Improvement of germination capacity of Sida hermaphrodita (L.) Rusby by seed priming techniques manuskript

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1 1 Improvement of germination capacity of Sida hermaphrodita (L.) Rusby by seed priming techniques manuskript Erika Kurucz 1* and Miklós Gábor Fári 1,2 1 Department of Plant Biotechnology, University of Debrecen AGTC, Debrecen; 2 Ereky Foundation, Hungary Summary *Corresponding author era.kurucz@gmail.com Sida hermephrodita or Virginia mallow is a perspective perennial herb in the Malvaceae family able to yield a biomass crop through the last two decades. Additionally, the plants have a lot of uses and benefits for instance it can use as a fodder crop, honey crop, ornamental plant in public gardens. It has favourable features like fast growing and resistance against the disease and climatic fluctuations, etc. Sida is in the beginning phase of domestication therefore it has a serious disadvantage: the low and slow germination as a big part of wild plants. Due to the expressly low germination percent the need of seed showing of driller is should tenfold, 200 thousand seeds/acre instead of thousand what is not available and expensive Therefore practical purposes of our research of seed physiology was to increase the seed germination percent in a available, basically wild Sida population. In the first stage of our experiments we examined two factors relating to seed germination percent and seed germination power during our research: the influence of hot water treatment and the effect of exogenous or endogenous infection of seed. However, in our germination tests, utilizing scarified seeds with hot water (65, 80 and 95 o C), from 29,3% to 46% germinated from those samples which were collected from the population of S. hermaphrodita in Debrecen. The average germination for all season was 5-10 % without treatment and rinsed using hot water up to almost 50%. When physically scarified used, the oldest seeds showed the best germination (46 %) after the hot water operation in spite of the previous studies. We discovered that there are close relationship between the seeds collecting time and the ration of seed infections, as well as germination percentage. Thus, 2009 season was the most favourable in case of contamination (control: 17,3% and 80 o C treatment: 0%) as well as germination percent. It could be concluded that, the best season for our findings was 2009 due to autumn harvest of Sida seeds. In our opinion, the autumn harvesting should be the best time to overcome the problem of the low germination and high infection percentage. We also discovered that apparently there are close relationship between the seed fresh weight or water uptake capability and the percentage of infection. Following these recognition we modified our technique, in such a way that we fractionated the seeds based on their fresh weight / or relative density before we carried out the treatment. When we filtered the floating seeds on the surface of water, the hot water treatment was performed considerably better on the sunk seeds after separation. Therefore, by this special priming process we were able to reach 80% germination capacity of Virgina mallow seeds under laboratory conditions (26 o C without illumination). Keywords Virgina mallow, Sida hermaphrodita, seed priming technique, seed germination

2 2 Introduction Sida (Sida hermaphrodita Rusby, Virginia mallow, Virginia fanpetal, Petemi) is a perspective perennial herbaceous semishrub plant originating from North America. This endangered species will probably receive more attention as a temperate climatic biomass crop. Based on European investigation results, the main value of Sida is that it can cultivate on marginal areas and the plants can develop under our warming climate conditions with biomass output around of DT ha -1. Besides this important point the water content of Sida decrease between 35 and 45% until November preceded the perennial rhizomatous grasses (PRG) and short rotation coppice species. Second (bio) generation energy plant are considered as excellent candidates for the future of bioenergy and they are going to able to serve the biomass supply chain programmes. The breeding of biomass plants can be economical and sustainable, if the cultivated plant is adaptable, and it possible to grow calculable and (as it is called), carbon dioxide - positive field cultivation way. Unavoidable the biotechnology and environmental research of Sida, which is in a base stage of domestication, before it is, took into cultivation for industrial biomass purposes. The aim of this and the future investigations are to detect and solve all of the problems linked closely to the propagation and growing. S. hermaphrodita is known in the literature as a plant with low seed germination potential. In Europe, many authors have dealt with this problem because of possible industrial uses of this plant (Chudzik et al., 2010). For this reason, in Europe this plant is reproduced mainly in a vegetative way from root cuttings. One of the limiting factor of this method is that currently not be well known the virological, phytophatological and pests background of the Sida. Because the lack of investigations of its phytopathological background, the farmers who planting Sida from root cuts take on considerable environmental and economical risks. Therefore, it has to give preference to propagate it from seeds than from root cuttings for industrial-scale bioenergy farms (Kurucz et al., 2012). In general, there are many reasons which can responsible for the low germination of seeds. For example, in case of Sida it was observed over 30% of the ovules remained in their juvenile stage at the time when pollen tubes reached the ovary. These ovules were probably aborted, did not attract the pollen tubes and underwent degeneration before or during fruit development (Chudzik et al., 2010). Under Polish conditions, other studies on the biology of flowering of S. hermaphroditain showed that as many as 95% of seeds were set. From the results of some previous author, it could be concluded that, the main cause of low germination is the strong seeds dormancy (Baskin, 2003; Barthodeiszky et al., 1980). Seeds of the majority of plant species in the world except tropical rainforest and tropical semievergreen forest are dormant at maturity (Smith et al., 2004). According to Smith et al. (2004) dormant seeds can be classified into one of five classes: physiological (low growth potential of the embryo); morphological (small undifferentiated or small differentiated, but underdeveloped, embryo); morpho-physiological (underdeveloped, physiologically-dormant embryo); physical (water-impermeable palisade or palisade-like layers of cells in seeds, or fruit coat); and combinational (water-impermeable seed or fruit coat + physiologically dormant embryo). Other authors divide seed dormancy into seven classes: undeveloped embryo, embryo is mechanically inhibited by the seed-coat, water-impermeable coats (hard

3 3 seed), gas-impermeable seed coats, endogen metabolism inhibitors, complex dormancy, secondary dormancy (Barthodeiszky et al., 1980). Species with water-impermeable seed or fruit coats, physical dormancy occur in some 15 plant families (Baskin et al. 2003) including the Malvaceae family. Barthodeiszky (1980) called this phenomenon as hard seed effect and this kind of seeds was called hard seed. This impermeability of the coat is caused by the presence of one or more palisade layers of lignified Malphigian cells (macrosclereids) tightly packed together and impregnated with water impermeable chemicals (Baskin et al., 2000). An anatomical structure in the impermeable layer(s) functions as the water gap, seven types of which have been described (Baskin et al., 2000). However, Dolinski (2009) have presented evidence that dormancy-break by heating and by chemical treatment of Sida seeds may occur through disruption of the seed coats. Immediately after the harvest, only 3% of control seeds germinated whereas after 6 months, their germination increased to 14.5% %, but after 1.5 years it is decreased (Dolinski, 2009). Fresh seeds had the best germination (73%) after immersing into the boiling water; germination capacity decreased along with the water temperature decrease. Water at 70 to 80 C temperature had the most positive effects on seeds (Dolinski, 2009). Spooner (1985) concluded that scarified seeds, 81% to 99% of the seeds collected from 10 populations of S. hermaphrodita in Maryland and Ohio germinated, respectively. The average germination for all 10 populations was 92% (Spooner et al., 1985). They collected the seeds in August and September. Whereas, the other studies did not mentioned the harvesting date, it is possible that these good results was owing to the autumnal harvest. It seems that boiling water has not damaged the germs of fresh seeds, but more and more seeds imbibed without germination in subsequent examination dates (Dolinski, 2009). The previous studies revealed that seeds of some species of Malvaceae family scarified using hot water or concentrated sulphuric acid could have positive effect on seed germination percent (Dolinski, 2009; Baskin et al., 2003). Even more, it has been stored for a long time with no fear of their fast loss of germination capacity (Dolinski 2009). The aim of our present investigation were (1) to study the effect of two hot water pretreatment priming methods and (2) to analyse the influence of seed harvesting time on S. hermaphodita seed germination percent and seed contamination ratio involving four growing years. Material and Methods Collection of seed samples Germination and, viability tests were conducted during February 2013 on seeds of Sida collected from our Plants for the Future Biomass Experimental Garden in different years, i.e., 2009, 2011, 2012 and In 2009, seeds were collected in autumn. In the following seasons, we gathered the seeds in spring, in order to ensure the low temperature influence which is considered necessary. Therefore, in the following seasons 2011, 2012 and 2013, the

4 4 seeds were collected in spring time. The matured dry fruit clusters were manually harvested and threshed. Hot water pre-treatment (HWT) Seeds of each HWT-samples (except the controls) were immersed into heated water regulated for the following temperatures and time regime: 65 and 80 C, for 2 minutes; 95 C for 30 seconds, non-scarified seeds constituted the control and then, part of seeds from every combination (3 50pcs) were sown on wetted filter papers put into Petri dishes for 26 o C without lighting, while others stored for further studies. Seedling counts were performed at 3 and 6 days later (Figure 1), and total percent of germination and contamination rate were calculated for each treatment. Floated seed priming technique (FSPT) The HWT method was complemented with a simple seed priming step to fractionate them based on their specific weight, and or imbibation / permeability characteristic. First, before we put the samples into heated water, the seeds were immersed into distilled water of o C for 30 minutes. After this, the hot water treatment was performed on the dipped seeds followed separation. The sunk but not treated seeds were considered as control. These FSTP seeds were sown on wetted filter papers and put into Petri dishes for 26 o C without lighting. Statistical analysis For the germination assays each treatment consisted pcs seeds and they were repeated at least three-times. The research results were calculated by Windows Office Excel software and evaluated statistically by the analysis of variance using the SPSS 14.0 programme. Results Germination assays The effects of various temperatures of water and influence of different growing seasons for overcoming seed dormancy and the seed contamination rate of Sida hermaphrodita Rushby are shown in Table 1. It could be notice that dipping seeds into boiling water, the HWT method significantly broke seed dormancy of Sida hermaphrodita. It was also observed that while the water temperature regime was higher, the contamination ratio was significantly lower. By the best combination of HWT, the average of germination ratio was elevated up to 80%. We also observed that from the untreated seeds only 4-5% germinated (Figure 1), except the growing year of 2009, in which the germination percent was 10%. The seeds of season 2009 have the best germination percent (46%) by means of HWT method. Figure 2. also

5 5 shows that 80 C HWT resulted the highest germination ratio, almost in each season, except It was noticed that there were no significant differences between HWT treatment made at 80 o C and 95 o C (Table 2). Visual observation of seed contamination Table 1. also clearly shows that the rate of contamination of seeds (or seed coat) is dramatically decreased by elevating the water temperature up to 65 o C and 95 o C. It could be noticed that the most contaminated season was 2012 in which the contamination ratio reach 52 % in the control (Figure 2). On the contrary, the seeds from 2009 were healthier (17.33% infected seeds in average). The statistical analysis clearly showed the significant difference between the control and the treated (65 o C, 80 o C, 95 o C) samples, meanwhile the difference between 80 o C and 95 o C was not significant. The infection ratio in the control seeds of all seasons fluctuated considerably; these discrepancies can be connected with seasonal and harvesting date differences. Results of FSPT-method Table 2 shows the germination and contamination percentage in water fractionated (dipped and supernatants) and control seeds. In this FSPT treatment we applied 80 o C, the most effective priming temperature. On the basis of our data we can say, that we were able to dramatically increase the germination percent from 11,3% to 80% in average, and we also were able decrease dramatically or entirely eliminate the seed contaminations from 32,6% to 0%, under laboratory conditions (Figures 1 and 2). Conclusion Hard seeds are common in a number of species in the Malvaceae family (Spooner, 1985). In the case of Sida spinosa L. it have been shown that water impermeability of the seeds is partly explicable due to a compact layer of integumentary palisade cells. A similar layer of cells occurs in seeds of Sida hermaphrodita L, which dormancy can be stopped by hot water treatment (Chudzik et al, 2010). Spooner et al. (1985) founded that the low germination percentages obtained apparently due to his failure to scarify the seeds. It should be note that the seeds was collected in autumn of However, in this work, germination tests utilizing scarified seeds germinated from 29,3 to 46% in the case of seeds collected from our S. hermaphrodita populations. The average germination for all season varied between 4,67% and 10% without hot water priming. When physically scarified, the oldest seeds (2009) showed the best germination percent (46%) after HWT treatment. Our study also revealed that Sida hermaphrodita seeds scarified using hot water treatment (HWT method) has a positive effect on S. hermaphrodita seeds contamination pattern. The highest percentage of contamination was observed in The infection ratio was reduced by 52% to 0%. The same effect was observed in all seasons. The most favourable temperature was 95 o C, but if we take account the germination percent, we should conclude from the data

6 6 that the most effective water temperature is 80 o C. However, the germination potential of S. hermaphrodita seeds showed variability, depending on the growing season. In many experiments the maximum number of germinating seeds was about 30-40%. For this reason, this plant actually is reproduced mainly by vegetative way in Europe (Chudik et al., 2010). We discovered that there are close relationship among the collecting time, the contamination percentage as well as germination ratio. Thus, 2009 season was the best in case of contamination as well as germination percent. It could be concluded that, the most favourable period of seed collecting for propagation is autumn, after the seed ripening. During these experiments it has been found, that the success of the Sida germination shows correlation not just with the amount of the endogenous and/or exogenous fungus contaminations, but their special weight and/or water imbibation / permeable characteristic. By the influence of these recognitions we modified our seed priming technique, in such a way that we fractionated the seeds based on their relative density (filtering out the floating supernatant seeds from distilled water) before we executed the treatment. The hot water treatment (HWT) was performed on the dipped / sunk seeds after separation; therefore we were able to reach 80% germination in Petri-dishes under laboratory conditions (26 o C without lighting). This method is named as FSPT (floated seed priming technique). However, in our germination tests, utilizing 80% of separated and scarified seeds collected from Debrecen can germinate, against our previous results in which the germination reached at maximum 46% by HWT method. Therefore, we can reduce the amount of sowing-seed requirements in case of Sida hermaphrodita from seeds ha -1 to seeds ha -1. Because of deficient knowledge of Sida pathological background, the farmers who planting Sida from root cuttings are confronted by considerable risk of human health and the hazard of environment. According to our opinion it has to give preference to propagation from seeds that from root cuttings. From the investigations presented in this work, it could not be concluded clearly that the features observed during the investigated stage of S. hermaphrodita seeds have physical or physiological dormancy, or both. Nonetheless, it should be cleared that how can we characterize and prevent the endogenous or exogenous seed infections. It should be underline that the present study is the first in the series of studies in which we plan to undertake the problem of the seed embryology of this promising energetic species. The aim of our further investigations is that we should obtain more information about seed biology of some other species of Malvaceae family (Sida hermaphrodita, Kitaibela vitifolia, Kitaibela balansae, Kitaibela x kovatsii, Althea canabina). If we can increase the seed germination percent of different mallow species, these achievements may be new complementary material for the second (bio) generation plants within the biomass supply chain. Acknowledgement This work is partly supported by the TAMOP A-11/1/KONV project and cofinanced by the European Union and the European Social Fund. Additional financial support is also gratefully acknowledged for the MOP Biotech Co Ltd. (Nyíregyháza, Hungary) and Ereky Foundation (Debrecen, Hungary).

7 7 References Barthodeiszky A., Czimber Gy. (1980): A magbiológia alapjai (in Hungarian) - ISBN Baskin C.C. (2003): Breaking physical dormancy in seeds focussing on the lens. New Phytologist 158: Baskin J. M. and Baskin C. C and Xiaojie Li (2000): Taxonomy, anatomy and evolution of physical dormancy in seeds. Plant Species Biology, 15(2): Baskin J. M. and Baskin C. C. (2003): When breaking seed dormancy is a problem: try a move-along experiment, Native Plant Journal 4(1): Chudzik B., Szczuka E., Domaciuk M. and P. Danail (2010): The structure of the ovule of Sida hermaphrodita (L.) Rusby after pollination. Acta Agrobotanica, 63 (2): 3 11 Dolinski R. (2009): Influence of treatment with hot water, chemical scarification and storage time on germination of Virginia fanpetals, Sida hermaphrodita (L.) Rusby seeds, Buletyn Instytutu Hodowli i Aklimatyzacji Roślin, 251: Kurucz E., Szarvas P., Fári M. G. (2012): Alternatives of the multiple use of Virginia mallow in Acta Agraria Debreceniensis, 46: Smith R., Dickie J., Linington S., Pritchard H. and Probert R. (2004): Seed conservation: turning science into practice - ISBN Spooner D. M. (1985): Observations on the distribution and ecology of Sida hermaphrodita (L.) Rusby (Malvaceae)

8 8 Legend Tables and Figures Table 1: Influence of hot water treatment (HWT) on germination (%) and contamination (%) of Sida hermaphrodita L. Rusby seeds (50 pcs seeds per each treatment) Growing Seasons 2009 Collection: autumn 2011 Collection: spring 2012 Collection: spring 2013 Collection: spring HWT treatment ( o C) Germination (%) Contamination (%) untreated control 8,00 17, ,00 0, ,00 0, ,67 0,00 untreated control 10,00 21, ,33 10, ,67 5, ,00 4,00 untreated control 4,67 52, ,33 18, ,00 0, ,67 0,00 untreated control 4,67 36, ,00 12, ,33 2, ,00 0,00

9 9 Table 2. Germination and contamination pattern of water-fractionated seeds with or without HWT priming (50 pcs seeds per each treatment) Treatments Repeat Measurement of germination Measurement of contamination FSTP method (waterfractionated and HWT-treated seeds at 80 o C) Supernatant seeds without HWT Control (untreated seeds) Number of germinated seeds Germination (%) Number of contaminated seeds Contamination (%) I 36,00 72,00 0,00 0,00 II 43,00 86,00 0,00 0,00 III 40,00 80,00 0,00 0,00 Mean 39,67 79,33 0,00 0,00 I 1,00 2,00 47,00 94,00 II 0,00 0,00 44,00 88,00 III 0,00 0,00 45,00 90,00 Mean 0,33 0,67 45,33 90,67 I 7,00 14,00 16,00 32,00 II 3,00 6,00 19,00 38,00 III 7,00 14,00 14,00 28,00 Mean 5,67 11,33 16,33 32,67

10 10 Figure 1: Effect of water-fractionation on the germination pattern of Sida hermaphrodita Rushby. Floated seeds= supernatant fraction; sunk seeds= imbibed fraction

11 11 Figure 2: Influence of hot water treatment (HWT) on germination ( %) of Sida hermaphrodita seeds harvested in four growing seasons 50,00 45,00 40,00 35,00 Germination % 30,00 25,00 20,00 15,00 10,00 5, Collection: autumn 2011 Collection:spring 2012 Collection:spring 2013 Collection:spring 0,00 Control Treatment ( o C)

12 12 Figure 3: Influence of hot water treatment (HWT) on contamination (%) of Sida hermaphrodita seeds harvested in four growing seasons 60,00 50,00 40,00 Infection % 30,00 20,00 10, Collection: autumn 2011 Collection:spring 2012 Collection:spring 2013 Collection:spring 0,00 Control Treatment ( o C)