Bjedov BREAKING OF JUDAS TREE SEED DORMANCY BY PLANT HORMONE TREATMENTS

The study deals with the influence of growth regulators on the germination of Judas tree (Cercis siliquastrum L.) seed which is double dormant. We simultaneously tested seeds prepared in the conventional procedure: scarification + stratification and scarified seeds treated with phytohormones from the groups of gibberellins, auxin and cytokinins. The results indicate a positive effect of gibberellic acid (GA), as well as some combinations of this phytohormone with others. Recommendations for practice are to combine the conventional procedure with GA. The procedure may shorten the duration of stratification; the application of GA should follow stratification because the temperature of 4°C does not provide growth regulators activity. The study results can serve as the base for easier generative reproduction of this valuable woody ornamental species which could have a wide use in changed climate conditions.


INTRODUCTION
In addition to the more widespread use of growth regulators in the vegetative propagation of ornamental trees and shrubs, which is reflected in the application of auxin for rooting cuttings of various types, as well as, micropropagation, plant growth regulators can be used for troubleshooting in the germination of species that have a dormant embryo or double seed dormancy (embryo + seed coat).The germination of tree seeds appears to be controlled by a variety of external and internal factors.Plant growth regulators figure prominently among these factors, but the range of mechanisms by which such control is edited may vary considerably-from physical to metabolic.
B e wl e y & Bl a c k (1985) reported a list of chemicals that have been proven to eliminate seed dormancy.It belongs to respiratory inhibitors, oxidants, nitrates, nitrites and growth regulators.
Levels of endogenous plant growth regulators are believed to play a major role in overcoming seed dormancy of different species with variable success.The most commonly used are gibberellins, cytokinins and ethylene.Gibberellins have the widest range of effect.Gibberellins (GAs) promote the induction of cell wall hydrolases and thereby promote endosperm weakening and endosperm rupture (micropilar endosperm in particular).Abscisic acid (ABA), acting as antagonist of GAs, inhibits the induction of cell wall hydrolases and thereby inhibits endosperm weakening and endosperm rupture.GA promotes and ABA inhibits the embryo growth potential (M ü l l e r et al., 2006).Work of Fe r n a n d e z et al. (1997) shows strong variations in endogenous GAs during dormancy release in beech seeds (Fagus sylvatica L.) and the capacity of non-dormant seeds to carry out metabolic conversions when labeled GA 20 was injected into the seeds.These findings support a dynamic role of GAs in the release of dormancy.Similar results obtained with Chionanthus virginicus L. seed where GA has shown helpful in overcoming endodormancy which is confirmed with species (beside Mechanical dormancy).Different concentrations of GA 3 had different influence.The lower one (100 mgL -1 ) has shown stimulative effect while the higher one (1000 mgL -1 ) has shown the inhibitor effects (Grbić et al., 2005).However, there are some species (Malus sylvestris Mill.) whose seed gibberellin is not affected, while cytokinins have a positive impact.Cytokinins reversed the inhibitory effect of ABA (similar with GAs when the ABA is in lower concentrations), but sometimes cause abnormal germination (inhibition of radicle emergence).It is known that the effect increases in phytohormone coactions with other factors or in the joint action with other phytohormones.Kinetin with low light intensity is more effective, and ethylene + gibberellin in the presence of light.Experiments with Lactuca sativa 'Grand Rapids' seeds indicates a positive effect of GA, negative when the GA and ABA together, and again positive when cytokinins added to the previous combination (K a h n , 1968).Reports of Ta m u r a et al. (2006) suggest that ABA and GA are involved in the control of seed germination by temperature, but how these hormones mediate the high-temperature signal remains unknown.
The aim of this study was to examine the effects of plant growth regulators (GA 3 , BA, and IBA), either single or in combination with other regulators and temperature (as one of the key factors in overcoming dormancy) on seed of Judas tree (Cercis siliquastrum L.).
Judas tree or redbud is a small tree to large shrub with several stems, 4-6 m tall and wide (10-13 m exceptionally) from Mediterranean Europe and Western Asia.Species belong to Caesalpiniaceae family.Habit is funnel-shaped becoming umbrella like: slowgrowing, occasionally with runners.
Leaves are up to 13 cm, often broader than long, round-reniform with blunt apex or occasionally with shallow notch, dull green; pale yellow from the end of October to November.The inflorescences are conspicuous with soft fragrance, crimson-pink flower clusters appear directly on the stem and branches (cauliflory), before leaves shoot, 2-3 weeks in April.The flowers are pollinated by bees, attracted by nectar.Fruits are brownish, 7-10 cm long flat leathery legume, lasting through the winter time.Nitrogen-fixing root is flat, forming runners when injured.
The species prefers deep, moderately dry, not too nutritious, well drained soil with high chalk content; demands a position in full sun or partial shade, hot and protected areas as sensitive to frost, and is good for urban areas (hardiness zone 7a).
Seeds of Judas tree show germination obstacle which is very frequent in species from Cesalpiniaceae family mainly caused by physical dormancy, but the seed of Judas tree possess double dormancy: hard test and physiological dormancy (S t i l i n o v i ć et al., 1983, 1985).The seeds of C. siliquastrum have a coat-imposed dormancy due, in part, to the hardness and impermeability of the seed coat (R i g g i o B e v i l a c q u a et al., 1985) and, in part, to the presence in the endosperm of ferulic acid as a chemical inhibitor (M a r t i n u c c i et al., 1985).Bound ferulic acid in the endosperm of Cercis siliquastrum L. Embryos in such genera as Cercis (Fabaceae) are still non-deep dormant and thus require a few weeks of cold stratification, i.e. after physical dormancy is broken and seeds imbibe water, before they will germinate (B a s k i n and B a s k i n , 2004).

MATERIALS AND METHODS
Legumes with seeds of Judas tree were collected from a green space of New Belgrade (44.815789° -44.814115° N;20.411993° -20.415791° E).Basic morphological characteristics and quantitative indicators of fruits and seeds collected in New Belgrade are shown in Table 1.The conventional way to eliminate complex forms of seed dormancy is a combined procedure of seed coat wounding, and, after that, seed exposure to positive temperatures close to zero, and high humidity (stratification) affects the dormant embryo.The seed of Judas tree, according to previous studies, corresponds to chemical scarification (H 2 SO 4 30 min) combined with stratification at +2°C for 30 days (S t i l i n o v i ć et al., 1983, 1985).The same conventional procedure was used for the control treatment in our studies.A study, individual and combined effects of tree growth regulators to speed up germination, was conducted in order to simplify the sowing pre-treatment.Mechanically scarified seeds were soaked for 24 hours in the following solution: GA 3 (500 mgL -1 /1.44 mM), BAP (200 mgL -1 /8.88 mM), GA 3 +BAP (500+200 mgL -1 /10.32 mM), GA 3 +IBA (500+500 mgL -1 /11.28 mM), and BAP+IBA (200+500 mgL -1 /18.72 mM).The seeds were placed in the sand at temperatures of +20°C and +4°C, after the treatment.Auxin IBA, although not in the group of phytohormones that affect overcoming seed dormancy is involved in the experiments due to the outstanding synergies BA/IBA often manifested in a balanced differentiation of tissues in vitro, and to prevent the potential impact of BAP on the formation of abnormal seedlings.
Since the Judas tree is not covered by the International Rules for Seed Testing by the International Seed Testing Association (ISTA, 1999), germination was carried out by the procedure for Gleditsia triacanthos L., species closest to Judas tree.The germination test was carried out in a cabinet germinator, in sand (4x100 seeds per treatment).The photoperiod was 16/8 (light -dark).The testing lasted 28 days, while germinative energy (GE), calculated on the 10th day based.
The result was presented by means of nine indicators of germination, some of which reflect only the quantitative value of germination (germinative capacity (GC), real germination (RG), and germinative energy (GE)), others only the dynamics (mean germination period (MGP), germination intensity (GI), coefficient of the rate of germination (CRG), and the coefficient of uniformity of germination (CUG), and still others refer to both groups (germination value by Czabator (GV (Cz) The conclusions on treatment efficiency were made on the basis of statistically analyzed obtained data using the Statgraphics program, version 5.0 (STSC Inc. andStatistical Graphics Corporation, 1994-2000, USA).The significance of differences between the mean values was determined by the analysis of variance (ANOVA, p <0.05) and the method of Duncan's multiple range test.

RESULTS AND DISCUSSION
The effect of phytohormones in combination with lower temperature (+4°C) did not affect germination, and all variants of phytohormone treatments have yielded negative results.Germination was completely absent (BA+IBA 0%, GA 3 +IBA 0%) or was below 2% (GA 3 1.5%, GA 3 +BAP 0.75%, BAP 0.25%).In our trials, low temperatures during stratification affected the removal of endogenous dormancy of seeds, if low temperatures are combined with phytohormones that showed an inhibitory effect.Exposure of imbibed seeds to low temperature promotes seed germination of many plant species.Experience with some species, such as Arabidopsis mutants, shows that some mutants are up-regulated and others are down-regulated by red light, which promotes seed germination.In addition to light, In Arabidopsis, expression of GA 20 -oxidase genes, GA 20 ox1 and GA 20 ox2, and GA 3 ox1 was reported to be up-regulated by low temperature in darkness, and germination of GA 3 ox1 mutant seeds are not stimulated by low temperature (Yamauchi et al., 2004).During germination of Arabidopsis seeds, GA 20 ox genes, GA 20 ox1, GA 20 ox2, and GA 20 ox3, and GA 3 ox genes, GA 3 ox1 and GA 3 ox2, are involved in GA synthesis (Ogawa et al., 2003).Suppression of all these GA 20 ox and GA 3 ox genes at supraoptimal temperature may be responsible for the suppression of GA synthesis.M i t c h u m et al. (2006) reported that seeds of the GA 3 ox1 GA 3 ox2 double mutant do not germinate in the light at 22°C and their germination is rescued by the application of bioactive GA.Therefore, the suppression of GA 3 ox1 and GA 3 ox2 genes at high temperature should be critical for thermo inhibition.Interestingly, the expression of GA 20 ox1, GA 20 ox2, and GA 3 ox1 has been reported to be up-regulated by low temperature (Ya m a u c h i et al., 2004).Thus, GA synthesis is likely to be regulated by a wide range of temperature through modulation of GA 20 ox and GA 3 ox gene expression.The expression of GA 3 ox genes in lettuce and Arabidopsis is well known to be photoreversibly regulated by light through phytochrome (To y o m a s u et al., 1998; Ya m a g u c h i et al., 1998).Understanding the cross talk between light and temperature signals on the regulation of GA 3 ox gene expression may give insight into how plants transduce multiple signals and how plants respond to complex environmental stimuli (To h et al., 2008).Studies with hormone-deficient Arabidopsis mutants have indicated that the release of dormancy can occur in the absence of GA-biosynthesis but this involves changes in the sensitivity of seeds to gibberellins (Karssen and Groot, 1987).
Studies of G a s t a l d o et al. (1982) conducted on Cercis siliquastrum seeds treated with kinetin confirm that this hormone does not interrupt dormancy in either whole seeds or those decoated at the radical pole.Seeds totally decoated or decoated at the cotyledon pole only demonstrated atypical germination linked to cotyledon growth, permitting the embryo to escape the inhibitory action present in the endosperm; this does not occur when the cotyledon surface is experimentally reduced.In our research, seed treated with BAP did not germinate, but in combination with GA 3 shows that relative germination (RG) does not differ significantly from the same indicator of stratified seeds.In combination with IBA it provides a much lower value, but in both cases there was no abnormal germination.
The results of experiments conducted at +20°C are shown in Table 2 and Figure 1 (non-scarified seed, scarified seed, and seed treated with BAP did not germinate, which is not shown).

Quantitative indicators of germination
Germinative capacity and Real germination clearly show the best results with seeds treated with gibberellic acid, and then followed by seeds treated with conventional procedure (scarification + stratification) and then, seed treated with the combination of GA 3 +IBA with or without significant differences from the conventional procedure, depending on whether the number of germinated seeds expressed on the basis of all seed placed on germination (GC), or on the basis just of sound ones (RG).Germinative energy (GE) of seeds treated with standard procedure is significantly higher than in other treatments (Tab.2; Fig. 1.).The results of germinative energy (GE) in comparison to GC and RG show differences.Lower values of GE compared with GC point out germination after the 10th day which is the base for GE.A longer period of inactivity during the test is a characteristic of a seed with partially overcome dormancy, so the standard procedure showed that GE was by more than 47% higher than the GE of seeds treated with GA 3 .In other treatments there were no germinated seeds after the 10th day of the test (Tab.2.).
G e b r e and K a r a m ( 2004) obtained similar results with various methods for breaking dormancy and enhancing the germination of Cercis siliquastrum seeds investigated.It was found that imbibed seeds that were treated with 1.4 mM GA 3 exhibited 48% germination, whereas those stratified for 16 weeks exhibited 77% germination.It was also reported that treating imbibed seeds with KNO 3 or thiourea failed to induce germination and imbibition occurred only when the seeds were scarified mechanically, with water at 80°C or with H 2 SO 4 , but none of the imbibed seeds germinated although seed viability was 80%.Significant difference between germination of treated seed with GA 3 and stratified seeds in our studies may be explained by the shorter duration of stratification (30 days), and genetic differences related to the provenance of the seeds, as well as different environmental factors during seed maturation.On the other hand, 60% (CG) and 63% (RG) germination is sufficient for it to be considered successful.Significant practical benefit is shortening of the presowing treatment duration by avoiding stratification.

Indicators of germination dynamics
Better results of MGP and GI were obtained with a conventional procedure also, indicating a better dynamic of germination then to hormone treated seed.Within these treatment order does not change.Treatment with GA 3 showed a significant difference compared to the GA 3 +BAP, GA 3 +IBA and BAP+IBA (Tab 2.).Better results with stratified seeds can be taken only in part because the first germinated seeds in this treatment received 31 days after the beginning of stratification, and in seeds treated with GA first germinated grains were observed 11 days after immersion in phytohormone solution.The germination intensity reflected identical tendencies as the previous parameter (Tab 2.).

Complex indicators
The coefficients of germination favour the conventional procedure, followed by GA 3 , and other procedures.The coefficient of uniformity of germination (CUG) did not show any statistically significant differences between the variants of the experiment (Tab.2.).

CONCLUSIONS
The effect of phytohormones on overcoming seed dormancy of Judas tree is expressed differently.
GA 3 can be a complete replacement for the traditional procedure for preparing Judas tree seeds (based on the quantitative indicators of germination).Recommendations for practice are to apply GA 3 only, or a combination of the conventional procedure with GA.The procedure may shorten the duration of stratification; the application of GA should follow stratification because the temperature of 4°C does not provide growth regulators activity.Continued research should be directed towards the choice of optimal concentration, due to the fact that H a r t m a n n et al. (1990) recommend using it for this purpose in the range of 200 to 1000 mgL -1 .In connection with this, need to continue research with GA 3 in combination with other phytohormones, including other GAs is necessary.BAP did not show a positive effect on germination, but no germination abnormalities too.Its combination with IBA exerts positive effects, which justifies the use of IBA in combination with the promoters of germination.
One more reason for additional trials are the findings of the research of post-germination events of Judas tree described by R a s c i o et al. (1998).It was found that the plantlets of Cercis siliquastrum that originated from GA 3 -supplied seeds were taller than those from stratified ones.Moreover, they produced a greater number of leaves but a reduced root mass and had some difficulty in maintaining a good water balance.Циљ рада био је да се испитају утицаји регулатора растења, било изоловано или у комбинацији са другим регулаторима и температуром (као једним од пресудних фактора на превазилажење дормантности).
) and germination value by Djavanshir (GV (MC)).The first five indicators are widely used and common, and their formulas are available in references.The coefficient of uniformity of germination and germination value by Czabator and Djavanshir have been calculated according to the formulas from the papers by B e w l e y & B l a c k (1985), C z a b a t o r (1962) and D j a v a n s h i r & Po u rb e i k (1976).

Diagram 1 .
Germination curves (GC) of Judas tree seeds treated in different ways Графикон 1. Техничка клијавост семена јудиног дрвета третираног различитим поступцима Mü l l e r K., Ti n t el n o t S., L e u b n e r-M e t z g e r G. (2006): Endosperm-limited Brassicaceae seed germination: Abscisic acid inhibits embryo-induced endosperm weakening of Lepidium sativum (cress) and endosperm rupture of cress and Arabidopsis thaliana.Plant and Cell Physiology 47: 864-877 R a s c i o N., M a r i a n i , P., D a l l a Ve c c h i a , F., L a R o c c a , N., P r of u m o , P., G a s t a l d o P. (1998): Effects of seed chilling or GA 3 supply on dormancy breaking and plantlet growth in Cercis siliquastrum L. Plant Growth Regulation 25: 53c, Kluwer Academic Publishers.Printed in the Netherlands.53-61 R ig g i o B e v i l a c q u a L., R o t i M i c h e l o z z i G., S e r r a t o Va l e n t i G. (1985): Barriers to water penetration in Cercis siliquastrum seeds.Seed Sci Technol 13: 175-182 С т и л и н о в и ћ С., Г р б и ћ М., Ђ о р ђ е в и ћ Д. (1983): Утврђивање правилних поступака за предсетвено третирање неких врста семена са тврдом и непропустљивом семењачом.Гласник Шумарског факултета, серија С 61:23-37 С т и л и н о в и ћ С., Г р б и ћ М., Ђ о р ђ е в и ћ , Д. (1985): Утврђивање правилних поступака за предсетвено третирање неких врста семена са тврдом и непропустљивом семењачом (II).Гласник Шумарског факултета 64:335-45 Ta m u r a N., Yo s h i d a T., Ta n a k a A., S a s a k i R., B a n d o A., To h S., L e p i n i e c L., K a w a k am i N. (2006): Isolation and characterization of high temperature-resistant germination mutants of Arabidopsis thaliana.Plant Cell Physiol 47 1081-1094 To h S., I m a m u r a A., Wa t a n a b e A., N a k a b a y a s h i K., O k a m o t o M., J i k u m a r u Y., H an a d a A., A s o Y., I s h i y a m a K., Ta m u r a N., I u c h i S., K o b a y a s h i M., Yam a g u c h i S., K a m i y a Y., N a m b a r a E., K a w a k a m i N. (2008): High Temperature-Induced Abscisic Acid Biosynthesis and Its Role in the Inhibition of Gibberellin Action in Arabidopsis Seeds.Plant Physiol.146(3): 1368-1385.doi: 10.1104 Toyo m a s u T., K a w a i d e H., M i t s u h a s h i W., I n o u e Y., K a m i y a Y. (1998): Phytochrome regulation gibberellin biosynthesis during germination of photoblastic lettuce seeds.Plant Physiol 118: 1517-1523.Ya m a g u c h i S., S m i t h M.W., B r o w n R.G., K a m i y a Y., S u n T.P. (1998): Phytochrome regulation and differential expression of gibberellin 3β-hydroxylase genes in germinating Arabidopsis seeds.Plant Cell 10: 2115-2126.Поред све шире примене регулатора растења у вегетативном размножавању украсног дрвећа и жбуња, која се огледа у примени ауксина за ожиљавање различитих типова резница као и у све комерцијалнијој микро пропагацији, регулатори растења могу се употребити и за отклањање сметњи у клијању врста које имају дормантан ембрион или двоструку дормантност (ембрион + семењача).

Table 2 .
Parameters of seed germination after different phytohormone treatments at +20°C