Propagation techniques for woody trees are used for producing plantlets and imply the culture of aseptic small sections of tissues and organs in vessels with defined culture and under controlled environmental conditions. These techniques have become an increasingly important tool for both science and commercial application in recent years (Kumar and Reddy 2011). The research on tissue culture of forest trees resulted in optimisation and increased efficiency of most of the culture techniques for trees, especially in the form of axillary shoots (or buds) regeneration and somatic embryogenesis (Wilhelm 2005). Therefore, propagation methods under specific conditions, gain ground over conventional vegetative propagation since they could be linked to the propagation of a great number of pathogen-free plants in a short time with high uniformity (e.g. Yasodha et al. 2004, Wilhelm 2005, Kumar and Reddy 2011).

The success of propagation involves several factors, such as the composition of the culture medium, culture environment, and genotype (Kumar and Reddy 2011). The propagation by leafy stem cuttings (axillary and adventitious shoot multiplication) has been mentioned as one of the most technically effective propagating techniques (Hartmann et al. 2002, Mateja et al. 2007). However, a key point of plant propagation from stem cutting is minimising the water stress caused from the cutting, until roots are formed (Mesén et al. 2001, Mateja et al. 2007). For overcoming this stress, propagation systems need to maintain an atmosphere with low evaporative demand, thus reducing water loss from transpiration and avoiding extreme tissue-water loss. For this purpose, the systems that were used ensured that the fog or mist on leaves allowed evaporative cooling, resulting in 90-100% humidity (e.g. intermittent mist and fogging systems of high-humidity, or non-mist polypropagators) (Harrison-Murray and Thompson 1988, Spethmann 1997, Hartmann et al. 2002).

The scenarios of climate change in Mediterranean regions bring back the need for the preference of tree species tolerant to drought and disease, for both timber production and for urban forestry purposes. The Cupressus sempervirens L. (Mediterranean cypress) is a coniferous tree species, which has a fundamental ecological, financial and ornamental role in the Mediterranean region (Giovanelli and de-Carlo 2007). Cupressus sempervirens is a medium-sized evergreen coniferous tree and is a monoecious wind-pollinated plant (Caudullo and de-Rigo 2016). Cupressus sempervirens is a pioneer species, growing quickly when young on most substrates but not on clay or waterlogged ones, and up to 35-40 m with trunks 1 m in diameter, rarely over 2 m (Eckenwalder 2009, Farjon 2010). Cupressus sempervirens has a long horticultural history in the Mediterranean region, and its natural distribution nowadays is the remnant of an extensive cypress forest of the Pliocene (López-Sáez et al. 2019). The present natural distribution of C. sempervirens occurs in the southwest Mediterranean basin over several geographically non-adjacent areas reaching, eastwards, Caucasus and southwestern Iran, on elevations from sea level (Crete) up to 2000 m (Turkey) (Eckenwalder 2009, Caudullo and de-Rigo 2016). The C. sempervirens is a light-demanding, drought- and heat-tolerant species, surviving in habitats with as little as 200 mm of annual rainfall (Caudullo and de-Rigo 2016). In winter it is quiescent, while in summer it remains dormant, quickly resuming after rainfall, owing to its extensive and shallow root system (Santini and Donardo 2000). The species favours rich, deep, moist and well-aerated soil with neutral pH, however also grows on rocky, dry and compact soils (Caudullo and de-Rigo 2016). It can live for longer than 100 years, while older individuals (of 200-500 year of age) are not uncommon in natural stands. The C. sempervirens was initially characterised by two varieties: (i) the wild type var. horizontalis (Miller) Aiton, widely represented in native populations, with a broad conical crown and the main branches forming a wide angle with the trunk and (ii) the fastigiated var. pyramidalis Nymann (= var. stricta Aiton), widely planted and cultivated, with a dense columnar crown and main branches growing upwards close to the trunk (Zacharis 1977, Tutin et al. 1993). However, according to the recent taxonomy rank, the variety relating to the growth form of fastigiated var. pyramidalis does not correspond to a taxon (Farjon 2010). Therefore, the columnar cultivated form is referred to as C. sempervirens f. sempervirens and the spreading form as C. sempervirens f. horizontalis (Farjon 2010, Korakis 2015).

As regards to the C. sempervirens timber, it has interesting characteristics of high natural durability and straightness. From ancient times its wood has been particularly appreciated for its resistance to fungi and insects, especially if immersed in water (Hadjikyriakos 2007). Despite the fact that C. sempervirens wood is suitable for small carpentry, exterior woodworks (doors, windows, garden furniture, etc.) and ship-building, the ecological importance of the species is more favourable for the climatic conditions nowadays. The species was used since Greek and Roman times as an ornamental tree for shading gardens, cemeteries, as a windbreak along roads, as well as for vegetable and fruit tree crops, and it has become a characteristic feature of the Mediterranean coastal and urban landscapes (Farjon and Filer 2013, Bagnoli et al. 2009). More recently, and owing to its ecological qualities, C. sempervirens was used in hot areas for forest protection against desertification and soil conservation, where soil is shallow and degraded and other forest tree species cannot grow (Caudullo and de-Rigo 2016). It is also considered as a valuable species for landscape rehabilitation, soil reclamation and quarry restoration (Korakis 2015).

Since the 1970s, and after the introduction of the serious disease of the “Cypress canker” in Europe (most likely from the USA), which is caused by the Seiridium cardinal fungus, recorded over large Mediterranean areas and leading to damage in forests, nurseries and ornamental plantations, the disease has become a limiting factor to cypress planting. Fungus management requires the removal of infested trees and the use of genetically resistant forms, and hence, clones have been recently patented (clonal selection in natural stands where the disease was present or by cross-breeding between selected trees in experimental fields) (Giovanelli and de-Carlo 2007). In addition, another fungus, the Diplodia pinea f. spp. cupressi causes cankers on the stem and branches of water-stressed cypresses.

Although a number of reports have indicated that propagation can be successfully applied to Cupressus species (Fossi et al. 1981, Hřib and Dobrý 1984, Franco and Schwarz 1985, Spanos et al. 1997, Giovanelli and de-Carlo 2007) in the past decades, the use of a more efficient propagation (and rooting) method for mass-selected genotypes or phenotypes of the targeted species can lead to increase of the conservation effort, as well as to commercial use for agroforestry (and urban forestry purposes) of C. sempervirens f. sempervirens. Hence, the current study aims to develop and present an effective protocol for the macropropagation of C. sempervirens f. sempervirens using axillary shoot multiplication by adopting both cutting propagation and intermittent mist methods. This protocol could be adopted to address the need for rapid propagation and cultivation of selected genotypes with commercial and ecological/silvicultural interest.

Sampling of shoots

Methodology

Statistical analyses

The T-test was used for comparing the mean value of successful rooting of the different cuttings of sampled shoots, between the two different rooting systems (bench and root trainer). This test detected statistically significant differentiation between rooting bench (M= 0.20; SD= 0.401) and root trainer (M= 0.17; SD= 0.37), with the mean difference being equal to 0.32 (t= 2.7, df= 2398, F= 18.7, p<0.01). Thus, the tested cuttings had slightly higher (but significant) amount of rooting in bench than in root trainer (Suppl. material 2). ANOVA was used to detect the differences among treatments (culture initiation and concentration of K-IBA) in successful rooting of shoot cuttings (Suppl. material 3). Statistically significant differences among the treatments were tested with the Tukey test at 0.95 confidence level, and the homogeneity of variance test was tested (showing non-significant level statistic). The ANOVA distinguished that the rooting percentage differed significantly among the three different parts of the shoot cuttings (basal, middle and terminal) (F= 15.348; p<0.05) with the basal cutting recording the highest rooting ability and the middle cutting recording the lowest percentage of rooting (Fig. 1). The concentration of K-IBA was also a critical point for the success of C. sempervirens f. sempervirens propagation. In addition, the ANOVA detected significant differentiation among the different K-IBA concentrations (F= 33.810; p<0.05) regarding the mean value of cuttings that rooted. More specifically, the concentration of K-IBA of 5500 ppm was the one with the highest presentence of rooting success compared to the other concentrations (Fig. 2).

Figure 1.  

Effectiveness of shoots rooting according to the sampled part from each shoot (basal, middle and terminal cutting).

Figure 2.  

Effectiveness of shoots rooting after dipping in different concentrations of K-IBA.

Remarkable is the fact that the survival percentage of new plantlets under the environmental adaptation (acclimatization) in growing conditions outside the green house was: (i) 99% for the first year and (ii) for the second year all (100%) of the survived plantlets of the first year. The acclimatization was carried out in Athalassa Forest Nursery, Cyprus Department of Forests, and followed all necessary maintenance steps for plants in a nursery (watering, manuring, etc.).

Today, the expanding of knowledge in the area of developmental tree physiology, in order to improve propagation systems, is important for several purposes in the context of propagation population, clonal forestry, and for conservation purposes, as well as for use οf tree species with specific shapes and growth characteristics in urban forestry (and in agriculture for windbreaks). In all these cases, the purpose of vegetative propagation is to produce clones of elite trees that have been selected from populations of genetically variable siblings (Wilhelm 2005). Thus, the vegetative propagation is essentially the reproduction of plant material in a way in which the offspring contains the genetically determined characteristics of the parent plant. For conservation efforts, propagation could support the maintenance and preservation of selected genotypes and/or phenotypes with proved resistance to disease, or with desirable crown shape. These arguments, as mentioned above, become more important in the case where serious constraints lead to the delay or failure of seeds to germinate in the nursery and/or where there is a need for mass production of plantlets for a specific plant species.

In the case of C. sempervirens previous studies reported protocols based on micropropagation methods of tissues (especially shoots) (e.g. Spanos et al. 1997, Giovanelli and de-Carlo 2007) for the mass-propagation of this target species. The current protocol adopted the most frequently used method for vegetative multiplication of the cutting propagation (macropropagation). Thus, the outcomes from this study come to support previous general guides about the propagation of plant species, and at the same time to argue that the macropropagation of C. sempervirens stem cuttings can be successfully implemented. The present sampling method allowed a flexibility with the handling of sampled shoots, because sampling was carried out during late winter to early spring, on an evergreen hardwood, and since cuttings were taken from stock plants from well-ripened, vigorous one-year-old shoots. In such case the shoot is dormant and thus there is considerably more flexibility with its handling (Wilhelm 2005). The semi-ripe wood and evergreen hardwood propagation is an important method for many conifers and broadleaved evergreen species for their cutting propagation (Wilhelm 2005).

A critical point of propagation is root formation, where for this formation, auxins have a central role. Despite the general assumption that different species exhibit varying rooting success for cuttings taken from basal, middle and terminal cuttings (parent stem) (Wilson 1993), more recent studies support more specific features of auxins. In practice, when a shoot is cut, auxins, which are produced in the shoot apex, accumulate at the shoot base to induce rooting (Wilhelm 2005). In this study the impact of this physiology mechanism is obvious, since cutting from the basal and the terminal of shoot showed higher ability of rooting than cutting from the middle. Hence, cutting from the basal and the terminal of a single shoot of C. sempervirens could hasten root initiation, increase the number and quality of roots and encourage uniformity of rooting. Besides, the observation from this study supports the argument that rooting percentage of cuttings was increased by specific auxin treatment (e.g. Assareh and Sardabi 2005, Kipkemoi et al. 2013), in this case the optimum concentration of K-IBA. On the other hand, the support of bench as rooting system (perlite with pitmus substrate in a 4:1 ratio), ensures the low cost for rooting, the high percentage and the quality of root cuttings, from this protocol. This observation is in accordance with the assumption that optimum propagation medium should provide sufficient porosity to allow good aeration and this ensures adequate oxygen availability for the developing roots system (Hartmann et al. 2002).

The protocol and workflow (Fig. 3) presented in this study, are directly applicable for nurseries interested in the quantitative reconstruction of mass-propagation of C. sempervirens f. sempervirens. It is particularly useful at smaller nurseries with limited funding and personnel, or those without laboratories or facilities for applying micropropagation (in vitro). Streamlining tasks and workspaces improved the speed at which specimens were being processed and increased the flow of data, while minimizing the perdition of cutting materials. In addition, simplifying the workflow improved outcomes, as this is supported by the survival of produced clones (plants) in the acclimatization stage (100% survival after the second year). The proposed propagation protocol could be adopted or used as the baseline knowledge for the massive propagation of other species of the Cupressaceae family.

Figure 3.  

Generalized workflow for the new propagation protocol of C. sempervirens f. sempervirens.

As mentioned above, C. sempervirens is resilent to extreme climate and soil conditions in the Mediterranean region. Thus, the species is highly preferred for several restoration purposes. In all cases, plants macropropagation provides a mass production of plantlets, whilst maximising efficient plantlets multiplication on site, operating in a non-aseptic environment, reducing labour requirements and overcoming a lack of plant stock material at the industrial site (Hartmann et al. 2002, Woods and Woods 2006). However, a key point for plantlets mass propagation by using the macropropagation method is the development of specific protocols with clear statements, as this is suggested in the present study for C. sempervirens.

The manuscript is part of the Master’s research of Mr C. Pericleous, referenced as: “Asexual reproduction of Cupressus sempervirens var. pyramidalis in Cyprus with cuttings” (2018), in the School of Pure & Applied Sciences, Open University of Cyprus. The authors thank Prof. I. Vogiatzakis and Dr. D. Sarris, supervisors of Mr. Pericleous for his MSc thesis. Warm gratitude is extended to the Cyprus Department of Forests for assistance with the mist system at Athalassa Forest Nursery. Particularly special thanks are extended to Dr. A. Christou (Chief Conservator of Forests at Department of Forests) and Mr. G. Kyriacou (Forest Officer 1^st Grade at Department of Forests). Many thanks to Assoc. Professor P. Mavrikiou (Frederick University) for his input on raw data analyses. Finally, the authors thank the reviewers for their insightful comments on a previous version of the manuscript.

C. Pericleous contributed to the fieldwork and the protocol development. N.-G. H. Eliades carried out the data analyses. Both authors contributed to the manuscript writing.