Long-term storage-dependent changes in seed physiological aspects of Hyoscyamusniger from a cold desert region of Trans-Himalaya

Henbane ( Hyoscyamus niger L.), an economically important medicinal plant, has an endangered status in Himachal Pradesh (India) that needs appropriate conservation interventions. We have examined seed physiological aspects of H. niger from Lahaul (Himachal Pradesh, India), a cold desert region. The freshly harvested seeds exhibited complete dormancy. Gibberellic acid (GA 3 ) and chilling treatment strongly promoted seed germination which was accompanied by increased α -amylase activity. KNO 3 , NaN 3 and sodium nitroprusside (SNP), an NO donor, also promoted germination. During storage, the seeds retained high viability even after a storage of 72 months under ambient conditions. However, they remained dormant during the entire storage period. The responsiveness of seeds to GA 3 and chilling treatment gradually declined with progression of storage period. Concomitantly, the triphenyl tetrazolium chloride (TTC) reduction ability of seeds was low-ered. The seed responsiveness to KNO 3 , NaN 3 and SNP during storage increased until one year and decreased thereafter. With the progression of the storage period, seeds exhibited elevated lipid peroxidation and reduced catalase activity implying a role of oxidative stress in observed changes. The involvement of phenolics in seed dormancy of H. niger was not evident. The findings are of significance for conservation and cultivation of H. niger through seeds in the arid mountain region. to the cultivation and conservation of H. niger . We report here the long-term storage-dependent changes in seed physiological parameters of such a population.


Introduction
Hyoscyamus niger L. (Solanaceae), commonly known as henbane, is an important multipurpose medicinal plant. It is used as a narcotic, anodyne, sedative, antiseptic and mydriatic, besides being used in nervous conditions, asthma, whooping cough, muscular pain and toothache. [1][2][3] Medicinal properties of henbane are principally due to diverse alkaloids (such as, hyoscyamin, hyoscine, scopolamine, bellado-nine, hyoscypikrin and atropine) present in most parts of the plant. The plant also contains certain non-alkaloidal components, e.g. grossamide and cannabisins that have been reported to exhibit moderate cytotoxicity in cultured human prostate cancer cells. 4 The plant grows well in regions with a semi-arid and cold climate; high temperature and humidity are detrimental to plant growth and alkaloid contents. Furthermore, the plants from higher altitude regions, as in the present study are notably richer in alkaloids than their lower altitude counterparts. 1 The Indian Himalayan Region (IHR) harbors many highly valuable medicinal plants.
H. niger populations are distributed in several areas of Himachal Pradesh in the IHR, particularly Kinnaur and Lahaul-Spiti. This species has an endangered status in Himachal Pradesh owing to unsystematic exploitation and habitat loss. 5 The mode of regeneration in H. niger is through seeds. Despite a large number of seeds produced, only a few seedlings are able to establish due to poor seed germination. 6 Obviously, an understanding of seed physiological aspects of H. niger could be expected to contribute to the efforts of seedbased in situ conservation as well as ex situ cultivation. In a previous study, we reported the stimulation of seed germination by GA 3 and KNO 3 in a H. niger population from Lahaul, a cold desert region in Himachal Pradesh. 6 The region remains devoid of any significant precipitation, because the main Himalayan ranges form a massive barrier which prevents most of the monsoon rain from reaching the area and creates a vast rain-shadow zone. 7 The scanty rainfall, low humidity, high altitude and cold climatic conditions of the region seem to be conducive to the cultivation and conservation of H. niger. We report here the long-term storage-dependent changes in seed physiological parameters of such a population.

Seed source
Seeds of H. niger were collected during August-September 2003 from the cold desert area of Lahaul (latitude 32° 8' and 32° 59' N, longitude 76° 49' and 77° 47' E and altitude 2,950 m above mean sea level), Himachal Pradesh, India. The seeds were collected manually, air-dried under shade and were stored in polyethylene jars at room temperature for subsequent studies (up to 72 months).

Seed viability
Seeds were subjected to 2,3,5-triphenyl tetrazolium chloride (TTC) test for viability evaluation shortly after collection and at regu-lar intervals during the subsequent storage period. The seeds were surface-sterilized and imbibed in distilled water for 24 h at 25±1°C. Thereafter, the seeds were cut at one end to facilitate the diffusion of TTC, incubated with 0.1% TTC solution in the dark for 24 h and examined for staining intensity. Seeds with completely stained embryo were considered viable. The TTC reduction by seed tissue was quantified by measuring the formazan formed. For this, the seeds, at the end of incubation with TTC, were homogenized with 5 ml MetOH and centrifuged at 10,000 g for 10 min; absorbance was recorded at 485 nm. TTC reduction was expressed as A 485 /50 embryos.

Seed germination assays
Seeds were surface sterilized with 0.1% HgCl 2 for 5 min, washed thoroughly under tap water and soaked in distilled water for 24 h at 25±1°C. Thereafter, they were transferred to Petri dishes lined with three layers of moist filter paper in a seed germinator at 25±1°C under continuous illumination (PAR: 40 μmol m -2 s -1 ). Seeds were considered germinated upon the emergence of 2-5 mm radicle and scored periodically for germination. The mean germination time (MGT) was calculated as fol- Application of physico-chemical and GA 3 treatments for dormancy removal/germination improvement Before subjecting to germination conditions, the seeds were treated with the following effectors: i) cold-stratification. The surface-sterilized seeds soaked in distilled water for 24 h were kept at 4°C for one month; ii) acid scarification. Seeds were treated with concentrated H 2 SO 4 for 1 min; this duration was decided on the basis of preliminary experiments in which various periods of time were considered. Thereafter, the seeds were washed thoroughly under tap water and allowed to imbibe in distilled water for 24 h and then transferred to germination conditions; iii) chemical treatments. The surface-sterilized seeds were soaked in aqueous solution of 0.2% potassium nitrate (KNO 3 ), 1 mM sodium nitroprusside (SNP), 0.1 mM sodium azide (NaN 3 ), respectively for 24 h, before subjecting to germination conditions; iv) GA 3 treatment. The surface-sterilized seeds were soaked in aqueous solutions of gibberellic acid (0.1 and 1.0 mM) for 24 h followed by germination on moist substratum; v) acid-scarification + GA 3 (1 mM). The acid-scarified seeds were imbibed in aqueous solution of GA 3 for 24 h and transferred to germination conditions. α-amylase activity α-amylase activity was assayed following the method described by Filner and Varner. 9 Seeds were homogenized with chilled Tris-HCl buffer (pH 7.2) and centrifuged at 10,000 g for 10 min at 4°C. The supernatant served as crude enzyme extract. Enzyme 1 ml was added to 1.0 mL substrate (0.15 % starch, 0.2 mM CaCl 2 in Tris-HCl buffer, pH 7.2) and incubated for 10 min at 25°C. The reaction was quenched by adding 3 mL IKI reagent (0.6% iodine in 6% KI; 1 mL of this diluted to 50 mL with 0.05 N HCl) and absorbance recorded at 620 nm. The amount of starch degraded was determined with the help of a calibration curve prepared with starch.

Measurement of lipid peroxidation
Lipid peroxidation was determined by measuring malondialdehyde (MDA) contents according to the method of Dhindsa et al. 10 In brief, the seeds were homogenized with 0.1% trichloroacetic acid (TCA) and centrifuged at 10,000 g for 10 min. One ml supernatant was reacted with 0.5% thiobarbituric acid (TBA) in 20% trichloroacetic acid (TCA) and incubated at 95°C for 30 min. The reaction was terminat-ed by cooling the reaction mixture and centrifuged. The absorbance was read at 532 nm and corrected by subtracting the absorbance at 600 nm. The MDA contents were measured using the extinction coefficient at 155 mM -1 cm -1 .

Catalase assay
Catalase activity was assayed polarographically by measuring the H 2 O 2 dependent oxygen evolution at room temperature with an oxygen electrode unit (Hansatech, UK). In brief, the seeds were homogenized with chilled Na-phosphate buffer (pH 7.4) and centrifuged at 10,000 g for 5 min at 4°C. The supernatant served as crude enzyme. Assay (1 mL) including 880 μL phosphate buffer (0.1 M) pH 7.4 and 100 μL of 0.1 M H 2 O 2 . The reaction was initiated by addition of 20 μL enzyme extract. The amount of O 2 evolved/min was recorded with the help of a chart recorder. The catalase activity was calculated through electrode calibration and slopes obtained on chart paper.

Protein contents
Protein contents were estimated according to the method of Lowry et al. 11 Total phenolic contents Total phenolic contents were determined according to the method of Goldstein and Swain. 12 The seeds were homogenized in 0.3 N HCl in methanol, and centrifuged at 10,000 g for 10 min. Supernatant was collected and pellet was again extracted in 0.3 N HCl in methanol and centrifuged. The supernatants were pooled and evaporated on a hot water bath. The residue obtained was dissolved in distilled water and final volume made to 5 mL. To this, 0.5 mL of Folin-phenol reagent was added and shaken vigorously. After 3 min, 1 ml 35% sodium carbonate solution was added, shaken and allowed to stand for 1 h. Absorbance was recorded at 630 nm and the amount of total phenols was determined with the help of calibration curve prepared with gallic acid.

Statistical analysis
All experiments were carried out in triplicate. Data are presented as arithmetic means and standard deviation. The statistical significance was tested using Student's t-test.

Seed viability
Freshly harvested seeds exhibited 100% viability. During storage, the viability status was maintained for at least 24 months. Thereafter, only a slight decline in viability was observed.
After 72-month storage, a 12% decrease in seed viability was observed ( Figure 1). Besides, the qualitative assessment of seed viability, the TTC reduction ability of seeds was also measured. This remained high for up to 24 months followed by a subsequent gradual decline ( Figure 1).

Seed dormancy/germination
Freshly harvested seeds exhibited dormancy as was apparent from a lack of germination in control. Of the various pre-treatments tested for their effectiveness to overcome dormancy/improve germination, GA 3 was found to be most effective. The GA 3 effect intensified with an increase in concentration. Thus, GA 3 0.1 and 1 mM induced 33 and 100% germination, respectively, within 10 d. Cold-stratification of seeds for 30 d was also effective causing 84% germination. GA 3 and chilling not only stimulated the seed germination but also effectively reduced the time required for germination as is evident from lower MGT values ( Figure 2).
Other seed pre-treatments tested, namely, potassium nitrate (KNO 3 ), sodium nitroprusside (SNP) and sodium azide (NaN 3 ), were effective in dormancy removal, only to a lesser extent than GA 3 and cold-stratification. Acid (H 2 SO 4 ) scarification of seeds was altogether ineffective in altering the dormancy/ germination status (Figure 2).  Storage-dependent changes in seed dormancy/germination

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During 72-month storage seeds generally remained dormant and showed no germination. During storage, the seed responsiveness to GA 3 (1 mM particularly) and cold-stratification remained unchanged for up to 24 months (Figure 3). Beyond this period, the seed responsiveness to these treatments declined slowly with the progression of storage period. After 72-month storage, 30 and 36% decline in seed response to GA 3 and cold-stratification was observed. Such a decline was not evident when seeds had been acid-scarified prior to the application of GA 3 (Figure 3).
The storage-dependent alteration in the responsiveness of seeds to other pre-treatments, namely, KNO 3 , SNP and NaN 3 in causing dormancy removal/seed germination improvement appeared to be biphasic. After storage of up to 12 months, the seed response to the above effectors increased by 200, 50 and 38%, respectively, as compared to the similarly treated freshly harvested seeds. In contrast, when storage exceeded beyond 12 months, the responsiveness to all above effectors decreased gradually; after 42 months, the effect was strongly diminished (Figure 3). Despite the changes in responsiveness of seeds to various pre-treatments during storage, the MGT was altered only slightly (data not shown).

α-amylase activity
The α-amylase activity in seeds increased until 24 months of storage and declined thereafter. GA 3 pre-treatment of freshly harvested seeds induced a 2-fold increase in α-amylase activity. This GA 3 -dependent enhancement, however, diminished with the progression of storage period. Chilling treatment also significantly increased the α-amylase activity but to a lesser extent as compared to GA 3 . KNO 3 and SNP treatment did not have any significant effect on α-amylase activity ( Figure 4).

Lipid peroxidation and catalase activity
During storage, the lipid peroxidation status (MDA contents) of seeds increased, particularly beyond 36 months; leading to a 54% increase after 72 months. Seeds pre-treated with GA 3 , KNO 3 and SNP showed higher MDA contents. For example, 20, 21 and 40% higher MDA contents were observed due to these treatments, respectively, in freshly harvested seeds. Moistchilling did not significantly alter the MDA contents of freshly harvested or differentially stored seeds ( Figure 5A).
The freshly harvested seeds exhibited a higher level of CAT activity as compared to stored seeds. The CAT activity declined with the progression of the storage period; a 52% decline in CAT activity was evident after 72 months. The activity was promoted by GA 3 but suppressed by KNO 3 pre-treatments in seeds stored up to 36 months. Moist-chilling did not considerably alter the CAT activity ( Figure 5B).

Total phenolic contents
Maximum total phenolic contents were found in freshly harvested seeds. During storage, the total phenolic contents of the seeds declined significantly, particularly beyond 24 months storage. After 72 months storage a 37% decline in seed phenolic content was observed. Chilling treatment also reduced the phenolic contents of the seeds. Various other seed pretreatments did not considerably alter the quantity of total phenolic content of the seeds (Table 1).

Discussion
H. niger, an economically important, multipurpose medicinal plant, has an endangered status in Himachal Pradesh, India. 5 Therefore, it requires an appropriate intervention for conservation. In addition, its in situ cultivation might reduce the pressure on wild populations as well as contribute to the economy of inhabitants. The mode of regeneration in the H. niger is through seeds. However, the seeds exhibit dormancy, 6 a situation unfavorable for seed based regeneration/multiplication. Obviously, a clearer understanding of seed physiological aspects including the storagedependent alterations thereof would be immensely valuable. The high degree of viability in freshly harvested seeds was generally maintained even after a 72-month storage of     seeds under ambient conditions as revealed in TTC reduction assay. This viability status was also reflected in germination observed in seeds treated with GA 3 alone or in combination with acid-scarification. The observed seed viability features under ambient conditions are desirable for seed based plant propagation. Despite being strongly viable, the freshly harvested seeds exhibited dormancy, which could be alleviated through seed pre-treatment with GA 3 ; 100% germination was achieved within a week. The seed germination stimulating effect of GA 3 in a H. niger population from Turkey was reported by Cirak et al., 13 where about 20% germination was induced. Thus, GA 3 effect seems to be population-specific. The GA 3 -induced seed germination accompanied by increased α-amylase activity suggests the role of GA 3 in enhancing the availability of sugars for embryo growth. GA 3 might similarly affect other hydrolytic enzymes too. GAs are likely to influence several other events associated with seed germination. For example, they are known to replace the requirement of afterripening, light and cold. 14 Furthermore, the GAs have been reported to alleviate mechanical restraints of the testa and endosperm allowing an easy protrusion of the radicle in Arabidopsis and tomato. 15,16 The effect of chilling treatment on seed germination and α-amylase activity in dormant seeds of H. niger were found to resemble those induced by GA 3 with essentially quantitative differences. Cold stratification is known to inhibit ABA synthesis, promote GA synthesis and/or increase the sensitivity of seeds to GA 3 and induce cell division in the shoot and root meristem of the embryo. 14,17 KNO 3 , NaN 3 and SNP significantly promoted the seed germination of H. niger. Nitrite and nitrates have long been known to break dormancy or stimulate seed germination in many plant species. 18 Hihorst 19 found that in seeds of Sisymbrium officinale, the nitrate content of the seeds from different habitats was directly proportional to their rate of germination. Nitrate provided during seed development via the maternal plant has been reported to lower dormancy. 20 However, the mechanism(s) involved in seed dormancy removal by nitrite and nitrates are not precisely known. It has been proposed that oxidized forms of nitrogen might promote seed germination by causing a shift in respiratory metabolism to the pentose phosphate pathway or through promotion of respiration by increasing oxidation of NADPH to NADP + , a limiting electron acceptor. 21-23 Such a relationship was, however, found missing in dormant seeds of Avena fatua. 24,25 McIntyre et al. 26 suggested the involvement of osmotic and nutritional effects of exogenous nitrate on seed germination of A. fatua.

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Sodium nitroprusside (SNP), a nitric oxide (NO) donor, promoted seed germination of H. niger in the present study. NO has emerged as a major signaling and effector molecule involved in many key physiological processes. [27][28][29][30] A few reports implying the involvement of NO in influencing seed dormancy/germination are also available. Thus, the application of NO donors was found to break the dark imposed seed dormancy in lettuce; the effect of NO donors was reversed in the presence of the NO scavenger cPTIO (carboxy 2-phenyl-4,4,5,5tetramethylimidazoline-1-oxyl 3-oxide). 31 Also, NO-dependent germination of wheat seeds under osmotic stress and stimulation of βamylase during early stage of germination has been shown. 32,33 Such effects of NO might explain the germination of dormant seeds of H. niger induced by SNP.
Quite remarkably, the H. niger seeds remained dormant even after 72 months of storage under ambient conditions. However, the responsiveness of seeds to GA 3 and chilling treatment began to decline consistently with the progression of the storage period, showing a correspondence with reduction in seed vigor (TTC reduction ability). In contrast, the storage-dependent alteration in the seed responsiveness to KNO 3 , SNP and NaN 3 treatments was biphasic with enhanced effect up to 12 months of storage followed by a gradual decline thereafter. Dry storage or after-ripening is known to widen the temperature range for germination, decrease ABA concentration, increase GA sensitivity, loss of light and nitrate requirement, and also increase the germination velocity. 14 All these factors might eventually lead to enhanced sensitivity of seeds to various effectors. However, a long-term storage is known to decrease the germination ability and sprouting of seeds which is strongly speciesspecific. 34 During seed storage, MDA contents increased particularly beyond one year which was accompanied by reduced CAT activity. These observations suggest the involvement, at least in part, of redox metabolic perturbation in the storage-dependent partial loss of seed viability. Sustained production of ROS due to prolonged storage of seeds leads to oxidative stress and related deteriorative events. 35,36 The decline in α-amylase activity with storage, particularly beyond 24 months, might also be ascribed to a certain extent to increased oxidative stress and related protein damage.
Total phenolic contents of seeds, measured as possible dormancy regulators, 20,37,38 decreased gradually during 72-month storage. Despite this, however, no germination was observed; apparently, there was no indication of involvement of phenolics in seed dormancy/ germination of H. niger.
In conclusion, the seeds of H. niger retained high viability even after prolonged storage and exhibited reasonably good germination through certain seed pre-treatments. The find-ings are of significance for conservation as well as for the possibility of commercial cultivation of H. niger through seeds in the arid mountain region.