Seed germination response of Indian wild pear (Pyrus pashia) to gibberellic acid treatment and cold storage

Knowledge of seed germination behaviour of different seed sources of tree species is useful in selecting the most responsive and adaptive ones for propagation and germplasm conservation. The wild Himalayan pear (Pyrus pashia Buch-Ham ex D. Don) produces highly nutritious edible fruits that are consumed by local communities. The populations of the species are threatened due to exploitation and lack of adequate conservation programmes. The study was conducted to examine the germination response of P. pashia seeds from two sources (S1-Champawat and S2-Pithoragarh) in Uttarakhand state of India, to different GA3 treatment and also to assess the viability and longevity of the seeds in cold storage (5 °C) for three years. In both sources germination percent (GP) increased significantly under GA3 treatment and speed of germination was also enhanced (reduction in mean germination time). In Source 1, GP increased under all GA3 application, highest being 94% under GA3 500 ppm treatment. In Source 2, GP of seeds doubled under 100 ppm GA3 treatment while its higher concentrations did not improve the germination. However, the differences observed in germination between the seed sources could be due to differences in the dormancy levels and/or sensitivity to dormancy breaking elements across their geographical range. Thus, exogenous application of GA3 is suggested for enhancing the germination in seeds of P. pashia. Seeds responded to cold storage by increased germination with duration, i.e. highest after three years in storage, indicating that the seeds got the required chilling treatment for overcoming dormancy.


Introduction Introduction Introduction Introduction
Pyrus pashia commonly known as Indian Pear, wild Himalayan Pear, belongs to the family Rosaceae. It is a moderate-sized deciduous tree with branches dark-brown in colour and often spiny. Leaves are simple, glabrous and ovate-lanceolate in shape (Kanjilal, 1928). Fruit of P. pashia is Pome, brown in colour, pulpy and contains approximately five seeds per fruit. Leaves of P. pashia are used as fodder, wood is utilised as fuel and for making small agricultural implements. Fruits are edible, highly nutritious and are included in the diet by

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Notulae Scientia Biologicae local people. They provide multiple ecosystem services as a source of nutrition, pollination, genetic variation for breeding programs, and even of valuable income for people living around them (Powell et al., 2013). The wild populations of P. pashia are distributed in India, Nepal, Bhutan, Afghanistan, Pakistan, Thailand, China, and Indochina at an altitude of 750-2500 m. In India, the species are found in Himalayan region and north-eastern regions (Troup, 1975). Pyrus species are threatened with about 85% pear varieties lost in 19 th century (Sindelar, 2002). Furthermore, due to various insect pests, pathogens and abiotic stresses, valuable and highly resistant pear germplasm has been lost (Fowler and Mooney, 1990). Therefore, more efforts to conserve the genetic resources of these species must be made. The genetic diversity can be best preserved by systematic collection for establishing field germplasm banks and long-term storage of seeds in Seedbanks.
Pyrus species are widely cultivated in many parts of the world but physiological dormancy associated with seed causes irregular germination in the species. Dormancy plays a major role in regulating germination in many tree species. Dormancy release is regulated by a combination of environmental and endogenous signal with both synergistic and competing effects. The balance of abscisic acid (ABA); gibberellin (GA) levels and sensitivity is a major, but not the sole, regulator of dormancy status (Finkelstein et al., 2008). Pear seeds belong to Rosaceae family usually show dormancy at maturity and require 2-3 months at 0-7 °C temperature in a moist, well-aerated medium to break dormancy (Hartmann et al., 1990), which is similar to the requirements for apple seeds (Lewak, 2011). Gibberellic acid (GA) activates the production of α-amylase, proteolytic enzymes, nucleases, etc., leading to loss of seed dormancy and promotion of seed germination (Dominguez et al., 2004;Mrva et al., 2006). Studies evaluating the exogenous application of gibberellic acid have shown to relieve certain types of dormancy, including physiological dormancy, thermo-dormancy and photo dormancy, and to replace partially or fully the necessary cold treatments in different plants (Hartmann et al., 1997). Seed viability can be extended by cold or dry storage at seed moisture content below 5% (Huang et al., 2003). Cold storage reduces or prevents the effects of ageing was indicated by the slower rate of damage caused by low temperature (Spano et al., 2006). The aim of our study was to examine the germination response of P. pashia seeds from two sources (Champawat and Pithoragarh) of Uttarakhand state in India, to different GA3 treatment (soaking in 100, 300, 500 and 700 ppm solutions for 24 hours) and also to assess the viability and longevity of the seeds stored at moisture content of 8% at 5 °C.

Materials and Methods Materials and Methods Materials and Methods Materials and Methods
Seed material Mature fruits of Pyrus pashia were collected in October 2017 from natural stands at two locations namely Champawat Forest Range (Source 1 or S1) and Pithoragarh Forest Range (Source 2 or S2) in Uttarakhand state of India. Geographical coordinates of the two sources of the species are presented in Table  1. Fruits were brought to Forest Tree Seed Laboratory of the Institute at Dehradun in permeable bags. The species produces pulpy fruits with seeds consisting of hard endocarp. Fruits were macerated, de-pulped and washed under running tap water to extract the seeds. Extracted seeds were shade-dried in ambient conditions for 3-4 days. Moisture content of the seeds The moisture content of the seed was determined by low constant oven dry method at 103 ±1 °C for 17 hrs (ISTA, 2010) in 4 replicates of 10 seeds each. Moisture content was determined on the fresh weight basis (%) using the formula: where MC is the moisture content, Fw is the initial fresh weight of the replicate and Dw is the dry weight.

Germination test
Germination test was conducted on fresh seeds from both seed sources. The experiment was designed in completely randomised block in four replications of 25 seeds each, which were placed on moist Whatman filter paper in sterile Petri dishes (15 cm dia.) and kept in a seed germinator (NSW New Delhi, India) at 25 °C temperature (ISTA, 2010) with 8 hours photoperiod. A seed was considered to have germinated when the radical was 1 cm long. The observations on seed germination were taken every day (except weekends) up to 28 days from the date of sowing (ISTA, 2010). Germination parameters such as germination percent; Mean Germination Time (Orchard, 1977) and Germination value (Djavanshir and Pourbeik, 1976) of the species were measured as per the standard methods. Germination percent of seed was expressed as: Germination percentage (G) = total number of seed germinated at end of germination test/total number of seeds placed for germination test ×100.
MGT -a parameter used for monitoring the speed of germination was calculated as: Mean germination time (MGT) = ΣFx/ΣF; where F is the number of seeds germinated on day x. Germination value was expressed as ∑DGS/N x (Final cumulative Germination Percent/10); where DGS is daily germination speed which is calculated by dividing cumulative germination percent by the number of days since beginning the test, N is number of counts and 10 is constant through germination test.

Gibberellic acid treatment
For observing the effect of gibberellic acid pre-treatment on the germination and its speed, seeds from both sources were pre-treated with GA3 at different concentrations; seeds were soaked in 100 ppm, 300 ppm, 500 ppm and 700 ppm GA3 solution for 24 hours before placing for germination experiment. For each test separate seed samples were used. Untreated seeds served as control.
Seed storage Fresh seed of P. pashia from both sources after quality evaluation (seed germination test) were desiccated to 8% moisture content from initial 11% using silica gel in 1:1 ratio. Desiccated seeds were thereafter stored at 5 ± 1 °C temperature in air-tight plastic boxes in a seed storage chamber and their viability was evaluated through germination test every six months, up to a period of three years (results only presented for yearly germination) to evaluate the germination pattern and longevity of seeds under storage conditions.

Statistical analysis
Statistical analysis of cumulative germination data was performed with SPSS Statistics for Windows, version 16.0' (SPSS Inc., Chicago, Ill., USA). Statistical analysis was performed using GLM multivariate ANOVA for testing the significance of treatment (4 levels for GA3 and 3 for storage duration), population (two levels) and their interactions on GP, MGT and GV. All tests were analysed at a significance level of α = 0.025.

Results Results Results
Germination response of P. pashia seeds to GA3 treatment Untreated seeds (control) exhibited a GP of 62 % in S1and 15 % GP in S2. This difference could be due to the degree of dormancy related to different seed sources. In S1, MGT was recorded as 12.37 days and in S2 MGT was recorded as 15.45 days (Figure 1) indicating significantly lower germination and slow speed of germination in seeds of Source 2. GV was observed as 12.02 in S1 and much lower (0.46) in S2 (Figure 2). GA3 treatment of seeds significantly increased the germination percent against that in untreated seeds. Highest GP (94 %) was observed in the seeds pretreated with GA3 500 ppm followed by the ones treated with 300 ppm (93 %), 700 ppm (92 %) and 100 ppm (86 %). Seeds pretreated with GA3 at 100 ppm resulted in highest GP (31 %) in Source 2 followed by 700 ppm (29 %), 300 ppm (25 %) and 500 ppm (23 %) (Figure 1). Mean germination time (MGT) which indicates the speed of germination ranged from 5.4 to 6.6 days in S1 and 7.4 to 10.6 days in S2. Germination was fastest (lowest MGT) in the seeds treated with 700 ppm GA3 in S1 and 500 ppm GA3 in S2. Highest MGT (slow speed of germination) was observed in the seeds treated with 100 ppm GA3 in both the sources (Figure 1) which means that seeds took longer to complete the germination. GV ranged from 42.6 to 60.4 in S1 and 2.4 to 4.8 in S2, which was significantly lower than that of seeds of S1 as the germination of seeds was significantly lower impacting the overall value of germination. Highest GV was recorded in seeds treated with 700 ppm GA3 in both sources ( Figure 2).   Study assessed the seed germination behaviour of stored seed for three years. In S1, germination percent of seed was 55 % after one year of storage, which increased to 64.5 % after second year and further to 82.5 % in third year while in case of seeds of other source (S2), GP was 12.25 %, 17.33 % after first and second year respectively, that doubled to 34.75 % after three years of storage. In both sources, highest GP was observed in third year of storage as compared to first year ( Figure 3) which clearly indicated that the seeds required cold treatment which they got and responded favourably by increased GP. In S1, lowest and highest MGT was 9.4 and 12.8 days. Lowest MGT in seed was recorded in the second year of storage that exhibited faster speed of germination in the seeds. In S2, lowest MGT of 1.4 days was recorded in the third year and highest was 8.9 days after the first year of storage ( Figure 3). Highest GV was recorded in seeds stored for three years in both sources ( Figure 4) as germination was also highest in them.        The effect of GA3 treatment (various concentrations) on germination percent and mean germination time of seeds of P. pashia was significantly different (p < 0.025), highest germination occurred in seeds presoaked in 500ppm GA3 solution in S1 and those in 100 ppm in S2. Similarly, the effect of population on germination percent and mean germination time of seed was significantly different (p < 0.025) but the effect of interaction between treatment and population on germination percent and mean germination time of seed was not significantly different (p > 0.025) ( Table 2). There was significant effect of treatments, population and interaction between treatment and population on germination value meaning that the germination value which is an index of expression of speed and totality of germination, under different treatments differed in the two populations significantly (p < 0.025).  Table 2. Summary of General Linear Model (GLM) multivariate analysis for testing significant effects of seed sources and treatment Treatment includes pre-soaking in 100 ppm; 300 ppm 500 ppm; and 700 ppm GA3 solution and cold storage for 1, 2 and 3 years on germination percent (GP), mean germination time (MGT) and germination value (GV) of P. pashia seeds from Source 1 (Champawat) and Source 2 (Pithoragarh) in Uttarakhand state in India. (P<0.025) denote significant difference on seed source and treatment independently and combined effect of both on GP, MGT and GV. Fresh seeds of P. pashia from both sources yielded lesser germination (without any pretreatment) as compared to GA3 treatment. It was under GA3 treatment, highest GP was exhibited in the seed pre-treated with 500 ppm in S1 while it was in 100 ppm in S2. Exogenous application of GA3 breaks dormancy and accelerates seed germination in many Rosaceae members. Gibberellins stimulate germination by inducing hydrolytic enzymes that weaken the barrier tissues such as endosperm, seed coat, inducing mobilization of seed storage reserves, and stimulating the expansion of embryo (Finkelstein et al., 2008). Kumar et al. (1988) studied germination in seeds of P. pashia using 0.5%, 1.2% and 5% ethanol and -3 log M, -5 log M and -7 log M GA3. Unstratified seeds of P. pashia responded with the germination of 60% in -7 log M GA3 and 90% in 5% ethanol treatment. Fogle (1958) reported that soaking seeds of sweet cherry for twenty-four hours in gibberellin at a concentration of 100ppm immediately after collection substitutes for two or three months of the after-ripening period. Pillay et al., (1965) reported that soaking seeds in gibberellin at a concentration of 100 ppm increases the rate of germination and partially replaces the chilling requirements in Prunus avium and Prunus mahaleb.
According to Donoho and Walker (1957) seed treatment with gibberellin could replace the low-temperature requirement to some extent in Prunus persica. Treatment of seeds with GA3 may substitute for cold stratification was also reported for Prunus persica, Myrica pensylvanica (Macdonald, 1993), Arbutus unedo (Smiris et al., 2006), Jasione supina subsp. supine (Güleryüz, 2021). In our study also, germination of seeds significantly increased under GA3 treatment exhibiting the replacement of chilling requirement of seeds for overcoming physiological dormancy which the seeds get during the winters in their natural habitat.
Germination percent of seeds increased from 55 to 82.5 % in accession 1 which was an increase of 27% and from 12.25 to 34.75% i.e. almost three times in S2 from first year to third year. It shows that seeds of P. pashia got the required chilling treatment during cold storage that resulted in subsequent increase in the GP and reduction in MGT. According to Baskin and Baskin (1988) some species can come out of dormancy during dry storage but a relative humidity of 30-40% is required for them to do so. Highest germination was observed in the seeds stored for 3 years in both sources. Storage of seeds in the air-dried state at low temperature for afterripening between 1 °C to 10 °C or 2 °C to 5 °C for periods of some months to several years have been reported to break dormancy for certain forest and fruit trees (Crocker, 1948;Stokes, 1965). Highest germination of P. pashia seeds after three years in stored conditions indicate that seeds in cold storage (at 5 °C storage temp, 8% seed moisture content) required several months to reach full ripening, breaking dormancy and accelerate germination. Generally, desiccation plays a crucial role in the after-ripening process, which prepares the seeds for germination. This suggests that one of the functions of the desiccation process is preparation for germination (Wawrzyniak et al., 2020).
Storage conditions and duration significantly affect germination percent has been reported in many species. Huang et al. (2003) reported that seed viability can be extended by cold or dry storage at seed moisture content below 5%. Temporary cold storage under high-moisture conditions enhanced germination in Arbutus unedo (Ana Vasques et al., 2014). Abdelbasit et al. (2012) observed that cold and dry storage increased seed germinability with increasing storage time in Acacia tortilis of three provenances. A comparative study on P. pashia seeds from two seed sources revealed a significant difference on GP, MGT of seed under similar pre-treatments (p < 0.025) but the effect of interaction between treatments and seed source is not significant in both seed sources under similar storage conditions which indicate the difference in quality of seeds collected from different natural ranges. Provenance /seed source studies in forest trees are very important for determining the quality, identifying the best and highly adaptable provenance and for screening the naturally available genetic variation to utilize the best material for maximum productivity and for further breeding programme (Suri, 1984;Shiv Kumar and Banerjee, 1986). Baskin and Baskin (1998) reported that fresh seed collected from various locations can alter the germination characteristics of seed due to different degree of dormancy. Seed from different accessions reported various depth of dormancy due to genotype-by-environment interactions in Arabidopsis (Donohue et al., 2005;Vidigal, 2016); Medicago truncatula (Renzi et al., 2020). In the present study also, the initial germination of seeds from Champawat (S1) was 62% which was more than 4 times that of seeds from Pithoragarh (S2). This could be because of varying dormancy levels in the seeds. Donohue et al. (2005) observed that depth of dormancy is determined genetically as well as by the ambient environment during seed formation. Genetic variation for germination can be detected when genotypes are compared in identical environments. This implies that not only the conditions of the germination test must be identical, but also growth conditions during seed development and storage conditions, including the time that the seeds are stored, must be the same (Bentsink and Koornneef, 2008). Germination responses of a seed vary according to geographical and environmental factors viz. altitude, elevation, soil moisture, soil nutrient, temperature, kind and density of plant cover, degree of habitat disturbance of the seed where the seed matures (Ginwal et al., 2005).

Conclusions Conclusions Conclusions Conclusions
In the present study, seed from both sources responded to exogenous GA3 applications as well as cold storage while higher seed viability and vigour was exhibited by seed collected from S1 in terms of all germination parameters. Seed source plays an important role for the contribution of germination percent; still further research should be extended to large provenance aligned in this direction for a better characterisation of effect of GA3 and long-term storage on seed germination of Rosaceae members especially species subjected to rapid environmental changes like in Pyrus pashia from different provenances/sources.

Authors' Contributions Authors' Contributions Authors' Contributions Authors' Contributions
Conceptualization: MT and NNK, Laboratory work and data curation: KR, Technical Supervision: MT and NNK Data analysis and interpretation: MT, NNK and KR, Manuscript writing and review: MT, NNK and KR. All authors read and approved the final manuscript.