Effect of Desiccation and Storage Environment on Longevity of Ehretia Cymosa Thonn. Seeds.

Background: Globally, forestry faces challenges in the availability of seeds due to limited knowledge on seed handling of various species. Forestry seeds are constantly being reviewed and classied as either recalcitrant, intermediate, or orthodox based on their storage behavior. It is essential to understand the tree seed storage behavior to maintain seed viability and thus minimize seed losses. There is scanty literature combining factors of seed moisture content (6%, 9%, 12%, 15%, and 20%), seed storage temperature (20 o C, 5 o C and -20 o C), seed storage duration (1, 4, 9 and 12 months), and germination in different sites with varying environmental variables. Ehretia cymosa is important in the Afromontane forestry landscape as a medicinal, rehabilitation, and conservation species. This study conducted desiccation and storage studies and their inuence on the viability of E. cymosa seeds. The study sought to determine the optimum conditions for the storage of Ehretia cymosa that maintains viability. Results: This study observed that E. cymosa dried to seed moisture content of 6%, stored for 12 months at 20 o C and sown in the laboratory had the highest germination performance (27.6 ± 3.18%) (p<0.05). Conclusion: This conrms that E.cymosa seeds exhibit orthodox storage behavior. The authors recommend longer storage studies (>12months) to determine the actual longevity of the seeds of this species. The signicance of these results would be useful for foresters and farmers that would need to use this species for various purposes. seed viability of stored orthodox seeds as germination increases with a decrease in seed SMC and a decrease in storage temperature (Dardengo et al., 2019; Reed, 2005). This study's observation on the effect of storage temperature and SMC disagrees with past studies as for E.cymosa had better germination for lower SMCs and higher storage temperatures. The present study, however, shows that for E.cymosa, the germinability increases with a decrease in MC and an increase in storage temperature under laboratory conditions.


Background
Trees seeds have in previous studies been classi ed as either orthodox, intermediate, or recalcitrant based on the moisture content level at storage (Bonner, 1990;. Orthodox seeds tolerate desiccation to a low moisture content of between 2-5% (Dardengo et al., 2019). Intermediate category of seeds have been observed from other studies that they can be dried to certain levels, but not as low as orthodox species, and often observed to fail to survive sub-zero temperatures (Ellis et al., 1990;Wyse & Dickie, 2017). Seeds that are not tolerant and cannot survive dehydration are categorized as recalcitrant (De Vitis et al., 2020; Wyse & Dickie, 2017). Variability in seed recalcitrance and desiccation tolerance has previously been studied in two ways: desiccation sensitivity and post-harvest behavior (Berjak & Pammenter, 1997;Costa et al., 2016;Gold & Hay, 2008). There are challenges in clearly de ning the seeds' storage behaviors of critical species leading to seed losses impacting seed availability Ndungú & Kimiti, 2017;Sivakumar et al., 2006; Thomsen, 2000).
There is scanty information on Ehretia cymosa Thonn . Past studies have focused on the medicinal values and distribution of E. cymosa, thereby highlighting a gap in seed collection and handling. There is scanty information focusing on desiccation tolerance and germination responses of E. cymosa (Ellis et al., 1990;. Ehretia cymosa has a crucial space in Kenyan communities for medicinal, conservation, and a species useful for rehabilitation of degraded areas (Bussmann & Lange, 2000;Ogundajo & Ashafa, 2017;Reshad et al., 2020). In order for the species to be utilized, there is an expectation for the availability of seed, but this has been a challenge exacerbating the demand (Albrecht, 1993;Bussmann & Lange, 2000). These shortages can be associated with the limited information on its handling.
This study sought to assess the effect of desiccation and storage environment on the longevity of Ehretia cymosa seeds. The speci c objectives were: i) to assess the effect of varying seed moisture content on germination E. cymosa seeds, ii) to determine the effect of storage temperature and seed moisture content on germination of E. cymosa seeds, iii) to determine the effect of seed moisture content and storage duration on germination of E. cymosa seed, and iv) to determine the effect of seed, storage temperature, storage period and site on germination of Ehretia cymosa seeds. The ndings from these objectives would guide organizations that collect, store, and distribute such seeds in a manner that maintains their quality.

Methods
The study was conducted between June 2009 and July 2010 at Kenya Forestry Seed Centre in Muguga, which is a Programme of the Kenya Forestry Research Institute (KEFRI). The seeds were collected from the Thogoto forest in the month of June 2009, which is the peak seeding season for the species (Bussmann & Lange, 2000). Thogoto forest is situated within Kiambu county at Latitude − 1.275495S Longitude 36.667082E and altitude 2,020 masl Seeds were collected from 5 randomly selected trees that had mature fruits. Mature fruits for E.cymosa turn colour from green to orange-red (Thomsen, 2000). Three kilograms of mature E.cymosa fruits were collected from each of the identi ed trees. The collected fruits were carried in cotton bags from the eld and transported to the laboratory at the Kenya Forestry Seed Centre, Muguga, for processing.
Fruits were taken from the bag and seeds extracted by squeezing the fruits by hand to remove the seeds, followed by surface drying the seeds with a dry towel . A sample weighing 5 g seeds was then subjected to moisture content test using an infrared moisture analyzer (Himmel-Changzhou scienti c instrument). The moisture content (MC) value obtained was assigned as the initial moisture content (control) for this experiment Thomsen, 2000) Schmidt 2000). Seeds from the rest of the fruits were extracted by de-pulping (gently rubbing with hand to remove the fresh pulp). Seeds were cleaned further in running water to remove the excess mucilage and dried by gently rubbing with a towel to remove the excess water on the seed surface (Hong and Ellis.1996). One subsample of 2,000 seeds was randomly drawn from the extracted seeds to act as control with ve other subsamples of 6,000 seeds each extracted for the ve MC levels Thomsen, 2000) This desiccation procedure involved the packing of ve lots (MC: 20%, 15%, 12%, 9%, and 6%) of the seed samples in porous cloth bags ( Table 1). These cloth bags were then put on blue silica gel and covered in a desiccator. During desiccation each bag was checked hourly by weighing and calculating using Eq. 1: where Wt s is seed weight at desired MC, Wt i is the initial weight of seed, MC i is the initial Moisture content, and MC t is the targeted Moisture content Thomsen, 2000).

Equation 1
From each bag, the seeds were further split into three groups (of 2,000/group) for storage temperatures: minus 20 0 C, 5 0 C, and 20 0 C (Sivakumar et al., 2006). Each storage temperature group for each MC was further split into ve storage period groups (400/group) for storage periods:1, 2, 4, 9, and 12 months ( Table 1). The seeds in each storage period group were packed in 75 airtight aluminum foil sachets for storage (25 @ minus 20 0 C, 25 @ 5 0 C and 25 @ 20 0 C).
Germination testing was conducted in both laboratory (Lab) and transparent glasshouse (G.H.) conditions. Each of the storage period groups after the expiry of the storage duration was split into two groups for the different germination sites: Lab and GH. the seeds in each site for each storage period group, for each storage temperature group and for each MC level, were nally divided into four replicates of 50 seeds each.
The seeds extracted earlier for the control were subjected to germination in 4 replicates of 50 seeds per replicate in both Lab and G.H. Seeds used for the control were germinated immediately when the targeted MC was attained.
During germination, all the seeds were placed in Petri dishes containing 1% Agar-agar solution and incubated in the germination chamber set at alternating temperatures 20 o C and 30 o C in 12 hours intervals (Ellis et al., 1990;ISTA, 2015). A seed was considered to have germinated when the radicle protruded (citation). In the glasshouse (mean rH 53.8% and mean temperature 27.3 o C) and seeds of E.cymosa were sown in germination boxes containing river sand that was sterilized by pouring a mixture of 450 ml,3.5% concentration. Sodium Hypochlorite solution in 20 litres of water.
Germinated seeds were counted three times per week on Mondays, Wednesdays, and Friday by counting all seeds that had germinated cumulatively for 30 days or until there was no further germination taking place within three consecutive counts, whichever came rst.

Data analysis
The data were tabulated in a data-sheet in M.S. excel and analysed for: effects of varying seed moisture content on germination, combined effects of storage temperature and seed moisture content on germination, combined effects of seed moisture content and storage duration on germination, and combined effects of seed storage temperature, storage period and experimental sites on germination of Ehretia cymosa seeds. This analysis was done with RStudio Version 1.2.1335. Post hoc analysis (Tukey's HSD) was used to determine the difference in means (95% CI) in the ANOVA with factors desiccation MC, storage temperature, storage period, and experimental site differences compared.
The highest mean germination percentage was observed from E. cymosa seeds dried to SMC 6% and stored in a 20 °C environment (21.1 ± 1.45%) (Fig. 2a). Seeds stored at 20 °C performed the best in germination for moisture content 6%, 9%, and 12% (Fig. 2a). Seeds stored at 5 °C performed highest in MC 15% and 20% (Fig. 2a). Generally, the mean germination of seeds stored at 5 °C and 20 °C in the laboratory performed better than in the greenhouse (Fig. 2b). It was also observed that in the laboratory seeds stored at 20 °C performing better in MC 6%, 9%, and 12% while seeds stored in 5 °C environment performed better in 15% and 20% (Fig. 2b). The mean germination of seeds in the greenhouse gave the highest results on seeds that were immediately sown at SMC 21% (20.5 ± 1.19%).
The highest mean germination was observed from seeds with SMC 9% stored for 12 months (24.4 ± 1.79%) (p < 0.05) (Fig. 3a). Similar observations were seen in SMC 6% and 12% for SMC 15% and 20%, where the storage duration of 1 month resulted in the best germination (Fig. 3a). Germination performance was highest under laboratory conditions; more so, the storage period of 12 months performed the best for SMC 6%, 9%, and 12%. Seeds stored for one month performed the best for the remaining SMC (15% and 20%) in laboratory conditions (Fig. 3b).

Discussion
Other studies focusing on the effect of moisture content on germination have shown lower SMC to be advantageous in the maintenance of Past studies on other species have shown that temperature and SMC affect seed viability of stored orthodox seeds as germination increases with a decrease in seed SMC and a decrease in storage temperature (Dardengo et al., 2019;Reed, 2005). This study's observation on the effect of storage temperature and SMC disagrees with past studies as for E.cymosa had better germination for lower SMCs and higher storage temperatures. The present study, however, shows that for E.cymosa, the germinability increases with a decrease in MC and an increase in storage temperature under laboratory conditions.
Previous studies on other species have shown that seeds with less moisture stored for longer periods resulted in higher mean germination (Rajjou & Debeaujon, 2008;Walters et al., 2005). This study on E.cymosa seeds shows that there is no deterioration with longer storage duration (12 months). There are studies that have shown there are environmental effects that prevent seed deterioration in longer storage durations at higher temperatures (Kalsa et al., 2011;Qaderi et al., 2005).
Few studies have focused on germination behavior under a combination of factors; seed MC, storage temperature, and duration, though on other species (Berjak & Pammenter, 1997;Dussert et al., 2018;Schmidt, 2000). This study looked at a combination of SMC, storage temperature, storage duration, and germination site conditions (temperature and relative humidity). The key factor of comparison was the two sites (greenhouse and laboratory conditions). The trends seen in the results section are similar to studies on other species focusing on low moisture content (< 10%) and longer storage periods (12 months) (De Vitis et al., 2020; Ellis et al., 1990;Li et al., 2020). The differences observed in this study came from storage and germination temperature that were higher compared to other studies for other species (Reed, 2005;Sivakumar et al., 2006).

Conclusion
This study observes that E.cymosa seeds had the highest germination when seeds were dried to 6% MC, stored at 20 0 C for 12 months, and germinated in the laboratory (relative humidity 70% and temperature range 20-30 o C). This con rms that E.cymosa seeds exhibit orthodox storage behavior. The authors recommend longer storage studies (> 12months) to determine the actual longevity of the seeds of this species.

Declarations
Ethics approval and consent to participate Not applicable

Consent for publication
Not applicable.

Availability of data and material
The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The Government of Kenya, through Kenya Forestry Research Institute, provided facilitation for the data collection and experimentation.  Figure 1 The effect of varying seed moisture content on mean germination of E. cymosa in both laboratory and greenhouse conditions (a and b) from each of the moisture content categories (6%, 9%, 12%, 15%, and 20%) Figure 1 The effect of varying seed moisture content on mean germination of E. cymosa in both laboratory and greenhouse conditions (a and b) from each of the moisture content categories (6%, 9%, 12%, 15%, and 20%) Figure 1 The effect of varying seed moisture content on mean germination of E. cymosa in both laboratory and greenhouse conditions (a and b) from each of the moisture content categories (6%, 9%, 12%, 15%, and 20%) Figure 2 Germination performance of E.cymosa seeds stored in different temperatures (control, 5°C, 20°C and minus 20°C) after being dried to different moisture content (6%, 9%, 12%,15%, and 20%). Germination performance of E.cymosa seeds stored in different temperatures (control, 5°C, 20°C and minus 20°C) after being dried to different moisture content (6%, 9%, 12%,15%, and 20%).