RO133900A2 - Device for ex-vitro acclimatization - Google Patents

Abstract

The invention relates to a device for the ex vitro acclimatization of in vitro rooted seedlings and/or for the simultaneous ex vitro rooting and acclimatization of sprouts obtained in the multiplication phase. The claimed device consists of a mini basin (1) made of transparent material, which has at its base a black strip (2) to prevent contamination of the water (3) in the mini basin (1) with algae (3), the mini basin (1) being provided with a removable cover (4), with ventilation windows (5), with a water supply orifice (6) provided with a valve and with a water drainage system (7) which is also provided with a valve, in the mini basin (1) there being a support-tray of polystyrene for vitroplants, made in two versions: a simple tray (8) of polystyrene, with strips (9) of a material with capillary properties, in which pearlite (10) is placed, or a tray (11) of polystyrene with orifices of preset diameter, through which the roots of the plants reach the water and some acclimatization devices which can be superposed, in which case they can be optionally connected to the water distribution network (12) and to the waste water collection network (13).

Description

Ex vitro acclimatization device

Description of the invention

1. The invention relates to a device for ex vitro acclimatization of in vitro rooted seedlings and / or for concomitant ex vitro rooting and acclimatization of shoots obtained in the multiplication phase, intended to be used for the final phase of plant micropropagation.

2. It is known that micropropagation allows the rapid production of high quality propagating material, free from diseases and pests and genetically uniform, regardless of season and weather. However, a major limitation in the widespread application of this technology is the loss of plants during transfer from the laboratory, from in vitro conditions to ex vitro conditions. During this transfer, vitroplants are exposed to abiotic stress (temperature, light intensity or humidity) but also biotic (microflora in the substrate), which is why they need acclimatization to survive in the new conditions (Deb and Imchen, 2010).

3. Ex vitro acclimatization is the final phase of plant micropropagation, a phase in which the seedlings are gradually transferred from artificial living conditions in in vitro culture under conditions of strict asepsis, controlled temperature and very high humidity to normal greenhouse living conditions. or field, close to natural conditions. This intermediate phase is necessary because seedlings grown in vitro cannot survive in the case of direct transfer under natural conditions, as they do not have the physiological characteristics necessary for this. During the acclimatization phase takes place the normal development of seedlings and the acquisition of physiological characteristics that ensure their survival in normal, artificially uncontrolled environmental conditions.

4. The rooted seedlings, resulting in the in vitro rooting phase or, in some cases, the non-rooted shoots from the multiplication phase are removed from the in vitro culture vessels, washed to remove traces of nutrient medium after which they are planted in special substrates. (soil, peat, perlite, vermiculite, etc. or mixtures thereof) then watered and placed in specially arranged places (vegetation rooms, greenhouses, solariums, tunnels) where they are provided

05/09/2018 of 2018 00649 controlled conditions of temperature, ventilation, light and especially humidity, the latter being close to 100% and, as the seedlings develop during the acclimatization phase, the atmospheric humidity is gradually reduced.

5. The main problem of seedlings grown in vitro is the lack of adequate formations to ensure their survival under normal conditions of humidity. Thus, there is no adequate cuticle and functional stomata to regulate sweating. Therefore, the main environmental factor that must be artificially controlled during the acclimatization phase is the humidity of the air, in order to prevent the dehydration of the seedlings.

6. Posposilovâ et al. (1999) show that due to the special conditions during in vitro culture result in morphologically, anatomically and physiologically abnormal plants that after ex vitro transfer, can be easily affected by sudden changes in environmental conditions so they need a period of acclimatization to correct anomalies.

7. As a result of the above, the efficiency of the seedling acclimatization process as well as the simultaneous rooting and acclimatization ex vitro of the shoots obtained in the in vitro multiplication phase aim to increase the percentage of acclimatized plants, reduce losses in the acclimatization phase and by consequently a lower cost of plants obtained by micropropagation.

8. In the patent literature there are data on the phases of in vitro cultures as well as some aspects related to ex vitro acclimatization, but there are no patents describing an acclimatization device.

9. U.S. Pat. No. 516,8059A discloses a process for improving the viability of plants in the transfer from in vitro micropropagation conditions to ex vitro conditions which consists in incorporating a concentration growth regulator from 0.001 to 10 in vitro culture medium. mg per liter of medium, the growth regulator being an inhibitor of the gibberellin pathway.

10. U.S. Pat. No. 4,854,252A discloses a process for improving the efficiency of plant micropropagation comprising treating plants during subsequent micropropagation and / or planting with an aqueous solution containing an amide or mixture of amides.

05/09/2018 of 2018 00649 substituted or a diamide or a mixture of substituted diamides to increase the survival rate of plants during acclimatization.

11. Also, in the specialized literature were carried out various studies on ex vitro acclimatization in which different culture substrates were tested for ex vitro planting or different culture media leading to obtaining vitroplants as suitable as possible for acclimatization but there are no data on the construction of an acclimatization device.

12. Najaf-Abadi and Hamidoghli (2009) reported an acclimatization of 85% to blackberry in the conditions in which they acclimatized seedlings rooted in vitro on media supplemented with IBA (indole butyric acid). The in vitro rooted seedlings were acclimatized in plastic containers mixed with peat and perlite (1/1, v / v), under glass, for three weeks then transplanted into pots. In comparison, in the acclimatization device described in the present patent, the blackberry shoots obtained in the multiplication phase were 98% acclimatized using water as substrate. Thus, the in vitro rooting phase, the ex vitro wire transplantation, the surface occupied by the plants during acclimatization, the plastic containers and the mixture of peat and perlite are eliminated.

13. Roy et al. (2001) describe a protocol for hop micropropagation that includes the in vitro rooting phase and acclimatization in a soil mixture that provides a 95% acclimatization percentage. In comparison, in the acclimatization device described in the present patent, in vitro rooted hop seedlings can be 100% acclimatized in polystyrene tray with perlite and unrooted shoots obtained in the multiplication stage can have a direct rooting and acclimatization percentage. in vitro polystyrene tray with 97.33% perlite, in which case the in vitro rooting phase is eliminated.

14. Lobna et al. (2008) developed a protocol for micropropagation in paulownia (Paulownia kowakamii) that proposes in vitro rooting and acclimatization of in vitro rooted plants in a mixture of peat moss and sand (1: 1) ensuring a 90% acclimatization percentage. By using the acclimatization device described in the present patent, it is possible to opt for an acclimatization protocol that excludes in vitro rooting so that the shoots obtained in the multiplication phase can be rooted and acclimatized simultaneously ex vitro in 90% using polystyrene tray with perlite. If 2018 00649 05/09/2018 opts for the protocol described by the authors, in vitro rooted plants can be acclimatized with the help of the acclimatization device even in 100%.

15. Aguilar et al. (2000) found that increasing endogenous ABA (abscisic acid) concentrations of plants in vitro (such as by ventilation or adding ABA to the environment) improves control of leaf water loss, ex vitro plant survival and plant vigor at Tagetes erect.

16. Da Silva et al. (2017) show that of the approximately 100 studies on micropropagation in Dendrobium reviewed, only 40% also present aspects related to ex vitro acclimatization. These studies detail aspects related to the substrate used for acclimatization and the environmental conditions in which the acclimatization was performed, in none of the studies was a device for acclimatization specified. Most of the acclimatizations were carried out in spaces with high humidity and in complex substrates formed by different mixtures, as follows:

- in substrate consisting of small pieces of coal and brick (1: 1), 75% shading, 95% acclimatization percentage (Vijayakumar et al. 2012);

- in the greenhouse, in a substrate consisting of small pieces of sterilized brick: coal: moss peat (1: 1: 0.5) 25-30 ° C, 60% -70% relative humidity, 80% acclimatization percentage (Hossain, 2013);

-in the greenhouse, in a substrate made of vermiculite: tree bark: soil (1: 2: 2), 100% acclimatization percentage (Luo et al. 2008);

- under plastic wrap, in a substrate consisting of coconut shell: perlite (1: 1), 92% acclimatization percentage (Rangsayatom, 2009);

17. Some ex vitro acclimatization methods in floating perlite or in hydroculture have been previously described by Clapa et al., 2013, 2017; Borsai et al., 2017; Fira et al., 2010, 2012, 2014a, 2014b, 2015). In contrast to these methods, the acclimatization device described in the present patent has a number of advantages: by using polystyrene trays with absorbent wick requires a smaller amount of perlite, which reduces the amount of waste (used perlite) that remains after transplanting rooted and acclimatized plants to the soil mixture and the use of polystyrene trays with holes ensures the contact of all vitroplants with the liquid substrate and a better aeration of them, avoiding the appearance of molds. Also, unlike these methods, acclimatization devices

05/09/2018 lj a 2018 00649 can be placed vertically in different spaces and can be very easily equipped with lighting installations if necessary and ensures higher percentages of acclimatization.

18. The technical problem solved by the invention is that the acclimatization device intended to be used for the final phase of plant micropropagation allows the acclimatization of vitroplants without the need to ensure a high humidity of the atmospheric air in the enclosure in which the seedlings are grown. of acclimatization and through the two variants of trays allows the acclimatization of the rooted shoots in vitro but also the acclimatization and rooting at the same time of the non-rooted shoots obtained in the multiplication phase. Unlike the classic, usual ex vitro acclimatization processes described above, our process is based on the culture of vitroplants in a layer of water 8-10 cm thick, in polystyrene trays provided with holes with a diameter of 10-15 mm, the above-ground parts of the above-mentioned vitroplants being in direct contact with atmospheric air and the roots or, as the case may be, the base of the shoots, in contact with water. The technical novelty of the invention consists in the use of water as a liquid substrate for acclimatization without the need to ensure a high humidity of the atmospheric air in the enclosure in which the vitroplants under acclimatization are cultivated.

19. In relation to the prior art, by applying the invention, the following advantages are obtained:

- The need for protected areas (greenhouses, solariums, tunnels, vegetation rooms) with special equipment for maintaining air humidity is eliminated, the device can be placed in ordinary constructions, for example greenhouses and solariums for general use, in vegetation rooms with simple endowments , similar to those for in vitro cultures or even in confined spaces, in the laboratory, for small-scale ad hoc tests.

- Does not require sophisticated technical equipment (eg devices for artificial fog, hygrostat) that break down easily and does not require the intervention of a professional. This increases the safety of the technological process of acclimatization, reducing the risk of losses due to technical failures.

- Due to the absence of sophisticated installations and devices, the acclimatization device presented is very easy to use and very safe, it does not run the risk of creating losses due to errors in adjusting the parameters necessary for acclimatization (atmospheric humidity, irrigation, drainage, temperature, ventilation), the process being passive and static, not dynamic.

05/09/2018 / l · a 2018 00649

- The need for irrigation is eliminated, because in the version of the acclimatization device with polystyrene trays with holes the acclimatization substrate is water itself and in the second version of the device the access of water to the acclimatization substrate (in this case perlite) is done passively , by absorption.

- The acclimatization device allows the application of the technological acclimatization process in a flexible, small-scale or large-scale manner. On a small scale a single acclimatization device or a smaller number can be used, and on a large scale a large number of acclimatization units assembled as a system of modules can be used (Figure 3). Theoretically, the module system can be as large, it can contain as many modules, and the minimum size of the system is a single unit (a single acclimatization device). Modules can be removed from the system or modules can be added as needed. The system can be assembled and disassembled quickly, can be relocated to another building or another room or greenhouse, can be reassembled into several subsystems, as the modules are easy to transport and handle.

- The acclimatization device allows the use of the available space in a flexible manner and in various ways. The modules can be placed horizontally and / or vertically in rooms, corridors, on shelves, on the floor or in greenhouses, solariums, even outdoors if the weather allows; can use natural and / or artificial light, can be very easily equipped with lighting fixtures. Thus, it has advantages over the usual, classic acclimatization methods that use spaces (greenhouses, solariums, tunnels, vegetation rooms) with high atmospheric humidity, natural light, with the possibility of supplementation with artificial light (greenhouses, solariums) or only artificial light ( vegetation rooms).

- Due to the low atmospheric humidity in the acclimatizer, the risk of diseases and pests is considerably reduced.

- Due to the fact that the water supply of the substrate (perlite) takes place passively, through capillarity, through the wicks made of textile material, there is no risk of flooding the substrate, thus eliminating the risk of damaging plant roots due to excessive moisture in the substrate.

- It is suitable for various simple and quick interventions, changing the water with solutions containing fertilizers and / or growth regulators, for example auxins, to manipulate the growth and development of plants and their nutrition.

20. The invention is presented in more detail below. The acclimatization device according to the invention consists of a mini-basin (1) made of polycarbonate or other type of transparent plastic, based on a black band (2) wide enough to prevent the penetration of light and contamination with algae. of water or solution in the basin (3),

05/09/2018 of 2018 00649 provided with removable lid (4), ventilation windows (5), orifice or more orifices for water supply (6) and tap or other system for draining water or the solution contained (7) . Inside the mini-basin, on the surface of the liquid there is a polystyrene support tray for vitroplants in 2 variants: simple polystyrene tray (8) with wick strips of cotton or other textile or non-textile material with capillary properties (9), in which it is located a layer of perlite with a thickness of 2-3 cm (10) or a polystyrene tray with holes (11) with a diameter determined according to the acclimatized species or according to the dimensions and particularities of the vegetal material transferred ex vitro for acclimatization (1-2 cm) . The acclimatization devices can be superimposed, in which case, optionally, they can be connected to a water distribution network (12) and a wastewater collection network (13) (Figure 1, Figure 2).

21. The acclimatization device may be used in the following cases:

- For ex vitro acclimatization of rooted seedlings obtained in the in vitro rooting phase. in this case the rooting of the shoots obtained in the multiplication phase is carried out in vitro, under sterile conditions, on the nutrient rooting media (rhizogenesis) in vitro. The rooted seedlings are removed from the culture vessels, washed under running water to remove residues from the culture medium and transplanted ex vitro under non-sterile conditions into the orifice tray (11) or perlite tray (8) provided with strips of wick, described herein. in the polystyrene trays with perlite layer the rooted seedlings are planted individually and in the trays provided with holes there are bundles of seedlings whose number is determined according to the vigor of the seedlings so that the package of plants is at the tray, the roots in contact with water and the aerial part in contact with the air. Inserting too few seedlings in a hole will encourage the seedlings to sink into the mini-basin and necroticize them. The plant trays are placed on the surface of the water in the mini-basin (1) ensuring the contact between the water and the wick strips. in 3-4 weeks the seedlings will be acclimatized and rooted in the acclimatization device resulting in plants suitable for transplanting into soil mixture.

- For direct ex vitro rooting and acclimatization, in a single phase, of the shoots obtained in the in vitro multiplication phase. In this case the in vitro rooting phase is removed from the protocol. Ex vitro transferred explants are axillary shoots regenerated in vitro in the multiplication phase. The seedlings in the in vitro multiplication phase have the morphological appearance of miniature bushes, with a few or a few dozen axillary shoots. These seedlings are transferred ex vitro, the axillary shoots are excised, which are then transplanted, one by one, individually, in the perlite layer (10) or grouped in small bunches of 10-20 shoots, depending on the species and size, and the respective bouquets are inserted into the holes in the tray

05/09/2018 of 2018 00649 of polystyrene with holes (11). In the acclimatization device takes root (rhizogenesis) at the level of axillary shoots transferred ex vitro, resulting in whole, rooted seedlings, which then grow in the device and acclimatize in 3-4 weeks, becoming suitable for transplanting into soil mixture.

- in the enclosures where the acclimatization devices are located, it is recommended that the air humidity is not high and the temperature is 20-25 ° C. crop care consists of regular replenishment of fresh water, removal of wastewater (burnished) if necessary and removal of any necrotic plants during the growing season. After the ex vitro acclimatization period, rooted and acclimatized plants are suitable for transplanting into pots or alveolar plates, in soil mixtures specific for classical horticultural crops, in greenhouses, solariums or shade.

22. The following are some examples of the use of the acclimatization device for different species. The acclimatization device tested had the following dimensions:

- minibasin volume: 15 1

- mini-pool dimensions: 35x25x15 cm

- volume of water:

- black band: 5 cm

- polystyrene tray with perlite: 22x14x3 cm, weight 8 g provided with a cotton wick strip with a length of 35 cm and a width of 2 cm (weight 6 g). The wet wick was placed along the tray, under the wet perlite layer, with the ends in contact with the water in the mini-basin.

- polystyrene tray with holes: 22x14x3 cm, weight 6g, with 32 holes with a diameter of 1.5 cm.

23. Ex vitro acclimatization to blackberries (Rubus fruticosus). The blackberry can be acclimatized with the help of the acclimatization device, in all variants:

- non-rooted shoots obtained in the multiplication phase acclimatized in a polystyrene tray with perlite;

- non-rooted shoots obtained in the multiplication phase acclimatized in a polystyrene tray with holes;

- in vitro rooted shoots acclimatized in polystyrene tray with perlite;

- in vitro rooted shoots acclimatized in polystyrene tray with holes.

In all cases the acclimatization percentages were over 98%. The fastest and most efficient option is to acclimatize the non-rooted shoots obtained in the multiplication phase in the tray.

05/09/2018 of 2018 00649 made of polystyrene with holes. In this case, the in vitro rooting step is eliminated from the micropropagation protocol and the labor requirement is very low, which reduces the costs of the propagating material obtained. in the acclimatization device with the dimensions described above, 640 non-rooted shoots obtained in the multiplication phase acclimatized in the polystyrene tray with holes could be acclimatized;

24. Ex vitro acclimatization to hops (Humulus lupulus L.). The acclimatization device allowed the ex vitro acclimatization of hops in the following variants:

- non-rooted shoots obtained in the multiplication phase acclimatized in a polystyrene tray with perlite;

- in vitro rooted shoots acclimatized in polystyrene tray with perlite;

- in vitro rooted shoots acclimatized in polystyrene tray with holes;

The percentage of ex vitro acclimatization in the polystyrene tray with perlite of the hop seedlings rooted in vitro was 100% and for those rooted in vitro and acclimatized in the polystyrene tray with holes was 90%. For the non-rooted shoots obtained in the multiplication stage, the rate of direct rooting and acclimatization ex vitro in the polystyrene tray with perlite was 97.33%. The non-rooted shoots obtained in the multiplication phase could not be acclimatized in a polystyrene tray with holes.

25. Ex vitro acclimatization to paulownia (Paulownia elongata). The acclimatization device allowed ex vitro acclimatization to paulownia in the following variants:

- non-rooted shoots obtained in the multiplication phase acclimatized in a polystyrene tray with perlite;

- in vitro rooted shoots acclimatized in polystyrene tray with perlite;

- in vitro rooted shoots acclimatized in polystyrene tray with holes.

The percentage of ex vitro acclimatization in polystyrene tray with perlite of paulownia seedlings rooted in vitro was 100% and for those rooted in vitro and acclimatizae in polystyrene tray with holes was also 100%. For the non-rooted shoots obtained in the multiplication stage, the rate of direct rooting and acclimatization ex vitro in the polystyrene tray with perlite was 90%. The non-rooted shoots obtained in the multiplication phase could not be acclimatized in a polystyrene tray with holes.

26. Ex vitro acclimatization to rhubarb (Rheum rhabarbarum L.). The acclimatization device allowed the ex vitro acclimatization of rhubarb shoots rooted in vitro in the tray from 2018 00649 05/09/2018 polystyrene with perlite. The root mini-rhubarb obtained in the multiplication phase could not be rooted and acclimatized in the device, necrotic in proportion of 100%. In vitro rooted mini-trees were 90% acclimatized in a polystyrene tray with perlite. Before planting in perlite, 1-2 leaves with half the length of the petiole (the longest) were removed for a better fixation in perlite and to reduce the leaf area during the acclimatization process. Rhubarb plants acclimatized to floating perlite and transferred to alveolar plates in peat substrate, in the greenhouse, survived in proportion of 98%. The acclimatization variant of the shoots rooted in vitro in a polystyrene tray with holes was also tested in other species with acclimatization percentages between 90-100%: Amelanchier canadensis, Drosera rotundifolia, Lycium barbarum, Nephrolepis sp, Ribes nigrum, Rosha hy.

27. The practical importance of the invention consists in reducing the need for labor for ex vitro transplantation, reducing the need for space for ex vitro acclimatization, eliminating the need to use a space with special equipment for ex vitro acclimatization (tunnel, acclimatization greenhouse with artificial fog devices) and removal of special mixtures of substrates. Acclimatization devices can be superimposed so that space is used vertically, they can be placed both in laboratory premises and in greenhouses or solariums, or even outdoors if the weather allows. The acclimatization device, through the two tray variants, allows both the acclimatization of in vitro rooted in vitro seedlings in the rooting phase but also the non-rooted shoots obtained in the multiplication phase, thus eliminating an expensive step in the micropropagation protocol. The acclimatization device has the advantage that it can be used successfully in laboratories for research but also for the production of propagating material obtained by micropropagation.

28. Cited literature:

1. US 5168059A

2. US4554252A

3. Aguilar, ML, Espadas, FL, Coello, J., Maust, BE, Trejo, C., Robert, ML, Santamaria, JM 2000. The role of abscisic acid in controlling leaf water loss, survival a 2018 00649 05 / 09/2018 and growth of micropropagated Tagetes erecta plants when transferred directly to the field. Joumal of Experimental Botany, 51 (352), 1861-1866.

4. Borsai O., D. Clapa, A. Fira, M. Hârța, A. Dumitraș, R. Pop, D. Pamfil. 2017. The Effect of Gelling Agent on the Micropropagation of Common Lilac (Syringa vulgaris L.). Agriculture - Science and Practice 3-4 (103-104): 63-71.

5. Clapa D., A. Fira, O. Borsai, M. Hârța, C. Sisea, R. Pop, D. Pamfil. 2017. Micropropagation of Roșa damascena Mill .: the Effects of Gelling Agents on the Multiplication Stages and Acclimatization. Agriculture - Science and Practice 3-4 (103104): 56-62.

6. Clapa D., A. Fira and N. Joshee. 2013. An Efficient ex Vitro Rooting and Acclimatization Method for Horticultural Plants Using Float Hydroculture, Hortscience 48 (9): 1159-1167.

7. da Silva, J. A. T., Hossain, Μ. M., Sharma, M., Dobrânszki, J., Cardoso, J. C., & Songjun, Z. E. N. G. 2017. Acclimatization of in Pzfro-derived Dendrobium. Horticultural Plant Joumal, 3 (3), 110-124.

8. Deb CR, Imchen T. 2010. An efficient in vitro hardening of tissue culture raised plants. Biotechnology 9: 79-83.

9. Fira, A., Clapa, D., Plopa, C. 2010. In vitro rooting and ex-vitro acclimation in apple (Malus domestica). Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Horticulture, 67 (1): 480.

10. Fira A., D. Clapa, L. A. Vescan. 2012. Direct ex Vitro Rooting and Acclimation in Blackberry Cultivar ‘Loch Ness’. Bulletin UASVM Animal Science and Biotechnologies 69 (1-2): 247-254.

11. Fira A., D. Clapa, V. Cristea, C. Plopa. 2014. In Vitro Propagation of Lonicera kamtschatica. Agriculture - Science and Practice 1- 2 (89-90): 90-99.

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12. Fira A., Clapa D., M. Simu. 2014b. Studies Regarding the Micropropagation of Some Blackberry Cultivars. Bulletin UASVM Horticultura 71 (1): 29-37.

13. Fira A., M. Simu, B. Vlaicu, D. Clapa. 2015. Aspects Regarding the In Vitro Propagation of ‘Royal Gala’ Apple Cultivated. Bulletin UASVM Horticultura 72 (2): 342-349.

14. Hossain, M.M., 2013. In vitro embryo morphogenesis and micropropagation of Dendrobium aggregatum Roxb. Plant Tissue Cult Biotechnol, 23: 241-249.

15. Lobna, S. T., Taha, Μ. M., Soad, I., Farahat, Μ. M. 2008. A Micropropagation Protocol of Paulownia kowakamii through in vitro culture technique. Australian Joumal of basic and applied sciences, 2 (3), 594-600.

16. Najaf-Abadi, A. J., and Hamidoghli, Y. 2009. Micropropagation of Thomless Trailing Blackberry (‘Rubus sp.’) By Axillary Bud Explants. Australian Joumal of Crop Science, 3 (4): 191-194.

17. Posposilovâ, J., Tichâ, I., Kadlecek, P., Haisel, D., Plzâkovâ, S. 1999. Acclimatization of micropropagated plants to ex vitro conditions. Plant Biology, 42 (4): 481-497.

18. Rangsayatom, N., 2009. Micropropagation of Dendrobium draconis Rchb. f. from thin cross-section culture. Sci Hortic, 122: 662-665.

19. Roy, A. T., Leggett, G., & Koutoulis, A. 2001. Development of a shoot multiplication system for hop (Humulus lupulus L.). In Vitro Cellular & Developmental BiologyPlant, 37 (l): 79-83.

20. Vijayakumar, S., Rajalkshmi, G., Kalimuthu, K., 2012. Propagation of Dendrobium aggregatum through the culture of immature seeds from green capsules. Lankesteriana, 12: 131-135.

Claims (4)

claims 1. Ex vitro acclimatization device characterized in that it consists of a mini-basin of polycarbonate or other transparent plastic material, based on a black band of sufficient width to prevent light penetration and algae contamination of water from basin, provided with removable lid, windows for ventilation, holes for water supply and tap or other system for draining water and inside the mini-basin, on the surface of the liquid there is polystyrene support tray for vitroplants in 2 variants: simple polystyrene tray with wick strips of cotton or other material with capillary properties, in which there is a layer of perlite 2 cm thick or polystyrene tray with holes with a diameter determined according to the acclimatized species or according to the dimensions and particularities of the transferred plant material ex in vitro for acclimatization. 2. Device for ex vitro acclimatization of non-rooted shoots from the propagation phase or of rooted vitroplants characterized in that the substrate used for acclimatization is water, the vitroplants having the above-ground parts (leaves and stems) in contact with atmospheric air, and the roots im using polystyrene support trays with holes and placed in a polycarbonate or other type of transparent plastic base, based on a black band of sufficient width to prevent light penetration and contamination with water algae, provided with a removable cover, ventilation windows, water supply holes and tap or other system for draining water. 3. Device for ex vitro acclimatization of non-rooted shoots from the propagation phase or of rooted vitroplants characterized in that the acclimatization is carried out in polystyrene support trays provided with cotton wick or other material with capillary properties, in which there is a layer of pearlite 2 cm thick and placed on the surface of the water in a mini-basin made of polycarbonate or other transparent plastic material, based on a black band of sufficient width to prevent the penetration of light and contamination with water algae , equipped with a removable cover, ventilation windows, water supply holes and tap or other system for draining water. a 2018 00649 05/09/2018 4. Ex vitro acclimatization method characterized in that the ex vitro acclimatization of the non-rooted shoots from the propagation phase or of the rooted vitroplants is made in a layer of water 8-10 cm thick, in polystyrene trays provided with holes with a diameter 10-15 mm, the above-ground parts of the above-mentioned vitroplants being in direct contact with atmospheric air and the roots or, as the case may be, the base of the shoots, in contact with water, not being necessary to ensure a high humidity of the atmospheric air in which vitroplants under acclimatization are cultivated.