6. Tuan_VOL2_2014.cdr Abstract Brunonia australis Rhodanthe(Goodeniaceae) and floribunda (Asteraceae) are two potential Australian native �ower species desired by �oricultural markets. However, the species are dif�cult to propagate. This study examined internal factors that constraint seed germination, seed viability and physiological dormancy. The study was conducted during April to May 2009 at the Gatton nursery, The University of Queensland Gatton Campus to identify the underlying reasons for poor germination and to provide recommendations for improving propagation effectiveness. Seeds of B. australis collected in 2003 germinated readily irrespective of germination media, whereas seeds collected in 2007 and 2008 with high proportion of viable seeds could only germinate with the presence of GA3 (100 mg.L ) in germination media though with relatively -1 low rates (8.75% and 10.00% respectively) of seed germination. Seeds of collected in 2003R. floribunda showed a signi�cant improvement in germination in agar media supplemented with 100 mg.L GA (67.5%) -1 3 compared to control treatment (10.0%). The results suggest that physiological dormancy occurs in both species. The use of GA is recommended for improving3 germination rates of the two species. Further studies on the effects of different GA concentrations to improve3 seed germination should be conducted. Keywords: Dormancy, germination, gibberellic acid, TZ, viability. Introduction Brunonia australis Rhodanthe(Goodeniaceae) and floribunda (Asteraceae) are recognized as economically important wild�ower species of Australia (Barker et al., 2002; Bunker, 1994; Joyce, 2005; Peacock and Smith- White, 1978; Stewart, 1996). Nonetheless, like other native �owers the two species have a poor ability to germinate and this is considered as a barrier for commercial development (Bunker, 1994; Johnston et al., 1999). Seed germination is controlled by both internal and external factors (Benech-Arnold and Sanchez, 2004; Khan, 1982), in which the former comprises physiological dormancy (genetic control of dormancy) and physical dormancy (a coat-induced dormancy) . In addition, seed(Bewley and Black, 1994) viability, i.e. the ability of seed to germinate, is also an internal factor that affects germination of seeds (Mayer and Poljakoff-Mayber, 1989). Gibberellic acid (GA), particularly GA has recently been3 proven to play an important role in regulating seed germination. GAs can replace the requirement for environmental factors that are required for germination of dormant seeds, including light and temperature (da Silva et al, 2005). According to Benech-Arnold and Sanchez an increase in the seed GA level can(2004) stimulate metabolism processes in the embryo as well as in the endosperm, leading to germination. Research carried out at The University of Queensland Centre for Native Floriculture (CNF) has shown that some samples of the two aforementioned species failed to germinate regardless of favorable external factors (temperature, humidity, light and nutrient) (Cave, 2009; Johnston, 2009; Robert, 2003). Consequently, the internal factors including seed dormancy and/or viability are assumed as the limitations for seed germination which need further studies. For this reason, the research direction was focused on i n v e s t i g a t i n g t h e m e c h a n i s m s l e a d i n g t o l o w germination rates and to formulate suitable technical methods to improve propagation ef�ciency. This is also in line with the recent strategies of the Queensland government and the CNF, a joint initiative of the government and the University of Queensland (UQ). The strategies focus on the discovery and development of potential �oricultural species to foster germplasms available to the industry via exploitation, selection, breeding and overcoming technical obstacles such as germination constraints, diseases problems and poor postharvest quality .(Johnston, 2003; Joyce, 2005) The overall objective of this research was to formulate reliable seed germination strategies for Brunonia australis Rhodanthe floribundaand based on understanding their physiological characters and germination responses. Seed viability (TZ) test and germination experiments with the use of GA at 1003 mg.L were carried out to determine whether or not seed -1 The Physiological Dormancy and Germination Responses of Brunonia and to Gibberellic Acid Treatmentaustralis Rhodanthe floribunda Tuan M. Ha Thai Nguyen University of Agriculture and Forestry Faculty of Agronomy, Thai Nguyen city, Vietnam Email: haminhtuan@tuaf.edu.vn Journal of Tropical Crop Science Vol. 1 No. , 20142 October www.j-tropical-crops.com 30 viability and/or seed dormancy are the limiting factors of seed germination in the two species. Materials and Methods Selecting and processing seeds Seeds of tested in this study consistBrunonia australis of three groups: (i) seeds collected from the wild (GPS coordinate: 27°42'748''S; 145°47'790”E) on 20 November 2003 (BA1); (ii) BA2 (19 November 2007) and (iii) BA3 (4 April 2008) collected at the UQ Gatton campus. Seeds of (RF) wasRhodanthe floribunda collected on 16 February 2003 from Site 3 (GPS coordinate: 27°57'748”S; 148°00'834”E) on the roadside of the Moonie Highway, Queensland. Seeds of each group were collected from at least 50 individual plants. Only mature seeds close to the point of natural dispersal were collected. Following collection, seeds were stored in paper envelopes at 15°C and 15 – 20% relative humidity in a cold room of the CNF laboratory until required. Experimental flow chart As discussed above, this study was carried out to test the two possibilities, dormancy and viability, as the internal factors that in�uence germination of the two species (Figure 1). Viability and germination tests Physical damages of the seeds were checked using a stereomicroscope. Only intact seeds were then selected for germination and viability tests. To a v o i d c o n t a m i n a t i o n p r o b l e m s d u r i n g t h e germination tests reported by previous studies on native �oricultural species (Johnston et al., 2004; Mullins et al., 2002), seeds were disinfected in chlorine (2000 mg.L ) -1 plus one drop of Tween 20 for 10 minutes then rinsed ® with sterile distilled water (DW) three times before being placed into sterile germination media. In this test, two treatments were used for each seed sample with four replicates of 20 seeds each sown into 9cm diameter plastic Petri dishes containing (i) 1% agar water (control) and (ii) 1% agar water and GA (100 mg.L ). Petri dishes3 -1 were sealed with plastic bands to avoid agar Figure 1. Experimental �ow chart to examine poor germination of (BA) and and seeds.Brunonia australis Rhodanthe floribunda The photo of TZ staining patterns of seeds was obtained from International Seed Testing Association (ISTA) Handbook on Flower Seed Testing (Ripka, 2008). Dotted arrows represent conclusions based on the test results. Journal of Tropical Crop Science Vol. 1 No. , 20142 October www.j-tropical-crops.com 31The Physiological Dormancy and Germination Responses of Brunonia.......... desiccation. Seeds of all treatments were placed in dark and air-conditioned room with temperature of 25°C. Germination, de�ned as radical emergence by at least 1 mm, was scored every week. Seeds that did not germinate by the end of the germination experiment was subjected to germination testing using TZ method. The seeds were imbibed for 24 hours in distilled water (DW) at ambient temperatures. Imbibed seeds were placed into 1.0% 2,3,5-triphenyl tetrazolium chloride (TZ) solution and held at 40°C in dark for 24 hours. After rinsing with DW, seeds were cut longitudinally to expose the embryo. Deep red to deep pink embryos were recorded as viable, whereas white to pale pink embryos were recorded as non-viable (Copeland and McDonald, 1995; Hampton and Tekrony, 1995). Experimental design and analysis The study was set up in a completely randomized factorial design with four replicates each for BA1, BA2, BA3 and RF seed group. Each replicate consists of 20 seeds, totaling 80 seeds for each seed group. The data of germination rates was analyzed by using balanced ANOVA of the IRRISTAT statistical package (IRRI Release 5, Manila, the Philippines, 2005) with least signi�cant differences (LSD) calculated at 5% level of signi�cance. GraphPad Prism (Release 6, GraphPad Software Inc., La Jolla, CA 92037 USA) was used to illustrate germination trend over time and compare germination rates amongst selected treatments. Results and Discussion Brunonia australis There were noticeable differences in germination rates of the three groups of (BA) seedsBrunonia australis (Table 1). BA1 seeds (seeds collected in 2003 from the wild) germinated readily on the �rst week, i.e. 70% germination on 1% agar medium plus GA (100mg.L )3 -1 and 78.8% on 1% agar without GA . The germination3 increased gradually until the �fth week, i.e. 87.5% and 80.0% for BA1 with and without GA respectively (Table3, 1). Table 1. Percentage of seeds germinated over �ve weeks.Brunonia australis Week 1 Week 2 Week 3 Week 4 Week 5 BA1 with GA3 70.00 78.75 85.00 85.00 87.50 BA1without GA3 78.75 78.75 78.75 78.75 80.00 BA2 with GA3 0.00 0.00 2.50 6.25 8.75 BA2 without GA3 0.00 0.00 0.00 0.00 0.00 BA3 with GA3 0.00 2.50 5.00 6.25 10.00 BA3 without GA3 0.00 0.00 0.00 0.00 0.00 Treatments Percentage of seeds germinated over time (weeks) (%) * P-value n.s * Note: BA1, BA2 and BA3 are seeds of collected in 2003, 2007 and 2008,Brunonia australis respectively; GA concentration: 100 mgL ; Date sown: 9 April 2009; n.s. not signi�cant; * P < 0.05.3 -1 However, there was no signi�cant difference in the germination rates between BA1 with and without GA ,3 suggesting that GA at 100 mg.L did not have3 -1 stimulating effect on germination of the BA1 seeds. BA1 seeds which were collected in 2003 might have experienced a suf�cient dormancy period which has enabled the seeds to germinate under the favorable temperature and humidity condition regardless of the GA treatment.3 In contrast, BA2 seeds (collected in 2007) and BA3 seeds (collected in 2008) sown on agar with 100 mg.L -1 GA did not germinate until week 2 and week 3,3 respectively. After that the germination rate rose slightly to 10 % and 8.8%, respectively, by the �fth week (Table 1). BA2 and BA3 seeds without GA did not germinate3 after �ve weeks (Table 1). GA treatment signi�cantly3 increased both BA2 and BA3 germination (P <0.05). Non-germinated BA2 and BA3 seeds were subjected to a viability test using the TZ staining technique. The results indicated high proportions of viable seeds, i.e. 82 % in BA2 and 81% in BA3. Therefore, it was concluded that the seeds of collected in 2007 (BA2) andB. australis 2008 (BA3) have a dormancy character, and that GA at3 100 mg.L had a stimulating effect on germination of -1 these seeds. These results demonstrated that the more recent harvested seeds (BA2 and BA3) experienced a certain dormancy period before being able to germinate under favorable environments. To break dormancy, addition of stimulants such as GAs is necessary. Journal of Tropical Crop Science Vol. 1 No. , 20142 October www.j-tropical-crops.com 32 Tuan M. Ha As described above, the seeds of collectedB. australis in 2003 (BA1) germinated readily without a need for addition of GA , whereas one- and two-year-old seeds3 (BA2, BA3) required GA for germination even though3 only a small percentage of seeds germinated (Figure 2 the same as Table 1). Further studies to test the effects of different GA concentrations on germination of the3 newly harvested seeds are recommended. Rhodanthe floribunda Rhodanthe seeds treated with and without GA started3 germinating at the �rst week with the percentages of 60 % and 5 %, respectively. The germination rates increased gradually until the �fth week, reaching 67.5% for GA -treated seeds and 10 % for control seeds.3 Therefore GA -treated seeds had a higher germination3 percentage compared to the control seeds (P <0.05) (Figure 3). Figure 2. Germination rates over time of (BA) seeds. Vertical bars representB. australis standard errors of means (SE). It can be be concluded that has dormancyR. floribunda character, and the use of GA at 100 mg.L can3 -1 overcome dormancy and stimulate seed germination. Conclusion The study has established that both Brunonia australis and seeds have dormancyRhodanthe floribunda character. The addition of GA at 100 mg.L to the3 -1 germination media can stimulate germination of R. floribunda collected in 2003 and one- and two-year-old B. australis seeds. For this reason, adding GA to3 germination media is recommended to increase propagation ef�ciency of the two species. Different concentrations of GA should be tested to determine the3 o p t i m u m G A c o n c e n t r a t i o n t o i m p r o v e s e e d3 germination. Acknowledgement The author would like to express sincere thanks to the staff of Gatton Seed technology laboratory (the University of Queensland, QLD, Australia) for conducting the viability test, Mr. John Swift for laboratory Figure 3 Germination rates of (RF) seeds over time. Seeds were collected on 16 February. Rhodanthe floribunda 2003. The seeds were sown on 9 April 2009. Vertical bars represent standard errors of means (SE). Journal of Tropical Crop Science Vol. 1 No. , 20142 October www.j-tropical-crops.com 33The Physiological Dormancy and Germination Responses of Brunonia.......... safety procedure, Mr. Allan Lisle for statistical advice, and Ms. Vishu Wickramasinghe for her assistance in germination media preparation. The author specially thank Dr Margaret Johnston for her valuable information on the research methods and constructive comments and advice for this manuscript. References Barker, J., Greig, J., Peate, N., Courtney, B., Salkin, E., Schaumann, M., Armstrong, J., and Thomlinson, G. (2002). "Everlasting daisies of Australia: identi�cation, propagation, cultivation". 196 p. C.H. Jerams and Associates. Australia. Benech-Arnold, R. L. and Sanchez, R. A. Eds. (2004). "Handbook of Seed Physiology: Application to Agriculture" 479 p. CRC Press.. Bewley, J. D., and Black, M., eds. (1994). "Seeds: Physiology of Development and Germination". 446 p. Springer. Bunker, K. V. (1994). Overcoming poor germination in Australian daisies (Asteraceae) by combination of gibberellin, scari�cation, light and dark. Scientia Horticulturae , 243-252.59 Cave, R. (2009). Pers.com. (M. T. Ha, ed.), Centre for Native Flowers, The University of Queensland, Australia. Copeland, L. O., and McDonald, M. B. (1995). "Principles of Seed Science and Technology“. 409 p. Chapman and Hall, Inc.Third edition. Da Silva, E.A., Toorop, P.E., Nijsse, J., Bewley, J.D. and Hilhorst, H.W.M. (2005). Exogenous gibberellins inhibit coffee ( cv. Rubi) seedCoffea arabica germination and cause cell death in the embryo. Journal of Experimental Botany , 1029 – 1038.56 Hampton, J. G., and Tekrony, D. M., eds. (1995). "Handbook of Vigour Test Methods". 117 p. The International Seed Testing Association, Zurich, Switzerland. Johnston, M. (2009). Pers.com. (M. T. Ha, ed.), Centre f o r N a t i v e F l o w e r s , T h e U n i v e r s i t y o f Queensland, Australia. Johnston, M., Kibbler, H., Fletcher, T. and Webber, J. 2000. The introduction to commercial �oriculture of recalcitrant Australian native plants. Acta Hort (ISHS) , 31-36.541 Johnston, M. E. (2003). The Centre for Native F l o r i c u l t u r e “ P r o c e e d i n g s o f t h e 5I n t h International Symposium on New Floricultural C r o p s ” ( A . F. C . To m b o l a t o a n d G . M . DiasTagliacozzo, eds.), pp. 165-169, Parana, Brazil. International Society for Horticultural Science. Johnston, M. E., Bauer, L. M., O'Brien, S. D., and Kochanek, J. (2004). Dormancy issues for Australian �oricultural species. "ProceedingsIn of the �fth Australian workshop on native seed biology" (S. W. Adkins, ed.), pp. 89-96. Australian centre for Minerals Extension and Research Brisbane, Queensland, Australia. Joyce, D. C. (2005). Queensland's Center for Native Floriculture. "Proceedings of the InternationalIn Symposium on Harnessing the Potential of Horticulture in the Asian-Paci�c Region“ pp. 47- 52. Khan, A. A., ed. (1982). "The Physiology and Biochemistry of Seed Development, Dormancy and Germination", 547 p. Elsevier Biomedical Press. Mayer, A. M., and Poljakoff-Mayber, A., eds. (1989). "The Germination of Seeds" 208 p. edition.. Third Pergamon Press, Jerusalem, Israel. Mullins, R. G., Koch, J. M., and Ward, S. C. (2002). Practical method of germination for a key jarrah forest species: Snottygobble (Persoonia l o n g i f o l i a E c o l o g i c a l M a n a g e m e n t &) . Restoration , 97-103.3 Peacock, W. J., and Smith-White, S. (1978). Cyto- geography of . , 31-Brunonia australis Brunonia 1 44. Ripka, Z., ed. (2008). "ISTA Handbook on Flower Seed Testing." ISTA, Bassersdorf, Switzerland. Robert, J. (2003). Pers.com. (M. T. Ha, ed.), Centre for Native Flowers, The University of Queensland, Australia. Stewart, A. (1996). Gardening on the wild side, the new Australian bush garden. ,Australian Plants 18 287-296. Journal of Tropical Crop Science Vol. 1 No. , 20142 October www.j-tropical-crops.com 34 Tuan M. Ha