Seed viability and optimal germination conditions of Cedrus atlantica (Manetti ex Endl.) Carrière

Atlas cedar (Cedrus atlantica (Manetti ex Endl.) Carrière) is an endemic Pinaceae of the Maghreb of great socio-economical and ecological value (M’hirit & Maghnouj, 1997). In Algeria, C. atlantica is estimated occupying an area of 2089 km² (Arar et al., 2020). The Atlas cedar is classified as an endangered species carrying the status of a quite rare plant (Yahi et al., 2008; Touati et al., 2021) and protected in Algeria by the Executive Decree No. 12-03 (J.O.R.A. 2012).

Cedrus atlantica is encountered in mountains of the Tellian and Saharan Atlas (from which its name has been derived, Atlas cedar), where it has a scattered distribution controlled mainly by climatic variables and edaphic traits (Quézel, 1998).

Currently, C. atlantica forests are subject to the harmful effects of global warming (Chenchouni et al., 2008; Cheddadi et al., 2017). According to the climate warming scenarios’ modulation, the Algerian cedarwood forests are expected to decline to 80.6% by 2070 with an altitudinal shift towards higher elevations and a disappearance at low altitudes (Arar et al., 2020).

In the Maghreb, the Atlas cedar is, in addition, under the threat of uncontrolled exploitation along with the phenomenon of dieback. The combined action of dieback factors as well as anthropogenic pressure (M’hirit & Benzyane, 2006) has resulted in the fragility and disruption in the equilibrium of cedar groves in some localities (Ezzahiri et al., 1994). These latter authors have emphasized that both challenges have generated the disturbance of several physiological and biological mechanisms particularly that of natural resilience. The low regeneration ability is rather exaggerated by climatic changes in the Mediterranean region in recent decades (Bentouati & Bariteau, 2006; Bentouati, 2008; Addar et al., 2016), in particular the increase in temperatures due to global warming and the decrease in precipitation resulting in a water deficit especially in summer which means that the young seedlings of C. atlantica cannot establish during this period (Linares et al., 2011).

The dieback problem in cedar groves is owing to multiple causes (M’hirit & Benzyane, 2006; Naggar, 2013), such as drought (Kherchouche et al., 2013; Abel-Schaad et al., 2018; Arar et al., 2020) and parasite attacks by some xylophages belonging mainly to the subfamilies of Buprestidae and Scolytinae (Talbi & Bouhraoua, 2015). This drought is affecting the distribution of populations of many species, such as the Atlas cedar, which are becoming vulnerable and subject to local extinction (Arar et al., 2020).

The preservation of the remaining Algerian cedar patches urgently requires appropriate scientific studies and the establishment of a relevant restoration project as short-term measures. The evaluation of the viability of seeds and their germination physiology are among the areas of research to be developed, knowing that germination is a crucial step in the establishment of plants in their natural environment.

Germination is often prevented by seed dormancy which is a physiological state of a viable seed that prevents it from germinating till the emergence of favorable environmental conditions. Underestimating seed dormancy and germination requirements when planning a conservation project can lead to high levels (> 90%) of plant establishment failure and seed wastage (Commander et al., 2013; James et al., 2013).

To overcome the seed dormancy of a large number of forest species and improve their germination performance, several types of pretreatments have been applied (Baskin & Baskin, 2014). These pretreatments may be applied before, during or after the seed storage period, and correspond to the methods by which seed dormancy can be released (Debroux et al., 1998). Various studies have shown that cold stratification is a useful pretreatment to stimulate seed germination in different Pinaceae species (Cooke et al., 2002; Ghildiyal et al., 2009). However, some studies found that stratification did not affect seed germination (Nelson, 2015; Shen & Cho, 2021). For example, eight weeks of cold stratification (prechilling at 5°C) is sufficient to significantly improve seed germination of Cedrus brevifolia (Hook. f.) A. Henry (90%) compared with untreated seeds (23%) (Daskalakou et al., 2015). The exogenous application of gibberellic acid (GA3) is another commonly used treatment to promote germination in physiologically dormant seeds (Kucera et al., 2005). Seed treatments with hot water had been described to improve germination in physically dormant seeds through uplifting water and O₂ permeability of the weakened seed shell (Aydin & Uzun, 2001). Proper pretreatment makes the seeds ready to germinate when all the necessary conditions are met.

In the context of C. atlantica conservation, and the given lack of data in its seed germination features, we have undertaken this work in order to fill the knowledge gap regarding the effect of temperature and water stress on seed germination in this species. Some selected physicochemical pretreatments were also tested on promoting seed emergence. The data obtained in this study might contribute to managing an urgent restoration project for this endemic.

Our study has showed that the seed viability rate of C. atlantica, assessed by the TZ test, differs from one provenance to another. This rate is important in the seeds of the cedar groves of Chrea and Thienet El Had (85% and 90%, respectively); while an average of viability with just 60% characterized the seeds of the Chelia cedar grove. The performance of the TZ test in assessing the physiological quality of seeds, in particular their viability rate, has been confirmed in the works of França-Neto & Krzyzanowski (2019) and Salazar-Mercado et al. (2020). The reducing reaction of the TZ salt solution under the action of dehydrogenase enzymes produces triphenylformazan, which shows a carmine red coloration in the living tissues of the seeds (França-Neto & Krzyzanowski, 2019). Completely stained seeds are viable; those that are not colored are not viable; while those which are partially colored will produce either normal or abnormal seedlings (Rao et al., 2006).

The need to restore the natural unpredictable habitats has set forth a rise in demand for seedlings pertaining to native forest species. Most of these species are spread via seeds and the success in seedling formation depends on the knowledge about the germination process of each utilized seed (Rego et al., 2009). This knowledge is missing in Atlas cedar, although it is available for two other cedars: Cedrus libani A. Rich. (Dirik, 2000) and C. brevifolia (Daskalakou et al., 2015). Our results showed that temperature and provenance have a highly significant effect on the germination capacity of seeds. However, analysis of variance revealed an insignificant interaction between temperature and provenance for the variables of three germination parameters (FGP, VC and LT). The VC seemed to be influenced only by the provenance factor. Furthermore, no effect of the two above-mentioned factors was demonstrated for the latency time. The best FGPs were noted at 20°C in the seeds of the three provenances. In fact, these FGPs were variable and remained low to medium with 7%, 32% and 52% for Chrea, Chelia and Thienet El Had, respectively. This heterogeneity in FGPs can be explained by genetic effects, harvesting conditions, treatments and storage conditions of seeds (Dirik, 2000).

Simultaneously, this author has obtained the same results on the cedar of Lebanon (C. libani) of Turkish origin. Indeed, he has noted that incubating C. libani seeds at 20°C, the germinability ranged from 36.2% to 63.4% depending on provenances. On the other hand, the highest germination percentage (80%) was obtained at 15°C in C. brevifolia seeds, while at 25°C almost full suppression of germination was observed (Daskalakou et al., 2015). The optimum temperature for germination in Atlas cedar is matched in nature with early spring, when climatic conditions are favorable for seedling survival, development and growth.

By comparing the results of the TZ test with those of the FGP, it appeared that many seeds of C. atlantica, although they were viable, failed to germinate. This prompted us to study the effect of some pretreatments on improving the germination performance of the most viable C. atlantica seeds from the cedar grove of Thienet El Had. In this context, the results obtained showed a significant effect of the physical and chemical pretreatments on seed germination behavior. As compared to the control, the treatment of the seeds with hot water (80°C) and with GA3150 ppm improved the germination capacity. We found that hot water allowed the softening of the seed coats and the elimination of the mucilage contained in them, which probably interfered with their germination. The work of Velempini et al. (2003) and Mohammadi et al. (2012) in the effect of pretreatments on seed germination of Corchorus olitorius (L.) and Abelmoschus esculentus (L.) Moench, respectively, showed that soaking the seeds of these two species in hot water at 80°C for 10 min and at 75°C for 5 min was the most effective treatment to improve germination. The beneficial effect of hot water treatment (60°C) on seed germination of Cupressus atlantica Gaussen has been also revealed by Arjouni et al. (2013). According to Budy et al. (1986), hot water is a form of thermal scarification which allows the removal of integumentary dormancy by causing cracks in the seed coats without altering the anatomy of the micropyle. These cracks allow water to percolate in order to trigger germination (Pritchard, 2000). The physiological role of GA3 as a promoter of seed germination has been reported by several authors in a wide range of plant species (Baskin & Baskin, 2004; Rogis et al., 2004; Sharma et al., 2020; El Hamdaoui et al., 2021). The favorable effect of GA3 and submersion in distilled water has also been demonstrated on Pinus gerardiana Wall. ex D. Don. (Kumar et al., 2014). It was noted in the work of Kabar (1998) that the advantageous effect of GA3 on seed germination of Pinus brutia Ten. and Thuja orientalis L. is explained by its ability to modulate the inhibitory effect of abscisic acid (ABA). The hormonal cross talk of GA with ABA has been well-documented in seeds of many plant species (Seo et al., 2009). In addition, soaking in distilled water allows the dissolution of germination-inhibiting substances, such as polyphenols in the seed covering (Bessam et al., 2010).

When the water potential decreases in the medium, the FGP and VC values weakened steadily and significantly (p < 0.05), especially at −1.16 and −2.20 bar; while the LT became shorter. Compared to many species which are tolerant to water stress and able to germinate at water potentials exceeding −8 bar, such as Salsola drummondii Ulbr. (Elnaggar et al., 2019) and Thymus fontenesii Boiss. & Reut. (Dadach & Mehdadi, 2021), C. atlantica seeds from Thienet El Had are water stress-sensitive species since their germination was completely inhibited beyond −2.20 bar. Similarly, the depressive effect of water stress, especially on FGP and VC has also been highlighted in some Mediterranean species belonging to different botanical families (Dadach & Mehdadi, 2018; Hamdini et al., 2021; El Hamdaoui et al., 2021). Consistently, Dirik (2000) on Cedar of Lebanon (C. libani) and Tilki & Dirik (2007) on P. brutia have found that in general the germination rate varies according to seed provenance and decreased steeply with increasing water stress in the soil. The decrease in FGP and CV values is a consequence of the reduction in the intensity of water uptake by the seeds due to the water deficit. This can inhibit respiratory pathways and enzymatic activity, causing oxidative stress, which stimulate the accumulation of various toxic by-products, such as reactive oxygen species (ROS). If the production of ROS overwhelms the scavenging capacity of cell, that can lead to lipids, proteins and carbohydrates denaturation (Rasheed et al., 2019), thus as a result the restriction of radicle emergence and seedling formation.

From the obtained results, it appeared that the viability of C. atlantica seeds varies according to the three studied provenances, with a moderate to high rate. However, their germination percentages were low to moderate at the optimal temperature of 20°C. This is certainly due to the seed coat dormancy combined with the potential existence of germination inhibitor substances that preclude seeds to emerge. Seeds’ germinative performance could be improved remarkably by treatment with hot water. Such easy-to-use pretreatment could be utilized to enhance the production of seedlings to rescue the remaining threatened natural populations of C. atlantica. The seed dormancy and the sensitivity of C. atlantica seeds to the water potential drop at the germination stage are assumed to be the limiting factors for a successful natural regeneration of this species.

Regarding the confirmed dreadful effect of water stress on seed emergence of C. atlantica, we recommend planting seedlings in habitats with high annual precipitation levels. We assume rains will mitigate the impact of global warming and hence increase the survival chances of this species in the field. As a complement to this work, it would be interesting to further elucidate, for supporting both in situ and ex situ conservation projects, the potentiality of adaptation and survival under water stress of several genotypes of Atlas cedar during their early establishment stages.