Georgios TSOKTOURIDIS and Nikos KRIGAS

Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization Demeter (ELGO-Dimitra), P.O. Box 60458, 57001 Thermi, Greece

Abstract

In search of new flower crops sourced from the endemic and native biodiversity of Greece, a consolidated strategy has been developed by the Institute of Plant Breeding and Genetic Resources (IPBGR) of the Hellenic Agricultural Organization – Demeter (ELGO-Dimitra). Local endemic rare-threatened plants with floricultural and/or aromatic-medicinal value originating from the unique Greek biodiversity have become through applied research protocols potentially new flower crops able to be commercialized in the agro-alimentary and ornamental sectors. Bringing plant material from the wild to the laboratory and nursery facilities with authorization for propagation and ex-situ conservation is a tedious procedure, especially for rare species with limited and range-restricted populations in nature and ill-known biological cycle. The identity of the valuable but limited wild germplasm (e.g., Greek tulips, Campanula spp., Helichrysum spp., Lamiaceae members, etc.) is consolidated with taxonomic and DNA barcoding system, thus obtaining unique accession numbers and/or genetic documentation. Propagation-wise, pretreatment methods are necessary for the plant material raised from stock mother plants using cuttings involving various plant growth regulators in combination with innovative nutrient products. Conservation-wise, acclimatization of the rooted microplants and/or massive production of the valuable germplasm is employed following various procedures and treatments. The propagated Greek endemic and native germplasm constitutes an established collection of great value which is maintained ex-situ in man-made environments at the premises of IPBGR. The strategy presented herein has been successfully applied through targeted research projects for >300 Greek endemic and >1500 native plant species of Greece during the last two decades, resulting in extensive living collections maintained as bulbs, corms, living plants in pots, stored seeds and microplants in vitro in IPBGR. The case of the native Aegean subendemic Helichrysum orientale is outlined as a candidate flower crop for commercialization. The use of synergistic products in combination with indole-3-butyric acid (IBA) for rooting is also demonstrated.

Keywords: native species, biodiversity, molecular markers, rooting, cuttings

INTRODUCTION

The development of new flower crops is a promising and dynamic area of study, by providing new products for the horticultural industry. The origin of the newly cultivated crops can often be sourced from the vast number of wild species found in nature, as part of any country’s plant biodiversity. The propagation of these unknown new plant species is required for the conservation and acclimatization of the wild-growing plants to a human-made artificial environment and the development of species-specific mass propagation protocols for the cultivation and exploitation of these new flower crops. In addition, several actions are required during multiplication to successfully provide elite, disease-free vegetative plant material. These complex processes have been successfully developed throughout the implementation of many research projects implemented at the premises of the Institute of Plant Breeding and Phytogenetic Resources, ELGO-Demeter (e.g., Maloupa et al., 2008, 2023; Krigas and Maloupa, 2021). These research projects have furnished to date rich living collections maintained as bulbs, corms, living plants in pots, stored seeds and microplants in vitro for >300 Greek endemic and >1500 native plant species of Greece during the last two decades at the grounds of the IPBGR. 

Conservation activities for threatened local endemic plant species of certain areas is of global concern. Plant conservation includes short-, medium-, and long-term strategies such seed banking and the application of advanced biotechnological tools like cryopreservation of plant genetic resources (Coelho et al., 2020) and an integration of in situ and ex situ conservation approaches (Crane, 2020). The importance of conserving rare or threatened wild plant species is also subject to the World Health Organization (WHO), accounting 80 percent of the world’s population to rely on traditional medicine and estimated 21,000 plant species have the potential for getting used as medicinal plants (Kadam and Pawar, 2020).

Nonetheless, the propagation of native, endemic, and generally unknown plants from a wild habitat is not an easy procedure. Although many investigations have been carried out and they are well described in the literature, there is absence of a unique guide of how to obtain genetic material from the wild and then follow a general application protocol for successful propagation of different species. The vegetative propagation method to produce ‘true to type’ and genetically identical to the wild plants meets additional constrains due to the unknown nature of these wild plant species, the associated microbiome and minimum habitat requirements. The development of a universal propagation protocol is very difficult due to the origin of initial plant material, the huge number of different species and limitations that must be handled in every stage during the species-specific propagation process. Although standard parameters applied during propagation have been extensively described (Hartmann et al. 2014), more investigations are currently carried out on the synergistic actions of various compounds with the auxins for rooting such as biostimulators (Kisvarga et al, 2022; Rajan and Singh, 2021), organic substances, microbial inoculants (Abbott et al., 2018) and natural rooting hormones such as coconut water (Abdel-Rahman et al., 2020), aloe vera, vermin wash or seaweed extracts, humic acid, willow leaf water, honey, etc. (Rajan and Singh, 2021).

The current study presents all fundamental steps for the integrated conservation and massive propagation of wild-growing plant species originated from the Greek flora, which are potential candidates for cultivation as new flower crops. A case study is illustrated for the ex situ conservation of Helichrysum orientale (L.) Gaertn. and its promotion as new flower crop including investigation for mass propagation using IBA synergistically with three different systemic copper fertilizers and trace elements. 

MATERIALS AND METHODS

Botanical expedition and plant material

The first step for a botanical expedition (BOTEX) was to obtain a special permit issued by the Greek ministry of Environment and Energy for wild material and plant collections. This application is annual and should be prepared during December every year in order to obtain the permission for the next year and includes the names of the people participating in the BOTEXs. In the end of the year, the ministry requires a full report for the plant species, the quantity and kind of plant material collected throughout the year of the collections. 

H. orientale wild material was collected in April 2015 from wild-growing plants dwelling on cliffs and rocky areas in the region of Lagada, Amorgos Island, Greece (Figure 1Β-C) and from the castle walls of Kythira in April 2021. All plant collections were implemented using a special permit issued by the Greek ministry of Environment and Energy (e.g., 26895/1527 of 21/4/2021). The next step was to organize the BOTEX. There are many things to consider or to estimate prior the implementation of a BOTEX, especially when the focal area is unknown. These parameters include: list of the targeted plant species, the stage of their biological cycle, GPS location, information about the habitat and the area of the BOTEX; group of participants with appropriate scientific expertise (biologist-botanist, horticulturist, etc.);

Figure 1. Overview of the integrated ex situ conservation of Helichrysum orientale: (A) Inflorescence of a wild-growing plant individual appreciated as potentially new flower crop; (B) Rock-dwelling plant individual in Agorgos island, Greece; (C) the same wild-growing plant after excision of plant material for propagation; (D) excised propagation material; (E) removal of old and infected leaves and preparation of the plant material for transportation; (F) placement of cuttings in a plastic box using water saturated floral foam blocks; (G) placement of cuttings using floral stem water tubes; (H) placement of cuttings using a 1 L honey jar with water saturated floral foam blocks; (I) placement of cuttings in a 50 ml Falcon tube using water saturated floral foam blocks; (J) cuttings immersed in THEOCOPPER for propagation and the initiation of the experiment; (K) cuttings placed in propagation tray using a 3:1 perlite/Terrahum substrate; (L) root induction of cuttings after 20 days on a bench fog system; (M) rooted plantlet ready for transplantation in 0.33 L pots; (N) transplantation of rooted cuttings in 0.33 L pots and acclimatization in an unheated greenhouse; (O) cultivation of acclimatized plants in soil.

availability of local people to guide the BOTEX if needed; the number of days required for the planned BOTEX; the kind of transportation required by special vehicles for mountainous off-road zones, or even by boat; booking of accomodation, ferry, air-tickets, etc; the flexibility to postpone or change the dates of a BOTEX due to weather conditions; the total cost of the planned BOTEX; estimation of the maximum quantity of wild plant material and plants that will be collected in respect to the size of the wild-growing plant population; shipping required for the collected wild plant material; trecking clothes, shoes, hat, skin protection from sun and mosquitoes required depending on season; a standard first-aid kit; labeling and packaging material (waterproof pens, pensils, label signs, paper bags, plastic zip-bags, simple or T-type plastic nursery tags) rope, tie-wraps and fine wire; transportation containers for plants and cuttings such as different size of plastic pots or boxes, floral foam blocks (Figure 1F), floral stem water tubes (Figure 1G), jars (Figure 1H), 50ml Falcon tubes (Figure 1H), water; pure ethanol, 15 or/and 50ml Falcon tubes for special sampling for microorganisms needed (https://tulips.gr/en/mycorrhiza); tools such as pruning shear, scissor, knife, snap-off knife, swiss army knife, standard plier, shovel, hoe, spade, pickaxe, battery hand compressor. Depending on the type and kind of wild plant material collection from nature and the BOTEX planned, the above described parameters were necessary to be considered. 

The wild plants brought in the IPBGR were placed in a well ventilated area of the nursery under shade. These wild potted plants were frequently observed for their acclimatization in the new man-made environment. Application with special nutrient products such as amino acids, vitamins and biostimulators was employed to speed up the acclimatization proccess; in addition the use of pesticides for plant protection was necessary.

Unique IPEN (International Plant Exchange Network) accession numbers (BBGK-15,5881-4 and BBGK-1-21,19-21) were assigned for H. orientale, after taxonomic identification by the plant taxonomist of ELGO-DIMITRA. During the cold winter months, the mother stock plants were maintained inside an unheated greenhouse at the IPBGR, (altitude 0 m) and later the pots of H. orientale were transferred outdoors in spring upon rise of temperature (>15^oC).

The wild plant material used for cuttings through vegetative propagation was maintained at low temperatures during the BOTEX (fridge), during transportation (mobile fridge, insulated containers, ice or ice packs etc.), and in the IPBGR (fridge) until propagation.

Vegetative propagation procedure

Shoots (rozettes of leaves) were exciced from mother stock plants and immersed for 15 min in a solution (0.5, 1 and 2%) of fertilizers (Theofrastos Fertilizers, Korinthos, Greece) following by a 10 sec soak in an indole-3-butyric acid (IBA) solution (0, 1000, 2000 and 4000 ppm) before planting in a propagation tray. In total, 52 treatments were administered using 13 cuttings per treatment, comprising various combinations of fertilizer product solution and IBA solution concentrations. Additionally, triple control treatments were applied, involving solely water, different concentrations of the fertilizer product solution, and different concentrations of the IBA solution. The fertilizers utilized were IXNO, GLOSTAR, THEOMIN, and THEOCOPPER, which were diluted to 0.5%, 1%, and 2%. The IBA solutions were prepared at 1000 ppm, 2000 ppm, and 4000 ppm of IBA. After the application of these treatments, the cuttings were placed in a multiple-slot propagation tray, in a substrate consisting of three parts of perlite (Geoflor, Perlite Hellas S.A., Volos, Greece) and one part of Terrahum peat (Klassmann-Deilmann GmbH, Geeste, Germany) and were placed on a bench-fog system located in an unheated greenhouse. The experiment took place in March 2024 and the results were evaluated three weeks after the establishment. The measurements employed the weight of total dry mass of the roots produced per cutting. The rooted cuttings were carefully removed from the substrate and the roots were immersed in tap water to remove the substrate carefully without damaging the roots. The roots were then removed from the aboveground part of the plantlet using a scalpel, placed in paper bags, and dried in an oven for 72 h at 65°C. The dry weight of the roots was weighed using a four-digit analytical balance. 

Statistical analysis

For the statistical analysis of the results, the normality of data distributions was evaluated using Shapiro-Wilk tests. Analysis of Variance (ANOVA) was then performed with significance level at a=0.05, followed by post-hoc Tukey tests to assess pairwise differences among groups. All analyses were conducted within the R computational environment (v. 4.2.2) utilizing the dplyr (Wickham et al., 2023), tidyr (Wickham et al., 2023), multcompView (Graves et al., 2023) and patchwork (Pedersen, 2022) packages. Data visualization was carried out using the ggplot2 package (Wickham, 2016).

Molecular analysis

Plant material was collected from wild-grown plants of H. orientale with the IPEN number BBGK-15,5883. Ten molecular markers were used for molecular barcoding of H. orientale; the oligonucleotide primer sequences have been described in similar work for T. sibthorpii by Tsoktouridis et al. (2019). Genomic DNA, PCR procedure, reagents, sequencing of PCR products, and analysis of DNA sequences followed the methodology described previously (Tsoktouridis et al. 2019; Samartza et al., 2024). BLAST searches were applied to all produced sequences using the available online databases (DDBJ/EMBL/GenBank). The pairwise genetic distance and the neighbor-joining tree were constructed using the Molec. Evol. Genetics Analysis (MEGA) 11.0 program (Tamura et al., 2021).

RESULTS AND DISCUSSION

Quality of the initial wild plant material for conservation and propagation.

Bringing an unknown wild plant species from nature into research setting with the plan to deliver it as a cultivated crop to be further utilized as a new product in the market is a very complex procedure. Such an endeavor requires a series of actions and contributions from scientists of various specialties; moreover, this process should follow the appropriate legislative framework. In Greece, to obtain wild plant material for research requires written permission from the Greek Ministry of Environment and Energy, with the obligation of an annual report in the end of the year, which accounts a list of plants and plant material collected during all authorized BOTEXs. Many Greek wild-growing plant species are nationally protected by: (a) the Greek Presidential Decree 67/1981 (“about the protection of the native flora and fauna and on the determination of procedures for the co-ordination and control of research”, Greek Off. Gov. J. F.E.K.A’ 23/30.1.1981, corrected by F.E.K.A’ 43/18.2.1981); (b) the law 1335/1983 (“Ratification of International Convention on the conservation of European wildlife and natural habitats”, Bern Convention 1979, OGG 32/v. Α’/14.03.1983); (c) the law 2005/1992 (CITES “The Convention on International Trade in Endangered Species of Wild Fauna and Flora”); and (d) the European Directive 92/43 ‘Habitats Directive’, (“On the conservation of natural habitats and of wild fauna and flora” that has been adopted by the Greek legislation with the Common Ministerial Decision K.Y.A. 33318/3028/1998, F.E.K.B’ 1289/28.12.1998). The organization of the BOTEXs to collect and bring the selected wild plant material from nature to the laboratory of IPBPR for conservation and propagation is very important procedure with many preparations required prior departure (Maloupa et al., 2008; Krigas and Maloupa, 2021). However, there are additional practical activities that have been developed during the last three years to improve the quality of the wild material brought in the laboratory of IPBPR which facilitate successful propagation and further conservation of these valuable plant materials in a man-made environment. As illustrated for H. orientale in Figure 1, wild plant material (cuttings) was collected from two islands of Greece during expeditions that lasted 4-6 days (Figure 1B-I). It was very important to keep the collected cuttings in the best possible condition during the BOTEXs until their transfer to the laboratory of IPBPR and therefore the whole step by step procedure was described in detail in the section of Materials and Methods. Emphasis should be given to the group of people participating in a BOTEX. Local people are useful for guidance to the unknown areas for the collection of wild plant material and agriculturists to guarantee the pest- and pathogen-free collected wild plant material; moreover, participants of different expertise obtaining practical experience in BOTEXs are required. The maintenance and transportation of the wild plant material throughout a BOTEX until the propagation of the focal species in the nursery of IPBGR (Figure 1J-N) was important and crucial for the successful production of new plants ex situ. The dehydration of the wild plant material should be avoided and therefore various practical measures (Figure 1F-I) were taken to keep the wild plant material fresh and suitable for propagation. When extreme weather conditions (heat) could not be avoided during a BOTEX, a special mobile refrigeration, insulated containers with a good seal to minimize heat penetration, common ice cubes and coolers were the best allies in the battle against hot weather.

Asexual propagation of endemic and native plant species

Most of the crops in the world are commonly propagated vegetatively by massively producing new plants, in a limited space, from a few mother stock plants (Hartman et al., 2014). This asexual type of propagation is favorable compared to the seed propagation method by providing true to type and early generating plants, which consequently produce homogeneous and high quality of products (Santoso and Parwata, 2014). This vegetative propagation method is also used during conservation for endemic and native plant species, to produce clonal elite plants of specific selected genotypes which are identical to their wild individuals in natural habitats. The quality of the wild plant material is very important for successful propagation and therefore it is necessary to have the participation of a specialist (e.g., a horticulturist) during the BOTEXs. This allows the selection of wild plant material (cuttings, bulbs, corms, rhizomes, etc.) from individual plants without infections from pests and diseases; this is very important to avoid transferring infections to the laboratory and nursery of IPBGR. A limited number of wild plant species which were transferred in IPBGR for conservation, were placed in a special isolated area of the nursery or greenhouse for acclimatization, and special treatments were applied for nutrition and plant protection. This procedure is also necessary to monitor pests and diseases on this new material and to avoid contamination of the rest of the plants conserved in the IPBGR. Having the best hygiene for the initial wild plant material, standard techniques were applied for the induction of rooting and plantlet production according to the methodology described by Hartmann et al. (2014).

Four clonal H. orientale lines from Amorgos, Greece and three from Kythira, Greece have been conserved in the IPBPR; vegetative propagation of these clonal mother plants has succeeded and managed to maintain 7-10 plants per H. orientale code in the IPBPR. Although the asexual propagation of H. orientale was successful by cuttings, producing a very good root system using IBA within 3-5 weeks on a table-fog system (Tsoktouridis et al., 2021), the synergistic effect of THEOCOPPER, THEOMIN, GLOSTAR, IXNO in combination with three different IBA concentrations (1000, 2000 and 4000 ppm) was investigated. The use of synergistic compounds besides IBA applications is nowadays a promising new area of research, aiming to omit or even reduce the application of high concentrations of synthetic hormones, to reduce the time required for rooting, to increase plant vitality and response of plants to abiotic stresses (Kisvarga et al., 2022; Rajan and Singh, 2021). The use of a 15-min pretreatment with 0.5, 1 and 2% of THEOCOPPER in combination with 1000, 2000 and 4000 ppm of IBA resulted to a mean number of 76.50, 55.83 and 54.38 mg dry mass of roots accordingly compared to 1000 ppm (3.01 mg), 2000 ppm (17.92 mg) and 4000 ppm (27.84 mg) of IBA (Table 1, Figures 2-4). All treatments of THEOCOPPER combined with the three concentrations of IBA resulted in the production of statistically significant high amounts of root dry mass and therefore is recommended for further investigations to other plant species as well (Figure 2). Similarly, the application of trace elements (IXNO) produced statistically.

Figure 2. Propagation of Helichrysum orientale using cuttings, three weeks after the initiation of the experiment: (a) control treatment, (b) IXNO 2% and without indole-3-butyric acid (IBA), (c) THEOCOPPER 2% and 1000 ppm IBA, (d) THEOCOPPER 1% and 2000 ppm IBA, (e) and (f) THEOCOPPER 0.5% and 4000 ppm IBA.

significant high amounts of root dry mass at the concentrations of 1% (50.29 mg) and 2% (53.50 mg) combined to 4000 ppm of IBA (Table 1, Figures 2-4). 

The application of GLOSTAR produced statistically significant high amounts of root dry mass for the concentration of 0,5% (66.90 mg) combined to 2000 ppm of IBA. The application of THEOMIN produced statistically significant high amounts of root dry mass for the concentration of 0,5% (66.85 mg), 1% (70.74 mg) and 2% (72.49 mg) combined to 2000 ppm of IBA. Synergistic actions were also referred in the literature using coconut water or seaweed extract in combination to 100 ppm IBA, when applied to Conocarpus erectus cuttings, which resulted to the best combinations of all treatments for rooting (Abdel-Rahman et al, 2020). However, the treatments of THEOCOPPER, THEOMIN, GLOSTAR, IXNO without the combination with IBA resulted in poor root dry mass, with best results only for IXNO at 0.5% (15.37 mg), 1% (10.33 mg) and 2% (12.72 mg) compared to the control (1.20 mg) or even to 1000 ppm of IBA (3.01 mg). The treatment of IXNO (a trace element fertilizer) could be considered as a successful hormone-free rooting compound for asexual propagation of H. orientale using adventitious shoots (Table 1, Figures 2-4). Natural root stimulating compounds are safe to use and represent a costless alternative for rooting in vegetative propagation industry; they are eco-friendly and can be utilized to replace synthetic hormones like IBA (El-Sherif et al., 2017). These substances are characterized as groups of biostimulants, organic substances, and microbial inoculants (Abbott et al., 2018). Biostimulants include various groups such as seaweed extracts, amino acids, chitosan, and humic substances (Kisvarga et al, 2022; Rajan & Singh, 2021). Natural rooting hormones are available as vermin wash, coconut water, willow leaf water, honey, humic acid, seaweed or aloe vera extract, cinnamon powder (Rajan & Singh, 2021) and have been extensively used to replace synthetic hormones like IBA and NAA in asexual mass vegetative propagation of horticultural crops. Plant growth promoting microbes can stimulate the formation and development of roots such as Agrobacterium spp., Bacillus spp., Pseudomonas spp. and Azospirillum spp. which are effective in the vegetative propagation of plants (Sarmast et al., 2019).

The most important result during the vegetative propagation of H. orientale was the rapid induction and development of rooting within three weeks; this allowed to release space in the bench-fog system and facilitated the prompt transfer of rooted cuttings to the next stage of acclimatization and plantlet development. Emphasis should be given to the application of fungicides every 7-10 days for the protection from potential pathogens which favor their development (ideal humidity and temperature) during the rooting process. When the massive production of plants utilized stock mother plants maintained in the IPBPR for conservation, treatment of these plants with pesticides for three months was necessary to secure the disease-free and best quality initial plant material for cuttings. The use of fungicides is part of the propagation procedure that has been followed routinely throughout root proliferation and development by dipping the cuttings first in a solution or powder fungicide prior the insertion of cuttings into the rooting substrate (Rajan and Singh, 2021) or spraying the cuttings weekly with fungicides (Assis et al., 2024). 

Acclimatization of vegetatively propagated endemic and native plants

Standard treatments and activities have followed for the acclimatization of the rooted cuttings in the nursery of IPBGR. Almost all wild plant material used for vegetative propagation in

Table 1. Average dry root mass (mg) and standard deviation across various combinations of four fertilizer types (at 0, 0.5, 1 and 2%) and three concentrations (0, 1000, 2000 and 4000 ppm) of indole-3-butyric acid (IBA) applied to cuttings of Helichrysum orientale. The different letters within each column denote statistically significant variances among means, as determined by Tukey post-hoc tests following ANOVA (p < 0.05).

Product		IBA
		0 ppm	1000 ppm	2000 ppm	4000 ppm
			Control: 1.20±0.64 d	Control: 1.20±0.64 f	Control: 1.20±0.64 f
0%		1.20±0.64 d	3.01 ± 2.49 d	17.92±9.61 e	27.84±19.77 cd
GLOSTAR	0.5%	1.88 ± 1.33 cd	16.89±3.88 abc	66.9±11.64 a	27.88±9.12 cd
	1%	1.76±0.43 cd	20.49 ± 9.33 abc	36.25±14.27 bcd	31.3±10.35 bcd
	2%	0.00 e	14.26±5.97 c	36.89±7.71 bcd	44.27±14.11 abc
IXNO	0.5%	15.37±4.12 a	30.31±14.81 abc	24.35±6.75 cde	37.01±18.25 abcd
	1%	10.33±3.79 ab	17.94±9.53 abc	21.74±4.59 de	50.29±10.52 ab
	2%	12.72±6.65 a	23.66±15.81 abc	39.66±15.6 bc	53.5±19.39 ab
THEOCOPPER	0.5%	2.75 ± 1.41 cd	33.61±14.12 ab	27.6±9.10 cde	54.38±13.63 a
	1%	0.00 e	38.78±14.58 a	76.5±16.64 a	46.54 ± 18.39 abc
	2%	3.55 ± 1.00 c	39.04±14.79 a	55.83±18.77 ab	33.76±8.00 abcd
THEOMIN	0.5%	2.32±2.11 cd	17.34±7.12 abc	66.85±10.88 a	27.74±13.09 cd
	1%	5.01±2.98 bc	30.2±11.07 abc	70.74±21.70 a	22.13±12.27 de
	2%	3.58±1.59 c	17.08 ± 9.94 bc	72.49±17.77 a	12.38±3.76 e

Figure 3. Helichrysum orientale propagation treatments using cuttings. Boxplots illustrating the distribution of root dry mass of H. orientale across treatments. The treatments examined herein include the controls and the best treatment per IBA concentration group. The box represents the interquartile range (IQR) with the median line inside. Whiskers extend to 1.5 times the IQR, with outliers plotted individually beyond the whiskers. Different letters above each boxplot indicate statistically significant differences as determined by Tukey post-hoc tests following ANOVA (p < 0.05).

IPBGR originated from plant species of the Greek flora; most of these species have only limited information on propagation available in the literature. Therefore, they are all treated according to the experience obtained in the IPBGR during the last 20 years, by monitoring the growth development of every new wild plant species in the nursery. The first stage of acclimatization took place in a high-humidity curtain-shaded greenhouse. The rooted plantlets remained for 10-15 days, avoiding the direct sunlight; the next 2-3 weeks the plants were transferred in a greenhouse with average humidity and direct sunlight until transplanted to 0.5-1 L plastic pots. An INM application of nutrients was supplied for the best growth of plants (Tsoktouridis et al., 2021). Depending on plant species and growth development the rooted cuttings, different groups of ex situ raised plants were ready to be transferred outdoors. The application of pesticides continued, but gradually was reduced until the hardening of the acclimatized plants. In case the growth and the development of some species 

was problematic, more intense care was required by changing the temperature, light, and humidity factors, by transferring these plants in controlled environments; there was also a choice of simple adjustable humidity dome plastic seedling trays for utilization. Cuttings originated from stock mother plants which have been already conserved in IPBGR presented no acclimatization problems. The type of substrate usually contained 15-30% soil, perlite, peatmoss, and pumice stones (5-30 mm), in proportions depending on the analysis of the soil-sample taken from the wild habitat of the plant species. The species-specific ecological profile

Figure 4. Helichrysum orientale propagation treatments using cuttings. Boxplots illustrating the distribution of root dry mass of H. orientale across the control, three concentrations of indole-3-butyric acid (1000, 2000 and 4000 ppm) in combinations with the fertilizers GLOSTAR, IXNO, THEOCOPPER and THEOMIN. The box represents the interquartile range (IQR) with the median line inside. Whiskers extend to 1.5 times the IQR, with outliers plotted individually beyond the whiskers. Different letters above each boxplot indicate statistically significant differences as determined by Tukey post-hoc tests following ANOVA (p < 0.05).

generated with geographical information systems (GIS) after linking the distribution points of natural occurrences with open-access data regarding temperature and precipitation data of the original wild habitats of plant species was also a valuable tool during the acclimatization process in the man-made environment.

Genetic documentation of endemic and native plants

To date, plant samples from various endemic and native wild plant individuals (genotypes) of the Greek flora were chosen for DNA analysis and genetic documentation in the IPBGR. The selection of plant individual was related to the feasibility and successful vegetative propagation to produce a reasonable number of clonal plants for conservation and future study in IPBGR. A number between 8-15 universal DNA molecular markers were usually applied for every new wild plant species conserved in the IPBGR (Maloupa et al., 2008). The DNA sequences were analyzed and submitted to the GenBank to obtain an exclusive accession number for every molecular marker. For the study in H. orientale, the 18S-26S (Lee et al., 2002), atpF/atpH (Yamane and Kawahara, 2005), psbK/psbI (Pennisi, 2007), trnK-matK (Fazekas et al., 2008), rpoB (Chase et al., 2007), petB/petD (Löhne and Borsch, 2005), trnH/psbA (Ledford 2008), rpoC1 (Chase et al., 2007), rpl16 (Fazekas et al., 2008), trnL/trnF (Fazekas et al., 2008) molecular markers were selected. The accession codes obtained were subject to: 18S-26S (407 bp, KT874493), atpF/atpH (555 bp, KT874491), trnK-matK (861 bp, KT874495), petB/petD (1,014 bp, KT874497), trnH/psbA (455 bp, KT874505), rpoC1 (509 bp, KT874503), trnL/trnF (930 bp, KT874489), rpoB (1,125 bp, KT874507), psbK/psbI (448 bp, KT874499) and rpl16 (1,078 bp, KT874501). The genetic documentation using an internationally accepted molecular barcoding system provided accurate genetic information at nucleotide level for H. orientale and other Greek species, along with useful information about the research team working on the focal plant species, its geographic location and wild habitat. This procedure provided the opportunity to the scientists to insert additional information as ‘notes’ which is open-accessed from the scientific community worldwide. It is worth mentioning that the GPS location of certain rare and threatened plant species was not submitted in full detail to not expedite illegal plant hunters.

Conclusions

The propagation of endemic and native species is not an easy procedure and requires the application of a wide spectrum of methods to achieve the multiplication of an unknown plant species for conservation and the production of plants in a man-made environment. The quality of the initial wild plant material obtained from nature and the methodology followed during a BOTEX is very important for successful propagation. The development of a standardized species-specific propagation protocol for the massive production of plants, which are indented for cultivation is depended to the wild species selection. The synergistic effect of THEOCOPPER in combination with IBA has increased the dry mass of roots of H. orientale cuttings and therefore is recommended to be applied to other endemic and native wild plant species. The application of the trace element fertilizer IXNO was also effective to the induction of rooting without the addition of hormones. Genetic documentation of an endemic or native plant species using universal molecular markers is required for species or genotype authentication, providing open access information worldwide.

Acknowledgements

The authors would like to thank I. Samartza and M. Parlamenta which have helped in various ways with different research lines related to the completion of the research described herein. G. Tsoktouridis also thanks the organizing committee of the Vth European Horticult. Congress, 12-16/5/24 in Bucharest for the invitation as Keynote speaker of. The research related to H. orientale has been co-financed by the E.U. and Greek national funds through the Ministry of Rural Development and Food, (M16SYN2-00419), entitled “Establishment of an Operational Group (EIP-AGRI) to promote new flower crops from rare seldom-seen autochthonous plants of Greece for cut flower production by precision fertilization”.

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