JP4701019B2 - Eucalyptus plant tissue culture method and clone seedling production method - Google Patents

Description

  The present invention relates to a method for producing a cloned seedling of a Eucalyptus plant by tissue culture.

Tissue culture technology that vegetatively propagates or propagates plant tissues in culture vessels to produce large numbers of cloned seedlings is a method for mass-producing not only vegetative florets and crops, but also plant individuals with excellent traits. In recent years, it has been widely used, and it has great effects such as homogenization of products and high yield, while woody plants, i.e. trees, mainly seeds that are difficult to cut, etc. Production and mass growth are expected. This is not an exception even in Eucalyputus, which contains tree species useful as pulp materials.

  In general, the production of cloned seedlings by tissue culture of woody plants is carried out through a regular bud formation process, an adventitious bud propagation process and a rooting process. That is, first, branches and the like collected from individuals growing outdoors or in a greenhouse are cultured using an appropriate medium for forming regular buds, and after forming buds such as top buds and buds, The adventitious buds are induced and grown by culturing these adventitious buds using a medium for adventitious bud growth. Subsequently, the proliferated adventitious buds are then rooted by transplanting to a rooting medium and culturing to obtain cloned seedlings.

  However, Eucalyptus plants have a constant bud formation rate in the above-described regular bud formation process, adventitious bud proliferation in the adventitious bud propagation process (proliferation rate and growth of adventitious buds), and / or rooting in the rooting process (indefinite). Many tree species are inferior in terms of the rooting rate from the buds and the number of roots per rooted adventitious bud. Therefore, for the purpose of increasing the productivity of eucalyptus plant clone seedlings, so far, for example, in order to improve the proliferation, rooting, etc. of adventitious buds, modification of the medium composition used for tissue culture (for example, Patent Document 1), control of light conditions during tissue culture (for example, Patent Document 2), control of carbon dioxide concentration (for example, Patent Document 3), and the like have been studied.

JP-A-6-133657 JP2000-60332 JP-A-8-252038

  The above studies for increasing the productivity of Eucalyptus plant clonal seedlings have yielded certain results. However, when it is assumed that these clone seedlings are applied to uses such as large-scale afforestation, the productivity is still not sufficient.

  Therefore, the present inventors aim to increase the productivity of Eucalyptus plants clone seedlings, the formation rate of regular buds in the regular bud formation process, the proliferation of adventitious buds in the adventitious bud propagation process, and rooting Research was conducted to provide a tissue culture method for Eucalyptus plants that can improve any of the rooting properties in the process.

  As a result of the above studies, the present inventors have achieved the above object when the fixed shoot forming step, the adventitious bud growing step and the rooting step are performed using a medium containing sodium chloride and / or magnesium chloride, respectively. As a result, the present invention has been completed.

  That is, the present invention provides a method for cultivating Eucalyptus plants, characterized in that the bud formation step of forming buds from Eucalyptus plant tissues is performed using a medium containing sodium chloride and / or magnesium chloride. A method of cultivating Eucalyptus plants, wherein the adventitious bud growth step of inducing and growing adventitious buds from Eucalyptus plant tissues using a medium containing sodium chloride and / or magnesium chloride, or The method for cultivating Eucalyptus plants, wherein the rooting step for rooting the tissues of the Eucalyptus plants is performed using a medium containing sodium chloride and / or magnesium chloride, or the formation of the above-mentioned buds A eucalyptus plant clone seedling comprising one or more of a step, an adventitious bud propagation step and a rooting step Production method for.

  ADVANTAGE OF THE INVENTION According to this invention, a fixed bud formation rate can be improved in the fixed shoot formation process of the tissue culture of Eucalyptus plant.

  Moreover, according to this invention, the proliferation rate and growth of adventitious bud can be improved in the adventitious bud propagation process of the tissue culture of Eucalyptus plants.

  Furthermore, according to the present invention, the rooting rate and the number of roots can be improved in the rooting step of Eucalyptus plants.

  Therefore, according to the present invention, the productivity of eucalyptus plant clone seedlings can be increased, and these clone seedlings can be applied to uses such as large-scale plantations.

  The present invention is described in detail below.

The plant which is the target of the tissue culture method or the clonal seedling production method according to the present invention is not limited as long as it belongs to the genus Eucalyptus. As plants belonging to Eucalyptus, for example, Eucalyptus Bikosutata (Eucalyptus bicostata), Eucalyptus camaldulensis (E.camaldulensis), Eucalyptus Shitoriodora (E.citriodora), Eucalyptus globulus (E.globulus), Eucalyptus grandis (E.grandis), Eucalyptus Maideni (E.maidenii), Eucalyptus Nitensu (E.nitens), mention may be made of Eucalyptus euro Philadelphia (E.urophylla) and the like.

  In one aspect of the tissue culture method according to the present invention, the sprouting process for forming sprouting from the tissue of the Eucalyptus plant is performed using a medium containing sodium chloride and / or magnesium chloride. Here, fixed shoots refer to shoots that are normally formed in plants, such as top buds and buds.

  In order to form buds from Eucalyptus plant tissues, for example, from an individual of an Eucalyptus plant growing outdoors or in a greenhouse, a branch containing the primordial or bud primordia is collected and the surface is sterilized. Then, it may be cultured under aseptic conditions by inserting it into a medium for bud formation. In about 2 to 4 weeks, apical buds and buds are formed from the primordium and extend.

  Surface sterilization can be performed by immersing the collected branches for 10 to 20 minutes in an aqueous solution of sodium hypochlorite having an effective chlorine concentration of 0.5 to 4% or an aqueous solution of hydrogen peroxide having an effective chlorine concentration of 5 to 15%. . The medium for forming shoots contains not only sodium chloride and / or magnesium chloride, which are essential components of the present invention, but also a carbon source, auxin and / or cytokinin as a plant hormone, and further a medium solid agent for plant tissue culture. A culture medium can be used.

  In addition, the amount of sodium chloride and / or magnesium chloride in the bud forming medium is preferably 5 to 250 mM, particularly preferably 10 to 200 mM. Here, the amount of sodium chloride and / or magnesium chloride, when sodium chloride or magnesium chloride is contained alone, is the amount of sodium chloride or magnesium chloride alone. When included, it is the total amount of both. Even if the amount of sodium chloride and / or magnesium chloride is less than 5 mM or more than 250 mM, an effect for improving the formation rate of buds cannot be expected. As a carbon source, sucrose 1 to 5 w / v%, as a plant hormone, benzyladenine (BAP) 0.02 to 1 mg / l, as a medium solid agent, gellan gum 0.2 to 0.3 w / v% or agar 0. 5 to 1.0 w / v% is preferable. As the plant tissue culture medium, Murashige-Skoog (MS) medium can be suitably used. As environmental conditions such as light conditions and temperatures, known environmental conditions can be adopted as conditions suitable for the growth of the Eucalyptus plant.

  In another aspect of the tissue culture method according to the present invention, the adventitious bud propagation step of inducing adventitious buds from the Eucalyptus plant tissue and growing is performed using a medium containing sodium chloride and / or magnesium chloride. . Here, adventitious buds refer to buds that are usually formed in areas where buds are not formed. In addition to buds that differentiate from branches, stems, leaves, roots, callus, etc., seedling primordia and multiple buds. Buds that differentiate from the body are also collectively referred to as adventitious buds. A multi-bud is a tissue obtained by differentiating a large number of adventitious buds, and can be induced by culturing adventitious buds or adventitious buds under appropriate conditions.

  Therefore, in order to induce and grow adventitious buds from the tissues of the genus Eucalyptus, it is necessary to use, for example, a medium containing sodium chloride and / or magnesium chloride as a medium for forming buds according to the above-mentioned bud forming process. No.) After forming shoots from the tissues of the Eucalyptus plant, this shoot is placed in an adventitious bud growth medium and cultured to induce multi-buds and grow them. Good. The multi-buds are induced in about 2 to 4 weeks after the fixed shoots are placed on the adventitious bud growth medium. In order to proliferate the induced multi-bud, it is only necessary to divide it into an appropriate size and place it on a medium for adventitious bud growth having the same composition as that used for the induction and culture it. The adventitious bud growth medium contains the sodium chloride and / or magnesium chloride, which are essential components of the present invention, and also contains a carbon source, auxin and / or cytokinin as a plant hormone, and a medium solid agent for known plant tissue culture. A culture medium can be used.

  The amount of sodium chloride and / or magnesium chloride in the adventitious bud growth medium is preferably 5 to 90 mM, and particularly preferably 10 to 80 mM. Here, the amount of sodium chloride and / or magnesium chloride, when containing sodium chloride or magnesium chloride alone, is the amount of sodium chloride or magnesium chloride alone, as described above. When both of magnesium is included, it is the total amount of both. Even if the amount of sodium chloride and / or magnesium chloride is less than 5 mM or more than 90 mM, the effect on the improvement of adventitious bud growth cannot be expected. As a carbon source, sucrose 1 to 5 w / v%, as a plant hormone, benzyladenine (BAP) 0.02 to 1 mg / l or kinetin (Kin) 0.02 to 1 mg / l, as a medium solid agent, gellan gum 0. It is preferably 2 to 0.3 w / v% or agar 0.5 to 1.0 w / v%. As the medium for plant tissue culture, an MS medium or a medium obtained by diluting the MS medium to about 2 times can be suitably used. As environmental conditions such as light conditions and temperatures, known environmental conditions can be adopted as conditions suitable for the growth of the Eucalyptus plant.

  In still another aspect of the tissue culture method according to the present invention, the rooting step for rooting the tissue of the Eucalyptus plant is performed using a medium containing sodium chloride and / or magnesium chloride.

  In order to root the Eucalyptus plant tissue, for example, the fixed shoots formed in the above-mentioned fixed shoot formation step or the adventitious buds induced and propagated in the adventitious bud propagation step are cut out and used for rooting. What is necessary is just to culture | cultivate by inserting in a culture medium. The buds or adventitious buds inserted in the rooting medium can be rooted in about 2 to 4 weeks to obtain cloned seedlings. As the rooting medium, a known plant tissue culture medium containing sodium chloride and / or magnesium chloride, which are essential components of the present invention, and carbon source and auxin as a plant hormone can be used. In this case, a medium solidifying agent may be used, but without using it, a support having an appropriate space made of phenol resin, rock wool, or the like is moistened with a liquid medium for rooting. Healthy roots can be obtained by rooting by inserting fixed shoots or adventitious shoots.

  The amount of sodium chloride and / or magnesium chloride in the rooting medium is preferably 5 to 350 mM, particularly preferably 10 to 300 mM. Here, the amount of sodium chloride and / or magnesium chloride, when sodium chloride or magnesium chloride is contained alone, is the amount of sodium chloride or magnesium chloride alone. When included, it is the total amount of both. Even if the amount of sodium chloride and / or magnesium chloride is less than 5 mM or more than 350 mM, the effect on the rooting improvement cannot be expected. As the carbon source, sucrose 1 to 5 w / v%, and as the plant hormone, indolebutyric acid (IBA) 0.5 to 5 mg / l or naphthalene acetic acid (NAA) 0.5 to 5 mg / l are preferable. Instead of using sucrose as a carbon source, carbon dioxide gas may be supplied into the culture environment. As the medium for plant tissue culture, a medium obtained by diluting MS medium or B5 medium 2 to 8 times can be preferably used. As environmental conditions such as light conditions and temperatures, known environmental conditions can be adopted as conditions suitable for the growth of the Eucalyptus plant.

  In the present invention, a eucalyptus plant clonal seedling is produced through one or more steps among the above-mentioned bud forming step, adventitious bud propagation step and rooting step. The clonal seedling produced can be made into a seedling that can be used for a predetermined purpose such as afforestation by being transplanted and grown in a seedling container or a nursery field after acclimatization. Conditions during this period, such as temperature and light intensity when growing seedlings, may be set as appropriate to suit the Eucalyptus plant.

[Action]
In the present invention, Eucalyptus plant tissues are cultured using a medium containing sodium chloride and / or magnesium chloride. Among known mediums for plant tissue culture, sodium chloride or magnesium chloride is included in the composition. There is nothing.

  In the first place, except for plants known as halophytes, even when ordinary plants are said to be quite strong in salt tolerance, when grown using a culture solution having a chloride ion concentration of about 300 mM, At most, it has a salt tolerance that can maintain a relative growth of about 70%. In addition, when grown using a culture solution having a chloride ion concentration of less than 300 mM, the growth of such a “strong salt-tolerant” plant is not promoted in the presence of the chloride ion concentration (“plant nutrition / Fertilizer Science, pages 150-151, 1993, published by Asakura Shoten Co., Ltd.). Eucalyptus plants are not halophytes, and are considered to be the same as ordinary plants in terms of salt tolerance.

  However, the present inventors have shown that the medium containing sodium chloride and / or magnesium chloride has the effect of improving all of the formation rate of adventitious buds, the proliferation of adventitious buds, and the rooting ability in the tissue culture of Eucalyptus plants. Found to have. It is not clear how such an effect is brought about, but it is presumed to be caused by some physiological property (for example, intracellular osmotic pressure etc.) common to Eucalyptus plants.

  Hereinafter, the present invention will be described based on examples.

[Example 1]
As a medium for bud formation, sodium chloride 10, 50, 100, 200 or 300 mM, or magnesium chloride 50 or 100 mM, sucrose 1.5 w / v%, BAP 0.1 mg / l, and gellan gum 0.25 w / v MS medium containing% was prepared.

On the other hand, the branch containing the bud primordium was collected from the current year branch of the 10th grade Eucalyptus globulus, soaked in a sodium hypochlorite solution having an effective chlorine concentration of 1% for 15 minutes, and then sterilized with water. A control medium having the same composition as the above-mentioned medium for forming regular buds (for the formation of fixed buds) except that it was washed and sterilized under the above-mentioned medium for forming regular buds or without addition of sodium chloride and / or magnesium chloride. And cultured at 25 ± 1 ° C., 16 hours in length, and light intensity of 40 μmol / cm ² / s.

  Four weeks after the start of the culture, the number of axillary buds formed on the branches subjected to the culture was investigated, and the results are shown in Table 1.

  As is apparent from Table 1, when a medium containing 10, 50, 100, or 200 mM sodium chloride or 50 or 100 mM magnesium chloride is used as the medium for forming regular buds, any buds exceeding 50% are used. The rate of formation, that is, the rate of regular bud formation was shown. On the other hand, the constant bud formation rate when the control medium (for constant bud formation) was used was only 36%, indicating that the above-mentioned constant shoot formation medium improved the fixed bud formation rate. It was.

[Example 2]
As a medium for adventitious bud growth, sodium chloride 10, 50, 80 or 100 mM, or magnesium chloride 50 or 80 mM, sucrose 2.0 w / v%, BAP 0.02 mg / l, and gellan gum 0.25 w / v% MS medium containing was prepared.

On the other hand, the axillary buds formed in Example 1 were cut off from the branches, and this was the same composition as the above-mentioned adventitious bud growth medium except that the adventitious bud growth medium or sodium chloride and / or magnesium chloride was not added. The cells were placed on a control medium (for adventitious bud propagation), cultured at 25 ± 1 ° C., 16 hours in length, with a light intensity of 40 μmol / cm ² / s to induce and proliferate multiblasts.

  Four weeks after the start of the culture, the raw weight of the induced and proliferated polyblasts and the length of adventitious buds differentiated from the multiblasts were investigated, and the results are shown in Table 2.

  As apparent from Table 2, when a medium containing sodium chloride 10, 50, or 80 mM, or magnesium chloride 50 or 80 mM was used as a medium for adventitious bud growth, the control medium (adventitious bud growth) was used. As compared to the case of using the adventitious buds, a multi-bud body having a heavy fresh weight and having good adventitious bud elongation was obtained. It was.

[Example 3]
As a rooting medium, a 4-fold diluted MS medium containing sodium chloride 10, 50, 100, 200, 300, or 500 mM, or magnesium chloride 100 or 300 mM, IBA 2.0 mg / l was prepared.

On the other hand, adventitious shoots were cut out from the multi-buds induced and propagated in Example 2, and the rooting medium or the addition of sodium chloride and / or magnesium chloride was omitted. Inserted into a foamed phenolic resin ("Oasis (registered trademark)" manufactured by Smither Oasis) moistened with a control medium (for rooting) of the same composition, carbon dioxide concentration 1000 ppm ± 200 ppm, 25 ± 1 ° C., 16 hours a day Incubation was performed at a long light intensity of 40 μmol / cm ² / s.

  Three weeks after the start of the culture, the number of adventitious buds with rooting and the number of adventitious buds with rooting were investigated, and the results are shown in Table 3.

  As is apparent from Table 3, when a rooting medium containing sodium chloride 10, 50, 100, 200 or 300 mM, or magnesium chloride 100 or 300 mM was used, regarding the rooting rate, the control medium ( The results were similar to or slightly inferior to those using rooting), but the control medium (for rooting) was used for the number of roots formed per adventitious bud rooted at this time. The rooting medium showed an improvement in the rooting ability of adventitious buds.

[Example 4]
As a medium for adventitious bud growth, an MS medium containing sodium chloride 10, 50 or 100 mM, sucrose 2.0 w / v%, BAP 0.02 mg / l, and gellan gum 0.25 w / v% was prepared.

On the other hand, a branch containing the bud primordium was collected from the current year branch of 10-year-old Eucalyptus globulus, and after surface sterilization and the like in the same manner as in Example 1, it was inserted into a control medium (for bud formation). Then, the buds were cultured to form buds. In the present example, the axillary buds thus formed were cut off from the branches, and this was used as a control medium (indefinite) with the same composition as the above-mentioned adventitious bud growth medium, or except that sodium chloride was not added. The buds were cultured at 25 ± 1 ° C., 16 hours in length, and light intensity of 40 μmol / cm ² / s to induce and proliferate multi-buds.

  Four weeks after the start of the culture, the fresh weight of the induced and proliferated polyblasts and the length of adventitious buds differentiated from the multiblasts were investigated, and the results are shown in Table 4.

  As is apparent from Table 4, when a medium containing 10 or 50 mM sodium chloride was used as a medium for adventitious bud growth, both were compared with the case where a control medium (for adventitious bud growth) was used. A multi-bud having a heavy weight and good adventitious bud elongation was obtained, and the adventitious bud growth medium showed that the adventitious bud proliferation was improved in this example as well.

[Example 5]
As a rooting medium, a 4-fold diluted MS medium containing sodium chloride 10, 50, 100, 200, 300 or 500 mM and IBA 2.0 mg / l was prepared.

On the other hand, a branch containing the bud primordia was collected from the current year branch of 10-year-old Eucalyptus globulus, surface sterilized, etc. in the same manner as in Example 1 and inserted into a control medium (for bud formation). Culture was performed to form axillary buds. Next, this axillary bud was cut from the branch, and placed in a control medium (for adventitious bud propagation) and cultured in the same manner as in Example 2 to induce and proliferate multi-buds. In this example, adventitious shoots were cut out from the multi-buds thus induced and propagated, and the control was the same as the rooting medium except that the rooting medium or sodium chloride was not added. Inserted into a foamed phenolic resin ("Oasis (registered trademark)" manufactured by Smither Oasis) wetted with a medium (for rooting), carbon dioxide concentration 1000 ± 200 ppm, 25 ± 1 ° C, 16 hours long, light intensity Culturing was performed at 40 μmol / cm ² / s.

  Three weeks after the start of the culture, the number of adventitious buds in which rooting was observed and the number of adventitious buds in which rooting was observed were examined for the number of roots, and the results are shown in Table 5.

  As is apparent from Table 5, when a medium containing sodium chloride 10, 50, 100, 200 or 300 mM was used as a rooting medium, with respect to the rooting rate, a control medium (for rooting) was used. However, the number of roots formed per adventitious bud rooted at this time greatly increased compared to the control medium (for rooting). It was shown that the rooting medium improved the rooting ability of adventitious buds in this example.

[Example 6]
An MS medium containing sodium chloride 10, 100 or 200 mM, sucrose 1.5 w / v%, BAP 0.1 mg / l, and gellan gum 0.25 w / v% was prepared as a medium for forming shoots.

On the other hand, branches containing bud primordia were collected from 6th grade Eucalyptus citriodora, and immersed in a sodium hypochlorite solution with an effective chlorine concentration of 1% for 15 minutes. After washing and aseptic conditions, this was inserted into the above-mentioned medium for bud formation or a control medium (for bud formation) having the same composition as the above-mentioned medium for bud formation except that no sodium chloride was added. Culturing was performed at ± 1 ° C., 16 hours of day length, and light intensity of 40 μmol / cm ² / s.

  Four weeks after the start of the culture, the number of axillary buds formed on the branches subjected to the culture was examined, and the results are shown in Table 6.

  As is clear from Table 6, in the case where a medium containing sodium chloride was used as the medium for forming regular buds, all showed a bud formation rate exceeding 40%, that is, a constant bud formation rate. In contrast, when the control medium (for constant bud formation) was used, the fixed shoot formation rate was only 24%, indicating that the above-mentioned constant shoot formation medium improved the formation rate of fixed shoots. It was.

[Example 7]
As a medium for adventitious bud growth, an MS medium containing sodium chloride 10, 50 or 80 mM, sucrose 2.0 w / v%, BAP 0.02 mg / l, and gellan gum 0.25 w / v% was prepared.

On the other hand, the axillary buds formed in Example 6 were cut off from the branches, and this was used as a control medium (adventitious buds) having the same composition as the adventitious bud growth medium, except that the adventitious bud growth medium or sodium chloride was not added. And was cultured at 25 ± 1 ° C., 16 hours in length, and light intensity of 40 μmol / cm ² / s to induce and proliferate multiblasts.

  Four weeks after the start of the culture, the raw weight of the induced and proliferated polyblasts and the length of adventitious buds differentiated from the multiblasts were investigated, and the results are shown in Table 7.

  As can be seen from Table 7, when the medium for adventitious bud growth was used that contained sodium chloride, the fresh weight was heavier than when the control medium (for adventitious bud growth) was used. In addition, a multi-bud body with good elongation of adventitious buds was obtained, and it was shown that the adventitious bud growth medium improved the proliferation of adventitious buds.

[Example 8]
As a rooting medium, a 4-fold diluted MS medium containing 50, 200, 300 or 500 mM sodium chloride and 2.0 mg / l IBA was prepared.

On the other hand, adventitious buds were cut out from the multi-buds induced and propagated in Example 7, and this was the rooting medium or a control medium having the same composition as the rooting medium except that sodium chloride was not added. Inserted into foamed phenolic resin ("Oasis (registered trademark)" manufactured by Smither Oasis) wetted with (for rooting), carbon dioxide concentration 1000 ± 200 ppm, 25 ± 1 ° C., 16 hours long day, light intensity 40 μmol Culturing was performed at / cm ² / s.

  Three weeks after the start of culture, the number of adventitious buds with rooting and the number of adventitious buds with rooting were investigated, and the results are shown in Table 8.

  As is apparent from Table 8, when a medium containing 50, 200 or 300 mM sodium chloride is used as the rooting medium, the rooting rate is about the same as when the control medium (for rooting) is used. Although the results were slightly inferior, the number of roots formed per adventitious bud rooted at this time increased greatly compared to the case of using the control medium (for rooting). It was shown that the culture medium improved the rooting ability of adventitious buds.

Claims (8)

The Eucalyptus Shitoriodora or Teime forming step of forming a Teime from Eucalyptus globulus tissue, sodium chloride and / or and performing using a medium containing magnesium chloride, tissues Eucalyptus Shitoriodora or Eucalyptus globulus Culture method. A eucalyptus citriodora or eucalyptus eucalyptus or eucalyptus eucalyptus eucalyptus or eucalyptus Globulus tissue culture method. A tissue culture method for Eucalyptus citriodora or Eucalyptus globulus , wherein a rooting step for rooting a tissue of Eucalyptus citriodora or Eucalyptus globulus is performed using a medium containing sodium chloride and / or magnesium chloride. Eucalyptus citriodora comprising one or more steps among the regular bud formation step according to claim 1, the adventitious bud propagation step according to claim 2, and the rooting step according to claim 3. Eucalyptus globulus clone seedling production method. Obtained in the regular bud formation process for forming regular buds from Eucalyptus citriodora or Eucalyptus globulus tissues, the adventitious bud propagation process for inducing and growing adventitious buds from the regular buds obtained in the regular bud formation process, and the adventitious bud propagation process A eucalyptus citriodora or Eucalyptus globulus clonal seedling production method comprising a rooting step of rooting the adventitious adventitious buds, wherein one of the above-mentioned adventitious bud formation step, adventitious bud propagation step, and rooting step A method for producing Eucalyptus citriodora or Eucalyptus globulus clone seedlings, which comprises performing the above steps using a medium containing sodium chloride and / or magnesium chloride. The eucalyptus citriodola or eucalyptus globulus clonal seedling production method according to claim 4 or 5 , wherein the bud formation step is performed using a medium containing sodium chloride and / or magnesium chloride at a concentration of 5 to 250 mM. . The method for producing Eucalyptus citriodora or Eucalyptus globulus seedlings according to claim 4 or 5 , wherein the adventitious bud growth step is performed using a medium containing sodium chloride and / or magnesium chloride at a concentration of 5 to 90 mM. . The eucalyptus citriodora or eucalyptus globulus clonal seedling production method according to claim 4 or 5 , wherein the rooting step is performed using a medium containing sodium chloride and / or magnesium chloride at a concentration of 5 to 400 mM .