1. Introduction
In the southwest Iberian Peninsula, Mediterranean oak woodlands occur as a particular anthropogenic habitat called “dehesas” in Spain or “montados” in Portugal [
1].
Dehesas are spaced oak woodlands in which a large part of the area is occupied by grassland managed as a savannah-like ecosystem [
2]. The evergreen oaks, such as holm oak (
Q. ilex L) and cork oak (
Q. suber L), are the main tree components of this agrosilvopastoral ecosystem and are of great socioeconomic and ecological importance in the SW Iberian Peninsula. Two subspecies of
Q. ilex, which differ in morphology, distribution and drought stress, have been identified:
Quercus ilex subsp.
ballota, synonymous with subsp.
rotundifolia, native to the southwest of the species geographic range, including the dehesas, and
Q. ilex subsp.
ilex, native to the north and east of the range [
3]. Animals are an essential part of the dehesas, in which hunting species coexist with livestock species, such as cattle, sheep, goats, and pigs, that transform the production of grass, acorns and branches into products of premium quality and high added value (e.g., meat, including the internationally renowned acorn-fed Iberian ham, and cheeses). Another by-product of holm oak dehesas is the highly valued truffle or edible fungus [
4]. The ecological importance of this ecosystem is indisputable, as it provides a multitude of advantages in terms of climate regulation, water provision, erosion control and carbon sequestration, among others [
5].
Oak decline syndrome is a phenomenon of great concern and affects oak stands worldwide [
6]. The sustainability of the dehesa ecosystem is threatened by oak decline syndrome (called “la seca” in Spanish). This severe disease has caused high mortality rates in holm oaks for several years and remains active at present. In most cases, disease episodes occur as the result of the interactions between different abiotic agents (severe drought, extreme temperatures, waterlogging, etc.) with and without the involvement of biotic agents. According to several authors,
Phytophthora cinnamomi is the main biotic agent causing the disease [
7,
8], although
P. quercina can also be involved [
9]. Another factor threatening the sustainability of the dehesa is the low natural regeneration capacity of the woodland, mainly due to aging of the trees. The highly variable production and germination of acorns and their high predation by livestock, along with their limited dispersion, also negatively affect the natural regeneration capacity of holm oak [
8,
10]. Moreover, land abandonment, land-use intensification, habitat fragmentation and transformation of the land into cultivated and urban areas may also affect the viability of the dehesas [
11,
12]. Numerous studies have highlighted the Mediterranean basin as potentially highly vulnerable to global change. A marked decrease in precipitation at the same time as the occurrence of very high-temperature episodes can cause decline or forest dieback across an area [
12,
13].
Attempts have been made to control the disease in the dehesas to reduce the risk of dispersion/infectivity of
P. cinnamomi, e.g., by applying limestone amendments to the soil [
14], injecting phosphonates in the trunk [
15] and biofumigating the trees [
16,
17]. However, limited success was achieved with all of these techniques. The use of genetically improved varieties to restore holm oak populations in affected areas is a possible alternative to chemical control of the disease [
18]. Nevertheless, long-term conventional breeding programs aimed at producing
P. cinnamomi-tolerant genotypes by controlled crossings have not been conducted [
19]. In the last few years, holm oak plants tolerant to
P. cinnamomi have been identified by the selection of surviving adult trees in oak decline hotspots. Acorns were collected from selected trees and germinated. Once the plants were established, they were repeatedly inoculated with the oomycete to confirm tolerance. Seedlings of different provenances of holm oak and cork oak collected in Huelva (Spain) were inoculated with
P. cinnamomi to test their tolerance to the oomycete [
20]. In addition, 100 pathogen- tolerant seedlings have been selected from 16 populations of
Q. ilex located in seven Mediterranean countries [
21].
Once the selected holm oak material is available, the next step involves the clonal propagation of this material to enable the capture of all gene combinations and provide strong genetic gains. Conventional clonal propagation of oaks, including holm oak, either by stooling [
22] or by root cuttings [
23] is very difficult and is clearly affected by the age of the ortet. Attempts to propagate the
P. cinnamomi-tolerant plants used in the present study by rooting cuttings were unsuccessful, regardless of the age of the mother plant (Dr A. Solla, pers. Comm.). The clonal propagation of mature elite individuals, threatened or endangered tree species, and genotypes with known resistance to pathogens is an important goal in forestry [
24]. Micropropagation is a potentially valuable alternative to conventional clonal propagation, although oaks (including holm oak) have always been considered recalcitrant to in vitro tissue culture [
25,
26]. Axillary shoot proliferation and somatic embryogenesis (SE) are the main procedures used for micropropagation and conservation of hardwood species [
27,
28]. Although propagation by axillary budding has been reported for several oak species [
25], to date few protocols have been developed for holm oak. In a very preliminary study, [
29] described a method of culturing shoots obtained from germinated acorns, although the procedure was not clearly explained. Recently, [
30] reported that the establishment and proliferation of shoot cultures from mature holm oaks, although rooting and acclimatization rates were very low.
Somatic embryogenesis is a powerful tool in clonal forestry, in the cryopreservation of valuable germplasm, and it also forms the basis of genetic transformation procedures [
28]. Somatic embryos of holm oak have been initiated from zygotic embryos [
31], floral tissues such as developing ovules [
26] and male catkins [
32], although low embryo induction rates have been obtained. There are several difficulties associated with the use of floral explants to induce SE, including the correct excision of the floral tissues to be cultured and the seasonal limitations to the availability of the material. In order to overcome these problems, [
33] highlighted the induction of somatic embryos from leaves and shoot apex explants excised from in vitro shoot cultures established from holm oak trees. The advantages of using shoot proliferation cultures as a source of explants for inducing SEs include the fact that sterilization of the explants is avoided, material at the same physiological stage can be produced throughout the year, and the growing conditions of the stock material can be controlled [
25].
In order to alleviate the ecological and economic impact of oak decline on holm oak populations in the Iberian Peninsula, the main goal of this study was to develop a method for the clonal multiplication of tolerant holm oak plants. Two micropropagation procedures were evaluated for this purpose: (1) Plant regeneration based on axillary budding, including optimization of the rooting step; and (2) induction of SE from explants excised from the same shoot culture lines generated in the axillary budding procedure. Conservation of shoot cultures of the valuable germplasm by cold storage was also considered.
4. Discussion
The main challenge of the present study was to evaluate the possibility of using biotechnological approaches to produce clonal plants of selected P. cinnamomi-tolerant genotypes of holm oak. Biotechnological techniques are potentially useful for propagating tolerant plants of this species, considering the lack of rooting response of cuttings isolated from the stock plants in conventional propagation procedures. Propagation of P. cinnamomi-tolerant Q. ilex trees is required to provide dehesa owners with appropriate plant material for restoring this valuable and currently threatened ecosystem.
This is the first study reporting axillary shoot proliferation and conservation by slow growth storage in holm oak plants that are tolerant to the root-rot pathogen
P. cinnamomi. Previous studies have addressed micropropagation by axillary budding of
Quercus species [
25]. However, very few studies have been carried out with
Q. ilex, and the results so far are rather inconclusive, illustrating the difficulties encountered regarding the micropropagation of holm oak [
29,
30].
As expected, contamination of initial cultures was higher (65.8–71.4%) in explants derived from plants forced to flush in a greenhouse than in those flushed in a growth cabinet (0.0–4.2%). In addition, induced sprouting in a climatic chamber enabled the use of a lower concentration of hypochlorite for a short time, leading to a decrease in the proportion of explants showing signs of contamination and browning. Initiation of in vitro cultures of holm oak under controlled conditions is, therefore, highly recommended. The percentage of responsive explants with sprouting buds after culture for eight weeks clearly depends on the genotype, but there were no differences between the two subspecies tested (
ballota and
ilex). The effect of genotype on different morphogenetic responses, including shoot culture initiation and proliferation, has been reported in a large number of woody species, including white oak [
39]. In the aforementioned study, axillary shoot cultures were established with six out of eight genotypes, of age 6–7 years, with marked genotypic differences in shoot initiation and proliferation rates [
39]. Although 0.5 mg L
−1 BA was necessary for initial stimulation and sprouting of nodal segments derived from mature holm oak trees [
30], this concentration was detrimental (producing hyperhydric shoots) to the tolerant holm oak material. A lower BA concentration (0.2 mg L
−¹) proved optimal for the development of vigorous shoots in the juvenile material used in the present study. Shoot proliferation cultures were stabilized in seven of the eight genotypes tested. Stabilization took longer than in other oak species [
39]. Unfortunately, genotype ES5-1, which produced a high percentage of responsive explants (73.2%), was lost in subsequent subcultures, as shoots acquired a succulent, hyperhydric appearance.
Axillary shoot proliferation was successfully promoted by reducing the BA concentration and by transfer of cultures to a fresh medium every two weeks over a six-week subculture cycle. A similar subculture regime has also been used for the proliferation of other Fagaceae species [
39,
40], including holm oak [
30]. In these studies, zeatin was used in combination with BA, but was not necessary for proliferation of the juvenile holm oak material in the present study (data not shown) or for
Cedrus libani [
41]. Total shoot number obtained in holm oak was higher in comparison to shoot proliferation cultures of different American oak species [
39], although the shoots were shorter. This finding may be related to the typical slow growth habit of holm oak, characterized by episodic growth and development of a large number of axillary shoots with short internodes.
Holm oak is considered a difficult-to-root species, which is one of the main constraints limiting vegetative propagation. Juvenile plants of woody species generally display a higher rooting ability than mature material. However, this is not the case in holm oak, in which rooting frequencies below 20% were recorded in conventional cutting propagation from very juvenile seven-month-old stock plants [
23]. In the present study, rooting was achieved in four out the five genotypes tested. Although tolerant stock plants were 4–7 years old, the rooting frequency was only acceptable in one genotype (96%), as it was below 50% in the other genotypes. However, better results were achieved than those reported by [
30] with mature material, probably due to the auxin treatment, inclusion of STS in the root expression medium and also the genotype effect. As observed in the culture establishment and shoot proliferation steps, the rooting process is clearly genotype-dependent, although there was no evidence of a relationship between the two subspecies studied. Genotypes PR8 and PR11, belonging to the subspecies
ilex, showed marked differences in rooting ability (20% and 96%, respectively). We also found that rooting frequency was higher when the microshoots were transferred (after auxin treatment) to a root expression medium containing 20 µM STS (
Table 4). Silver ions in the form of STS can modulate the deleterious effects of ethylene—a gaseous plant regulator that can be synthesized during in vitro tissue culture [
42], and that negatively affects different morphogenetic processes, including shoot proliferation and genetic transformation ability [
43,
44,
45]. The role of ethylene in the rooting step is unclear, as it seems to be species-dependent [
46], and can promote [
47,
48] or reduce [
49] rooting capacity. Regardless of the role of this plant regulator, the application of STS to holm oak microcuttings seems to improve rooting rates, particularly in the PR11 and E2-1 genotypes, although a larger number of genotypes should be tested to clarify this aspect. By contrast, the addition of AC to the root expression medium in holm oak had a detrimental effect on the rooting ability. However, the addition of AC promoted the rooting frequency in avocado shoots [
50] and in pedunculate oak [
51]. Activate charcoal adsorbs inhibitory compounds present in the culture medium, such as toxic metabolites, phenolic exudates, metal ions, and PGRs [
52]. Unfortunately, only 20% of rooted shoots survived as viable plants after hardening off, thus limiting the commercial application of the procedure. Handling and technical requirements should be improved in order to increase the efficiency of the acclimatization step.
The findings of the rooting experiments showed the difficulty in optimizing the procedure. Specific modifications to the general protocol described are necessary to produce whole plants in non-responsive and recalcitrant genotypes of the species.
Somatic embryogenesis has recently been studied in holm oak to define a reliable system for mass clonal propagation, as well as for the use of somatic embryos as target material in cryopreservation and genetic transformation experiments [
19,
53]. In the present study, SE was induced in two out the three genotypes evaluated. Interestingly, the two responsive genotypes, PR9 and E2-1, belong to the subspecies
ballota. Genotype, explant type and PGRs added to the culture medium are considered crucial factors in SE induction [
28].
Studies on SE induction in conifer species [
54] seem to indicate that the induction rate is a highly heritable trait, thus leading to the possibility of producing families with a reasonably high SE initiation capacity through conventional breeding [
55]. This type of study is scarce in hardwood species. In one of the few reported studies on the subject, [
56] tested SE induction in 13 open-pollinated families of eucalypts, concluding that the induction step is under the control of additive genetic effects, and that a large number of maternal parents should be screened for use in controlled pollination. This approach could be tested in unresponsive hardwood genotypes. On the other hand, indirect evidence of the effect of the genotype on SE process in hardwoods is well documented [
28]. However, genotypes can respond differently under different culture conditions. For example, NAA has been considered the standard PGR for inducing SE in immature zygotic embryos of
Eucalyptus globulus [
57]. Application of the same protocol to shoot apex and expanded leaf explants from two genotypes of mature
E. globulus trees did not induce SE, whereas a positive response was obtained after modification of the culture medium, by replacing NAA with picloram [
58]. The lack of response of holm oak genotype PR11 observed in the present study may be due to inappropriate culture conditions, regardless of the genetic profile. This may also apply to the unresponsive genotypes in in vitro axillary shoot establishment and rooting mentioned in the present study.
Somatic embryos induced from non-zygotic embryos, leaf, shoot apex and nodal tissues could be used as responsive explants, regardless of whether they are excised from seedlings or mature hardwood trees. We have extensive experience in using shoot tips (comprising the apical meristem and three to four leaf primordia with their axillary zones) and the two most apical expanding leaves to initiate SE in different
Quercus and other hardwood species [
28]. However, only shoot tips responded as suitable explants for initiating SE in holm oak. Shoot tips contain pluri- or toti-potent stem cells (the precursors of plant organs), which can favour SE induction [
59,
60]. The lack of response of holm oak leaf explants may be related to the tendency of the excised leaves to undergo rapid necrosis after culture for 48 h [
33].
High concentrations of auxin with or without a cytokinin are generally used for initiating SE in non-zygotic explants [
28]. The presence of auxins in the culture medium and the concentrations required to induce SE seem to be related to the level of cell differentiation of the initial explant. When these tissues contain predetermined embryogenic cells, somatic embryos are frequently initiated on medium containing a weak auxin, such as IAA, or on medium devoid of PGRs. The results reported here are consistent with this approach, as SE was induced in medium supplemented with IAA and also medium without PGRs. This appears to corroborate the low level of differentiation of holm oak shoot apex, together with the excision stress of the explant tissues, which may be sufficient for redirecting the embryogenic pathway. According to [
61], stress induces remodeling of the plant cell fate, with wound stress being a primary trigger for SE induction [
62].
Maintenance of embryogenic competence in holm oak is considered problematic, as isolated somatic embryos at torpedo and cotyledonary-stages are not capable of generating new somatic embryos by secondary embryogenesis [
19,
26,
32]. In the present study, several embryogenic lines from the two responsive genotypes were multiplied indefinitely and maintained in culture by using NSs as the explants to be subcultured for embryo proliferation. The use of NSs to generate somatic embryos was also investigated in embryogenic systems initiated from shoot apex explants derived from
Q. ilex trees [
33] and other related species, such as
Castanea dentata [
63],
C. sativa [
64], and
Q. rubra [
65]. Histological studies performed in NSs of white oak and red oak [
65] revealed the presence of embryogenic cells capable of detaching from the surface layers and of proliferating as proembryonic aggregates, which may explain the high productivity of the NSs.
After a stratification period and subsequent transfer to germination medium, about 46% of somatic embryos converted into viable plants, indicating that SE is an additional method for micropropagating P. cinnamomi-tolerant Q. ilex plants. Further research is required specifically in the maturation and germination steps to improve the conversion rates obtained.
In conclusion, the results of the present study clearly show that the application of biotechnological approaches is a realistic possibility for the vegetative propagation of selected holm oak trees tolerant to
P. cinnamomi. The two proposed procedures, i.e., axillary shoot proliferation and SE, enable the propagation of viable plants, although several problems must be solved. It is not clear which is the most appropriate procedure, although axillary shoot micropropagation can be considered technologically easier than SE. In addition, in order to initiate SE following the procedure described here, axillary shoot cultures are required as the original source of explants. However, once the embryogenic cultures are established, their multiplication capacity is generally higher than that of axillary shoots. In addition, two results derived from the two procedures used in the present study should be highlighted. First, plants obtained from the germination of somatic embryos have a pivotal root system (
Figure 3D) similar to that of plants obtained from the germination of zygotic embryos (and acorns). By contrast, the rooted microshoots exhibit the typical arrangement of adventitious roots around the shoot (
Figure 1E). Given the limited water availability in Mediterranean areas, a deep and pivotal root system may provide oak trees with a key advantage to survive drought conditions during summer [
66]. Second, the medium-term conservation of holm oak germoplasm has been demonstrated in axillary shoot cultures. However, cryopreservation under liquid nitrogen is ideal for long-term germplasm conservation. Our experience in cryopreservation of embryogenic cultures of different Fagaceae species [
67,
68,
69,
70], including
Q. ilex [
19], highlights the good potential offered by this technique for the long-term conservation of tolerant holm oak germplasm [
67,
68,
69,
70]. After the successful cryopreservation of embryogenic lines derived from
Q. ilex mature trees, a positive response of holm oak somatic embryos developed from tolerant genotypes (being studied, at present) is also expected. Cryopreservation of embryogenic lines will be a valuable tool during the field testing of regenerated plants following a similar model to that of multi-varietal forestry defined for conifer species and the use of tested tree varieties in plantation forestry [
54,
71].
The micropropagation procedures defined in this study should also be tested by applying temporary immersion systems for large-scale propagation of these species, which would enable the production of
P. cinnamomi-tolerant holm oak plants for use in future restoration programs in areas affected by the pathogen. In recent years, temporary immersion systems, such as RITA®, have been successfully applied in the proliferation of axillary shoots of chestnut [
72], as well as in the proliferation of both pedunculate oak [
73] and cork oak [
74] embryogenic cultures.