Study of effect gibberlic acid and chilling on dormancy breaking and germination of Alstroemeria(Alstroemeria Ligtu hybrid) Seed in Vitro and in vivo conditions
F. Nasri[*],N. Ghaderi**, S. N. Mortazavi***
Department of Horticultural Science, University of Zanjan, Iran
Abstract
The dormancy characteristics and optimum conditions for seed germination of Alstroemeria Ligtu have not been explained. The seed of Alstroemeria Ligtuhybrid were tested separately for study their germination behavior. In vitro and in vivo seed germination tests were conducted at two different treatments (gibberlic acid 0, 100, 200 and 400 mg.l-1 with and without chilling in 5±1 °C). During experiment course shoot and root length, number of root and leaf, root fresh and dryweight, seed germination percentage, germination velocity and mean germination time were recorded. Chilling had a significant effect on seed germination of Alstroemeria ligtu hybrid (p < 0.05). Soaking for 24 h in 100 mg.l-1gibberlic acid (GA3) supplemented with chilling under in vitro and in vivo conditions increased germination to 76.67%, 70%, respectively. Mean germination time (MGT) decreased with duration of chilling and concentration of GA3. Any of three concentrations of GA3 had positive effects on seedlings length under in both condition in vivo and in vitro. According to the results found in this study, A.ligtu hybrid species probably exhibits a combination of physiological dormancy (pd). Non-chilled seeds fails to germinate, whereas seeds stratified for 21 days gave 36.66%, 40% germination (respectively under in vivo and in vitro). Results show that chilling is successful in breaking seed dormancy. chilling at 5±1 °C for 21 days or 100 mg/l GA3+ 21days of chilling overcame seed dormancy and increased the germination percentage of A.ligtu hybrid seeds. In higher concentration(200 and 400 mg.l-1) germination rate decreased. In general, in vivo germination rates were lower than in vitro rates.
Key words: Alstroemeria Ligtu hybrid, GA3(Gibberellic acid),Germination, Dormancy, Chilling
Introudaction
The genus AlstroemeriaL.(Alstroemeraceae) is a south American genus with two main centersof distribution, one Chileand the second throughout the eastern of brazil and contiguous Paraguay and Argentina(Bayer, 1987; Aker and Healy, 1990). Alstroemeria is an important source of commercial cut flowers throughout the world(Gonzalez-Benito and Alderson, 1992). All species are herbaceous, perennial and rhizomatous plants with big flowers, living in a wide range of habitats from rainy forest to desert areas and from the mountains to the coast(Munoz and Moreira, 2003).This plant is cultured in greenhouse for cut flower production and is propagated vegetatively by rhizome division. Seed propagation is uncommon due to variability in the percent germination and the time required for germination variability may be caused by inviable seedsor improper techniques(King and Bridgen, 1990).The dormancy characteristics and optimum conditions for seed germination of this species have not been explained. Thus of some information about effective factors on dormancy breaking and optimal conditions of seedling growth are necessary for recovery of seed germination in this plant. The erratic and unpredictable nature of Alstroemeria germination is undesirable for commercial growers who tend to higher and more synchronous germination. Poor Alstroemeria germination is attributed to its seed dormancy(King and Bridgen, 1990).
Seed dormancy is a block to the completion of germination of an intact viable seed under favourable conditions (Hilhorst, 1995; Bewley, 1997). This block to germination has evolved differently across species through adaptation to the prevailing environment, so that germination occurs when conditions for establishing a new plant generation are likely to be suitable (Hilhorst, 1995; Bewley, 1997; Baskin & Baskin, 2004).Dormancy is an innate seed property that defines the environmental conditions which seed is able to germinate. It is determined by genetics with a substantial environmental influence which is mediated, at least in part, by the plant hormones such as abscisic acid and gibberellins(Finch-Savage and Leubner-Metzger, 2006). Two major forms of physiological seed dormancy have been described, namely embryo and coat dormancy(Kucera et al., 2005). Physiological dormancyis the most abundant form and found in seeds of gymnosperms and all major angiosperm (Finch-Savage and Leubner-Metzger, 2006). Physiological dormancy can be divided in to three levels: deep, intermediate and slight dormancy (Baskin and Baskin, 2004). Genotypic and physical constraints, morphologically immature embryos, and may be physiological inhibitors in the seedcoats appear to cause a combined dormancy in Alstroemeria seeds(King and Bridgen, 1990).
In physiological dormant seed, it is thought that temperature and gibberellins can both release dormancy and promote germination(Kucerna et al., 2005, Baskin and Baskin, 2004). Gibberellins play a key role in dormancy release and promotion of germination(Kucerna et al., 2005, Nago and Furutani, 1986).GA3 is widely used to break dormancy of seeds of various plant species. Dormant seeds which require chilling, dry storage after ripening and light as a germination stimulator, are often treated with GA3to overcome their dormancy(Gupta, 2003). In case Alstroemeria increased germination of the warm-cold treatment suggests that there are physiological factors in the seedcoat that are responsive to cold stratification or that time is required for sofening of the seedcoat(King and Bridgen, 1990). The embryo of many seeds fails to germinate because oxygen dose not diffuse through the seed coat. At low temperature more oxygen dissolves in water and therefore more oxygen is prepared for embryo(Young and Young, 1992). Dormancy and germination are complex phenomena that are controlled by both developmental and environmental factors(Bewley, 1997; Koornneff et al., 2002). When triggered, the receptors then initiate a signal transduction cascade, perhaps involving synthesis of or sensitization to germination-promoting gibberellins that lead to the completion of germination(Bewley, 1997). Imbibitions stimulates GA secretion from embryo. Secreted GA increases synthesis of hydrolytic enzymes located under aleuron layer.Synthesized enzymes are transported to endosperm via scutulum and are used for decomposing of stored food to supply the energy required for germination(Cirak et al., 2004).
The aim of the present study is to find a practical method to promote A.ligtu hybrid seed germination and the subsequent seedling growth by mean of chilling and GA3application. Therefore, we examine the effect of some treatments on A.ligtu hybrid seed germination.
Materials and method
This investigation was carried out in the Department of Horticultural Science, Agriculture Faculty, University of Zanjan, in Iran. Seeds of Alstromeria ligtu hybrid were immediately washed with tap water and then divided in to three groups(each group was divided in to four replicates) and subjected to one of the following treatment: soaking in to tap water only for 24 h(control); soaking in a gibberellic acid(GA3) solution at 0, 100, 200 and 400 mg.l-1 for 24 h supplemented with chilling at5±1ċ and soaking in a gibberellic acid(GA3) solution at 0, 100, 200 and 400 mg.l-1for 24 h without chilling.Each group ofseeds were sterilized individually by 70% ethanol(1 min), 3% sodium hypochlorite solution(20 min) and then rinsed with sterilized water(10 min each).All these treatments before transfer to the chilling were out separately at 21ċ in 16h light conditions(1-week).
In vivo germination
The seeds were sowing directly in the soil(peat/sand/perlite mixture) at a depth approximately 0.5-0.7cm in pot(10 seed per pot). Watering was done every 3 days. Each pot was containing 10 seeds. Germination of seeds was recorded at daily interval. After three-weeks keeping seeds in the chilling conditions, transferred to the growth chamber, and cultures were placed under 21 0C and 16h light.
In vitro seed germination
The seeds were incubated in 250 ml jars containing half strength MS medium(Murashige & Skoog, 1962), supplemented with 1% sucrose and 0.7% agar, and pH was adjusted to 5.8. All of the jars after three-weeks keeping in the chilling conditions, transferred to the growth chamber and kept at 210Cand in of 16h light and 8h darkness photoperiod. Each jar was containing 10 seeds. Seed germination were recorded at daily interval. The progress of seed germination was recorded for a period of 30 day.In in vivo and in vitro experiments, Daily measurements was recorded after pre-chilling start. The oven-dried weight was obtained by drying seedlings at 700Cto constant weight. Radicle length of 2 mm was scored as germinated seed(Kaya et al., 2006). Germination percentage was recorded every day. Mean Germination Time(MGT) was calculated to assess the rate of Germination(Ellis and Roberts, 1981). The data for germination percentage (%),germination rate and mean germination time (MGT) was recorded during the course of study.
Statistical analysis
Experiments were arranged in a randomized complete design with 4 replications and 10 seeds per replicate. The statistical analysis was made using theGLM procedure of SAS. The differencesbetween the means were compared using theDuncan’s multiple test (p < 0.05).
Result and discussion:
In vivo experiment
Chilling had a significant effect on seed germination of Alstroemeria ligtu hybrid (p < 0.05). Non-chilled seeds gave only 13.33% germination, whereas seeds stratified for 21 days gave 36.66% germination (Table. 1). chilling might act simply to lower the rate of enzymatic reactions taking place in the seed, and might cause differential changes in enzyme concentrations or in enzyme production (Bewley and Black, 1994).Imbibed dormant seeds are metabolically very active and in this state can receive external signal(e.g., light, chilling, alternating temperatures, and chemical or hormonal treatment) that can stimulate germination (Bewley, 1997). Chilling alone or in combination with GA3improved significantly seedling characteristics including seedling length, root dry weight, shoot dry weight and has a larger effect than the GA3 treatments applied in this study(Table1).Our results show that chilling is successful in breaking seed dormancy, though the duration of treatment may vary with the species. Germination percentage was affected by gibberllic acid application (Table. 1).Mean germination time (MGT) decreased with duration of chilling and concentration of GA3 (Figure.1), indicating that dormancy can be suppressed by moist chilling and exogenous GA3 application. When, the seeds were soaked at 100 mg.l-1GA3 solution for 24h, the mean germination time(MGT) reached 16.44 (Fig.1).Chilling has been reported to induce an increase in GA3 concentration (Bretzloff and Pellett, 1979). Application of GA3 during and after chillingon Pistachio seed, increased the length, trunk diameter, internodes length, leaf area and fresh and dry weight of seedlings(Rahemi and Baninasab, 2000). In present study, the application of GA3(100 mg.l-1) resulted inhigher germination percentage and rate,shoot and rootfresh weight, shoot and root dry weight than those of seeds treated with 200 and 400mg.l-1GA3(Table1). Seeds treated with 100 mg/l GA3 without chilling gave 13.33% germination, whereas seeds treated with 100 mg/l GA3 + 21 day chilling gave 70% germination. It has been reported thatgermination can be induced by gibberellic acid in Vaccinium myrtillus L. (Giba et al., 1993) and Fagus sylvatica (Nicolás et al., 1996) seeds. These results confirm that GA3 treatment enhances seed germination. GAs are known to obviate the requirement of seeds for various environmental cues, promote germination, and counteract the inhibitory effects of ABA, frequently in combination with cytocinins(Bewley and Black, 1982, 1994). In ourexperiment, application of GA3 stimulated the seed germination ofA.ligtu hybrid. This response was dependent on the concentration ofapplied GA3. At lower concentrations, germination was higher. There was not significant differences between 100 and 200 mg-1GA3 treatments. Control treatment resulted at the end of the test in table.1 (without germination) indicating a high level of dormancy. As seeds readily imbibed water there can be no physical dormancy present (Willan, 1987; Schmidt, 2000). The chilling process appears to enhance the production of some types of growth-promoting substances such as GA (Powell, 1987). Giba et al. (1993) reported that the inhibitory effect of retardants was overcome by gibberellic acid. GA3 improved germination percentage which could indicate the presence of chemical dormancy, as application of gibberellic acid has shown effect in overcoming dormancy caused by inhibitors (Bewley and Black, 1994). Three successful treatments involve gibberellic acid (100, 200 and 400 mg.l-1), indicating presence of chemical dormancy, as gibberellic acid has shown effect in overcoming dormancy caused by inhibitors (Bewley and Black, 1994).Physiological dormancy in seeds is dependent on the ratio of the levels of abscisic acid (a growth inhibitor) and GA (a growth stimulator) (Hilhorst and Karssen, 1992). The results of this experiment confirmed that the A.ligtu hybrid seeds were in a dormant state. chilling at 5±1°Cfor 21 days or 100 mg/l-1GA3 + 21days of chilling overcame seed dormancy and increased the germination percentage of A.ligtu hybridseeds. These findings, except for the scarce response to GA3, firmly support the hypothesis that A.ligtu hybrid seeds fit the characteristics a non-deep physiological dormancy according to the dormancy classification of Baskin and Baskin (2004). Further, physiological barriers to germination in embryos have been overcome by cold stratification in a number of rose species (Zhou et al., 2009). Results obtained in this study present strong evidence that the pericarp, the testa, and the embryo play important roles in regulating seed dormancy.
The negative effect of the testa on germination can be attributed to some inhibitory substances in the testa and not to its role as a mechanical barrier or in restricting access to water (Bo et al., 1995). Baskin et al. (2001), Baskin and Baskin, 2001 reported that 5 °C is often the optimum cold stratification temperature that alleviates dormancy in temperate woodland herbs. They hypothesized that cold temperatures (6–10 °C) alternating with cool temperatures (15–20 °C) alleviate physiological dormancy. King and Bridgen (1990) reported that may be physiological inhibitors in the seed coats appear to cause a combined dormancy in Alstroemeria seeds . El-Refaey and El-Dengawy (2005), shown that chilling of seeds at 4-5°C or treatment of seeds with GA3 was successfully over come dormancy in Eriobotrya japonica seeds. Cold stratification, generally in the range of 1-10 °C, can break seed dormancy for a number of species (Bewley and Black, 1994), and the data presented here show that this includes A.ligtu hybrid. The current study demonstrated that seed germination of A.ligtu hybrid is promoted by an 3-week cold period, which suggests that A.ligtu hybrid seeds may have a similar primary endogenous dormancy.
Generally, pre-sowing GA3 treatments improved the germination rate and uniformity, and early seedling growth in both under in vitro and in vivo. Therefore, maximum seedling fresh and dry weights(roots and shoots) were recorded from seed treatment with 100mg/l-1GA3.
In vitro experiment
The seeds treated withGA3+ chilling germinated after of 21days, while those without pre-chilling not germinated after 45 days. These results showedthat pre-chilling treatment was more benefit for A. Ligtuhybridseed germination (Fig.1).GA3 treatments increased germination speed in compare to those did not treated with GA3. In the present study, seed germination rate was depended to GA3 concentration. The germination percentage in half strength MS medium(in both under chilling and without chilling) were higher than in vivo rates (Table1). This could have been due to the effect of various elements used in the medium. In vitro germination condition is a nutrient medium containing macro and micro elements and sucrose (1/2MS) that has a positive effect on A.ligtu hybrid seeds germination.Seeds didn’t germinate without GA3 and chilling treatments, Butthe seedstreated withgibberellicacid,without chilling, germinated after 45, 49, 51 days,respectively, at 100, 200 and 400mg l-1 (Figure-1).
Little effect of GA3in stimulatingseed germinationcanprobablybe attributed tofollowing possibilities: a negative effect of GA3 on the level of some enzymes activity (glutamate-oxaloacetate transaminase, pyruvate kinase and malate dehydrogenase) and consumption of nucleotides in the synthesis of nucleic acid (El-Dengawy, 1997) and/or the production of a proteinaceous germination inhibitor. Further,GA3 is effective in breaking the slight physiological dormancy, but it does notovercome the deep physiological dormancy (Baskin and Baskin, 1990). Result for pre-treatments with GA3 and chilling for seed A.ligtuhybrid under in vitro conditions are given in table 1.Seed germination percentage in control treatment(only chilling) after 3-week of chilling, which produced 40% germination(Table 1).The highest germination percentage after 3-week of chilling was foun in seed treated with 100 mg.l-1GA3(76.67%). Any of three concentrations of GA3 had positive effects on seedlings length under in both condition (in vivo and in vitro), showing that 100 mg.l-1 was better than other concentrations. In higher concentrations(200 and 400 mg.l-1) germination rate decreased, Further,mean germination times(MGT) decreased in chilled compared to non-chilled treatments. According to the results found in this study, A.ligtuhybrid species probably exhibits a combination of physiological dormancy(pd). Chilling was able to break dormancy. ApplicationGA3 (supplemented with or without chilling) affected total germination in this experiments(Table1). Soaking in 100 ppm GA3(supplemented with chilling) resulted in 76.66% germination, which was higher than that in control, but further increasing of GA3concentration did not have any effect. However, application higher concentrations, stimulate early germination, but maximum germination percentage was observed 21 days after culture. The seeds without GA3 and chilling treatments (control) didn’t have germination. These results may be related to seed dormancy in present study.
Table 1-Effect of Gibberellic acid (GA3) and Chilling treatments on germination parameters in Alstroemeria Ligtu hybrid seeds.
D.W.R / D.W.S / F.W.R / F.W.S / Root Length / Shoot Length / No.Leaf / G.R
(in day) / G.P
(%) / Treatmaent / Type Culture
GA3×Chilling
3.4cd / 6.43cd / 34cd / 82d / 2.91d / 3.81d / 2.47 cde / 0.17g / 36.66c / * / 0 / In vivo
0i / 0i / 0i / 0h / 0h / 0i / 0 J / 0m / 0.0e / - / 0 / In vivo
1.6ef / 3.39efg / 14.67f / 27fg / 0.65g / 2.32e / 1.55fjhi / 0.17g / 40c / * / 0 / In vitro
0i / 0i / 0i / 0h / 0h / 0i / 0J / 0m / 0.0e / - / 0 / In vitro
8.8a / 17.77a / 63.5a / 339.3a / 5.17a / 8.89a / 5.04a / 0.31e / 70ab / * / 100 / In vivo
1.1fgh / 1.4hi / 9.1fgh / 28fg / 0.95fg / 1.85efg / 1.97defgh / 0.025k / 13.33d / - / 100 / In vivo
3.4cd / 6.3cd / 31.67d / 71de / 4.03c / 5.5c / 3.34b / 0.37a / 76.67a / * / 100 / In vitro
0.52hi / 0.45i / 5hi / 9.2gh / 0.57g / 0.91h / 0.97i / 0.027k / 16.67 / - / 100 / In vitro
4.57b / 13.8b / 42.67b / 237b / 4.81ab / 7.97b / 2.71bcd / 0.35c / 67b / * / 200 / In vivo
1.4efg / 1.9ghi / 11fg / 27fg / 1.2ef / 1.97ef / 1.82efgh / 0.019l / 10d / - / 200 / In vivo
2.97d / 4.87de / 38bc / 58.67de / 4c / 4.96c / 2.30cdef / 0.36b / 73ab / * / 200 / In vitro
0.58hi / 0.41i / 6.9gh / 7.1gh / 0.91fg / 1.1gh / 1.22h / 0.034j / 15d / - / 200 / In vitro
4bc / 8.03c / 33cd / 152c / 4.61b / 5.6c / 3bc / 0.30f / 66.67b / * / 400 / In vivo
1.6ef / 2.5fgh / 14f / 32fg / 1.4e / 2.1e / 2.1defg / 0.032j / 16.67d / - / 400 / In vivo
2.1e / 3.9ef / 21e / 51ef / 3.86c / 3.97d / 2.41cde / 0.32d / 70ab / * / 400 / In vitro
0.62jhi / 0.66hi / 5.3ghi / 11gh / 0.86fg / 1.3fgh / 1.33ghi / 0.044i / 17 d / - / 400 / In vitro
Means in a column followed by the same letter are not significantly different at the 5% level as determined by Duncan’s. *: with chilling -: without chilling.