Introduction

Medicinal and aromatic plants (MAPs) have properties that are highly valued in the pharmaceutical, cosmetic, fragrance, food, and flavor industries, and many are widely cultivated, e.g., Lavandula spp. (lavender), Origanum vulgare L. subsp. hirtum (Link) Iestw. (Greek oregano), Rosmarinus officinalis L. (rosemary), Origanum majorana L. (marjoram), Matricaria chamomilla L. (chamomile), Salvia spp. (sages), and Melissa officinalis L. (lemon balm). However, to date, the production of MAPs still relies to a large degree on wild collection (Lange 1998; Kupke et al.2000), such as in Albania (Lange 1998), Hungary (Bernáth and Németh 2000), Slovakia (Kupke et al.2000), and Turkey (Özhatay 1997). In Europe alone, the vast majority of > 1300 different MAPs used are still collected from the wild, while only 3 to 6% are exclusively sourced from cultivation. The practice of harvesting wild plants coupled with land conversion or habitat loss is currently threatening with extinction about 20 to 25% of MAPs (Lange 1996; Murch et al.2004; Schippmann et al.2006).

The uncontrolled harvesting in the Mediterranean, which dates back to antiquity, has been identified as a major threat for the populations of the MAPs, and it has led to several species being on the verge of extinction, such as the rosemary in Sardinia (Mulas and Mulas 2005), and Gentiana acaulis L. and Arnica montana L. in Croatia (Šatović 2004). At least 30% of the rare and threatened plants of Greece, many of which have medicinal and/or aromatic properties, actually suffer from uncontrolled wild collection (Krigas et al.2014a). For example, the wild populations of the local Balkan endemic Sideritis scardica Griseb. (Greek mountain tea, ‘Tsai Olympou’) are already assessed as “Near Threatened” by the IUCN (International Union for the Conservation of Nature) Global Red List; Sideritis sipylea Boiss. (endemic to Greek East Aegean Islands and adjacent part of Anatolia, Turkey) and Sideritis euboea Heldr. (single-island endemic of the island of Evia, Greece) are both nationally assessed as “Endangered”; Sideritis raeseri Boiss. & Hledr. subsp. attica (Heldr.) Papan. & Kokkini (local endemic of Sterea Hellas) is assessed as “Vulnerable” (Phitos et al.2009); and the populations of the Cretan endemic Sideritis syriaca L. subsp. syriaca (Cretan Mountain tea or Malotira) have severely declined during the last decades, which has led to a population monitoring program launched recently in Crete. Out of ten wild perennial taxa (species and subspecies) of the genus Sideritis traditionally traded in Greece for the preparation of mountain tea, only a couple of them are cultivated on a small scale (S. scardica, Sideritis raeseri Boiss. & Heldr. subsp. raeseri), while cultivation of S. syriaca subsp. syriaca and Sideritis clandestina (Bory & Chaub.) Hayek subsp. clandestina has only recently started.

Furthermore, there is an increase in the electronic trade of range-restricted single-country endemic plants, among which the majority are MAPs and many of them are threatened with extinction. Examples include the Greek endemics Origanum dictamnus L. and Origanum calcaratum Juss. (Krigas et al.2014b), and the Cypriot endemics Origanum cordifolium (Montbret & Aucher ex Benth.) Vogel, Scutellaria cypria Rech. f. subsp. cypria, and Sideritis cypria Post (Krigas et al.2017).

Although domestication and cultivation of MAPs is the most sustainable method to exploit their valuable properties (Schippmann et al.2006), knowledge regarding wild species of MAPs is still limited. For the latter, species-specific propagation and cultivation protocols are necessary for the industry and ornamental market to avoid draining wild populations. Therefore, sexual propagation is needed to raise new plants for conservation or breeding purposes, while asexual propagation is required for the production of genetically identical elite plants with high value. Multiplication using cuttings can be slow and labor intensive, rooting is often difficult (Gonçalves and Romano 2013), and domestication of rare and threatened plants could be problematic, especially when the availability of initial propagation material is usually restricted for these plants (Grigoriadou et al.2011). In vitro propagation methods could help overcome these limitations and increase the probability of success (especially when threatened species are targeted), which would facilitate rapid multiplication of selected and valuable plant material for various uses without seasonal restrictions (Zuzarte et al.2010).

In general, in vitro techniques for plant propagation (micropropagation) have major advantages over conventional methods: (i) For initial plant establishment, only a small amount of explant material is needed in a limited working space; (ii) propagation in a sterile environment ensures pathogen-free plants; (iii) large numbers of identical clones are raised, with genetic and physiological uniformity, and chemical consistency for commercial purposes, independent of seasonal variations (e.g., from one explant, it is possible to produce over 1,000,000 plants in 1 yr); (iv) stock material can be securely maintained almost perpetually; and (v) the maintenance of mother plants is easier and safer given that one plant may be enough to take the explants needed for the establishment of the culture (George and Debergh 2008).

In this review, the current knowledge is summarized regarding the in vitro propagation of selected MAPs, herbaceous perennials, bulbous, subshrubs, and trees, which are native to Greece and are also assigned with conservation priority. The studies reviewed were sourced from (a) Scopus database, using the “in vitro” and “conservation” keywords in article titles and/or using the names of known scientists working with in vitro technology and Greek native plants; and (b) from other sources of grey literature such as faculty repositories, books, and conference proceedings. Among the in vitro studies retrieved, it was cross-checked whether each of the vascular taxa (species and subspecies) mentioned in these studies were assigned with (i) native status in Greece (Dimopoulos et al.2013, 2016), (ii) conservation concerns due to restricted distribution range such as single-country Greek endemics and other range-restricted taxa (Dimopoulos et al.2013, 2016); (iii) threatened or near threatened status, either nationally (Phitos et al.1995, 2009; Tsiftsis and Tsiripidis 2016) or globally (see Global IUCN Red List website); (iv) MAP potential such as specific medicinal and aromatic properties, or specific ethnobotanical uses; and (v) national (Greek) protection status. After this strict selection procedure, only the cases of range-restricted species and subspecies (taxa) of Greece were included in the review, such as single-country endemics or endemics to Greek phytogeographical regions, or parts thereof, Balkan endemics or East-Mediterranean endemics, most of them threatened or near threatened and/or protected, which are currently considered as MAPs with phytochemical or ethnobotanical evidence. This included five additional Orchidaceae taxa (some with wider distribution range), with medicinal properties and ethnobotanical uses that are protected by the Greek legislation (Table 1, n = 22 taxa).

Table 1 List of selected conservation priority medicinal and aromatic plants native to Greece reviewed in this study (alphabetically according to the family they belong to)
Full size table

In vitro propagation of Greek native MAPs with conservation priority

Micropropagation can provide a rapid method for the multiplication of conservation priority species. In this case, only a few plants may be available as stock plants, or the collection of plants and seeds from the wild may be minimized. Many institutes, organizations, and botanic gardens involved in the ex situ conservation of native plants apply micropropagation procedures, when needed, for maintenance and clonal reproduction of important species (Hartmann et al.2002; Maloupa et al.2008). Without efficient conventional propagation methods for such species, or due to limited available material, or decline of wild populations, or original anthropogenic habitat alterations, the micropropagation procedure may facilitate future needs to reinforce wild populations by raising adequately selected plants from different stocks (neopopulations). Currently, reintroduction programs in Greece are only performed on a pilot scale in Crete; these include conservation translocation actions following the guidelines of the International Union for the Conservation of Nature (IUCN) and exclusively rely on plants raised in vivo by seeds to ensure enriched species’ genetic diversity (see http://www.care-mediflora.eu/).

In most MAPs, micropropagated plants are usually generated by the development of axillary buds or adventitious shoots (Gonçalves and Romano 2013), which was usually a five-stage procedure (Debergh and Maene 1981; George and Debergh 2008). In this section, in vitro propagation of 17 cases of Greek native MAPs with conservation priority, which includes herbaceous perennials, bulbous, subshrubs, and trees, was examined (Tables 2, 3, and 4).

Table 2 Most optimal conditions for the in vitro establishment of selected native Greek medicinal and aromatic plants with conservation priority (alphabetically, excluding Orchidaceae plants)
Full size table
Table 3 Overview of the best media and plant growth regulators (PGRs) tested for proliferation (rate calculated in 4-wk periods) and shoot length reported for in vitro studies related to native Greek medicinal and aromatic plants with conservation priority (alphabetically, excluding Orchidaceae plants)
Full size table
Table 4 Overview of media and plant growth regulators (PGRs) used for rooting (in 4-wk subculture) and ready-plant acclimatization (calculated in 6 wk) as reported in in vitro studies related to native Greek medicinal and aromatic plants with conservation priority (alphabetically, excluding Orchidaceae plants)
Full size table

Stage 0: Mother plant selection and preparation

The selection of initial plant material is important for any culture establishment and to avoid infected stock mother plants. For the Greek native MAPs with conservation priority, a specific protocol was reported regarding research on their sustainable use (Maloupa et al.2008). In this process, healthy target plants were collected from the natural environment using a special collection permit issued by the Greek government for specialized botanical expeditions in various regions of the national territory, during which appropriate propagation material was collected (seeds, cuttings, rhizomes, tubers, and/or whole plants). Plant material was transferred to the Laboratory for the Conservation and Evaluation of Native and Floricultural Species (LCENFS), and the Balkan Botanical Garden of Kroussia (BBGK; which is part of the Institute of Plant Breeding and Genetic Recourses, Hellenic Agricultural Organization-DEMETER), where samples were sorted and each one assigned a unique “accession code.” The specimens were taxonomically identified, and some were also documented with DNA barcoding. To allow ex situ maintenance of previously unknown MAPs, a geographic information systems (GIS) ecological profiling was used (Krigas et al.2010, 2012). The ecological information extracted can illustrate the soil and climate profiles in the wild habitats of the targeted plants quantitatively and qualitatively. This information is used to keep mother plants in appropriate environmental conditions so that any differences in their behavior during the later stages will be primarily due to genotype expression. Both LCENFS and BBGK apply this protocol and specialize in ex situ conservation of the native plants of Greece. Mother plants of the collection of LCENFS and BBGK were widely used for the establishment of tissue culture for Greek native species with conservation priority (Grigoriadou et al.2011; Samaras et al.2012; Grigoriadou et al.2014; Sarropoulou and Maloupa 2016a, b), while others were initiated by plant material collected directly from the wild (Kartsonas and Papafotiou 2007; Martini and Papafotiou 2013).

Stage I: Establishment of in vitro cultures

Greek native MAPs with conservation priority are often rich in essential oils and phenolic compounds (Panagouleas et al.2003; Hanlidou et al.2012; Hanlidou and Lazari 2013), and glandular hairs or multicellular glands containing essential oils, which makes disinfection and effective establishment difficult (Grigoriadou et al.2011; Martini and Papafotiou 2013; Sarropoulou and Maloupa 2017a). Explants were disinfected using 1.5 to 4% (v/v) sodium hypochlorite (NaOCl), shaken on a rotary shaker for 20 min and rinsed 3 times with sterile distilled water, depending on the period of the year (Kartsonas and Papafotiou 2007; Martini and Papafotiou 2013), and/or on the type of explant (Grigoriadou et al.2011, 2014; Sarropoulou and Maloupa 2017a; Table 2). Pretreatment with ethanol and/or fungicides was usually effective in three difficult cases (Sarropoulou and Maloupa 2017a, b).

For culture initiation of the studied taxa, shoot tips or nodal segments from fresh annual growth was typically used (Table 2). Seeds were also used, after disinfection using 1.5 to 4% (v/v) NaOCl with the addition of three drops of Tween® 20 (Öz et al.2008; Papafotiou et al.2017). Special pretreatments were sometimes needed to overcome seed dormancy. This was the case of Amsonia orientalis Decne. in Jacquem., in which the use of 100 μM sodium nitroprusside (SNP) was necessary for successful in vitro germination (Öz et al.2008).

Murashige and Skoog (MS) medium (Murashige and Skoog 1962) was used for shoot tips and nodal segments during the initiation phase of most Greek native MAPs with conservation priority, and was supplemented with 6-benzyladenine (BA), either alone or in combination with auxins such as indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and α-naphthaleneacetic acid (NAA) (Table 2). Woody Plant Medium (WPM; Lloyd and McCown 1980) was used for Quercus trojana Webb subsp. euboica (Papaioannou) Chr. in Strid & Tan, which is a woody plant with hyperhydricity problems (Kartsonas and Papafotiou 2007).

Establishment success of Greek MAPs with conservation priority in vitro varied from 13 to 90%. Even lower rates were considered sufficient for this stage, because the objective was not a high success rate, but to have pathogen-free cultures readily available. In cases of limited and valuable initial material, even one plantlet could be sufficient and after continuous subcultures, enough stock material (identical) could be produced (George and Debergh 2008); however, this is at the expense of the concomitant species genetic diversity, thus compromising conservation efforts.

Stage II: Proliferation in vitro

For the proliferation of Greek native MAPs with conservation priority, MS proved to be the preferred basal medium (Table 3). In some cases, the used medium was supplemented with extra iron (Fe; Minas 2001; Grigoriadou et al.2011). This need was observed primarily for species that naturally grow at high altitudes, which have been maintained at lower altitudes where cultivation temperatures are generally higher, even in in vitro studies. Under those elevated temperatures, sometimes Fe is not efficiently absorbed by microplants (Monteiro et al.2000). The MS medium contains Fe in the chelate form of Fe-ethylenediaminetetraacetic acid (EDTA). However, given that the most effective form of chelated Fe is Fe-ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) (EDDHA), substitution with Fe-EDDHA could provide better results (Molassiotis et al.2003). For the woody species studied, hyperhydricity problems were observed when WPM was used (Kartsonas and Papafotiou 2007). Woody plant medium contains one-third to one-fourth the level of macroelements compared with MS, but it is a medium with a very good balance of macroelements and is often used for difficult cases of micropropagation, such as micropropagation of species of the Fagaceae family (Ostrolucká et al.2007).

In the studies of Greek MAPs with conservation priority, different plant growth regulators (PGRs) were used. Use of cytokinins either alone or in combination with auxins (Table 3), with BA being the most common, was also reported for other MAPs (Gonçalves and Romano 2013). The use of BA was effective in most cases and resulted in high proliferation rates, as in A. orientalis (Öz et al.2008), and Campanula incurva A. DC. in DC. (Grigoriadou et al.2014) (Table 3). In other cases, such as Achillea occulta Constantin. & Kalpoutz. (Grigoriadou et al.2011), Malus florentina (Zucc.) C.K. Schneid. (Martini and Papafotiou 2013), and Sideritis perfoliata L. subsp. athoa (Papan. & Kokkini) Baden in Strid & Tan (Papafotiou and Kalantzis 2009a), BA gave new shoots that were < 1.5 cm high, which were not capable of advancing to the next stage of rooting (Table 3). In those cases, stage II should have been followed by a special treatment in different media for shoot elongation (stage IIIa, Debergh and Maene 1981), which would result in inevitably increased production costs (George and Debergh 2008). Zeatin (zea) was used in the cases of S. raeseri subsp. raeseri and Lithodora zahnii (Halácsy) I. M. Johnst. when other PGRs were not effective enough. The combination of 2 μΜ of zea with 11 μΜ of α-tocopherol for S. raeseri subsp. raeseri reduced the severity of hyperhydricity problems that appeared when zea was used alone (Sarropoulou and Maloupa 2017b). Hyperhydricity was also observed in L. zahnii cultures when a high concentration of zea was used (Papafotiou and Kalantzis 2009b). This zea effect was counteracted by using high concentrations of sucrose and agar.

Somatic embryogenesis and regenerated plants from mature organs have been reported for endangered species, allowing genetic improvement and supporting conservation efforts (Litz et al.2008; Martínez et al.2019). However, this has not yet been reported in Greek native MAPs with conservation priority, probably due to limited research in this area in Greece.

Stage III: Rooting in vitro

Before rooting in vitro, it may be necessary to allow time for shoots to grow to a suitable size (usually > 1 to 1.5 cm), depending on the species (Debergh and Maene 1981). In many Greek native MAPs with conservation priority, it seems that expanding the elongation treatment would have been probably useful leading to length increases > 1.5 cm (Table 3).

For the rooting of taxa reviewed in this research, MS was the usual medium, sometimes with macronutrients reduced by half, which was a typical procedure reported for other MAPs (Dias et al.2002; Gonçalves and Romano 2013). For an effective micropropagation protocol, the addition of auxins (IBA in most cases) in concentrations of 2 to 10 μM proved to be effective, and resulted in a high percentage of rooting (Table 4). Achillea occulta, M. florentina, and S. perfoliata subsp. athoa showed poor rooting results. The addition of activated charcoal enhanced rooting of M. florentina (Martini and Papafotiou 2013). Even though these MAPs had a sufficient proliferation rate, the shoot length was short, which was probably the reason for their limited root induction. As previously highlighted, an elongation stage could prove to be beneficial (increasing the length to > 1.5 cm) for these taxa.

Stage IV: Acclimatization

The acclimatization of Greek native MAPs with conservation priority mainly depended on the previous in vitro rooting stage (Table 4). For C. incurva, the micropropagation procedure proved to be the ultimate multiplication method, as it is estimated that > 1,000,000 plants can be produced, starting from one explant in a 1-yr period (Grigoriadou et al.2014).

In the cases of conservation priority Greek native MAPs, in which root induction was > 50%, acclimatization success of the rooted plantlets was > 80% in a period of 4 wk, which was considered to be sufficient for an effective massive micropropagation protocol (Table 4). Only for A. occulta 33% survival was reported, which was due to limited in vitro rooting (Grigoriadou et al.2011).

Assessment of protocol effectiveness

An in vitro propagation protocol that can be broadly applied should generate thousands of acclimatized plants in a short period of successive subcultures (e.g., Firoozabady and Gutterson 2003; Kaur and Sandhu 2015). In order to be able to compare the effectiveness of the different protocols produced in an integrated way, the number of successfully acclimatized plants for the Greek native MAPs with conservation priority that can be generated in 1 yr from one explant (Np) was calculated in the present study (Table 4), based on information reported in original sources (or calculated from data reported in them). To do this, the basic parameters that were taken into account were (i) best average proliferation rate (PR) per studied species, as reported from different number of replicates, including reported losses due to hyperhydricity; (ii) proliferation subcultures achieved (n) per studied species in 1 yr (calculated in 4-wk periods), allowing the routinely needed time for successful establishment (n-1); (iii) average rooting percentage (R) per studied species (as reported from different number of replicates, calculated in 4-wk periods); and (iv) average acclimatization success rate per studied species (AC), according to cited references (calculated in 6-wk periods). All the abovementioned parameters (i, ii, iii, iv) represent specific rates with no units, and all refer to different numbers of plant individuals that potentially can be proliferated, rooted, and acclimatized, respectively, from a single explant per given species, if the best treatments reported in the reviewed studies are followed. Therefore, if these parameters are combined, the potential number of successfully acclimatized plants (Np) of a given Greek native MAP species with conservation priority obtained from a single explant within 1 yr can be calculated using the following equation:

$$ {N}_p={\mathrm{PR}}^{n-1}\times R\%\times \mathrm{AC}\% $$

where

PR:

average best proliferation rate among treatments according to cited references (Table 3), including reported losses due to hyperhydricity;

n:

number of proliferation subcultures achieved in 1 yr (calculated in 4-wk periods); n−1 is given, which allows the average time needed for successful establishment;

R:

average rooting percentage (%, not in decimal values) according to cited references (calculated in 4-wk periods; Table 4);

AC:

average acclimatization success (%, not in decimal values) according to cited references (calculated in 6-wk periods; Table 4).

The calculation of Np was introduced to allow quantitative comparison of the propagation protocols reviewed in terms of conservation purposes and mass production. The Np assumes that there are no space limitations, meaning that all explants produced during subsequent subcultures will proceed to rooting with no time intervals, and those rooted plants will be immediately transferred for acclimatization. With these limitations in mind, in this study, a protocol was arbitrarily considered as “effective for conservation purposes” when > 100 plants yr−1 could be reproduced and as “effective for massive production” when 10,000 plants yr−1 could be reproduced. These threshold values reflect in an empirical way the amount of plants needed when conservation is the objective (e.g., 100 individuals yr−1 raised from a limited and difficult to access initial material for selective population re-enforcement), or when routine massive production is considered for a given species (e.g., 10,000 plants yr−1 from an elite material with desired characteristics for small scale commercial cultivation). With this perspective, effective protocols for conservation purposes were designated for all 17 Greek native MAPs with conservation priority reviewed in this study. For 13 MAPs, the protocols reviewed were also considered as effective for mass reproduction. However, for four of them (A. occulta, O. dictamnus, Q. trojana subsp. euboica, S. raeseri subsp. raeseri), the described methods were characterized as “not effective” in terms of mass reproduction, due to the long time required to generate a high number of acclimatized plants (Table 4).

Micropropagation is argued to be an expensive method of plant propagation, as it requires the use of specialized personnel and expensive infrastructure. When it is used for commercial mass propagation, the protocols followed should produce several hundreds of thousands of plants within a year (De Paoli et al.1994). For example, cost-effective protocols are considered to be those resulting in 1,850,000 acclimatized plants for the industrial crop Saccharum officinarum L. (sugarcane; Kaur and Sandhu 2015), and 850,000 for Ananas comosus (L.) Merr., the pineapple crop (Firoozabady and Gutterson 2003). Therefore, if the above-described Np is > 100,000, then the protocols could be applied commercially at low cost, especially for conservation important MAPs. If the Np is < 100,000, these would result in high-cost reproduction schemes, while intermediate costs (balancing costs and benefits) could be attributed to Np > 15,000 (Table 4). Following this perspective, it seems that five taxa could be multiplied at high cost (A. occulta, M. florentina, O. dictamnus, Q. trojana subsp. euboica, S. perfoliata subsp. athoa), eight taxa at intermediate cost (Anthyllis splendens Willd., Calamintha cretica (L.) Lam., L. zahnii, Lomelosia hymettia (Boiss. & Spruner) Greuter & Burdet in Greuter & Raus, S. raeseri subsp. raeseri, S. scardica, S. sipylea, S. syriaca subsp. syriaca, Staehelina petiolata), and only four of the taxa (A. orientalis, C. incurva, Muscari macrocarpum Sweet, S. scardica) could be massively multiplied at low cost (Table 4). However, given the conservation priority assigned for all the taxa of Table 4, their maintenance and proliferation in vitro are considered an important steppingstone towards conservation.

In vitro propagation of Orchidaceae MAPs of Greece with conservation priority

Agroalimentary and medicinal uses of orchids, for making salep or salepi (a flour made from the tubers of orchids) in particular, date back to ancient traditions in Greece and several species are involved (Kreziou et al.2015), such as Anacamptis spp. (including Anacamptis laxiflora (Lam.) R.M. Bateman, Pridgeon & M.W. Chase subsp. laxiflora, Korakis and Vidakis (2016)), Dactylorhiza spp. and Orchis spp., especially Orchis mascula (L.) L. subsp. mascula (Kreziou et al.2015). Additionally, flower parts of Orchis apifera Huds., Orchis argolica H. Fleischm., and Orchis scolopax Cav. subsp. cornuta (Steven) E. G. Camus may be rich in flavonoids and could be used as antioxidants (Karioti et al.2008).

In Greece, the Orchidaceae family is currently represented by 141 taxa (species and subspecies), according to Dimopoulos et al. (2013, 2016), or by 193 taxa according to Tsiftsis and Tsiripidis (2016), and taxa in this family are nationally protected (Presidential Decree 67/1981, law 1335/1983, and law 2005/1992, which enacts CITES, known as the Convention on International Trade of Threatened Species).

There is a very limited amount of published data on the in vitro propagation of European terrestrial orchids (Valletta et al.2008). Compared with tropical orchids, temperate ones are harder to propagate and cultivate (Ponert et al.2011), and the few reports that exist lack detail on the laboratory techniques used (Katsalirou et al.2017). In this context, propagation of Mediterranean orchids is poorly documented. There are just a few reports from Greece, although Greece is the most orchid-rich country in Europe based on the number of taxa per unit area (Georghiou and Delipetrou 2010).

Seed germination and callus formation

Seed treatments using various disinfecting agents prior to establishment represent a vital step during micropropagation of orchids and serve a dual purpose: to disinfect the seeds and to break embryo dormancy (chemical scarification). The literature is contradictory as to the recommended duration of disinfection/scarification that leads to seed germination in various species (Katsalirou et al.2017). A scarification time of only a few minutes in 1% (v/v) NaOCl seems optimal for germination of seeds with rather permeable coats, such as those of A. laxiflora subsp. laxiflora (Table 5). Due to dormancy induced with maturation of Orchidaceae seeds, non-mature seeds are preferable for in vitro culture (Malmgren 1996; Steele 1996). Kitsaki et al. (2004) reported different reactions of immature and mature seeds of bulbous Orchidaceae MAPs of Greece with conservation priority (Table 5). For MAPs of Greece of the genus Ophrys with conservation priority, mature seeds and a coconut-enriched medium were reported as a proper treatment for in vitro germination, although there were cases in which using immature seeds resulted in better germination results and callogenesis (Table 5).

Table 5 Overview of materials, techniques, optimal media, plant growth regulators (PGRs), and substances used during the establishment phase and protocorm formation, as reported in in vitro studies on native Greek medicinal and aromatic Orchidaceae with conservation priority (alphabetically)
Full size table

Different culture media have been used for seed germination of bulbous Orchidaceae MAPs of Greece with conservation priority, and the addition of organic substances such as coconut milk, pineapple juice, or a cube of potato seem to be critical (Kitsaki et al.2004; Valletta et al.2008; Katsalirou et al.2017). However, the reproducibility of these trials could be questioned due to the variable composition of these supplements. The duration of the germination/callus formation stage from seeds was long, from 3 to 6 mo, and occasionally, the success was low (Table 5).

Protocorm formation and plantlet development

In the native Greek Orchidaceae MAPs with conservation priority studied, protocorm formation compared with callogenesis was reported to be considerably lower, and this was true for both mature and immature seed cultures, especially on pineapple-enriched media (Kitsaki et al.2004). Once the protocorms developed from callus tissues, they could easily continue to plantlets. Thus, the most crucial stage remains the differentiation of callus to form shoot and root meristems. This, for the native Greek Orchidaceae MAPs with conservation priority, was influenced by the genotype, varying from 0 to 55% in a 3-mo period, and was not proportional to the frequency of callogenesis (Table 5).

In the native Greek Orchidaceae MAPs with conservation priority studied, protocorms developed into plantlets with leaves and roots. Next, they were transferred to the same growing medium for 10 mo, which allowed minituber formation and were then successfully transferred for culture in pots (Kitsaki et al.2004).

Conclusions

This review focused on 22 native MAPs of Greece with conservation priority (mostly range-restricted Greek endemics), for which there was available information regarding their in vitro propagation. The study reflects the current state of knowledge on this topic.

Regarding the effectiveness of in vitro propagation of Greek native species of Orchidaceae family with MAP potential and conservation priority, germination protocol improvements must be further investigated to decrease the time required to reach plantlet development. Although these data are rather fragmentary, they can facilitate and serve the future development of species-specific in vitro propagation protocols including shoot formation, rooting, and acclimatization. By that time, although the ex situ conservation can be investigated for these taxa, their sustainable exploitation and possible commercialization will still be compromised.

Despite the limited number of cases reviewed in this study, the protocols produced to date show that in vitro culture techniques are efficient not only for increasing the number of plants in cases in which other methods are insufficient or inadequate, but also for the ex situ conservation of Greek native species with conservation priority.

The protocols reviewed were assessed in terms of effectiveness either for conservation purposes or mass production needs with an equation produced in this study. While all of them were assessed as effective for conservation purposes, it seems that in more than half of the cases (n = 13), these are also effective for mass production, allowing their possible sustainable exploitation and commercialization. However, this can be done with high cost in five cases, with intermediate cost in nine cases, and with low cost only in three cases (A. orientalis, C. incurva, M. macrocarpum).

Undoubtedly, a large majority of the Greek native MAPs with conservation priority has not been studied yet. However, at least for those taxa reported here, selected plant material in excellent hygienic condition can become readily available either for conservation purposes, reintroduction into the wild or reinforcement of wild populations, or for breeding trials and cultivation programs.