Warm stratification improves embryos development and seed germination of Cycas revoluta

The broad objective of this research is to study the effect of warm stratification on Cycas revoluta zygotic embryos length, seed germination and plant development. Four warm treatments were applied to seeds. Our results showed that seeds storage at room temperature or 30°C improved significantly zygotic embryos length. Moreover, time of germination was significantly reduced with the warm stratification. The highest percentage of germination was obtained with seeds warm treated at 30°C for 2 months while only 25% of seeds were able to germinate in the control. Regarding seedlings development, our results demonstrated that warm stratification did not affect plant develop­ ment. No significant differences have been recorded in all the evaluated para­ meters except for root length. Taken together, these results underlined the beneficial effect of warm stratification on Cycas revoluta seed germination and plant development and proposed a new method to improve seed germination of Cycas revoluta.


Introduction
The sago palm (Cycas revoluta L.) is one of the widespread ornamental trees, grown in temperate, subtropical and tropical regions more precise ly in Miyazaki and Kagoshima Prefectures in Kyushu District down to the Ryukyu Islands, Okinawa Prefecture in Japan (Zarchini et al., 2011). Described as one of the most primitive species among the living cycads, Cycas revoluta has been used as an indoor and outdoor landscape plant for centuries (Stevenson, 1990;Jones, 1994).
Cycas revoluta is propagated either from seeds, which remain viable for only a short time, or from vegetative offshoots (Demiray et al., 2017). Germination of seed of Cycas revoluta is hard and time consuming (Zarchini et al., 2011). Seeds can take 3 to 9 months to initiate germina tion before they can continue to germinate for periods of a year or more. C. revoluta seeds also demonstrates rapid loss of viability and low mor phogenic potential, which hinder its conservation (NADERI et al., 2015).
Breaking dormancy is the main problem faced by all Cycas revoluta (Frett, 1987). Several attempts have been made to overcome with Cycas revoluta seed dormancy problem. Priming treatments of seeds seems to offer a new way to increase seeds germination percentage. Mechanical and chemical scarification has been described widely as an efficient way to improve germination of the hardseeds species of Cycas and some other species known for their hardcoated seeds (Frett, 1987;Rouhi et al., 2010). Indeed, several studies have reported a great responsiveness of Cycads seeds to various pretreat ments, namely, scarification, depulping, exposure to chemical substances like potassium nitrate, gibberel lic acid or sulfuric acid or soaking in hot water for specific period of time (Zarchini et al., 2011;Millaku et al., 2012). Warm stratification was also used to improve seed germination of many species such as Sambucus and Symphoricarpos (Baskin et al., 2002). The present study aimed to improve the germination of Cycas revoluta seeds through different warm strat ification treatments as a way to develop an efficient in vivo germination protocol for this ornamental species.

Plant material
Freshly harvested seeds collected from 50 years old female mature plants grown in Faculty of Sciences garden, University Mohammed V in Rabat (Morocco) were used in this study.

Zygotic embryos length measurement
Seeds were soaked in water for 48 hours in order to soften the sacrotesta; the orange external layer. The sacrotesta was then removed mechanically with a knife. Seeds were then flamed with ethanol for 2 minutes. Sclerotesta layer was mechanically eliminat ed. The megagametophytes were surface sterilized for 20 minutes by soaking in 30% dilution of NaOCl containing 23 drops of Tween20, followed by 34 rinses with sterile distilled water. After surface steril ization, megagametophytes were pooled, longitudi nally bisected and the zygotic embryo (ZE) was excised from each megagametophyte. ZEs length was measured and the mean was calculated from at least 20 biological replicates.

Warm stratification treatments and seeds cultivation
After removing the sacrotesta mechanically, equal samples of seeds were subjected to different treat ments. Treatments consisted in seed storage at room temperature (1820°C) or 30°C for 2 or 4 months depending on the treatment (Table 1). The warm stratification temperatures were chosen based on previous reports that underlined the beneficial effect of seed storage at 18°C and 30°C on seed's germina tion (Roh et al., 2004;Baldos et al., 2014;Keun et al., 2016). Untreated seeds were cultivated immediately and referred as control treatment (T 0 ). Seeds of Cycas revoluta were then planted in bins containing steril ized soil at 25 cm depth. Cultures were incubated at 25±2°C, with a photoperiod of 16 hours of light and 8 hours of darkness and watered daily depending on soil moisture.

Germination and plant growth parameters recording
Weekly observations were performed and seed emergence was recorded after 10 months of cultiva tion. The data for the kinetic of germination and time of germination (days) were recorded. Percentage of germination was calculated after ten months of cul ture. Number of leaves, stip height and width, root length and the length of the most developed leaf were determined at the end of the experiment.

Statistical analysis
Zygotic embryos length, time of germination, number of leaves, stip height and width, root length and the length of the most developed leaf were com pared using a fixed model of analysis of variance (ANOVA). For each parameter and condition, means were calculated based on at least thirty biological replicates. In case of significant difference between groups, a Tukey test was used for means separation, at risk of 0.05.

Effect of warm stratification on zygotic embryos length
Zygotic embryos (ZEs) length was investigated in the different warm pretreatments. Our results showed that all the applied treatments (T 1 , T 2 , T 3 and T 4 ) influenced ZEs length compared with the untreat ed seeds (T 0 ) (Fig. 1). ZEs length of seeds subjected to T 1 , T 2 , T 3 and T 4 was significantly enhanced, but no significant difference was observed between T 3 and T 4 seeds. Indeed, ZEs length increased by 134%, 276%, 300% and 342% in T 1 , T 2 , T 3 and T 4 warm treat ed seeds respectively when compared to untreated seeds.

Effect of warm stratification on seed's germination
As mean to gain more insight on the effect of warm storage on seed germination, time of germina tion (calculated starting from the first day of seeds cultivation), percentage of germination and its kinetic were determined for each treatment. The results were summarized in figures 2, 3 and 4.

Effect of warm stratification on the time of germination
Minimum time of germination (163.33 days) was recorded in seeds stored at 30°C for 4 months fol lowed by those stored at 30°C for 2 months (198.19 days) and those stored at room temperature for 4 months (202 days). Note that the difference between these three treatments was statistically insignificant. Untreated seeds (T 0 ) and those stored at room tem perature for 2 months (T 1 ) took the maximum time duration for germination with an average of 294.4 days and 256.75 days respectively (Fig. 2).

Effect of warm stratification on the percentage of germination
Investigating the percentage of germination of seeds from the four different treatments and the untreated ones revealed that seed germination response varied among the different warm treat ments. The highest percentage of germination (49.33%) was recorded in seeds stored for 2 months at 30°C while the lowest value of 8% was observed for seeds stored at room temperature for 4 months (Fig. 3). Untreated seeds showed although a percent age of germination around 25%.

Effect of warm stratification on the kinetic of germination
Investigating the kinetic of germination revealed a high variability between the different treatments applied (Fig. 4). Our data showed that seeds storage at 30°C for two or four months (T 2 ) allowed seeds to germinate faster. Indeed, seeds started to germinate  after four months of culture whereas, the first germi nated seeds appeared after five months and 6 months respectively for T 1 and T 0 . Seeds storage at room temperature for four months (T 3 ) delayed the germination by one month compared to untreated seeds (T 0 ). These data joined those related to time of germination.

Effect of warm stratification on plant's growth and development
Seeds pretreatment with temperature affected zygotic embryos length and their germination. These results prompted us to see whether the pretreat ment can influence seedlings growth and develop ment. Several growth parameters namely, number of leaves per plant, stip height and width, root length and the length of the most developed leaf were eval uated. Table 2 summarized the results.
Data analysis had shown that seeds pretreatment did not affect the number of leaves per plant. Seeds storage for 2 months at 30°C gave a maximum num ber of leaves per plant (1.50±0.59) while the lowest value was obtained with T 4 treatment (1.14±0.37). Note that these differences remained insignificant. Leaf length was also not significantly affected by the priming treatments. It was found that maximum leaf length was 57.33±3.05 cm in seedlings subjected to T 3 treatment while a minimum of 48.22±4.23 cm was recorded with T 1 treatment.
Stip height showed no significant difference between the different treatments. Stip width, on the other hand, displayed significant variations between the different treatments. A significant increase in stip width was observed in seedlings subjected to T 1 and T 2 as compared to the control (T 0 ) while nonsignifi cant changes was observed between the remaining treatments(T 3 and T 4 ).
Regarding root development, root length showed significant variations between the different treat ments. Seeds storage for 2 months at 30°C resulted in a significant increase in root length compared to untreated seeds while a decrease in root length was observed with T 3 and T 4 treatments.

Discussion and Conclusions
Cycas revoluta is an ornamental tree that has been widely used as an indoor and outdoor land scape. However, despite its importance in landscape design, it is facing problems regarding its germination mainly due to the hardcoat (Ullah et al., 2020). Different methods have been tested to overcome hardseededness. These include mechanical, chemi cal, and physical scarification treatments to make the seed coat permeable for water uptake. Several reports have shown that seeds pretreatment with sulphuric acid (H 2 SO 4 ), potassium nitrate (KNO 3 ) or GA 3 results in a better germination results. Indeed, Zamia furfuracea and Cycas revoluta germinations were assessed by Schutzman, using a chemical scari fication with concentrated H 2 SO 4 and then soaking them in gibberellic acid (GA 3 ) (Schutzman, 1984(Schutzman, , 1989. However, these pretreatments are not always reliable with cycads (Dehgan and Yuen, 1983). For instance, Zarchini et al. (2011) have found that the use of sulphuric acid even at lower concentrations seems to affect negatively seed's germination while combined with hot water seems to improve Cycas revoluta seed germination.
It is well admitted that seeds storage in a warm environment usually results in relatively rapid embryos development (Baskin et al., 2002;Merritt et al., 2007;Razavi and Hajiboland, 2009). However, no previous work has been conducted to study the effect of seed storage in warm conditions on Cycas revoluta germination and seedlings development. Thus, we investigated the effect of warm storage on zygotic embryos development, seeds germination and growth of young seedlings of Cycas revoluta. Our results showed that warm storage at 30°C for 2 months or 4 months speeded the germination when compared to the untreated plot (Fig. 2). Seeds stor age at 30°C for 2 months improves also the germina tion percentage. Meanwhile, the prolonged storage at 30°C for 4 months reduced the germination per centage by half compared to the untreated plot which suggest that prolonged storage have an inhibi tive effect rather than stimulating the germination (Fig. 3). Chen et al. (2007) have found that Prunus campanulata seeds required 46 weeks of warm fol lowed by 8 weeks of cold stratification for maximum germination percentage. This finding was explained by the accumulation of high amounts of GA as a result of the cold stratification while GAs in warm stratified embryos were significantly low. Thus, Prunus warm stratified embryos failed to germinate since GAs content was very low. In H. salicornicum and S. imbricate, seeds germination was significantly improved when seeds were stored at 40±2°C for three months (ElKeblawy, 2013). Warm stratification for at least 1 month appeared to be essential for the germination of Japanese snowbell (Styrax japonicus) (Baskin, 2009). Indeed, Roh andBentz (2003) found that without warm stratification, seeds were not able to germinate. Seed's dormancy in some orchids, mainly Epipactis palustris and Goodyera pubescens, could be overcame by a warm incubation of seeds followed by cold storage. This was explained by the fact that warm and cold stratification increased seeds permeability to water thereby softening the testa (Roh and Bentz, 2003). Thus, the increase observed in germination percentage could be attributed to the increase of seed's hard coat permeability caused by the warm storage which allow the removal of the physical barrier to water absorption. Besides the improvement of seeds germination percentage and the reduction of the germination time, warm storage improved significantly zygotic embryos length which suggest that warm scarification result in a better development of zygotic embryos. Regarding seedlings development, our data showed that warm scarification did not affect plant development (Fig. 5). Indeed, no significant differences have been record ed in all the evaluated parameters except for root length. Taken together, these results suggest that warm scarification improved seeds development and germination by prompting zygotic embryos length, increasing germination percentage and reducing time of germination. This study aims to study the effect of seeds stor age at different temperatures on Cycas revoluta seeds germination and development in order to develop an efficient in vivo germination protocol that can be used for mass production of this ornamental tree. Based on our results, we found that seeds storage at 30°C for 2 months or 4 months reduced time of germina tion. However, the highest percentage of germination was only assessed when seeds were stored at 30°C for 2 months. Taken together, this protocol repre sents a useful and potential method to improve com mercial mass propagation of Cycas revoluta.