ES2386990B1 - METHOD OF PRODUCTION OF MICORRIZED SCARLS WITH DESERT TRUFFLE. - Google Patents

Abstract

Method of production of plants of the family Cistaceae mycorrhized with the different species of desert truffle. The method uses the cultivation of seeds / explants in photoautotrophic conditions. The plant material is subjected to a progressive adaptation to a decreasing relative humidity to environmental values. # Inoculation of the mycorrhizal fungus is based on spores and / or mycelium. The inoculum is made at room temperature or cold to promote adhesion of the propagules to the inert porous matrix after homogenization. The application of the inoculum is carried out during the pot transplant phase. # The acclimatization phase is 7 to 12 days. 95% survival values are obtained. From 30 to 40 days after acclimatization the percentage of mycorrhization is set at values of 70 to 80%.

Description

OBJECT OF THE INVENTION The present invention falls within the technical field of in vitro and ex vitro production of mycorrhized plant with the different species of desert truffle.

It is a method of plant production that ensures the establishment of mycorrhizal synthesis of plants from seed and micropropagated explants of species of different genera of the Cistaceae family (gen. Milk, Fumana, Helianthemum, Crocanthemum, Hudsonia, Tuberaria, Halimium and Cistus according to Guzmán and Vargas of 2009) with the mycorrhizal fungal genera that form the so-called group of desert truffles (Gen. Terfezia, Tirmania, Picoa, Ba / Samia, Delatreopsis, De / as / Ria, Leucangium, Mattirolomyces and Tuber).

BACKGROUND OF THE INVENTION Desert truffles are edible hypogeous fungi that are symbiotically associated with species of cystaceae and Pinus. Mycorrhizal symbiosis is a nutritional strategy that most plants and some species of fungi have developed to ensure their mutual benefit. The former receive the nutrients and water that the latter have taken from the prospected soil. In turn, the plants close the symbiotic contract with the contribution of carbohydrates to the symbiote fungus (Harley and Smith, 1983). The type of mycorrhiza established by the fungal species included in the desert truffle group together with the cystaceae species is variable. The presence of an external mantle of fungal cells next to the Hartig network (ectomycorrhiza) has been described in culture in vi / ro, while in the greenhouse and / or field phase colonization is mainly intercellular and intracellular (endomicorrhiza). This strategic mycorrhizal diversity does not depend on the fungal species, but rather on the growing conditions (Fortas and Chevalier, 1992; Gutiérrez et aL, 2003). The cystaceae species associated with desert truffles are plants that respond to marked xerophytic conditions, areas where water scarcity, in the form of rainfall, is normal (50-380 mm). This fact highlights the more that

Extensible application in unproductive semi-arid lands to date, allowing to revalue socio-economically rural environments (Honrubia el al. ~ 2007; Murcia el al., 2002; Murcia el al., 2003)

The biogeographic distribution of desert truffles is limited to the arid and semi-arid areas of the different countries that make up the Mediterranean basin, the Arabian Peninsula, Iran, Iraq., Syria, South Africa, North America, Japan and China (Merte el al ., 2008)

The interest aroused by desert truffles lies in its balanced content in proteins, essential amino acids, minerals and fiber, complemented by a high concentration of antioxidant compounds (Honrubia el al., 2007).

The wild collection of desert truffles is in continuous regression due to the gradual loss of their natural habitat, as a consequence of human activities (Morte el al., 2008). These circumstances raise the need to conserve truffle habitats, together with the establishment of viable production protocols and methods to respond to possible habitat restoration plans, as well as the previously mentioned implementation of plantations that revalue rural areas.

The biotechnological aspects involved in the production of the mycorrhized plant are associated with the changing ecology of the host plants. A series of critical factors are contemplated for the establishment of mycorrhizal symbiosis. The characteristics of the soil are very varied, paying special attention to its texture, type of pH, conductivity, organic matter content and C / N ratio.

Currently there are high mortality rates that occur during the acclimatization process in the in vi / ro cultivation protocols, therefore there is no large-scale and economically viable method that allows transfer of Cislaceae family plants from the in cultivation Potted vitro.

The fact is that both seedlings from seed, and explants, after cultivation in in vi / ro conditions, are exposed to biotic (microflora) and abiotic (light, temperature, environmental humidity) stress conditions. Therefore, they need an acclimatization phase for their establishment and subsequent survival (Deb and Irnchen, 2010). In general, plants grown in in vi / ro conditions have non-functional stomata, weak root system and poor development of waxes and cuticles (Mathur el al., 2008). The acclimatization process favors the necessary adaptations at the morphological and metabolic level of the plants to the external environment (Pospósilová el al., 1999).

Those abiotic factors involved in the growth of the plant in vitro and that directly influence the quality of acclimatization would be the following: an unsuitable light intensity causes photoinhibition and bleaching of chlorophylls, as it does not have a functional photosynthetic device ( Van Huylenhroeck, 1994; Van Huylenbroeck al., 1995); Environmental humidity is directly implicated in the delay in the development of waxes, cuticle, non-functional stomata, causes of the high rates of perspiration that occur when the plants are removed from the culture containers (Chen and Chen, 2002; Pospósilová et al. , 1999). To alleviate this rapid drying, the use of antiperspirant and growth retardant compounds has been proposed to improve the survival rates associated with acclimatization (Chandra el al., 2010). In general, the intake of water and solutes by the plant is very limited by the poor ability to drive the roots (Fila el al., 1998; Short el al., 1985). The origin of the high relative humidity value is due to the low rate of exchange of gases with the outside, which takes place inside the culture boats (Chen and Chen, 2002; Huang and Chen, 2005); The concentration of carbohydrates, present in a common way in the composition of the in vitro culture media of seeds and explants, negatively affects due to the fact of changing in a short time from a heterotrophic to autotrophic nutrition (Wainwright and Scrace, 1989) . To overcome this problem, some in vitro cultures have been developed in a photoautotrophic way (Kozai, 1991); Abscisic acid levels are a parameter that plays an important role in the water balance of the plant, as well as in its adaptation to different stress situations (Finkelstein and Gibson, 2002; Hetherington, 2001). This plant hormone is transported via xylem to the stems where the mechanism of regulation of water loss by perspiration is launched, as well as the regulation of leaf growth (Hronková el al., 2003).

Biotic factors can generate stress situations caused by the microbial communities present in the soils naturally, but absent in in vitro cultures. This fact highlights the maladaptation of seedlings to the external biotic environment (Chandra el al., 2010). Work begins on the use of microorganisms during in vitro culture. This process takes the name of bio hardening (Srivastava al., 2002). Through these emerging protocols endophytic bacteria and vesicular-arbuscular fungi are used to improve survival rates during acclimatization (Chandra al., 201 O) of mycorrhizal plants.

DESCRIPTION OF THE INVENTION The following invention describes a method for the production of plants of the species of the family Cistaceae mycorrhized with the different species of desert truffle. The method is based on the in vitro and ex vitro photoautotrophic production of seeds and explants of plant material that adapt during their cultivation to a gradual hardening derived from the changing conditions to which they are subjected until the definitive transplant to a pot. Thanks to this progressive hardening, the acclimatization phase is successfully overcome. The potted plant is contacted with a spore and / or mycelial nucleus embedded in a porous matrix that facilitates the adhesion of fungal propagules and the development and colonization of the roots, not yet suberized of the seedlings. The application of this method of plant production definitely solves the problem that carries with it the high mortality of plants of the Cistaceae family during the acclimatization phase, as a consequence of the various biotic and abiotic stresses to which the plant grown in is subjected vitro. The production method solves the morphological and / or metabolic deficiencies that the plant grown in vitro carries due to the conditions of photomixotrophic growth. In the first place, the saturating relative humidity associated with the delay in the development of waxes, cuticle and functional stomata ceases to be a problem because the production method allows a progressive decrease thereof, up to values present in the natural growth environments. Therefore, antiperspirant or growth retardant compounds are not necessary. Secondly, photoinhibition and / or chlorophyll bleaching as a result of high irradiance has no place, because the seedling / explant grow and develop from the beginning of the crop, in the absence of an external carbon source, allowing the plant to establish an autotrophic nutrition from the beginning. Likewise, the intensity can be established at high values (95-295 micromol * m <2 * s'l) from the beginning of the crop or carry out a progressive adaptation, as required by the cystacea species, thus respecting its ecological adaptation in natural conditions The absence of a carbon source does not favor the growth of unwanted microbiota. Third, the root apparatus as well as its stem connections are functional from the germination of the seed or

from the growth of adventitious roots in the explants. A complete method of plant production of the mycorrhized Cistaceae family with desert truffle has been characterized as a complementary alternative to production through explants and agar seed plants. Through the production method we carry out the replacement of the agar with perlite. This substrate has a porous matrix that favors the aeration of the crop and promotes the development and growth of functional roots.

In autoclavable containers a volume equivalent to 20-30% of the container, of the inert perlite substrate is deposited. This volume of perlite is irrigated with a nutrient solution of MS medium (Murashige and Skoog, 1962), without a hydrocarbon source. The irrigation medium is established at a variable pH and dependent on the species to be cultivated. The pH values will range between 5 and 8. Irrigation of! substrate will depend on the granulometry of perlite used. For horticultural perlite e! irrigation will be done in such a way that e! water potential of the irrigated substrate ranges between ° and -2 KPa. A perforable and disposable plastic lid that fits its autoclave is fitted to the mouth of the container. After autoclaving the porous matrix, based on perlite irrigated with nutritive medium and under sterile conditions, the adjustable lid is removed, the seeds previously sterilized on the surface are deposited by the usual means, as well as the previously selected explants. The density of the crop will depend on the surface available in the container, preventing them from overlapping in e! container areas of! canopy of each seedling. During the development of the crop we take advantage, first, the plastic cover and second, the canopy area to eliminate losses by evaporation of moisture from the substrate avoiding increasing the salinity of the medium and therefore, situations of osmotic stress.

Once we have established the seeds / explants, the containers are placed in growing conditions: 16-26 oC; 30-65% relative humidity; 350-400 ppm of C02; 95295 ~ mol * m · 2 · s · '; 0.1 -0.7 my wind speed. In order to carry out the gradual hardening, work has been actively carried out on the control of the relative humidity of the environment in relation to that present inside the sealed container and the initial hydration of the substrate expressed as water potential, determining factors in the acclimatization of the plant.

In comparison with US20031l0687-A1 (Kozai el al., 2003), where they use sugar cane as a plant material - C4 plant - and an atmosphere enriched in CO2, the cultivation conditions proposed by this method do not use an atmosphere enriched

Based on the phenology of each plant species, it is estimated that the period of stay in the vessel is defined by the conservation of the juvenile state of the roots, a decisive factor in the establishment of mycorrhizal symbiosis. The choice of the appropriate container should be assessed according to the growth in height of the plant in relation to a good root system with juvenile characteristics.

Once the emergence and development occurs from the embryo of the essential organs, root system, hypocotyl, epicyclic and cotyledons, we proceed to perforate the plastic shed. The size of the perforations will range from 0.5 to 1 mm on the surface of the perforable plastic cover that will facilitate the loss of relative humidity. Your number will be set based on the volume of the container. The balance of the gas exchange established in the container is given by the external conditions of the growing area. The moisture loss obtained with this method is described by a decreasing exponential curve.

The opening of the perforations influences the exchange rate of gases: CO2, ethylene and water vapor. The progressive destabilization of the leaf boundary layer, since the opening of the first perforation, facilitates the bidirectional diffusion of the gases at the leaf level. Thanks to a limited, but necessary wind speed inside the growing areas, we favor the renewal of the gaseous environment inside the container. The gradual decrease in the concentration of water vapor inside the container favors the vapor pressure gradient between the leaves and the atmosphere of the container. This progressively reaches a more negative value of water potential compared to that present in plant tissues, generating a leaf-air gradient. Thanks to the progressive adaptation to states of decreasing relative environmental humidity, the plant adapts stomatic control gradually and not at the time of acclimatization. Associated with a correct regulation of foliar perspiration, the roots are functional from the moment of their emergence.

The use of perforated plastic caps against the usual in vitro plant culture containers avoids the low concentrations of CO2 present in the light phase and the high concentrations during the dark. CO2 can be a limiting factor for plant growth during the IUI ~: Iinosa phase. Through the passive diffusion that takes place between the perforated vessel and the atmosphere, the concentrations of the different

ES 2 386 990 Al

gases are balanced until usual in the external environment described above. In the face of biotic stress associated with this method, the use of culture media

Carbohydrate-free limits the development of unwanted microbial flora. Once the in vi / ro growth phase is finished, the transplant is carried out in

5 previously autoclaved culture substrates. The texture of said substrate mixtures should be established in such a way that it facilitates drainage of the culture container in order to avoid the growth and proliferation of pathogens that displace mycorrhizal symbiosis. Texture and water load requirements of the soil are established in Table 1. The physical-chemical profile of the substrate must be established between the values of the soil.

10 Table 2.

Table 1. Car-aCleristics of transplant substrates. Where, CC: field capacity; PMP: permanent wilting point; AV: useful water

Soil texture

Moisture content (% dry weight)

Franco-Sandy

ce 6-18 PMP 2-8 AV 5-8

Franco-Arcillosa

18-31 81.5 8-12

Table 2. Table 2_ Physico-Chemical Characteristics of the transplant substrates.

Physical-Chemical Determinations

pH (in water 1: 2.5)

6.8-8.7

EC. 1: 5 (microS / cm)

123.1-302

Assimilable sodium (meq / lOO g)

0.15-1, 20

Assimilable potassium (meq / lOO g)

0.28-1.5

Assimilable calcium (meq / lOO g)

4.94-23.38

Assimilable magnesium (meq / l 00 g)

0.95-3.7

Chemical Determinations

Organic material (%)

058-3.92

Total orgflnioo carbon (%)

0.34-2.28

Total Nitrogen (%)

0.058-0.267

C! N ratio

2.8-10.15

Total carbonates (%)

5.1-80.1

Active limestone ("'lo)

3.4-24.76

Assimilable Phosphorus (ppm)

7.5266.4

Chlorides (meq / IOO g)

0.05-0.09

Sulfates (meqllOO g)

0.01-0.32

Assimilable iron (ppm)

1.79-79.5

Assimilable Copper (ppm)

0.31-2.73

Asimi lable manganese (ppm)

3.03-57.12

Assimilable Zinc (ppm)

0.3-3.12

Adhesion of mycorrhizal propagules is carried out with a short

cold treatment A partial cold drying procedure has been developed (5

8 OC) of the mixture to favor the adhesion of the spores and hyphae to the pores and

perlite cavities once the water has been absorbed by the perlite and

5

evaporated by cooling action in a refrigerated chamber, for a time not

greater than 60 minutes and less than 30 minutes.

In the transplant phase the roots of the plant are contacted with

a porous matrix containing the spore and / or mycelial inoculum grown in vi / ro.

The inoculum percentage will be 5 -10% of the volume of the final container.

\OR

Then the containers are exposed to the variable conditions

nursery, between wide thresholds of external conditions: <40 "'C;> 20% humidity

relative; 350-400 ppm of C02; 295-1000 ~ mol · m-2 · s-1; variable wind speed.

Irrigation will be carried out in such a way that the water tension measured in the area of

Maximum root density ranges from -15 to -30 KPa. Irrigation management is vital

fifteen

importance to prevent the proliferation of pathogens that compete with symbiosis

mycorrhizal

After the first 30-40 days after transplant, proceed to

quality control of mycorrhization by differential staining of the structures of

fungal chitin and their count. With this production method you get

twenty

mycorrhization percentages of 75-85% at the end of container growth.

DESCRIPTION OF THE DRAWINGS

Figure 1. Phases of progressive hardening inside the container, of the

Helianlhemum almeriense, H violaceum and H canariense species. Moisture in the

Inside the container has been recorded as absolute humidity (gr / cm 3) and humidity

25

relative (%). These humidity values belong to the growing conditions: 18-22

oC; 30-35% external relative humidity; 360 ppm C02; 150-170 micromol + m-2 · s1;

0.3 -0.5 my wind speed. It has been recorded that the absolute moisture loss

(gr / cm3) to match the present in the culture environment is described by a

exponential equation during Phase 11: y = 16,927 x (e -0.02 + day). In the case of

30

relative humidity (%), is described by an exponential equation during Phase 11:

y = '88,302 x (e -0.02 day).

Figure 2. Foliar area of the three species of Helianlhemum spp. studied. The columns belonging to each culture medium, within each species and that do not share the same letter are significantly different (ANOVA; p <O, 05). (HA = Helianthemum almeriense; HC = H canariense; HV = H violaceum; FA = Photo autotrophic; FMA = Photomixoautotrophic).

Figure 3. Stem length of the three species of He / ianthemum spp. studied, The columns belonging to each culture medium, within each species and that do not share the same letter are significantly different (ANOVA; p <O, 05). (HA = Helianthemum almeriense; HC = H canariense; HV = H. violaceum; F A = Photoautotrophic; FMA = Photomixoautotrophic).

Figure 4. Root length of the three species of Helianthemum spp. studied. The columns belonging to each culture medium, within each species and that do not share the same letter are significantly different (ANOVA; p <O, 05). (HA =

Helianthemum almeriense; HC = H. canariense; HV = H. violaceum;

F A = Autotrophic photo; FMA = Photomixoautotrophic).

Figure 5. Total chlorophyll concentration, estimated for the three species of Helianlhemum spp. The columns belonging to each culture medium, within each species and that do not share the same letter are significantly different

(ANOV A; p <0.05). (HA ~ Helianthemum almeriense; HC ~ H. canariense; HV ~ H. violaceum; FA = Photo autotrophic; FMA = Photomixoautotrophic).

Figure 6. Concentration of the chlorophyll a fraction, estimated for the three species of Helianthemum spp. The columns belonging to each culture medium, within each species and that do not share the same letter are significantly different (ANOV A; p <O, 05). (HA = Helianthemum almeriense; HC = H. canariense; HV = H. violaceum; FA = Photo autotrophic; FMA = Photomixo autotrophic)

Figure 7. Concentration of the chlorophyll b fraction, estimated for the three species of Helianthemum spp. The columns belonging to each culture medium, within each species and that do not share the same letter are significantly different

(ANOVA; p <O, 05). (HA = Helianthemum almeriense; He = H. canariense; HV = H violaceum: FA ~ Photoautotrophic; FMA ~ Photomixoautotrophic).

Figure 8. Concentration of the xanthophyll and carotenoid fraction, estimated for the three species of Helianthemum spp. The columns belonging to each culture medium, within each species and that do not share the same letter are significantly different (ANOV A; p <O, 05). (HA = Helianthemum almeriense; HC ~ H. canariense; HV ~ H. violaceum; FA = Photo autotrophic; FMA = Photomixoautotrophic).

Figure 9. Total chlorophyll concentration estimated by SPAD 502, of the three species of Helianthemum spp. studied. The columns belonging to each culture medium, within each species and that do not share the same letter are significantly different (ANOVA; p <O, 05). (HA = Helian / hemum almeriense; HC = H. canariense; HV = H. violaceum; FA = Photo autotrophic; FMA = Photomixoautotrophic)

PREFERRED EMBODIMENT OF THE INVENTION Here is an example of the application of the production method to three species of the genus Helian / hemum, which is not intended to limit its scope. The selected species are: H almeriense, H violaceum and H canariense. For all species, we work with explants and seeds, in each case the characteristics of their processing are detailed. The process description is detailed below:

1. Seed and micropropagation

11. Inoculation and acclimatization

III. QA

1. Seed and micropropagation

to. Obtaining the seed.

Obtaining plant material begins with field collection, classification and labeling of Helian / hemum species. The aerial part is collected and

Press for later storage in herbarium. Then, the

maturation level with the help of a stereo microscope. Finally it is labeled and

Store plant material in darkness at room temperature.

b. Preparation of culture media.

5

For the micropropagation of the explants and the sowing of seeds of the

different species of Helianthemum in medium with agar and hydrocarbon source

We follow the established protocols (Morte and Honruhia, 2009) For the cultivation of

seeds and explants we work with 580 ml autoclavahle glass containers where

a volume of 150 horticultural perlite is deposited. This volume of perlite is irrigated with

!OR

100 ml of MS nutrient solution, pH 6.5 (Murashige and Skoog, 1962) without source

hydrocarbon Here are two ways to follow:

Track 1. The autoclavable transparent plastic cover fits into the container

perforable in the mouth of the container and proceeds to autoclave sterilization

(20 minutes, 121 0C) ~

fifteen

Via 2. Containers containing the culture medium are deposited in bags

autoclave and autoclave sterilization (20 minutes, 121 ° C).

In sterile environment it is opened to deposit the

explants / seeds inside the containers with sterile culture medium.

Then the perforable transparent plastic cover fits into the

twenty

bowl mouth

C. Seed preparation and seed planting:

Scarification and sterilization according to established protocols (Morte and Honrubia,

2009). The adequate density of explants ranges from 4-6 explants and 5-10 seeds

by boat

25

d. Placement in the culture chambers:

Under the following culture conditions: 18-22 oC; 30-35% relative humidity

external; 360 ppm C02; 150-170 ~ mol * m-2 * s-1; 0.3 -0.5 my wind speed.

and. Opening of the perforations after germination / adventitious roots:

From 7 to 10 days after the laying of the seeds and 18-25 days in case of

30

explants proceed to drill the first holes on the surface of the

plastic.

We have optimized the process with the opening of 7-12 holes / boat. Each hole

It is 0.5 to lrnm in diameter. In Figure 1 we reflect the effect of the opening of

said holes on gas exchange rates, in particular humidity

absolute (gr / m3) and relative humidity (%). This matches with the present in the

external environment of the culture chamber. Registered moisture loss rates

They are governed by an exponential curve (Figure 1). The application of the

S

production method of mycorrhized plant, in the case of species of

Helianlhemum, by establishing a moisture loss rate at

measure, which allows the development and growth within the culture vessels

during its progressive adaptation to external conditions. As depicted in the

Figure 1, during Phase 1 there is an emergency and development from the embryo of

10

The essential organs, root system, hypocotyl, epicotyl and cotyledons, is the

moment we proceed to drill the cover plastic. This fact gives way to

Beginning of Phase 11, the graph represents an exponential response (R2 = 1) for the

loss of absolute and relative humidity, with respect to the initial one kept in the container

without perforations Phase III begins by removing the cover plastic from the container,

fifteen

at this time the absolute humidity values of the

container, to match those present in the external environment. This is the time to

begin the transplant to the final substrate.

Below are the results where the

significant differences between photoautotrophic culture (AF) AND

twenty

photomixotrophic (FMA) of seedlexplants of the species H. almeriense, H.

violaceum and H. canariense. We have selected a series of parameters that we

They estimate the quality of the plant to be acclimatized. These are: leaf area,

stem and root length, photosynthetic pigments, total chlorophyll estimation

by the measure of SPAD 502 (Mino Ita. Tokyo. Japan).

25

After carrying out the different determinations of the physical parameters,

notes that there are significant differences (p: SO, 05) between FA treatments and

FMA

In the three species, the physical variable, foliar area is approximately 50%

greater in FA treatment compared to FMA (Figure 2), these differences are

30

significantly different for the three species of Helianlhemum,

The leaf area is a variable that defines the quality of the crop. In FA crops,

a larger foliar area has higher initial photosynthetic rates and, therefore, rates

of greater growth. Also, low relative humidity pressures inside

of the FA culture boats also favor the development of leaf areas and lengths of the stems higher than those grown in FMA crops. This behavior is reflected in the data represented in Figure 2, where the leaf areas of the FA cultures are superior.

The length of the stems (Figure 3) registers superior growth in the three species, as was the case with the leaf area.

Thanks to a greater length in the stems and therefore greater bearing of the plant, we see shortened the subculture time of the crop plants for the FA treatment, in approximately two weeks. This time saving is essential for the viability objectives of any nursery company, which boasts of producing mycorrhized plant.

At the root level (Figure 4), we find a significantly higher development in those plants that have been developed in FA culture medium, for all species. The substitution of the conventional agar gel with porous materials considerably affects the root zone and therefore its anatomical characteristics.

A good development of the root system helps the intake of water and nutrients, promotes the correct growth of the seedlings and improves their health, resulting in higher survival rates at the time of transplantation ~ in environmental conditions (Afreen et al. , 1999). It has been successfully verified in other woody species, such as eucalyptus (Eucalyptus camaldulensis Dehnh.), The correlation of greater survival in ex vitro conditions associated with better root development (Kirdmanee al., 1995).

As far as photosynthetic parameters are concerned, a significant increase in the concentration of photosynthetic pigments (p '::; O, 05) has been observed for AF treatments with respect to FMA. We observe that the results of total chlorophyll and the majority chlorophyll a, are superior (p: SO, 05) for the three species of jarilla. A proportion of one third of chlorophyll b, with respect to chlorophyll a, is observed for all treatments and species (Figures 5-7). In FA culture, photosynthesis is the only source for the production and accumulation of carbohydrates.

As can be seen in the results of Figures 5 through 8, the FA-type culture, which has favored gas exchange with the culture environment, shows a significant increase in photosynthetic pigments.

In the case of xanthophylls and total carotenoids (Figure 8), we observed an increase in the three species of jarilla, but not significant in the case of H. canariense. The function of this family of pigments lies in providing mechanisms for dissipation and extinction of light energy (Rivas, 2008). The data obtained inform us that those plants that grow in the FA medium have higher values of these pigments and is related to a better adaptation to higher light intensities, once transplanted.

In parallel to the measurement of the concentration of photosynthetic pigments, non-destructive arrest of total chlorophyll is provided through the use of SPAD502 (Figure 9). When comparing the values of SPAD502 with those of total chlorophyll (Figure 5), we observed an identical behavior for each species and within each treatment.

11. Inoculation and acclimatization

to. Substrate Preparation:

For the preparation of the mixtures, substrates for horticultural use are used - (1) perlite, (1) peat, (1) sand-adjusting the physico-chemical and texture requirements described above. Once the mixture is done, it is autoclaved (60 minutes, 121 o C). The mixture is deposited in 220 cm culture containers) for use in transplantation.

b. Obtaining inoculum:

The collection is carried out in the field during the fruiting period. Thereafter, the level of spore ripening, drying, crushing, labeling and storage is established (Honrubia et al., 2007). In order to obtain mycelium in sufficient quantity, it is isolated in vitro culture for subsequent growth in bioreactor (Morte and Honrubia, 2009).

C. Inoculation

For the previous preparation of the inoculum, either spore or mycelial produced in bioreactor we proceed with the established protocols (Mars and Honrubia, 2009). Once prepared, it is carried out by mixing one or the other with an inert porous matrix - perlite - in a proportion of 1 to 10% by volume, of the inoculum with respect to the porous matrix.

After homogenization, the preparation of the inoculum can be carried out at room temperature or by means of a partial cold drying process (5-8 OC) of the mixture to favor the adhesion of the spores and hyphae to the pores and cavities of the perlite once that the water has been absorbed by the perlite and evaporated by cooling in a refrigerated chamber, for 45 minutes.

d. Transplant

Plants from seed and / or explant are put in direct contact with the inoculum prepared in Il.e. when the transplant was performed. The percentage of inoculum that is deposited is 5 -10% of the total volume of the pot.

Irrigation is carried out in such a way that the water tension measured in the area of maximum root density ranges between -25 and-3D KPa. Special attention should be paid to these values of water potential, since they prevent the proliferation of pathogens in the root zone and favor the establishment of mycorrhiza.

and. Acclimatization

This phase is shortened to the first 7-12 days after the transplant. The nursery conditions that must be maintained for these three species are: <30 ° C; > 50% relative humidity; 350-400 ppm of C02; 295-1000 ~ mol · m · 2 · s'; variable wind speed. The survival rate of acclimatized plants after the first 7-12 days is 95%.

1II. QA

After forty days in nursery conditions <40 ° C; ambient relative humidity ~ 350-400 ppm of C02; 295-1000 ~ mol · m · 2 · s'; variable wind speed. the quality control of the mycorrhization is carried out. Fungal colonization is visualized by staining the roots in Chinese ink, according to the method of Phillips and Hayman. The percentage of mycorrhized roots is estimated according to the method of Gionvannetti and Mosse (Giovannetti and Mosse, 1980; Phillips and Hayman, 1970). This method ensures that mycorrhization percentages are higher than 80% while the nursery conditions can exceed the previously indicated values.

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one.

Claims (3)

l. Production method of plants of the Cistaceae family (gen. Milk, Fumana, Helianthemum, Crocanthemum, Hudsonia, Tuberaria, Halimium and Cistus) mycorrhized with desert truffle species (gen. Terfezia, Tirmania, Picoa, Ba / samia, Delalreopsis, From / asria, Leucangium, Mattir% myces and Tuber) characterized by presenting two stages. The first stage is called seed and micropropagation, in this the plant material comes from seed and / or explant; its cultivation is carried out in photoautotrophic conditions; The plant material is exposed to decreasing conditions of relative humidity described by a decreasing exponential curve. The term of the first stage is defined by the conservation of the juvenile state of the root system together with an adequate bearing of the seedling for transplantation. The second stage is called inoculation and acclimation, in this the spore nodule and / or mycelial coinciding with the moment of transplantation of the seedlings that come from the first stage. The inoculum is subjected to a short cold treatment to favor the adhesion of the mycorrhizal propagules to the pores and cavities of the perlite used as an inert support. The progressive adaptation of the seedlings to a decreasing relative humidity favors the acclimatization being significantly shortened. At the end of this stage, plants of the mycorrhized Cistaceae family are obtained with desert truffle species with a high percentage of mycorrhization. 2. Method of production of plants of the family Cistaceae (gen. Milk, Fumana. He / ianthemum. Crocanthemum, Hudsonia, Tuberaria, Ha / imium and Cistus) mycorrhized with desert truffle species (gen. Terfezia, Tirmania. Picoa, Balsamia, Delatreopsis, Delaslria, Leucangium, Mattirolomyces and Tuber) according to claim 1 which presents a first stage of seed and micropropagation characterized by being carried out in autoclavable containers that penetrate the passage of photosynthetically active light where an equivalent volume of perlite is deposited 20-30% of the container. This volume of perlite is irrigated with a nutrient solution of Murashige and Skoog medium without a hydrocarbon source with a pH ranging between 5 and 8 depending on the species to be cultivated. The irrigation of the perlite is carried out in such a way that the water potential of the irrigated substrate oscillates between O and -2 KPa. A perforable and disposable plastic lid is fitted to the mouth of the container. After autoclaving the set, container, watered substrate and lid, it is removed, depositing previously disinfected seeds or previously selected explants. The density of the crop depends on the area available in the container, preventing the canopy areas of each seedling from overlapping in the container. The lid is then adjusted to the mouth of the container to start the cultivation of the seeds and / or explants. Containers with seeds / explants are placed under growing conditions: 16-26 oC; 30-65% relative humidity; 350-400 ppm of CO .; 95-295 ~ mol'm- "sl; 0.1-0.7 my wind speed. Next, a progressive adaptation of the seedlings / explants to the external environmental conditions is carried out thanks to the perforations of 0, 5 to 1 mm in diameter that are carried out on the surface of the perforable plastic lid, once the emergence and development of the root, hypocotyl, epicotyl and cotyledon systems occurs.The number of perforations is established according to the volume of the container. Loss of moisture obtained with this method is described by a decreasing exponential curve.Depending on the phenology of each species of the Cistaceae family, it is estimated that the period of stay in the vessel is defined by the conservation of the juvenile state of the root system together with a adequate height for your transplant, factors that limit the choice of the culture vessel. 3. Method of production of plants of the Cistaceae family (gen. Milk, Fumana, Helianthemum, Crocanthemum, Hudsonia, Tuberaria, Halimium and Cistus) mycorrhized with desert truffle species (gen. Terfezia, Tirmania, Picoa, Ba / samia, De / atreopsis, De / astria, Leucangium, Mattirolomyces and Tuber) according to claim 1 presenting a second stage of inoculation and acclimatization characterized by proceeding to the transplantation of the seedlings that come from the first stage to the cultivation substrate. The texture of said substrate is established in such a way that it facilitates drainage and maintains the water potential in the area of maximum root density between -15 and -30 KPa. The preparation of mycorrhizal inoculum is done from two types of propagules in between liquid, spores and / or mycelium hyphae. It is carried out with a short cold treatment (5 to 8 'C; 30 to 60 minutes) between perlite and possible mycorrhizal propagules, to favor the adhesion of the propagules to the pores and cavities of the perlite once the phase liquid is absorbed by the perlite and evaporated by cooling action. During the transplant the juvenile roots of the seedling are contacted with the mycorrhizal inoculum prepared previously, the percentage to be applied is 5 to 10% of the volume of the final container. The acclimatization phase takes place in seed / explant culture conditions for 7 to 12 days with a 95% survival of the 10 seedlings inoculated and transplanted. After acclimatization the seedlings are exposed to the following conditions: <40 ° C; > 20% relative humidity; 350-400 ppm of CO2; 295-1000 ~ mol · m-2 · s-l; variable wind speed. After the first 30 to 40 days after inoculation and transplantation, percentages of mycorrhization are obtained from 70 to 80% in the roots.