Effects of seed treatments and storage period on seed dormancy

Effects of seed treatments and storage period on seed dormancy, viability and germination of Michelia champaca L.

THESIS
BY
CARRYMEWELL SURONG
(Roll no.1606090010)
Submitted in partial fulfilment of the requirement for the
Degree of
MASTER OF SCIENCE
IN
FORESTRY AND BIODIVERSITY

TRIPURA UNIVERSITY
(A Central University)
SURYAMANINAGAR, AGARTALA, TRIPURA– 799022
INDIA
2018
INTRODUCTION
Michelia champaca L. is belonging to the family Magnoliaceae. The tree has conical and spherical crown. The seed surface is wrinkled. The immature seeds are brownish in color and when the seeds gets matured, the seeds turn black in color. Follicles are elliptical in shape. Five to eight seeds are enclosed in a follicle. This species bears seeds twice a year. Fruits of Michelia champaca are edible by birds.

Dormancy classification in seeds is based on the permeability of seeds to water and on the physiological response to temperature (Baskin and Baskin 1998). Seed dormancy can be overcome by treating with some growth hormones. However, the use of growth hormones in breaking the seed dormancy could be as a result as of many other factors such as tree species, types of hormone and climatic factors (Christopher et al., 2015). Some other methods have also been used to overcome the seed dormancy including scarification, stratification, temperature treatments, and light. There are chances that the seeds of any particular species might have two kind of dormancy. One study reported that the seeds of Magnolia punduana has both morphological as well as morphophysiological dormancy (Martin 1946; Zhou et al, 1990). There are number of studies addressed to class of dormancy present in different tree species. Seeds of Fabaceae exhibited physical dormancy because of impermeability of seeds to water and gases (Shaik et al., 2008). Seed dormancy plays a significant role in the conservation of the tree species as the failure of germination due to seed dormancy could lead to extinction of any particular species.
Several environmental factor such as light, temperature, pH, and soil moisture have been known to have an influence on germination of seeds (Koger et al., 2004). Soil moisture is an important factor that that has an influence on the seed germination and seedling emergence (cardwell, 1984).

Viable seeds are seeds which have the ability to germinate under favorable environmental condition. There are several factors having an influence on the viability of seeds. Seed moisture content and temperature are the major factors influencing storage of seeds. The slight increase in the temperature and moisture content make the seeds more susceptible to fungal attack. Seed viability is best retained at low temperature and high concentration of CO2 (Hadidi, 1996). Seed viability varies with different tree species. Seed viability of Aquilaria agallocha and Aesculus assamicus is 1-2 months and 10-12 months, respectively (Deb et al., 2013). Seed storage also plays a significant role in the conservation of the tree species with low viability.
The seeds of Michelia champaca have low viability as well as germination. More over some of the studies which had carried out on the seed dormancy as well as viability of Michelia champaca by Bahuguna et al (1987and 198) and Thilina et al (2013) failed to enhance the germination as well as the viability of this species. Therefore, the present study was undertaken with the following objectives:
To determine the kind of seed dormancy in Michelia champaca.

To study the effects of different treatments on seed dormancy, viability and germination in Michelia champaca.

To study the effects of storage period on seed dormancy, viability and germination of seeds in Michelia champaca.

REVIEW OF LITERATURE
The chapter gives an overview of literature available on the types of seed dormancy, effect of different treatments on seed dormancy and germination; and effects of storage period on viability and germination. The same has been reviewed under the following heads:
2.1. Types of dormancy
2.2. Effect of different treatments on seed dormancy and germination
2.3. Effects of storage period on viability and germination
2.1. Types of dormancy
Seeds of Michelia champaca were classified as having physiological dormancy based upon enhanced germination (up to 80%) with Gibberellic acid (GA3) (Bahuguna et al. 1988). Seeds of Magnolia punduana were classified as having physiological dormancy evidenced by enhanced germination with application of GA3 (Bahuguna et al., 1998). Seeds of Fabaceae were classified as having physical dormancy because of the impermeability of seeds to water and gases (Shaik et al., (2008).The most effective method of pre-treatment for the enhancement of germination in Albizia falcataria and Albizia procera was physical scarification followed by soaking in flowing water for 24 hours. This indicated that the seeds of this species have physical dormancy (Sajeevukumar et al., 1993).The application of Gibberellic acid (GA3) at the concentration of 1,000 mg·L-1 had an effect in breaking seed dormancy of Cupressus atlantica. This indicated that the seeds of Cupressus atlantica have physiological dormancy (Youssef et al., 2012).Seed scarification with H2So4 for a duration of 1 hour, 50, 45, and 30minutes or sandpaper enhanced seed germination of Ormosia arborea. This indicated that the seed of this species has physical dormancy (Edilma et al., 2011).Satisfactory percent germination of 97.2% was obtained in Innula racemosa followed by Carum carvi (93.4%), Rheum webbianum (95.1%), Saussurea lappa (90.01%) and Bunium persicum (81.4%) when seeds were pretreated with acid H2SO4 for 5 minutes. This indicated that the seeds of Innula racemosa, Rheum webbianum, Carum carvi and Saussurea lappa had physical dormancy (Warghat et aal., 2016).Seeds of Tamarindus Indica after being treated with fifty (50%) percent sulphuric acid concentration within sixty (60) minutes soaking period showed satisfactory germination percentage (Abubakar et al., 2013).Treatments involving seed coat removal and application of H2So4 for a duration of 45 up to 150 min were effective on the enhancement of germination percentage of Gleditsia caspica. This concluded that the seeds of Gleditsia caspica have physical dormancy (Kazemet et al., 2016)
2.2. Effect of different treatments on seed dormancy and germination
2.2(a). Mechanical scarification
The germination of the seeds of Robina pseudoacacia increased after being mechanically scarified (Sadhu and Kaul 1989).

2.2(b). Sulphuric acid treatment
The germination of the seeds of Cassia fistula L increased after being treated with H2S04 for 5 to 11 minutes from 4 to 67% (Randhawa et al. 1986).Seeds of Leucaena glauca after being soaked in concentrated H2SO4 for a duration of 20-30 minutes increased germination of this species (Ramdeo, 1971).Chemical scarification of the seeds with H2So4 exhibited highest germination percent in Albizia procera and Albizia falcataria (Sajeevukumar et al. 1995).

2.2(c). Gibberellic acid
The application of GA3 at various concentrations enhanced germination and growth of seedlings of 15 tropical tree species including, Albezzia lebbek ,Acacia spp., and Leucaena glanea (Shanmugavelu, 1970).The application of GA3 at the concentration of 500ppm enhanced the germination of Michelia champaca (Bahuguna et al., 1988).The application of GA3 enhanced the germination percentage of the seeds of Ducrosia anethifolia (Ashtari (2003).Seeds of Carissa carandus L. after being treated.with GA3 at the concentration of 25 ppm exhibited 67 per cent germination (Banker, 1987).

2.2(d). Water treatment.

Seeds of different species of Pinus, Picea, Larix, Cunninghamia, Ptatychladus, Hippophae and Vitex exhibited better germination after being soaked in water for 15 to 24 hours. Seeds of teak after being soaked in running water for a duration of 96 h before sowing in the nursery exhibited better germination (Mioma, 1986).

2.2(e). Hot water treatment
The germination of the seeds of Albizia falcataria increased after being soaked in hot water for a duration of 1.5 minutes at 83?C and 100?C (Koffa ,1983).Hot water pre-treatment (85-90o C) for a duration of 40 min was found effective for the enhancement of germination in Dichrostachys cinerea L. (Roy et al., 1984).Seeds of Prosopis chilensis after being soaked in boiling water for a duration of 10 minutes exhibited 60-80 per cent germination in comparison to control (52%) germination (Ibrahim et al,.1987).Seeds of Ficus bengalensis, F. glomerosa and F.religiosa after being soaked in hot water at a temperature of 60 0C exhibited satisfactory germination (Rai et al., 1988).

2.2. (f). Acid scarification
The satisfactory germination percentage in seeds of Acrocarpus fraxinifo1ium was observed after being treated with concentrated H2S04 for 10 minutes (Rai, 1976).The seeds of Ehretia occuminata after being soaked in concentrated H2So4 acid for a duration of 10 minutes gave 87.6 per cent germination (Sagnala, 1986). The acid scarification for different duration of time was found effective in breaking of Acacia auriculiformis, Albizia procera, Casssia glauca, Cassia siamea and Peltoforum ferrugineum (savitha Kandya, 1990).Maximum germination of 97.2% was observed in species such as Innula racemosa followed by Rheum webbianum (95.1%), Carum carvi (93.4%), Saussurea lappa (90.01%) and Bunium persicum (81.4%) after the seeds were being pretreated with acid H2SO4 for a duration of 5 minutes (Warghat et aal., 2016).

2.3. Effects of storage period on viability and germination
The germination percentage of the seeds of Michelia champaca stored in sealed PB, perforated polybag, Markene bag and moist blotter after a period of 1 month of storage were 42.5%, 52.5%, 56.0% and 49.0% respectively (Bahuguna et al., 1987).The seeds of Michelia champaca stored at room temperature in open tray lost the viability even after one month of storage (Bahuguna, 1987).Seeds of M. champaca are also short-lived (about 2 – 3 weeks), but the viability can be retained by moist storage especially at low temperatures (5 °C) up to 9 months (Troup, 1921; Campbell, 1980; Bahuguna et al., 1987; Beniwal & Singh, 1989; Murali, 1997; Bisht & Ahlawat, 1999). Seeds of Magnolia punduana stored at 25?C exhibited viability percentage of 66.66% after 180 days of storage period (Irahu et al., 2016).The seeds of Dalbersia sissoo stored in polythene bags under ambient condition retained viability for up to 2 or more years with a slight decline in germination (87 to 70%) after 2 years (Robbins, 1988). The seeds of Quercus alba stored in po1ythene bags produced significantly lower shoot and in all cases, germination decreased significantly with increase in storage time (Rink and William, 1984).The viability of the seeds of Azadirachta indica could be retained for a short duration under normal room temperature (26-280C) (Ezumah, 1986).Dry storage of seeds of four tropical arborescent species (Shorea roxburghii, Hopea odorata, Mangifera indica, and Symphomia was effective in enhancing the viability than wet storage (Corbineau et al., 1988). Viability of the seeds of Shorea roxburghii and Hopea odorata was 5-10 days when stored in open air at 25oC (Carbineau et al., 1989). In Pongamia pinnata seeds lost viability after one year under ambient room temperature (Chacko et. al., 2002).The viability of the seeds of Saraca asoca (CABI, 1998) could be retained for a period of six months under low temperature.

MATERIAL AND METHODOLOGY
The present study on ” Effects of seed treatments and storage period on seed dormancy, viability and germination of Michelia champaca L..” was undertaken in the experimental area of Tripura University by collecting seeds from selected superior trees in an around the Tripura University Campus, Suryamaninagar. The details of the study and methodology has been detailed under the following heads:
3.1. Study area:
3.2. Seed source
3.3. Moisture content
3.4. Imbibition test
3.5. Germination test
3.6. Viability test
3.1. Study area
The experiment was carried out on the experimental area of the Tripura University. It has a warm and humid climate. It has an average temperature of 35? C during summer season and 10? C during winter season. It is situated at an elevation of 12.80m from the Mean Sea Level. It has sandy soil.

3.2. Seed sources
Matured fruits of M. Champaca were collected during August from the campus of Tripura University and adjoining areas for the purpose of this study. Seeds were soaked in water for 24hrs till the arils got soften. The seeds were washed manually with water to remove the arils. The seeds were then dried and stored in a zip lock plastic bag and subjected to different storage treatments.

3.3. Moisture content
To determine the moisture content, the initial weight of seven samples, each with ten seeds were measured. The samples were oven dried for 3hrs at 100 ° C and the final weight of each sample was measured (ISTA, 2008).

3.4. Imbibition test
Scarification of 50 seeds was done by removing a small portion of seed coat using a sand paper and the initial weight of each seed was measured as well as the weight of other 50 non scarified seeds. The scarified as well as the non-scarified seed which was of the same weight was placed on moist blot paper in Petri dishes and kept in the laboratory. Each scarified as well as non-scarified seed was re-weighed after 3, 20 and 44 hrs. The percentage change in mass was then calculated.

3.5. Germination test
Three experiments were carried out to study the germination (i.e. appearance of radicles).In the first experiment, a sample of 520 seeds were pretreated with different treatments before sowing in a seed bed consisting of a mixture of soil of silt and sand in the ratio of 2:1 in an opened green house, soon after depulping the arils, without any storage. In this experiment, for each treatment, four replicates of 40 seeds were maintained. For sowing, the seeds were kept on the surface of the soil and they were partially covered with the soil in order to study the germination (appearance of radicles).The treatments involved pretreatments of mechanical scarification, KNO3 and the use of growth hormones (GA3, IAA, and MnSo4) to overcome dormancy. In this experiments, a sample of four replicates of 40 seeds were also sown without given any treatment to the seeds. The untreated seeds were used as control. Watering was done twice a day.

The details standardized treatments applied for the enhancement of germination were as follows:-
T1: IAA (100ppm) for 24hrs
T2: Acetone (500ppm)
T3: Scarification with seed puncturing + IAA (100ppm) for 24hrs.

T4: Scarification with seed puncturing + Acetone (500ppm)
T5: MnSo4 (2000ppm) for 24hrs
T6: Gibberellic acid (1000ppm) for 24hrs
T7: Scarification with seed puncturing + Gibberellic acid (1000ppm) for 24hrs
T8: KNO3 (50%) for 24hrs+ Gibberellic acid (1000ppm) for 24hrs
T9: Scarification with seed puncturing + Mnso4 (2000ppm) for 24hrs.

T10: (Control) without any treatment
T11: KNO3 (50%) for 24hrs+ Acetone (500ppm)
T12: KNO3 (50%) for 24hrs + Mnso4 (2000ppm) for 24hrs
T13: KNO3 (50%) for 24hrs + IAA (100ppm) for 1hrs
T14: Hot water treatment + Polymer seed coating
Observations recorded were:
Germination per cent
Mean germination time
Seedling vigour index
In the second experiment, the seeds were stored in a zip lock plastic bag in laboratory under normal temperature. A Sample of 520 seeds was repeatedly drawn from a storage and pretreated with different treatments before sowing in a seed bed consisting of a mixture of soil and sand in the ratio of 2:1 in an open green house, after 10, 20 and 35 days of storage. In this experiment, for each treatment, four replicates of 40 seeds were maintained. For sowing, the seeds were kept on the surface of the soil and they were partially covered with the soil in order to study germination (appearance of radicles). Also a sample of four replicates of 40 seeds were also sown after 10, 20 and 35 days of storage period without given any treatments to the seeds. The untreated seeds were considered as control. The treatments involved pretreatment of scarification, KNO3 and the use of growth hormones (GA3, IAA, and MnSo4) to overcome dormancy. The same detailed treatments as in the first experiment were used in this experiment. Mean of four replicates was taken for total germination and was expressed as a percentage.

Observations recorded were:
Germination per cent
Mean germination time
Seedling vigour index
In the third experiment, the seeds were stored in different storage treatments. Then, after 40, 80 and 120 days of storage period, a representative sample was drawn from each storage treatment and were pretreated with IAA (100ppm), KNO3+GA3 and GA3 and sown in four replicates of 40 seeds. Thereafter, the seeds were sown in seedbed filled with a mixture of silt and sand in the ratio of 2:1 in open green house.

The detail storage treatments were as follows:
S1 – Storage of seeds in zip lock plastic bag at -20 ? C after the moisture content of the seeds had been reduced to 10-12%
S2 – Storage of coated seeds in petridishes after being coated with polymer seed coating and Carbendazim
S3 – Storage of seeds in zip lock plastic bag at 5?C without any substrate after the moisture content of the seeds had been reduced to 10-12%.

S4 – Storage in zip lock plastic bag at 5 ?C with moist sand as a substrate after the moisture content of the seeds had been reduced to 10-12
S5 – Storage of seeds in vacuum after being coated with Carbendazim
The details treatments were as follows
T1- IAA
T2- KNO3 + GA3
T3- GA3
Observations recorded were:
Germination %
Mean germination time
Data analysis
For each replication in each treatments, the germination percentage, mean germination time, germination index and the seedling vigour index were calculated as follows:
Germination (%) = n/N x 100
Where, n is the number of germinated seeds and N is the total number of seeds.

Mean germination time was calculated using the formula:
n1+n2+n3/ N
Where, n1, n2, n3…. = the number of days taken by individual seeds to germinate,
N= Total number of seeds.

The Seedling Vigour Index (SVI) Iqbal and Rahmati (1992)
VI = (mean root length + mean hypocotyls/shoot length) x Percent germination.

The seed germination experimental design was completely randomized. The experimental data obtained on various parameters in the first, second and third experiment were subjected to analysis of variance (ANOVA) at 5% significance level. Mean and standard error of germination percentage, mean germination time and seedling vigour index were calculated.

3.6. Viability test
Viability test carried out by using a colorless solution of 2, 3, 5-triphenyl tetrazolium chloride. The seed coats were manually scarified by using a sand paper before soaking in water for 24 hours. At the end of the soaking period, hard seed coat got soften and then the embryo was bisected into two halves to expose the cotyledon. One half of the bisected embryo was immerged in a beaker containing a colorless solution of 2, 3, 5-triphenyl tetrazolium chloride of concentration 0.5% and soaked for a time period of 24 hrs and the other half was discarded. A beaker was then kept in an oven at 35 degree Celsius for a time period of 20 hrs. At the end of the treatment period, embryos were transferred in a Petri dish and washed with water and then examined by visual observation. The live embryo, cotyledons and other tissue stained pink to red color showing that the seeds were viable.

Two experiments were conducted to understand the viability characteristics and identify a suitable storage conditions. In the first experiment, fresh, clean and disinfected seeds were stored in a zip lock plastic bag under normal temperature. The viability test for the seeds stored under normal temperature was carried out after 10, 20 and 35 days of storage period. In the second experiment, the seeds were stored in different storage treatments. The viability test for the seeds stored in different treatments was carried out after 40, 80 and 140 days of storage period. Seed viability was determined using TTZ assay (Enescu 1991). For both experiment, four replicates of 40 seeds were maintained.

The detailed storage treatments were as follows:
S1 – Storage of seeds in zip lock plastic bag at freezing point after the moisture content of the seeds had been reduced to 10-12%
S2 – Storage of coated seeds in petridishes after being coated with polymer seed coating and Carbendazim
S3 – Storage of seeds in zip lock plastic bag at 5 ?C without any substrate after the moisture content of the seeds had been reduced to 10-12%
S4 – Storage in zip lock plastic bag 5 ?C with moist sand as a substrate after the moisture content of the seeds had been reduced to 10-12
S5 – Storage of seeds in vacuum after being coated with Carbendazim.

The experimental data obtained were subjected to analysis of variance at 5% significance level. Mean and standard error of viability % were recorded.

RESULTS
The results on the effects of growth hormones to overcome the seed dormancy as well to enhance the germination of the seeds of Michelia champaca and the effects of storage treatments for maximum retention of viability were detailed under the following heads:
4.1. Moisture content
4.2. Imbibition test
4.3. Germination.

4.4. Seedling vigour index
4.5. Viability test
4.1. Moisture content
Result in Table 1 showed that the moisture content of the seeds were in the range of 14.27 – 23.46%.

Initial weight (mg) Final weight (mg) Moisture content (%)
0.876 0.71 18.95
0.807 0.647 19.83
0.817 0.7 14.32
0.813 0.697 14.27
0.993 0.76 23.46
0.857 0.724 15.51
0.926 0.761 17.82
4.2. Imbibition test
The result in table 2 showed that both the non-scarified seeds as well as the scarified seeds imbibed water after a period of 3, 20 and 44 hours at different rates. The increase in weight between scarified and non-scarified seeds was not significant (critical value > observed value). It was also found that there was no difference in weight after 44hrs. The increase in weight of scarified and non-scarified seeds after 44 hours was 1.83% and 1.53%, respectively.

Table 2. Imbibition test of the seeds of Michelia champaca.

Description Initial weight of seeds (%) Weight of seeds after 3hrs (%) Weight of seeds after 20hrs (%) Weight of seeds after 44hrs (%) Weight of seeds after 48hrs (%)
Mean weight of scarified seeds 7.08 7.93 8.67 8.91 8.91
Mean weight of non-scarified seeds 7.08 7.67 8.35 8.61 8.61
Critical value 4.414 4.414 4.414 4.414 4.414
Observed value 0 0.167 0.315 0.286 0.286
Fig1. Imbibition test of the seeds of Michelia champaca.

4.3. Germination.

In the first experiments, results from ANOVA in Table 3 showed that there were significant difference on the effect of different treatments on germination of seeds without any storage period (observed value > critical value). In Table 4, the result from ANOVA showed a significance difference in the number of days taken by the seeds without any storage period to germinate after treated with different treatments (observed value > critical value). In Table 3, the seeds without any storage period after treated with KNO3 (50%) for 24hrs and GA3 (1000 ppm) for 24hrs showed satisfactory germination percentage of 100.00±.00 among all treatments followed by KNO3 (50%) for 24hrs + MnSo4 (2000ppm) for 24hrs and KNO3 (50%) for 24hrs + IAA (100ppm) for 24hrs with a germination percentage of 97.5±2.5% and 87.5±4.79% respectively. In Table 3 and 4, seeds without any treatments (control) showed a germination percentage of 60.0±4.08% with a mean germination time of 14.5±.65 days. The seeds did not show any response to Acetone (500ppm). In Table 4, the seeds without any storage period after treated with Mechanical Scarification + GA3 (1000ppm) for 24hrs took less number of days to germinate with a mean germination time of 8.55±.37 days followed by seeds treated with KNO3 (50%) for 24hrs + GA3 (1000ppm) for 24hrs and GA3 with a mean germination time of 8.8±.33 and 9.72±.19 days, respectively.

In the second experiment, result from ANOVA in Table 3 showed that there were significant difference on the effects of treatments on germination after 10, 20 and 35days of storage period (observed value > critical value). In Table 3, the results showed that after 10 days of storage period, the seeds treated with GA3 (1000ppm) for 24hrs and KNO3 (50%) for24hrs + GA3 (1000ppm) for 24hrs showed a highest germination percentage of 80.00±4.08% and 80.00±.00% respectively. Seeds without given any treatments showed a lowest germination percentage of 50.00±4.08 % after 10 days of storage period. After 20 days of storage period, the seeds treated with IAA (100ppm) for 24hrs showed a highest percentage of 50.00±10.80% .Germination percentage of seeds decreased with increasing storage period of which the seeds treated with GA3 (1000ppm) for 24hrs and IAA (100ppm) for 24hrs after 35 days of storage period showed a highest germination percentage of 12.50±4.79% and 10.00±4.08%, respectively among all treatments. The Seeds without any treatments (control) showed a germination percentage of 50.00±4.08 %, 20.00±4.08% and 7.50±2.50% after 10, 20 and 35 days, respectively. The seeds did not show any response to Acetone (500ppm) after 10, 20 and 35 days of storage period. In Table 4, the result from ANOVA showed that there were a significant difference on the number of days taken by seeds to germinate only after 10days of storage period (observed value > critical value).The seeds treated with GA3 and KNO3 (50% for 24hrs) + GA3 (1000pm for 24hrs) took less number of days to germinate with a mean germination time of 8.68±.26 and 9.44±.50 days, respectively after 10 days of storage period. However, there was no significant difference in the number of days taken by seeds to germinate after 20 and 35 days of storage period (critical value > observed value).

Table 3. Effect of different treatments as well as storage period on mean germination % of the seeds of Michelia champaca.

Treatments Mean Germination percentage without any storage period (%) Mean Germination percentage after 10days of storage period (%) Mean Germination percentage after 20days of storage period (%) Mean Germination percentage after 35dyas of storage period (%)
T1 82.50±10.31 72.50±8.54 50.00±10.80 10.00±4.082
T2 .0000 .0000 .0000 .0000
T3 70.00±14.72 70.00±4.082 17.50±2.50 .0000
T4 .0000 .0000 .0000 .0000
T5 77.50±8.54 67.50±6.29 22.50±6.29 5.00±2.89
T6 82.50±10.31 80.00±4.08 12.50±2.50 12.50±4.80
T7 70.00±17.32 60.00±8.16 12.50±7.50 .0000
T8 100.00±.00 80.00±.00 17.50±4.79 2.50±2.50
T9 77.50±13.15 52.50±15.48 15.00±2.89 2.50±2.50
T10 60.00±4.08 50.00±4.08 20.00±4.082 7.5000±2.50
T11 .0000 .0000 .0000 .0000
T12 97.50±2.50 72.50±7.50 20.00±4.08 7.50±4.79
T13 87.5000±4.79 75.00±2.89 7.50±2.50 2.50
T14 32.50±12.50 65.00±9.57 17.50±2.50 .0000
Critical value 1.96 1.96 1.96 1.96
Observed value 15.69 20.39 7.46 2.62
Fig2. Effect of different treatments as well as storage period on the mean germination % of the seeds of Michelia champaca.

Table 4. Effect of different treatments as well as storage period on mean germination time of Michelia champaca.

Treatments Mean germination time without any storage period (days) Mean germination time after 10days of storage period (days) Mean germination time after 20 days of storage period (days) Mean germination time after 35 days of storage period (days)
T1 10.30±.79 10.29±.36 13.17±.86 13.33±.33
T3 11.42±.67 10.36±.26 11.73±.91 .-
T5 10.55±.16 10.73±.62 16.25±1.49 15.00±2.00
T6 9.72±.19 8.68±.26 14.25±1.44 13.17±1.36
T7 8.55±.37 9.81±.47 12.45±.05 .-
T8 8.80±.32 9.44±.50 13.88±.97 13.00±0.00
T9 9.98±.52 10.96±1.18 14.40±2.31 11.00±0.00
T10 14.50±.65 16.22±.74 15.02±.43 20.00±1.73
T12 10.85±.68 11.62±.71 15.00±1.06 11.75±2.25
T13 10.73±.68 12.38±.38 13.00±1.73 14.00±0.00
T14 12.05±1.97 14.18±1.28 15.88±.83 .-
Critical value 2. 14 2. 13 2.16 3.5
Observed value 5.67 9.99 1.17 3.10
Fig 3. Effect of different treatments as well as storage on the mean germination time of the seeds of Michelia champaca.

In experiment three, the results from ANOVA in Table 5, showed that there was significant different on the effects of different storage treatments on the germination of seeds after 40, 80 and 120 days of storage period (Observed value > critical value). In Table 5, the results showed that the seeds stored in zip lock plastic bag at – 20 ?C failed to germination even after 40 days of storage period. The result also showed that the seeds stored in zip lock plastic bag at 5? C without any substrate after the moisture content of the seeds had been reduced to 10-12% showed a satisfactory germination percentage of 95.00 ± 2.89% after being treated with IAA (100ppm for 24hrs) after 40 days of storage period among all storage treatments. The germination of the seeds stored in zip lock plastic bag at 5?C with moist sand as a substrate after the moisture content of the seeds had been reduced to 10-12 after 40 days of storage period also showed a satisfactory germination percentage of 92.50 ± 2.50 % after being treated with IAA (100ppm for 24hrs). The seeds stored in vacuum after being coated with Carbendazim also showed a satisfactory germination percentage of 87.50 ± 9.46 % after being treated with IAA (10 ppm for 24hrs) after 40 days of storage period. The seeds stored in petridishes, after being coated with polymer seed coating and carbendazim failed to germination after 80 days of storage period. The germination percentage of the seeds stored in zip lock plastic bag at 5 ? C declined to 75.00 ± 10.40 % after 120 days of storage period. Whereas the germination percentage of the seeds stored in zip lock plastic bag at 5 ?C with moist sand as a substrate showed a rapid decline to 50.0 ± 10.80 % after being treated with IAA (100ppm for 24hrs) after 120 days of storage period. But the germination percentage of the seeds stored in vacuum after being coated with Carbendazim steadily declined to a germination percentage of 82.50 ± 4.79 % after being treated with IAA 120 days of storage period.

In Table 6, the result from ANOVA showed that there was significant difference on the effect of different storage treatments on the mean germination only after 40 and 120 days of storage period (observed value > critical value), but there was no significant difference on the effects of different storage treatments on the mean germination time after 80 days of storage period (critical value > observed value). The results in Table 2 showed that the seeds stored in vacuum after being coated with Carbendazim took less time to germinate with a mean germination time of 12.73 ± .45 days after being treated with IAA (100ppm for 24hrs) in comparison to other treatments after 120 days of storage period.

Table 5. Effects of different Storage treatments on mean Germination % of seed of Michelia champaca after 40, 80 and 120 days of storage period.

Storage treatments Germination percentage after 40 days of storage period (%) Germination percentage after 80 days of storage period (%) Germination percentage after 120 days of storage period (%)
S1T1 0 0 0
S1T2 0 0 0
S1T3 0 0 0
S2T1 42.5 ± 7.50 0 0
S2T2 20.0 ± 10.80 0 0
S2T3 20.0 ± 10.80 0 0
S3T1 95.00 ± 2.89 70.0 ± 9.13 75.00 ± 10.40
S3T2 75.00 ± 17.32 55.0 ± 8.66 60.0 ± 14.71
S3T3 65.00 ± 12.58 47.50 ± 4.78 65.0 ± 10.40
S4T1 80.0 ± 10.80 52.50 ± 4.78 50.0 ± 10.80
S4T2 92.50 ± 2.50 40.0 ± 4.08 40.0 ± 4.08
S4T3 77.50 ± 4.79 35.0 ± 2.89 47.5 ± 4.79
S5T1 87.50 ± 9.46 87.50 ± 2.50 82.50 ± 4.79
S5T2 80.00 ± 4.08 85.00 ± 2.89 80.00 ± 7.07
S5T3 82.50 ± 12.58 80.50 ± 4.08 s 82.50 ± 8.54
Critical value 1.918 1.918 1.918
Observed value 24.41 68.68 25.06
Fig 4. Effect of different storage treatments on germination percentage of the seeds of Michelia champaca after 40, 80 and 120 days of storage period.

.Table 6. Effect of different storage treatments on mean germination time of the seeds of Michelia champaca after 40, 80 and 120 days of storage period.

Storage treatments along with treatments Mean germination time after 40 days of storage period (days) Mean germination time after 80 days of storage period (days) Mean germination time after 120 days of storage period (days)
– – –
S1T1 – – –
S1T2 – – –
S1T3 16.13 ± .79 – –
S2T1 22.93 ± 2.03 – –
S2T2 25.53 ± 0.29 – –
S2T3 23.85 ± 0.38 22.35 ± 1.28 16.27 ± 1.02
S3T1 26.00 ± .81 22.22 ± .63 16.78 ± 1.64
S3T2 24.73 ± .32 22.11 ± 1.56 14.92 ± .97
S3T3 25.81 ± .47 24.42 ± .89 18.63 ± 1.43
S4T1 26.10 ± .33 23.93 ± 1.36 17.22 ± .95
S4T2 25.41 ± .33 22.44 ± 1.07 15.75 ± .96
S4T3 25.68 ± .31 22.416.2 ± .51 12.73 ± .45
S5T1 26.05 ± .39 22.08 ± 1.17 13.75 ± 1.24
S5T2 25.99 ± .92 21.76 ± .73 14.58 ± 1.33
S5T3 2.08 2.31 2.31
Observed value 20.60 0.76 2.50
Fig 5. Effect of different storage treatments on mean germination time of the seeds of Michelia champaca after 40, 80 and 120 days of storage period.

4.4. Seedling vigour index
In table 7, result from ANOVA showed that there were significant difference on the effects of different treatments on the seedling vigour index of seeds without any storage period (observed value > critical value). The maximum seedling vigor index was observed 1270.0000±99.79061, when the seeds without any storage period were treated with KNO3 (50% for 24hrs) + GA3 (1000ppm for 24hrs) whereas the minimum seedling vigor index was observed 349.07±61.79, when the seeds were treated to hot water (40° C) + seed coating with polymer seed coating. In the second experiment, the result from ANOVA in Table 3 showed that there were significant difference on the effects of different treatments on seedling vigour Index after 10 and 20 days of storage period (observed value > critical value). However there were no significant difference on the effect of different treatments on seedling vigour index after 35 days of storage period (critical value > observed value). The seeds treated with GA3 (1000ppm for 24hrs) showed the maximum seedling vigour index of 937.18±84.83 after 10 days of storage period. The seeds without given any treatments (control) showed the minimum seedling vigour index of 436.20±45.06 after 10 days of storage period. After 20 days of storage period, the seeds treated with IAA (100ppm) for 24hrs showed a highest seedling vigour index of 564.60±115.92.

Table 7. Effect of different treatments as well as storage period on seedling vigour index of Michelia champaca.

Treatments Mean Seedling vigour index without any storage periods Mean Seedling vigour index after 10 days of storage period Mean Seedling vigour index after 20 days of storage period Mean Seedling vigour index after 35 days of storage period
T1 884.23± 58.93 872.22±105.39 564.60±115.92 128.83±37.65
T3 634.98±139.84 773.55±19.64 160.88±34.91 T5 822.06±115.21 841.40±111.67 212.25±84.27 90.00±25.00
T6 1031.38±174.40 937.18±84.83 101.25±32.20 170.00±34.60
T7 883.03±232.10 651.55±95.57 309.50±62.50 T8 1270.00±99.791 905.40±15.07 156.63±43.19 59.00±0.00
T9 908.30±400.16 562.13±176.80 109.00±37.01 60.00±0.00
T10 612.83±113.10 436.20±45.06 193.35±38.93 109.00±8.33
T12 1028.28±257.54 754.95±84.58 181.70±48.32 184.50±109.50
T13 870.00±102.17 790.18±27.66 53.67±16.90 87.00±0.00
T14 349.07±61.7976 656.30±83.72 162.00±19.42 Critical value 2.14 2.13 2.16 3.50
Observed value 5.67 9.99 1.17 3.11
Fig 6. Effect of different treatments as well as storage period on seedling vigour index of Michelia champaca.

4.5. Viability test
In table 8, the result from ANOVA showed that there were significant difference on the effect of storage period on the seeds stored under normal temperature in a zip lock plastic bag. The results showed that the viability percentage of the seeds after 35 days of storage period was 30.0 ± 4.08%.

Table 8. Effect of storage period under normal temperature on viability percentage of the seeds of Michelia champaca.

Storage period Viability (%)
After 10 days of storage period 82.50 ± 4.79
After 20 days of storage period 50.00 ± 4.08
After 35 days of storage period 30.00 ± 4.08
Critical value 4.26
Observed value 37.44
Fig 7. Effect of storage period under normal temperature on viability percentage y of the seeds of Michelia champaca.

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In Table 9, the results from ANOVA showed that there was a significance difference on the effects of different treatments on the viability % of the seeds after 40, 80 and 120 days of storage period ( observed value > critical value). The result from table 3 showed that the maximum viability percentage of 95.00 ± 2.89 % was observed on the seeds stored in zip lock plastic bag at 5°C without any substrate after the moisture content of the seeds had been reduced to 10-12%. And the minimum viability percentage of 0 % was observed on the seeds stored in zip lock plastic bag at – 20°C after the moisture content of the seeds had been reduced to 10-12% even after 40 days of storage period. The viability percentage of the seeds stored in zip lock plastic bag at 5?C with moist sand as a substrate after the moisture content of the seeds had been reduced to 10-12 was 92.50 ± 4.79 % after 40days of storage period. Whereas the viability of the seeds stored in vacuum after being coated with carbendazim was 92.50 ± 4.79% after 40 days of storage period. The viability percentage of the seeds stored in zip lock plastic bag at 5?C without any substrate after the moisture content of the seeds had been reduced to 10-12% declined to 80.00 ± 4.08% and 77.5000 ± 4.79% after 80 and 120days, respectively. Whereas the viability percentage of the seeds stored in zip lock plastic bag at 5?C with moist sand as a substrate after the moisture content of the seeds had been reduced to 10-12 declined rapidly to 67.50 ± 2.5% and 60.00 ± 4.08 % after 80 and 120 days, respectively. But the viability of the seeds stored in vacuum after being coated with Carbendazim showed a steady declined to 82.50 ± 4.79% and 80.00 ± 4.08% after 80 and 120days, respectively.

Table 9. Effects of different storage treatments on viability % of the seeds of Michelia champaca.

Storage treatments Viability after 40 days of storage period (%) Viability after 80 days of storage period (%) Viability after 120 days of storage period (%)
S1 0 0 0
S2 67.50 ± 4.79 35.00 ± 2.89 0
S3 95.00 ± 2.89 80.00 ± 4.08 77.50 ± 4.79
S4 92.50 ± 4.79 67.50 ± 2.5 60.00 ± 4.08
S5 92.50 ± 4.79 82.50 ± 4.79 80.00 ± 4.08
Critical value 3.49 3.49 4.26
Observed value 8.73 35.23 6.33
Fig 8. Effects of different storage treatments on viability % of the seeds of Michelia champaca.

DISCUSSION
The following discussions were on the effects of different treatment to overcome dormancy as well as to enhance the germination percentage and storage treatments on viability of the seeds of Michelia champaca
Although the seeds of M. champaca were depicted as very hard and impermeable to water :;( Norman, 1971; Bahuguna et al., 1988; SID, 2008). The results showed that both scarified as well as non-scarified seeds imbibed water (fig.1). Thus, the seeds of this species are not physically dormant.

In Table 3, the result showed that the seeds without any storage after treated with GA3 (1000ppm) and IAA (100ppm) for 24hs exhibited a germination percentage of 82.50±10.30 and 82.50±10.30 respectively. The application of growth hormones, such as GA3 is effective in enhancing the germination in freshly collected seeds with physiological dormancy (Commander et al. 2009; Turner et al. 2009).Moreover, physiological dormancy was overcome by the use of certain growth hormones such as Indole 3-acetic acid (IAA), Gibberellic acid (GA3) and in some cases cytokinin (Phillips, 1962; Wright, 1968). This support the conclusion of Bahuguna et al., (1988) that the seeds of Michelia champaca have physiological dormancy. In Table 3, the result also showed that the combined effect of KNO3 (50% for 24hrs) with other growth hormones such as GA3 (1000ppm), Mnso4 (2000ppm) and IAA (100ppm) for 24hrs showed satisfactory germination percentage of 100.00±.00, 97.50±2.50 and 87.50±4.79 respectively. Moreover, the combined effects of KNO3 (50% for 24hrs) + GA3 (1000ppm for 24hrs) showed the highest germination percentage of 100.0000±.00000 among all treatments. This could be attributed to the fact that nitrates reduces the composition of abscisic acid (ABA) in the seeds, whereas GA3 helps in promoting germination (Hilhorst and karssen 1992). Also, the results showed that the seeds treated with growth hormones i.e. GA3 (1000ppm), IAA (100ppm) and Mnso4 (2000ppm) after being mechanically scarified failed to increase the germination with a germination percentage of 70.00±17.32%, 70.00±14.72% and 77.50±13.15% respectively. This result supports the conclusion of Fernando et al., 2013 that the seeds of Michelia champaca do not have physical dormancy.

In Table 3, the result showed a decline in germination percentage of the seeds stored under normal temperature in a zip lock plastic bag after 10, 20 and 35 days of storage period. The germination of the freshly collected seeds after 10 days of storage period under normal temperature in a zip lock plastic bag declined to a germination percentage of 80.00±.00% after being treated with KNO3 (50% for 24hrs) + GA3 (1000ppm for 24hrs). After 35 days of storage period, the germination percentage of the seeds stored under normal temperature in a zip lock plastic bag declined to a germination percentage of 12.50±4.79% after being treated with GA3 (1000ppm for 24hrs). This result supports the conclusion of Troup (1921); Dent (1947) that the seeds of this species have low viability. In Table 4, the result showed that the mean germination time of the seeds stored under normal temperature in a zip lock plastic bag after 10 days of storage period was 8.68±.26 days after being treated with GA3 (1000ppm for 24hrs) which increased to 11.73±.91 days after the mechanically scarified seeds were treated with IAA (100ppm for 24hrs) after 20 days of storage period. This indicated that with the increasing storage period, the number of days taken by the seeds to germinate also increased.

The three major factors affecting storage are seeds temperature, moisture content, and oxygen concentration (Robert 1972). In Table 5, the result showed the seeds stored in zip lock plastic bag at – 20 ? C after the moisture content of the seeds had been reduced to 10-12% had no effect on germination of the seeds of Michelia champaca. The seeds stored in ziplock plastic bag at 5?C after the moisture content has been reduced to 10-12 % showed a germination percentage of 75.00 ± 10.40% after 120 days of storage period. This supported the conclusion of Fernando et al. (2013) that the seeds of Michelia champaca are identified as intermediate based on the conclusion of Hong and Ellis (1996) that the seeds that can be able to survive after the moisture content has been reduced to 10 -12% but died when stored at – 20 ? C. For the seeds stored in zip lock plastic bag at 5?C with moist sand as a substrate after the moisture content of the seeds had been reduced to 10-12 % and in petridishes after being coated with polymer seed coating and Carbendazim, progressive and significant reduction in the germination was observed with increasing duration of storage period. Among all storage treatments, the seeds stored in vacuum after being coated with Carbendazim showed a steady decline in the germination after 80 and 120 days of storage period. This supported the conclusion of Ghive et al (2007) that reduction in oxygen concentration may enhance viability, which is either accomplished by vacuum or by raising partial pressure of CO2, Nitrogen, argon and Helium. Also the seeds stored in zip lock plastic bag at 5?C without any substrate after the moisture content of the seeds had been reduced to 10-12% showed a steady decline in the germination percentage. This supported the conclusion of Bahuguna (1987) that the process of deterioration is adjourned if the seeds is stored at 5 ?C due to the reason that low temperature stop rapids changes in the seed. In Table 6, the result also showed that with the increasing storage period, the seeds stored in vacuum after being coated with Carbendazim after 120days took less number of days to germination in comparison to other storage treatments. The result of this investigation exhibited that the seed of Michelia champaca does not require aeration for respiration during storage. Moreover, the results revealed that the seeds stored in different treatments after being treated with IAA (100ppm for 24hrs) before sowing showed satisfactory germination percentage in comparison to other treatments.

In Table 8, result showed that the seeds of Michelia champaca stored under normal temperature in a ziplock plastic bag lost their viability after 35 days of storage period with a viability percentage of 30.0000 ± 4.08248%. This test also supports the conclusion of Troup (1921); Dent, (1947) that the seeds of this species has low viability
In Table 9, result showed that the seeds stored in vacuum after being coated with Carbendazim and in zip lock plastic bag at 5?C without moist sand as a substrate after the moisture content of the seeds had been reduced to 10-12% retained viability of the seeds up to 120 days of storage period with a viability percentage of 77.5 ± 4.79% and 77.5 ± 4.79 %, respectively. This supported the conclusion of Ghive et al (2007) that reduction in oxygen concentration may enhance viability, which is either accomplished by vacuum or by raising partial pressure of CO2, Nitrogen, argon and Helium and Bahuguna (1987) that the process of deterioration is adjourned if the seeds is stored at 5?C due to the reason that low temperature stops rapids changes in the seed. The germination test, exhibited that the seed of Michelia champaca does not require aeration for respiration during storage.

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Fig 9. Effect of different treatments on germination without any storage.

Fig10.Effect of different treatments on germination after 35 days storage.

Fig 11. Effects of storage treatments after 40 days of storage.

Fig12.Effects of storage treatments after 120 days of storage.

Fig 13.Viable seeds

Fig1. Non viable seeds
SUMMARY
This study was undertaken to find the kind of dormancy responsible for low germination of the seeds of Michelia champaca as well as to determine the storage treatments which could retain the viability for long term storage. Michelia champaca L. belongs to the family Magnoliaceae. The kind of dormancy present in this species was physiological dormancy as reported by Bahuguna (1988). The Seeds of M. champaca were also short-lived (about 2 – 3 weeks), but the loss of viability could be minimized by moist storage especially at low temperatures (5 °C) for up to 9 months (Troup, 1921; Campbell, 1980; Bahuguna et al., 1987; Beniwal & Singh, 1989; Murali, 1997; Bisht & Ahlawat, 1999). This study involved the use of different growth hormones to determine the kind of dormancy present on the seeds of the species. Thirteen standardized treatments were used in this study so as to enhance the germination of the seeds of Michelia champaca. The imbibition test was carried out to determine whether the seeds had physical dormancy or not. Five storage treatments were used so as to enhance the viability of the seeds of Michelia champaca. The viability test using a colorless solution of 2, 3, 5-triphenyl tetrazolium chloride was carried out to determine the viability of the seeds stored under normal temperature as well on different storage treatments after certain days of storage period. The experimental result showed that the seeds without any storage after treated with GA3 (1000ppm) and IAA (100ppm) for 24 hrs exhibited a germination percentage of 82.50±10.31 % and 82.50±10.31 %, respectively. This supported the conclusion of Bahuguna (1988) that the seeds of Michelia Champaca had a physiological dormancy. Acetone had no influence on germination of Michelia champaca. The seeds treated with KNO3 (50% for 24 hrs) + GA3 (1000 ppm for 24 hrs) showed a satisfactory germination percentage among all treatments with a germination percentage of 100.00 ±.00%. The seeds stored in vacuum after being coated with Carbendazim as well as in zip lock plastic bag at 5?C retained germination up to 120 days of storage with a germination per cent of 82.5000 ± 4.787 and 75.00 ± 10.40, respectively after being treated with IAA before sowing. This supported the conclusion of Ghive et al (2007) that reduction in oxygen concentration may increase longevity, which is either achieved by vacuum or by raising partial pressure of carbon dioxide, Nitrogen, Argon and Helium, as well as the conclusion of Bahuguna (1987) that the process of deterioration is delayed if the seeds is stored at 5 ?C due to the fact that low temperature prevents rapid changes in the seed.

CONCLUSION
On the basis of the results, it is recommended to treat the seeds of Michelia champaca with KNO3 (50% for 24hrs) + GA3 (1000ppm for 24hrs) before sowing for satisfactory germination without any storage period. The seeds of Michelia champaca have physiological dormancy. The seeds of Michelia champaca are short lived i.e. loses viability within few days. It is also recommended to store the seeds in vacuum after being coated with Carbendazim and in zip lock plastic bag at 5?C without any substrate after the moisture content of the seeds had been reduced to 10-12% for maximum retention of viability.

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