-144. Czabator, F.J. 1962. Germination value: an index combining speed and completeness of pine seed germination. Forest Science, 8: 386-396.  5 Esnault, M.A., Legue, F. and Chenal, C. 2010. Ionizing radiation: advances in plant response. Environmental and Experimental Botany, 68: 231-237. Hegazi, A.Z. and Hamideldin N. 2010. The effect of gamma irradiation on enhancement of growth and seed yield of okra (Abelmoschus esculentus (L.) Monech) and associated molecular changes. Journal of Horticulture and Forestry, 2 (3): 38-51. Ibrahim, R., Mondelaers, W. and Debergh P.C. 1998. Effects of X-irradiation on adventitious bud regeneration from in vitro leaf explants of Rosa hybrida. Plant Cell Tissue Organ Culture, 54: 37-44. ISTA 2014. International Rules for Seed Testing 2014. Ordinary General Meeting 2013, Antalya, Turkey. Online ISSN 2310-3655  http://www.seedtest.org/seedhealthmethods Melki, M. and Marouani A. 2010. Effects of gamma rays irradiation on seed germination and growth of hard wheat. Environmental Chemistry Letters, 8: 307-310. Macovei, A., Garg, B., Raikwar, S., Balestrazzi, A., Carbonera, D., Buttafava, A., Bremont, F.J., Gill, S.S., Tuteja N. 2014. Synergistic Exposure of Rice Seeds to Different Doses of -Ray and Salinity Stress Resulted in Increased Antioxidant Enzyme Activities and Gene-Specific Modulation of TC-NER Pathway. BioMed Research International, Volume 2014, Article ID 676934, 15 pages. Kahrizi, Z.A., Kermani, M.J., Amiri, M., Vedadi, S. and Hosseini, Z. 2013. In vitro radio-sensitivity of different genotypes and explants of rose (Rosa hybrida). Journal of Horticultural Science & Biotechnology, 88: 47-52. Pipino, L. 2011. Improving seed production efficiency for hybrid rose breeding. PhD thesis, Ghent University and University of Turin, 189 pp. Pipino, L., Scariot, V., Gaggero, L.,  Mansuino, A., Van Labeke, M.C. and Giovannini, A. 2011. Enhancing seed germination in hybrid tea roses. Propagation of Ornamental Plants, 11(3): 111-118. Pipino, L., Leus L., Scariot V. and Van Labeke M.C. 2012. Embryo and hip development in hybrid roses. Plant Growth Regulation, 69: 107-116. Sharma, L.K., Kaushal, M., Gill, M.I.S. and Bali, S.K. 2013. Germination and survival of Citrus jambhiri seeds and epicotyls after treating with different mutagens under in vitro conditions. Middle-East Journal of Scientific Research, 16 (2): 250-255. Vilenchik, M.M. and Knudson, A.G. 2000. Inverse radiation dose rate effects on somatic and germ-line mutations and DNA damage rates. Proc. Natl. Acad. Sci. USA, 97: 5381-5386. Zlesak, D.C. 2006. Rose. Rosa x hybrida. Chapter 26. In N.O. Anderson, ed. Flower Breeding and Genetics: Issues, Challenges and Opportunities for the 21st Century. Springer Dordrecht. pp. 695-738. Tables  Table 1. Identification code (ID), mother plant and pollen donor of the six crosses of Rosa hybrida. Cross ID Mother plant Pollen donor 8 2364 yellow code 3523 yellow code 51 3951 red code  4100 red code  72 3418 yellow code 3523 yellow code  187  3940 orange code  1225 orange code  325 3947 cream code  15 red code  331 2466 red code 3471 red code   6 Table 2. Effects of treatments (Non Treated, 50, 100 and 200 Gy) on the germination process (final germination percentage, FGP; first emergence, Em; half-time of germination, T50; germination period, GPD; germination energy, Ge) and on the percentage of flowering seedlings (Fl).  Cross ID Treatment (Gy) FGP (%) Em (days) T50 (days) GPD (days) Ge (%) Fl (%) 8 NT 32 60aβ 67 81 28 50 50 44 30b  67 81 34 68 100 32 30b  74 81 18 37 200 46 60a  74 81 34 52 P ns * ns  ns  ns ns 51 NT 14 30b 74a 81a 8 86 50 10 60a 74a 81a 6 100 100 18 30b 60b 60b 8 100 200 16 60a 60b 60b 10 75 P ns * * ** ns ns 72 NT 44 60a  67 81 22 68 50 44 60a  67 81 32 59 100 46 30b  74 81 32 57 200 34 30b  74 81 18 53 P ns * ns ns  ns ns 187 NT 18 60a 67 81 14 67 50 24 30b 67 81 20 75 100 24 60a 67 81 18 75 200 28 30b 67 81 22 86 P ns * ns ns ns ns 325 NT 26 60a 60 60b 14 62 50 36 30b 67 81a 18 67 100 20 30b 60 81a 10 50 200 40 60a 67 81a 24 45 P ns * ns ** ns ns 331 NT 14 60b 67b 81a 8 57 50 8 30c 60b 81a 4 75 100 14 60b 67b 60b 6 43 200 2 81a 81a 81a 2 - P ns ** * * ns ns βMean values followed by the same letter are not statistically different at p ≤ 0.05 according to the REGW-F test. The statistical relevance of ‘Between-Subjects Effects’ tests (ns = non significant, * = p < 0.05, ** = p < 0.005, *** = p < 0.001).  Table 3. Changes in percentage (%) of seed germination observed for each Rosa hybrida cross in comparison with the untreated sample (NT) at different radiation doses. Dose Cross ID  8 51 72 187 325 331       50 Gy +12 -  4     0 + 6 +10 - 6    100 Gy    0 + 4  + 2 + 6 - 6   0    200 Gy + 14 + 2 - 10 + 10 + 14 - 12  

Mutation breeding using gamma rays to increase seed germination in Rosa hybrida.

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Mutation breeding using gamma rays to increase seed germination in Rosa hybrida.

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