SEED GERMINATION 6.1. INTRODUCTION CHAPTER 6

Transcription

1 SEED GERMINATION CHAPTER INTRODUCTION Botanically, the seed is a mature ovule containing an embryo, storage tissue and a protective outer covering which is the result of sexual fertilization (Hartmann and Kester, 1983; Hartmann et al., 1997; Esau, 1997; Fahn., 1982). The activation of the metabolic machinery of the embryo leading to the development of a new seedling plant is known as seed germination (Hartmann et al., 1997). Seed germination is the process of continuation of life and assuring of plant species survival in nature (Copeland and McDonald, 1994; Hartmann et al., 1997). Most of the researchers and authors defined seed germination differently and in the present study a seed is said to have germinated with the emergence of the radical (Copeland and McDonald, 1994). Hippophae rhamnoides is a fascinating plant species known for its multifarious properties i.e., food, fuel, fodder, medicines, cosmetics, etc. and also being a nitrogen fixer it has been considered a very useful for rehabilitation of degraded land particularly in high altitude areas of the Himalaya. On the other hand the plant is being planted for raising orchards for commercial purpose in several countries. In China the plant species is also used for afforestation programmes for greening the hilly tracks and terrains (Rongsen, 1992). It is commonly propagated by mature seeds, which are collected after the maturation of fruit berries during the winters particularly in the months of November to December. Though few studies are available on seed germination of this species (Sankhyan et al., 25; Singh and Gupta, 23; Fefelov and Selekhov, 23) but in-depth studies considering various aspects are almost lacking. Therefore, the aim of this study was to investigate seed viability, imbibition and germination of Seabuckthorn seeds under different treatments collected from five valleys of Uttarakhand. 72

2 6.2. MATERIALS AND METHODS Seed collection and processing Mature and dry seeds of Hippophae rhamnoides were collected manually (hand picked) from natural pockets of five different valleys namely Mana, Niti, Bhuyandar, Yamnotri and Gangotri in the first week of November 23 and 24 (Table 6.1). The seeds were collected by squeezing out the fruit pulp and juice. Table 6.1. List of natural pockets of H. rhamnoides sampled for seed collection Valley Place District Altitude (m amsl) Habitat Mana Bhyundar Nti Yamunotri Gangotri Hanumanchatti Chamoli Lambaggarh Chamoli Radhang Bend Chamoli Pulna Chamoli Bhyundar Chamoli Jumma Chamoli Along the river, road side, steep slopes of mountains. Along the river Road side Along the tributaries Along the tributaries Along the river, road side, steep slopes of mountains Along the river, road side Jhelam Chamoli Gwar Chamoli 22 Along the river Jankichatti Uttarkashi 26 River bank, mountain area Phoolchatti Uttarkashi 25 River bank, Naradchatti Uttarkashi 24 River bank Harsil Uttarkashi 25 River bank Dharali Uttarkashi 25 River bank 73

3 Experimental design, data collection and analysis All experiments related to seed germination were carried out in the laboratory at G.B.Pant Institute of Himalayan Environment and Development, Garhwal Unit-Srinagar Garhwal. Seed germination of Hippophae rhamnoides was tested at constant temperature regimes at 25 ± 2 C in 16 hours light and 8 hours dark. Each observation unit (constant temperature with light and dark conditions) consisted of three replicate petri dishes (9 cm in diameter) with 25 viable seeds. The seeds were placed in two layers of Whatman No.1 filter papers and prior to sowing they were moisten with distilled water. In the dark treatment, the petri dishes were wrapped in double layer of aluminum foil according to Baskin and Baskin (21) in order to ensure % darkness. The petri dishes were inspected daily throughout the germination period. Parametric statistical analysis, ANOVA (Analysis of Variance) was performed using the data on germination percentage and the level of significance was subsequently determined Seed storage and viability Seeds were transferred to transparent polythene bags and stored under ambient conditions in the laboratory of the institute. Seed samples were tested for viability at 365 days intervals for three consecutive years. Seeds of Hippophae rhamnoides were soaked in distilled water for 24 hours and the seed coat was carefully removed. Decoated seeds were then completely submerged in 1,2,3 triphenyl tetrazolium chloride (1%, w/v; ph 7, 24 h, 25 o C, dark), percent viability was determined by counting the total number of stained (Red) portions of embryo and cotyledons in each replicate (I.S.T.A., 1993). To get an idea of viability the seeds were stored for two consecutive years from Imbibition capacity Seeds of each of population were grouped, and their initial fresh weight was recorded. These were allowed to imbibe water (25 o C, dark) in beakers and seeds were removed at one-hour intervals, wiped dry with blotting paper and their fresh weight determined. Once the weight was recorded, the seeds were transferred back into beakers containing water, the seed weight was recorded till the fresh weight became constant and attain 74

4 plateau (Hartmann and Kester, 1989). An imbibitions curve was drawn by plotting percent increase in seed weight over initial weight against time Germination test The fresh harvested seed were dipped in Bavistin (.5%, w/v) solution for 3 min to remove fungal infection and then twice washed in running tap water. The seeds were then wiped clean and air-dried for 1hr in room temperature at 2 o C. The germination study was conducted under laboratory condition, in 16 hours light and 8 hours dark at 25 C±2 C in seed germinator. Emergence and / or growth of the radical through the seed coat by more than 2 mm were used as criterion for germination (Omokaro et al., 1999). Each treatment contained 2 seeds with three replicates (2+2+2=6). The number of germinated seeds was recorded daily Chemicals and plant growth regulators Chemicals (Potassium Nitrate (KNO 3 ) and Thiourea; Sisco Research Laboratories (SRL) Pvt. Ltd., India and Qualigens Fine Chemicals Pvt. Ltd., India) and plant growth regulators viz. Gibberellic acid (GA 3 ) and 6-Benzylaminopurine (BAP); both from Sigma Chemical Co. St. Louis USA) were used to observe their effects on seed germination. Stock solutions of all chemicals were prepared in water or in 5 % ethanol (v/v and stored at 4 C until used; these were diluted with water just before treatment. The final concentration of ethanol in test solutions never exceeded 2.%. The chemical treatments included KNO 3 and Thiourea (.1%,.5% and 1. %; v/v while PGR treatment included GA 3 and BAP (, 25 and um). The seeds were treated with above mentioned chemicals or PGRs by placing them in beaker (25 ml) containing appropriate solutions (24 h, 25 C, dark) with occasional shaking Mechanical scarification (Effect of physical treatments) The following physical treatments were carried out in batches of 6 seeds with three replicates: (i) Seed coats were cut through chalazal end using a sharp scalpel, (ii) The seed were immersed in water and stored for 7, 14 and 21 days at -4 o C and (iii) Seeds were immersed in hot water at 5 C for 5, and 15 min. 75

5 Control Sets of seeds were not subjected to any kind of PGRs, Chemicals, and physical/mechanical treatments are served as control (Table 6.2). Table 6.2. Different treatments applied in seed germination study of H. rhamnoides. S.No. Treatments Concentration Soaking time 1 GA 3 µm 24 Hours 2 25µM 24 Hours 3 µm 24 Hours 4 BAP µm 24 Hours 5 25µM 24 Hours 6 µm 24 Hours 7 KNO 3 25µM 24 Hours 8 5µM 24 Hours 9 µm 24 Hours Thiourea 25µM 24 Hours 11 5µM 24 Hours 12 µm 24 Hours 13 Hot Water 5 C 5 min 14 5 C min 15 5 C 15 min 16 Chilling -4 C 7 days 17-4 C 14 days 18-4 C 21 days 19 Scarification - 24 hours 21 Control - 24 Hours 6.3. RESULTS Seed storage and viability The fresh seeds collected in November 23 readily stained with TTC (2,3,5-Triphenyl tetrazolium chloride) after few days of collection and lost their viability with a very slow rate (Fig. 6.1). The maximum percentage of viability was recorded 98.89±1.11 % for the seeds of H. rhamnoides collected from Gangotri valley whereas, the seeds of Mana valley exhibited minimum 93.33±3.33 % viability (Fig. 6.1). After storage of one year (365 days) the viability % in the seeds of Gangotri valley declined up to 3.33 % followed by 7.78 % (Yamunotri valley), 8.89 % (Bhyundar valley), % (Mana valley) and % (Niti valley). The seeds stored for two years (73 days) revealed that the viability of 76

6 Gangotri and Yamunotri seeds reduced up to % and remained 87.78±2.94 % and 85.56±7.29 % respectively. On the other hand maximum viability loss (17.78 %) in the second year of collected seeds was found in the seeds from Bhyundar valley followed by Niti (15.56 %) and Mana valley (15.55 %). Viability (%) days 365 days 73 days Storage period Fig.6.1. Percent viability of seeds after storage of consecutive three years Imbibition capacity The seeds collected from five valleys, indicated a slow rate of water imbibition. The imbibition experiment was observed for 36 hrs till it reaches the plateau. The imbibition took long time of 24 hrs (Gangotri valley) to 3 hrs (Bhyundar valley) to initiate and develop a plateau of percent increase over initial weight of seeds. The seeds of Gangotri valley imbibed water at a faster rate than the other valleys while, the seeds collected from Niti valley showed a slower rate of imbibition. The seeds of Gangotri valley attained 5 % of increase over initial weight of seeds in between 84 to 96 hrs and % in between hrs and finally increased up to % in 24 hrs of experiment. Seeds collected from Yamunotri valley took more then 12 and 168 hrs to reached 5% and % of increase over initial weight. Ultimately, the % increases over initial weight of seeds collected from Yamunotri were reached up to 113 % in 276 hrs. However, seeds from 77

7 Bhyundar valley imbibed the 5% and % of water in less then 4 and 144 hrs, respectively. The final weight of 13 % in the 3 th hrs of experiment was recorded for seeds collected from Bhyundar valley. The Fig. 6.2 showed that in the 8 th hrs of experiment the seeds of Mana valley took only 39 % water and within next 48 hrs (2 days) it crossed 2.67 % and reached a plateau of total 121 % increased over initial weight of seeds in 276 th hrs. The minimum imbibition of total 8 % in 228 th hrs of experiment was observed for the seeds collected from Niti valley. 14 % Increase Over Initial Weight Imbibition Time (h) Fig Percent increase in fresh weight over initial weight of H. rhamnoides seeds collected from five valleys, at various times after the seeds were allowed to imbibe water at 25 C in the dark Seed Germination Experiments (i) Effect of Plant Growth Regulators (PGR) on seed germination The seeds collected from five different valleys were treated with GA 3 and BAP resulted in good percentage of germination. The seeds from Bhyundar valley exhibited the maximum germination potential followed by Mana, Niti, Yamunotri and Gangotri valleys. The highest percentage (93.33±4.41 %) of germination was estimated for the 78

8 seeds treated with GA 3 (µm) collected from Bhyundar valley whereas, the minimum germination percentage (43.33±6.1 %) was recorded for the seeds of Gangotri valley treated with GA 3 (µm). In the Gangotri valley the maximum number (81.67±8.33 %) of seeds germinated under the BAP (25µM) treatment whereas, the minimum germination (5.±7.64 %) was observed when treated with BAP (µm). In Yamunotri valley the germination percentage was found maximum (93.33±4.41 %) when seeds treated with BAP (µm), which was % more than the control (5.±12.58 %). The minimum seed germination percentage (58.33±7.26 %) was observed for Bhyundar valley with BAP (µm) treatment. The treatment of BAP (25µM) resulted maximum seed germination (85.±5. %) in Mana valley whereas, GA 3 (25µM) treated seeds of the same valley showed only 63.33±11.67 % of germination. The seeds of Niti valley showed maximum germination percentage when treated with GA 3 (µm) while, minimum of 53.33±.14 % was observed in the control seeds of same valley. There were significant differences (p<.5) in percent germination was observed among the seeds of all five valleys, within PGRs and also within different concentrations (Fig. 6.3, 6.4). Germination (%) µm 25µM µm Concentration Fig Effect of GA 3 (PGR) concentrations on seed germination in different valleys. 79

9 Germination (%) µm 25µM µm Concentration Fig Effect of BAP (PGR) concentrations on seed germination in different valleys (ii) Effect of nitrogenous compounds on seed germination Seabuckthorn seeds were tested and treated with various concentrations of KNO 3 and Thiourea for assessing the effects of nitrogenous compounds on the seed germination percentage. The overall results of seed germination showed maximum germination for Bhyundar valley followed by Gangotri, Mana, Yamunotri and Niti valleys. The seeds treated with KNO 3 (µm) and Thiourea (5µM) respectively, of Yamunotri and Mana valleys showed same with maximum percent of germination (93.33±4.41 %). However, the minimum germination percentage (6.±5.77 %) was found for the seeds treated with Thiourea (5µM) of Niti valley. The results obtained from the seeds of the Gangotri valley varied from minimum 75.±8.66 % (KNO 3, 25µM) to maximum 85.±8.66 % in µm concentration of Thiourea. The seeds soaked in the concentration of µm of Thiourea resulted the minimum (6.±2.89 %) percentage of germination for Yamunotri valley. Though the seeds treated with various concentrations of KNO 3 showed more than 9% germination for the seeds of Bhyundar valley (Fig. 6.5). Seeds collected from Mana valley exhibited germination between (75.±5. % in µm of KNO 3 ) to (93.33±4.41 % in 5µM of Thiourea). The minimum and maximum germination percent was found 6.±5.77 (treated with 5µM of Thiourea) and 78.33±8.82 (treated with 25µM of

10 KNO 3 ), respectively for Niti valley. The ANOVA results showed that there were significant differences (p<.5) in percent germination amongst all five valleys, within both nitrogenous compounds (KNO 3 and Thiourea) and also within under different concentrations of KNO 3 and Thiourea (Fig. 6.6). Germination (%) µM 5µM µm Concentration Fig Effect of KNO 3 (Nitrogenous compound) concentrations on seed germination in different valleys. Germination (%) µM 5µM µm Concentration Fig Effect of Thiourea (Nitrogenous compound) concentrations on seed germination in different valleys. 81

11 6.3.3.(iii) Effect of physical/mechanical treatments on seed germination The results obtained from physical/mechanical treatments of seeds reveals that there were significant differences (p<.5) amongst all five valleys, within physical and mechanical treatments and also within different time periods. The average seed germination results indicated that seeds of Gangotri valley showed maximum percentage followed by Bhyundar, Yamunotri, Niti and Mana valleys. The maximum percentage (93.33±3.33) of germination was found in the seeds of Gangotri valley, treated with hot water at 5 C for 15 minutes whereas, seeds of Mana valley treated with Chilling at 4 C for 7 days exhibited minimum 5.±13.23 % germination. The hot water treatment resulted better germination among the seeds collected from all five valleys as compared to the seeds placed under chilling and scarification experiments (Fig. 6.7). The results of chilling experiments were varied from maximum 71.67±4.41 % (14 days chilling) for Niti valley and minimum 5.±13.23 % (7 days chilling) for Mana valley (Fig. 6.8). However, in the Gangotri valley the maximum percentage of seed germination was observed when seeds treated with hot water at 5 C for 15 minutes while, minimum was obtained for the seeds treated with hot water at 5 C for 5 minutes. The seeds treated with hot water at 5 C for 15 min represented maximum (81.67±4.41 %) germination and minimum (51.67±8.33) was found when seeds treated with chilling at 4 C for 14 days for Yamunotri valley. In Bhyundar valley the seed germination was increased from 55.±5.77 % (scarification) to maximum 81.67±7.26 % (treated with hot water at 5 C for min) (Fig. 6.9). The seeds collected from Mana valley showed relatively better results in terms of germination as compared to other valleys with maximum (78.33±8.82 %) value was obtained when seeds treated with hot water at 5 C for min and minimum (5.±13.23 %) with chilling (for 7 days) treatments. Hot water at 5 C for min showed maximum (78.33±12.2 %) germination in the seeds of Niti valley whereas, the chilling at 4 C for 7 days resulted minimum (55.±5.77 %) percentage of seed germination. 82

12 Germination (%) min min 15 min Time Fig Effect of Hot Water (Physical Treatment) at 5 C with time intervals on seed germination in different valleys. Germination (%) days 14 days 21 days Duration Fig Effect of Chilling (Physical Treatment) at -4 C with days intervals on seed germination in different valleys. 83

13 Germination (%) Scarification Fig Effect of Scarification (physical treatment) cut made near the chalazal end, on seed germination in different valleys. Germination (%) Control Fig. 6.. Effect of Control (physical treatment) on seed germination in different valleys Effetct of PGRs, chemicals and physical/mechanical treatments on Mean Germination Time (MGT) of H. rhamnoides seeds The various treatments/ concentrations of Plant Growth Regulators and Nitrogenous compounds (Chemical and Physical/mechanical) showed significant (p<-5) results on 84

14 the Mean Germination Time (MGT) of seed germination within all treatments and concentrations/time periods amongst all five valleys. The over all results of minimum MGT (6.43±.25 days) were obtained for the seeds treated with PGRs of Yamunotri valley whereas, the average of MGT was recorded maximum (8.42±.13 days) for the seeds of Mana valley (Fig. 6.11). Similarly, the overall result of MGT exhibited that when seeds treated with chemicals (KNO 3 and Thiourea) showed minimum (6.12±.3 days) for the seeds of Gangotri valley and maximum (8.31±.38 days) for Mana valley. Physical treatments revealed that the minimum MGT (6.44±.2 days) of seeds was found in the samples of Gangotri valley and maximum (8.77±.18 days) in Mana valley. The scarification (cut made near chalazal end) reduced the MGT of seeds with 5.92 days (minimum) observed in the seeds of Gangotri valley and it tooks 8.67 days (maximum) of MGT for the seeds of Yamunotri valley. The seeds those were not treated with any kind of PGRs, Chemicals, physical/mechanical indicated the significance of these treatments for reducing the Mean Germination Time (MGT) of seeds. The results of seeds kept under control condition showed minimum (7.7 days) MGT for Gangotri valley and maximum (9.8 days) MGT for Niti valley. The MGT for other treatments for different valleys is given in the Fig MGT (days) PGRs Chemicals Physicals Treatments Fig Overall average of MGT of seeds under different treatments collected from five valleys. 85

15 MGT (Days) MGT (Days) µm 25µM µm µm 25µM µm GA 3 Concentrations BAP Concentrations MGT (Days) mM 5mM mm KNO3 Concentrations MGT (Days) mM 5mM mm Thiourea Concentration MGT (Days) Min Min 15 Min Hot Water (Duration) MGT (Days) Days 14 Days 21 Days Chilled (Duration) Scarification Control Gangotri Yamunotri Bhyundar M ana Niti Gangotri Yamunotri Bhyundar M ana Niti Fig Mean Germination Time (MGT) of seeds collected from different valleys under different Treatments. 86

16 A B C D E F Plate Seed germination: (A) Series of experimental treatments adapted for seed germinator, (B) Initial sprouting phase, (C) Seed of H. rhamnoides, (D) emergence of radical (E) fully germinated seed, (F) developmental stages of seedling establishment. 87

17 6.4. DISCUSSION Natural regeneration of a plant species depends on the production of viable seeds, subsequent germination and successful establishment of seeds. A number of physiological and biochemical processes are essential for retention of seed viability. Viability can be lost through the denaturation of water binding proteins and other stored metabolites if seeds are dehydrated too rapidly during early stage of germination (Roberts et al., 1973). In present experiments it was observed that seeds of Hippophae rhamnoides (Seabuckthorn) remained viable for longer durations. The TTC (1,2,3 triphenyl tetrazolium chloride) test indicated that the seeds collected from different valleys remained viable up to two years (73 days). The present study highlighted that the highest percentage of viability in the seeds can be achieved if seeds stored for one month whereas, there was a gradual decline in viability observed on long storage of seeds. After storage period of 365 days (One year) there was only 3.33 % of viability loss observed in the seeds of Gangotri valley whereas, under the same storage condition the maximum (17.78 %) viability loss was found in the seeds of Bhyundar valley. The study on seed germination of H. tibetana indicated that the seeds of control conditions (Untreated) produced 98.5 % germination after 6 months of storage and 99. % germination after one year of storage (Sharma et al., 24). The earlier studies on H. rhamnoides revealed that the seeds of this species do not possess any kind of dormancy after six months or one year of ordinary storage conditions and retain their viability for many years (Sankhyan, H.P. and Sehgal, R.N., 26). Generally, at room temperature, the viability of seeds remains quite high (85- %) for 2 years, but it declines rapidly (Singh and Gupta, 23) and almost similar results were observed in the present study. However, Fefelov and Eliseev (1986) estimated 92 to 94 % germination after 3.5 years under laboratory conditions (Singh and Gupta, 23). The present results and its comparison with earlier studies indicated that on an average of about 3 years of storage period for Hippophae seeds under room conditions may provide better results. The seed storage at low temperature for long period does not affect the viability, however, storage under high humidity badly affects the seed germination (Smirnova and Tikhomirova, 19; Singh and Gupta, 23). 88

18 The study on imbibition indicated that there was a slow rate of water absorption in the seeds of H. rhamnoides and it took maximum time of 36 hrs to attain final plateau. The result exhibited minimum (24 hrs for Gangotri valley) and maximum (3 hrs for Bhyundar valley) time to reach up to the level of plateau. However, there was a proper imbibition of minimum8 % to maximum 13 % (increased over initial weight of seeds) for the seeds of Niti and Bhyundar valleys, respectively. Lot of studies available on seed germination aspect of H. rhamnoides (Sharma et al., 24; Sankhyan and Sehgal, 25; 26; Sankhyan et al., 25; Singh and Gupta, 23; Fefelov and Selekhov, 23) but studies focussed on seed imbibition in the Indian context are least undertaken. In the present investigation, germination response of seeds subjected to different treatments showed significant variation (p<.5) among the seeds collected from the five valleys. The effect of Plant Growth Regulators (PGRs), namely GA 3 and BAP (with different concentration) on seed germination was also examined. The seeds of Bhyundar valley revealed the maximum germination potential with PGRs followed by Mana, Niti, Yamunotri and Gangotri valleys. Treatment with GA 3 (µm) showed highest and significant improvement in seed germination of Bhyundar valley (93.33±4.41 %) and same results were obtained for the seeds of Yamunotri valley when treated with BAP (µm). Avanzato et al. (1987) have reported better seed germination, when seeds treated with ppm of GA 3 for 24 hrs. Application of GA 3 is known to promote germination by breaking dormancy in a wide range of seeds (Nickell, 1982; Bradbeer, 1988) and the efficacy of GA 3 to enhance germination has been demonstrated in several studies conducted by various workers time to time (Nagveni and Srimathi, 19; Singh and Murty, 1987; Fox et al., 1994; Nadeem et al., 2; Pandey et al., 2). It is quite interesting to observe that the two nitrogenous compounds (KNO 3 and Thiourea) used in the present case markedly enhanced the germination in H. rhamnoides. KNO 3 has been used as a growth regenerating and germination-stimulating substance with marked success on germination in many species (Ozturk et al., 1994). In Avena fatua seeds, KNO 3 (5 and µm) was found to enhanced germination of dormant caryopses by accumulating in the embryo where it acted osmotically to increase water uptake (McIntyre et al., 1996; Pandey et al., 2).Seeds treated with KNO 3 (µm) showed maximum percentage of germination collected from Bhyundar valley while, 89

19 Thiourea (5µM) enhanced seed germination (93.33 %) of Mana valley (Fig. 6.5). It was observed that the nitrogenous compounds (KNO 3 and Thiourea) resulted into % to % higher germination as compared to seeds kept under control conditions. The results obtained in the present case showed significant at p<.5 level for enhancing seed germination with the application of nitrogenous compounds under various concentrations. Amongst various physical treatments applied in this study, hot water (5 C) treatment yielded best results for germination. However, the overall results of hot water treatments were not relatively better as compared to PGRs treatments. Only seeds of Gangotri valley showed highest germination (93.33 %) otherwise seeds from other valleys germinated in between % (Niti valley) to % (Yamunotri and Bhyundar valleys). The earlier study on H. rhamnoides indicated that the seeds treated with hot water at C for min produced 6 % of germinated seeds while the other species such as H. salicifolia germination enhanced up to % under same treatment (Sankhyan and Sehgal, 26; Sankhyan et al., 25). Jhuree et al., 1988, observed that boiling treatment had a very harsh effect on some species e.g., Calytrix tetragonal and had little or no effect on some seeds with hard seed coat. However, Bhatt et al. (2) reported significant improvement in germination after treating the seeds of Myrica esculenta in hot water at C for 1 min. The chilling treatment was not found suitable for seed germination as it reduced germination percentage of some seeds as compared to control condition (Fig. 6.). The maximum % germination was found in the seeds of Niti valley which were treated with chilling at 4 C for 14 days whereas, seeds of Yamunotri valley showed lowest percent of increment (6.67 %) treated with chilling at 4 C for 21 days. The seeds collected from Gangotri, Bhyundar and Mana valleys showed the declining of germination percentage when treated with chilling at 4 C as compared to control. However, on the other hand the study carried out by Sankhyan et al. (25) revealed that seeds treated with cold water for 6 days improved germination up to % in H. rhamnoides and 5 % in case of H. salicifolia. In their experiment they could not mentioned the temperature of the cold water and thus it is difficult to say at which temperature the seeds produced better result. Simultaneously, in the present study it was 9

20 observed that under control conditions seeds performed better germination instead of chilling treatment at 4 C for 7, 14 and 21 days. Similar results were observed for the scarified seeds in which germination improved with the range of 55 % to %, respectively, for seeds of Bhyundar and Gangotri valleys. The overall results of scarified seeds were not found significant (p>.5) for the improved germination in comparison to seeds kept under control condition. The Mean Germination Time (MGT) of Seabuckthorn seeds was also affected by various treatments applied in the present study. The PGRs (GA 3 and KNO 3 ) have a significant impact over the MGT of seeds collected from different valleys. Amongst all five valleys, the seeds of Gangotri required minimum time to germinate under laboratory conditions. While, the seeds from Yamunotri germinated faster then other valleys under different chemical treatments (KNO 3 and Thiourea) as well as physical treatments (hot water, chilling and scarification). It was estimated that the control (untreated) seeds of H. rhamnoides required on an average of 7.7 days (for Gangotri valley) to 9.8 days (for Niti valley) of MGT to germinate. However, if treated with KNO 3 (25µM) then the mean germination time reduced with 5 days (for Gangotri) to 7.73 days (for Niti valley). The results indicated that there was a significant relation of different treatments (PGRs, Chemicals and Physicals) with mean germination time of the seeds. 91