JP4868812B2 - Cutting method of cuttings - Google Patents

Description

  The present invention relates to a method for cultivating cuttings to obtain a plant individual, in particular, a method for cultivating cuttings that can improve the rooting rate from cuttings, and rooting plants that are difficult to root. It relates to a suitable method.

  When a plant is used industrially, a step for multiplying a large amount of a homogeneous seedling having a trait suitable for the purpose is always required. This is the same whether breeding or planting. At this time, the traditional cutting method and the tissue culture method born by the development of biotechnology in recent years are useful as a means for mass propagation of seedlings. According to these methods, not only can a large amount of seedling be grown, but also a plant individual having the same genetic properties, that is, a cloned seedling, can be rapidly and rapidly grown.

  In the cutting method, branches, or in some cases, buds, leaves, etc., are cut out from plant individuals to be proliferated to form cuttings, which are inserted into an appropriate culture soil and rooted to produce seedlings. On the other hand, when a large amount of woody plants are to be proliferated in the tissue culture method, they often go through multi-buds or shoot primordia. Specifically, buds and stem vertices are cut from the individual plant to be grown, cultured, and after generating shoots and shoot primordia, adventitious buds extending from these are cut, and this indefinite. The buds are inserted as roots and inserted into an appropriate support to cause rooting. That is, regardless of which method is used to produce a clonal seedling, in the end, it often goes through a process of rooting from the cutting head.

  However, the rooting of cuttings varies greatly depending on the type of plant. In general, woody plants are less rooted than herbaceous plants. In the same woody plant, for example, willow and cypress are easy to root from cuttings, but eucalyptus and pine are extremely difficult to root from cuttings.

  Such methods for improving the rooting ability of difficult-to-root plants can be broadly divided into approaches that focus on external factors and those that focus on internal factors. In this case, the approach that focuses on external factors is most suitable for rooting by examining environmental conditions such as temperature, humidity, oxygen, carbon dioxide concentration, light conditions, and culture medium when cultivating cuttings. Find environmental conditions and try to achieve this.

  On the other hand, in the approach focusing on the internal factors, the cutting head itself is examined to obtain a high rooting ability. As methods for obtaining such cuttings, for example, the cuttings are germinated in a dark state (yellowing treatment), the cuttings are treated with various auxins, the transpiration from the cuttings of the cuttings is suppressed, and the young are young Using cuttings from the parent tree, treating the parent tree with a gibberellin biosynthesis inhibitor such as B-nine or paclobutrazol (Patent Document 1), applying a negative voltage to the cuttings (Patent Document 2), etc. This method has been reported so far.

JP 2001-231355 A JP-A-2005-13163

  In order to improve the rooting ability of difficult-to-root plants, not only external factors of cuttings but also internal factors need to be in a state most suitable for rooting. However, it cannot be said that sufficient studies have been made on the improvement of rooting, focusing on the internal factors of cuttings.

  In consideration of such problems, the present invention has been made in order to improve the rooting property by focusing on the internal factors of cuttings.

  As a result of diligent research, the present inventors have found that, when the cuttings are sonicated, this sonication affects the internal factors of the cuttings, and the formation of adventitious roots is promoted, completing the present invention. I let you.

  That is, the present invention forms adventitious roots by sonicating at least a part of the portion of the cutting head in contact with the medium and / or culture soil when cultivating the cutting using the medium and / or the culture soil. The present invention relates to a method for cultivating cuttings.

  According to the present invention, adventitious root formation from cuttings is promoted.

  Therefore, according to the present invention, the rooting rate from the cutting head is improved, and the number of roots generated at this time is also increased. And such an effect is exhibited also in a hard-to-root plant which is said to be difficult to root from cuttings.

That is, according to the present invention, by the cutting method or the tissue culture method of a clone seedling of a hardly rooted plant having a useful trait such as Eucalyptus globulus (hereinafter simply abbreviated as E. globula). Mass growth is industrially possible.

  There is no restriction | limiting in particular in the kind of plant which can be used as a cutting head in this invention. The present invention can be applied to herbaceous plants such as chrysanthemum and carnations as well as woody plants such as eucalyptus, pine, acacia, bayberry, kunugi, grape, apple, cherry, rose, camellia and ume. However, the present invention is particularly effective when applied to difficult-to-root plants such as eucalyptus and pine.

  These cuttings may be obtained from individual plants growing in a greenhouse or outdoors, or may be obtained by a tissue culture method. When cuttings are obtained from a plant individual, branches, stems, buds or leaves may be cut out and used as cuttings. In the case of woody plants, green branches (current year branches) and mature branches (branches extending before the previous year) are usually used, and in the case of herbaceous plants, buds and leaves are usually used. When branches are used as cuttings, it is also effective to excise part of the leaves in order to suppress the transpiration of the leaves attached to the branches and promote the formation of adventitious roots. In the present invention, the adventitious root means a root formed in a portion where a root is not normally formed, such as a branch, a stem, or a leaf.

  When obtaining cuttings by tissue culture, it is possible to induce multi-buds and shoot primordia, cut adventitious buds extending from these tissues from their roots, and use them as cuttings . The multi-bud or shoot primordia can be induced in each plant using a known method. For example, in order to obtain the cuttings used in the present invention by forming multi-buds from the above-mentioned woody plant, it is generally performed as follows.

  First, tissues such as apical buds and axillary buds are collected from a plant as a material, and the collected tissues are subjected to sodium hypochlorite aqueous solution having an effective chlorine content of 0.5 to 4% or peroxidation having an effective chlorine content of 5 to 15%. Surface sterilization is performed by immersing in an aqueous hydrogen solution for 10 to 20 minutes. Next, this is washed with sterilized water, inserted into a solid medium to open the buds, and the elongated shoots are subcultured in a medium having the same composition to form multi-buds. When Eucalyptus or Acacia sprouts are used, the solid medium is 1 to 5% by weight of sucrose, benzyladenine (hereinafter abbreviated as BA) as a plant hormone, 0.02 to 1 mg / l, gellan gum 0.2 A Murashige scoog (hereinafter abbreviated as MS) medium containing ~ 0.3 wt% or agar 0.5-1 wt% or a modified MS medium in which the ammonium nitrate component and potassium nitrate component of this MS medium are halved is used. preferable. Since the adventitious buds are actively differentiated from the multi-buds thus formed and extend, in the present invention, the adventitious buds that have been extended may be cut out and used as cuttings. The multiblasts themselves can be maintained and proliferated by appropriately dividing them and culturing them in a medium having the same composition as the medium used for the formation of the multibuds.

  In the present invention, the cuttings thus obtained are cultured in a medium or culture soil. The medium and / or culture soil used at this time may be those suitable for rooting each plant. For example, as a culture medium, a Murashige-Skoog (MS) medium, a Rinsmeier-Skoog medium, a Gamborg B5 medium, a white medium, a niche niche medium, or the like is generally well known as a tissue culture medium for plants. If necessary, one or more auxins as plant hormones and / or 5 to 30 g / l of sucrose as a carbon source can be added to the basic medium or a diluted one thereof. Auxins are not particularly limited, but indole butyric acid (IBA), naphthalene acetic acid (NAA) and the like are easily available and easy to use. In the present invention, the medium may be a liquid medium or a solid medium. When used as a solid medium, agar or gellan gum is further added to the above ingredients and solidified for use. In addition, when using the culture medium which added sucrose etc. which are also a carbon source of microorganisms as a carbon source, cuttings are cultured in a sterile environment. Instead of using carbohydrates such as sucrose as a carbon source, carbon dioxide can be cultured in a culture environment with a concentration of about 300 to 1500 ppm. In this case, it is necessary to cultivate the cuttings in a sterile environment. Absent.

  As the culture soil, common culture soil used for cuttings such as red soil (red bean soil), river sand, mountain sand, kanuma soil, vermiculite, perlite, peat moss, and water can be used. As other rooting materials, “Oasis (registered trademark)” manufactured by Smither Oasis, “Florialite (registered trademark)” manufactured by Nisshinbo Industries, Ltd., and the like can be used.

  In order to form adventitious roots from cuttings, usually the cuttings are inserted into the medium or culture soil and cultured. That is, when branches, stems, buds or adventitious buds are used as cuttings, these cuttings cut from the original individual or tissue are used as the solid medium, water, commercially available liquid fertilizer or the liquid. When using leaves as culture heads moistened with culture medium and using leaves as cutting ears, leaves are cut out from the original individuals or tissues, and the leaves are cut into the same solid medium or the above culture soil. Culture. The culture soil may be moistened with water or the like after the insertion of the cutting head.

  In the present invention, ultrasonic treatment is performed on at least a part of the portion of the cutting head that forms the adventitious root in this way and in contact with the medium and / or the culture soil. The frequency of the ultrasonic wave used at this time is preferably in the range of 10 to 100 khz, and particularly preferably in the range of 20 to 30 khz. The ultrasonic treatment time is preferably 30 to 300 seconds, particularly preferably 60 to 180 seconds. When the ultrasonic frequency used is less than 10 khz, or when the ultrasonic treatment time is less than 30 seconds, the effect of promoting the formation of adventitious roots cannot be expected. On the other hand, when the ultrasonic frequency exceeds 100 khz, or when the sonication time exceeds 300 seconds, the moisture in the solid medium or the culture soil in which the cuttings are inserted generates heat and the cells of the cuttings In addition to the possibility of indirect damage, the cells in the cuttings may be directly damaged by the ultrasonic treatment.

  Specifically, in order to perform ultrasonic treatment on at least a part of the cutting head medium and / or the portion in contact with the culture soil, for example, in a suitable container, solid medium, water, commercially available liquid fertilizer or the liquid Prepare the culture soil moistened with the medium, insert the ears into this soil, and then put this container in the ultrasonic treatment tank of the ultrasonic cleaning machine that is filled with water so that water does not enter the container. It is only necessary to operate the ultrasonic cleaner. At this time, the ultrasonic wave reaches the tissue of the cutting head inserted in the solid medium or the culture soil through the water stretched in the ultrasonic cleaning tank and the medium and / or the culture soil. Sonication can be performed on at least a portion of the portion in contact with the culture soil. As the ultrasonic cleaner, a commercially available ultrasonic cleaner with an output of about 10 to 100 w can be used. However, in the present invention, ultrasonic treatment may be applied to the cutting head before being inserted into a solid medium or culture soil. Even in this case, adventitious root formation from the cuttings is promoted.

  In the present invention, it is preferable to cultivate the cutting spike by inserting its base into a solid medium, or water, a commercially available liquid fertilizer, or a culture soil moistened with the liquid medium. Plants inherently recognize the top and base, and adventitious roots are usually formed from the base, so by inserting the spikes in this way, the formed adventitious roots remain as they are and the necessary nutrients are added. It is because it can extend into the contained solid medium or culture soil.

  Moreover, if the cutting spike has a cut surface or a cut portion at a portion in contact with the medium and / or culture soil, adventitious root formation from the cutting spike is promoted. This is thought to be due to the fact that the cells at the site were damaged by the cutting and cutting of the cutting head, which caused physiological stimulation and promoted adventitious root formation from the nearby site. For this reason, artificially, one or more small scratches may be made on the insertion part of the cutting head to the medium or the like with a cutter or the like. It is obtained by cutting leaves or adventitious buds, etc., and has a cut surface at the base thereof. Usually, this effect can be obtained simply by inserting the base of the cutting head into a medium or the like. it can. At this time, if the cutting head is cut obliquely, the stimulation due to cell damage increases, and the contact area between the cut surface and the medium and / or culture soil also increases, so that adventitious root formation is further promoted.

  Furthermore, adventitious root formation from cuttings can be promoted by preliminarily treating the cuttings before culturing with auxin. The auxin treatment may be carried out by immersing the cuttings in an auxin solution having a concentration of 5 to 100 ppm, or by applying a powder mixed with 1 to 20 mg of auxin per 1 g of talc and applying it to the cut surface of the cuttings.

  It should be noted that the cutting head after being inserted into a solid medium, or water, commercially available liquid fertilizer or culture soil moistened with the liquid medium, preferably has a temperature of 23 to 28 ° C., a wavelength component of 650 to 670 nm and 450 to It is preferable to culture under a light irradiation containing a 470 nm wavelength component in a ratio of 9: 1 to 7: 3. Adventitious roots are usually formed in 2 to 5 weeks from the base of the cuttings thus cultured.

  The cuttings in which adventitious roots are formed are continuously cultured for a certain period of time to enrich the roots, and then transplanted to a seedling container or a nursery field as rooting seedlings to grow them. It can be set as the seedling which can be used for the purpose. What is necessary is just to set suitably the conditions at the time of growing a seedling, conditions, such as temperature and light intensity, so that it may be suitable for the plant. When adventitious buds derived from cultured tissues such as multi-buds or shoot primordia are used as cuttings, it is usually necessary to undergo an acclimatization process before transplanting to a seedling container or the like.

[Action]
When the cuttings are cultured in a suitable medium and / or culture soil, components such as the medium are absorbed into the cuttings from the portion in contact with the medium and so on, and adventitious roots are formed. At this time, if this part of the cutting head is subjected to ultrasonic waves, cells are activated, cell division is promoted, and adventitious root formation is promoted. In particular, difficult rooting plants take a long time to root, and the ears often die before the adventitious roots are formed. Acts very advantageously in plants.

  In addition, sonication is thought to cause ionization of water contained in the culture medium and culture soil and reduction of the molecular weight of the water cluster. Therefore, when sonication is performed on the cuttings through the medium and / or the culture soil, not only water but also the components of the culture medium and / or the culture soil dissolved in the water, the cell membrane of the cuttings and its Since it becomes easy to permeate the organelle membrane present in the cells and nutrients, plant hormones, and the like are effectively utilized by the cells, the formation of adventitious roots is further promoted.

  Hereinafter, the present invention will be described in more detail with reference to examples.

[Example 1]
E. Buds are obtained from the current year branches of two globulae strains, and using these buds as materials, the method described in JP-A-8-228621 is used to obtain two multi-buds derived from these two lines (line 1 and line 2). Induced. About one month after the start of induction of multi-buds, adventitious buds extending to a length of 2 to 5 cm were cut out from the two obtained multi-bud lines and used as cuttings. In addition, relatively large leaves attached to adventitious buds were cut in half to suppress transpiration, and when cuttings were densely planted, the leaves of adjacent cuttings were not overlapped. And used as cuttings.

  On the other hand, “Oasis (registered trademark)” manufactured by Smither Oasis (vertical 1 cm × width 1 cm × depth) wetted in a 4-fold diluted MS liquid medium supplemented with 2 mg / l of IBA as a plant hormone and 2 μM of silver thiosulfate (STS). 3 cm), a cube with a 1 cm diameter circular opening with a polytetrafluoroethylene membrane (Millipore's “Milliseal”) with a pore diameter of 0.45 μm attached to the top surface and a slightly protruding body 16 pieces are prepared per case in a culture vessel made of polycarbonate (maximum dimension: 8.4 cm long x 8.4 cm wide x 5.4 cm high) and inserted as described above. One ear was inserted for each one. At this time, STS in the liquid medium is added to suppress the generation of ethylene and prevent the cutting heads from dying.

  After insertion, the cutting head is placed in the culture vessel in the ultrasonic washing tank of the ultrasonic washing machine (Honda Desktop Electronics W-113MKII) filled with water. In a culture chamber adjusted to a carbon dioxide concentration of 1000 ppm, a temperature of 25 ° C., and a humidity of 60% after sonication for 60, 180 or 300 seconds at a frequency of 31 kHz and an output of 100 w. The culturing was performed under light irradiation containing a wavelength component of 650 to 670 nm and a wavelength component of 450 to 470 nm at a ratio of 8: 2.

  Three weeks after the insertion, the state of rooting was visually observed, and the number of rooting cuttings and the number of roots generated per cutting head at that time were investigated. The results are shown in Table 1.

[Comparative Example 1]
Two systems of E. coli were performed in the same manner as in Example 1 except that the ultrasonic treatment was not performed. The cuttings obtained from two buds derived from globulas were inserted into “Oasis (registered trademark)” and cultured, the rooting situation was observed, the number of rooting cuttings, and then The number of roots produced per cutting head was investigated. The results are shown in Table 1.

  As is clear from Table 1, E.I. In both globulae lines, adventitious root formation was promoted by sonication, and the rooting rate improved compared to the case without sonication, and per rooted shoot. There were also a lot of roots.

[Example 2]
An apple ( Malus pumila var. Domestica ) 2-5 cm of the current branch was cut out and used as an ear. At this time, the relatively large leaves on the branch of the current year are cut in half to suppress the transpiration, and when the cuttings are densely planted, the leaves and leaves of adjacent cuttings overlap. It was used as a cutting head after there was no such thing.

  On the other hand, “Oasis (registered trademark)” manufactured by Smither Oasis, which was moistened with a 4-fold diluted MS liquid medium supplemented with 2 mg / l of IBA as a plant hormone, was placed in a polycarbonate culture vessel in 16 cases per culture vessel. One piece was prepared, and the cutting heads obtained as described above were inserted one by one. At this time, “Oasis (registered trademark)” and a polycarbonate culture vessel having the same dimensions and shape as those used in Example 1 were used.

  After insertion, the cutting head is placed in the culture vessel in the ultrasonic washing tank of the ultrasonic washing machine (Honda Desktop Electronics W-113MKII) filled with water. In a culture chamber adjusted to a carbon dioxide gas concentration of 1000 ppm, a temperature of 25 ° C., and a humidity of 60% after sonication at a frequency of 31 kHz and an output of 100 w for 60 seconds, 650 to 670 nm And a wavelength component of 450 to 470 nm were cultured under light irradiation containing a ratio of 8: 2.

  Three weeks after the insertion, the state of rooting was visually observed, and the number of rooting cuttings and the number of roots generated per cutting head at that time were investigated. The results are shown in Table 2.

[Comparative Example 2]
Except that no sonication was performed, in the same manner as in Example 2, the cuttings obtained from the current branch of the apple were inserted into “Oasis (registered trademark)” and cultured, and the rooting situation was observed. Then, the number of rooted cuttings and the number of roots generated per cutting head at that time were investigated. The results are shown in Table 2.

  As is clear from Table 2, E.I. Like globulas, apple cuttings promoted adventitious root formation by sonication, and improved rooting rate compared to the case without sonication, and per rooted cuttings. An increase in the number of roots produced was observed. In particular, in this case, the effect on the improvement of the rooting rate was remarkable.

Claims (4)

When cultivating cuttings using a medium and / or culture soil, at least part of the portion of the cuttings in contact with the medium and / or culture soil is subjected to ultrasonic treatment using ultrasonic waves with a frequency of 20 to 100 kHz for 30 to 300 seconds. A method for cultivating cuttings, wherein adventitious roots are formed by culturing.   The method for cultivating cuttings according to claim 1, wherein a base of cuttings is present in a part in contact with the culture medium and / or culture soil.   The method for cultivating cuttings according to claim 1 or 2, wherein the cuttings have a cut surface or a cut part at a part in contact with the medium and / or culture soil. Inserted through the media and / or culture soil are cultured ear, characterized by sonication at least part of the portion in contact with the media and / or culture soil cuttings, claim 2 or 4. The method for cultivating cuttings according to 3 .