AU781465B2 - Method for producing rooted cutting - Google Patents

Description

S&F Ref: 536798

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Nippon Paper Industries Co., Ltd.

4-1, Oji 1-chome Kita-ku Tokyo Japan Actual Inventor(s): Akira Murakami, Toshiaki Tanabe, Kunichika Murakami

S..

Address for Service: Invention Title: Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Method for Producing Rooted Cutting The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c

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METHOD FOR PRODUCING ROOTED CUTTING BACKGROUND OF THE INVENTION i. Filed of the Invention The present invention relates to a technology for commercial production of clone plantlets having excellent characters on a large scale according to a cutting method.

2. Brief Description of the Background Art Cutting is a traditional vegetative propagation method wherein a part of an artificially cut plant tissue (a cutting) is inserted in a rooting bed and rooted in the rooting bed to produce a whole plant body. The method is widely used for herbaceous plants to woody plants as a method for producing and propagating plants having the same genetic characters as the parent plant, clone plantlets, on a large scale.

This method is convenient and suitable for obtaining a large amount of plantlets at a time. Since plants having excellent characters can be produced on a large scale at a low cost, the method is commercially advantageous but all the plants cannot always be propagated according to the method. Even when the method is used, there are many species and varieties which cannot be propagated because their cuttings are not rooted at all or the rooting rates are very low.

oo o oo oo °ooo .11 i oooo go o o 1 Moreover, rooting from a cutting is carried out in the open air or in a greenhouse, frame or the like. On the other hand, the environment should be maintained at relatively high humidity until a cutting is rooted to form a sound plantlet. The reason is that low humidity causes withering and weakening of the cutting owing to transpiration from its leaves. However, even in the case that rooting from a cutting is carried out in a greenhouse or a frame, and needless to say, in the case of the open O air, it is considerably laborious to maintain an environment of high humidity.

Under these circumstances, there are still many plants having excellent characters but impossible to propagate at a level which permits their commercial production.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing a rooted cutting, applicable even to a ***plant which is impossible or difficult to propagate by cutting.

Another object of the present invention is to provide a method for producing a rooted cutting, capable of *maintaining an environment of high humidity until the rooting and of improving the productivity of the plantlet.

2 Furthermore, an object of the present invention is to provide a method for propagating a clone plantlet on a large scale, which is applicable to many plants and is commercially advantageous.

As a result of extensive studies, the present inventors have found that the above objects can be achieved by culturing a cutting in a culturing vessel with feeding nutrients necessary for the growth of a plant, and therefore, have completed the present invention.

\O Namely, these and other objects of the present invention have been achieved by a method for producing a rooted cutting comprising: preparing a rooting bed wetted with a carbon source-free liquid medium which comprises nitrogen, Sphosphorus and potassium as essential elements in a culturing vessel, 0•co inserting a cutting therein to culture it, and rooting the cutting while regulating the concentration of CO 2 gas in the culturing vessel.

eeee DETAILED DESCRIPTION OF THE INVENTION S. The present invention will be explained below in detail.

*The kinds of plants to which the present invention is applicable are not particularly limited. Specifically, it is also possible to apply the present invention to woody 3 plants, such as eucalyptuses, acacias, myricas, oaks, vines, apple trees, cherry trees, rose bushes, camellias, Japanese apricot trees, and the like, and herbaceous plants, such as chrysanthemums, carnations, and the like. The present invention is equally applicable to plants previously regarded to be difficult to propagate according to a cutting method and plants not regarded so.

As the cutting, in addition to a green branch (a current shoot) and a hard branch (a branch grown last year O or before), a sprout or a leaf can be used. It is usual to use a green branch or a hard branch in the case of a woody plant and a sprout or a leaf in the case of a herbaceous plant. In the conventional method for producing a rooted cutting, in the case of using a branch as the cutting, it is necessary to cut off a part of a leaf in order to suppress transpiration through the leaves attached to the branch. However, in the present invention, the cutting is stood under high humidity, so that the transpiration is suppressed without subjecting to such treatment. Therefore, 0 such pretreatment is not necessary.

In the method of the present invention for producing a rooted cutting, the maintenance of humidity of the environment surrounding the cutting is facilitated by carrying out the operation in a culturing vessel using a L5 rooting bed wetted with a liquid medium. For example, when a wide-mouthed flask or the like having an opening at a r d r r u 4 position higher than the height of the rooted cutting to be produced in the culturing vessel is utilized, the culturing environment is spontaneously maintained at relatively high humidity until the rooting. Moreover, in not only such vessel but also various types of vessel, the environment in the vessel can be maintained at high humidity by adjusting the size and position of the opening of the vessel.

Therefore, in the present invention, the culturing vessel can be selected from various types of vessels in consideration of operability and the like. From the viewpoint of maintenance of the environment in the culturing vessel at high humidity, a tightly closed vessel is more preferably used. In this case, however, CO 2 gas should also be supplied to the culturing vessel. For example, covering the opening of the vessel with a CO 2 gaspermeable membrane can be adopted for that purpose.

The rooting bed can be one which is substantially homogeneously wetted with a liquid medium and can hold the cutting to be inserted therein in the inserted state. For 2.Q example, natural soil sand, attapulgits, etc. artificial soil vermiculite, perlite, glass beads, etc.), a porous molded product foamed phenol resin, rock wool, etc.), or the like, placed in a culturing vessel can be used as the rooting bed.

In the present invention, the liquid medium contains nitrogen, phosphorus and potassium as essential 5 elements. The concentration of each element is adjusted according to the kind of a plant to be prepared as the cutting. As such liquid medium, a commercial mixed fertilizer for home gardening or a known liquid medium for plant tissue culture can be used as it is or with suitable dilution. For example, a solution of "Hyponex liquid 5-10- (registered trademark)" (manufactured by Hyponex Japan) diluted 250-fold to 500-fold as a mixed fertilizer for home gardening or a solution of Murashige-Skoog medium (Murashige and Skoog (1962), hereinafter referred to as "MS medium") diluted 4-fold to 16-fold can be used as common liquid medium in the present invention.

Also, known media for plant tissue culture including the above-mentioned MS medium contain mineral salts and vitamins such as thiamine, pyridoxine and nicotine which comprises, in addition to nitrogen, phosphorus and potassium, hydrogen, carbon, oxygen, sulfur, S; calcium and magnesium as major elements, and iron, manganese, copper, zinc, molybdenum, boron and chlorine as .0 minor elements. Therefore, a medium containing these elements as mineral salts or vitamins in addition to nitrogen, phosphorus and potassium can be used as the liquid medium of the present invention.

00 Furthermore, at least one plant growth regulator can be added to the liquid medium in the present invention.

For example, the rooting of a cutting can be accelerated by 6 adding an auxin, which accelerates formation of adventitious roots from plant tissues, such as indoleacetic acid, indolebutyric acid (IBA), naphthaleneacetic acid, or the like, or a combination of two or more of them, in an amount of 0.1 to 10 mg/L to the liquid medium of the present invention.

On the other hand, the liquid medium of the present invention is free of any carbon source such as sucrose and the like. A carbon source is an energy source common to many living things. In the present invention, however, since operations under an aseptic condition is not an essential requirement as in a usual method for producing a rooted cutting, when a medium containing a carbon source is used, bacteria attached to a cutting and those in a culturing environment also proliferate using the carbon source in the medium as the nutrient to cause withering of the cutting and the plantlet to be formed therefrom.

Actually, not only a plantlet to be formed from the *0 0000 cutting but also the cutting has an ability to perform o photosynthesis by itself, and therefore, can use CO, gas as an energy source by assimilating the gas upon irradiation 0 of light at a strength suitable for the growth of the plant.

:Therefore, the cutting of the present invention is also expected to root without feeding a carbon source as the 0 3* 2 nutrient by utilization of CO 2 gas in the atmosphere. In the present invention, however, the cutting to which 7 nutrients such as nitrogen, phosphorus and potassium are fed through a liquid medium conducts photosynthesis actively, so that it becomes necessary to regulate the concentration of C02 gas artificially. Namely, since the 3 active photosynthesis of the cutting lowers the concentration of CO 2 gas in a culturing vessel, it is necessary to supply the gas artificially. Otherwise, even when the nutrients other than CO 2 gas are supplied sufficiently, the ability of photosynthesis lowers in a short time, and thereby, rooting from the cutting, i.e., the formation of a plantlet is to be inhibited.

The concentration of CO 2 gas necessary for active photosynthesis of a cutting in a culturing vessel and for enhancement of the rooting rate thereof varies with the kind of the plant to be prepared as the cutting. In general, it is preferable to regulate the concentration of

CO

2 gas in a culturing vessel to the range of 300 to 1500 oooo S" ppm. When the concentration of CO 2 gas in a culturing vessel is lower than 300 ppm, remarkable enhancement of the ability of photosynthesis and the rooting rate of the cutting cannot be expected. And, when the concentration of 0coo ro ~CO 2 gas in a culturing vessel is higher than 1500 ppm, the ability of photosynthesis and the rooting rate of the cutting are not enhanced to an extent which corresponds to the increase of the concentration of CO 2 gas. The concentration of CO 2 gas can be regulated in each culturing 8 vessel separately but it is convenient and advantageous in view of the cost to regulate the concentration of C02 gas in the culturing vessel to a predetermined concentration by regulating the concentration in the environment itself in which the culturing vessels are placed. At that time, the culturing vessel can be a vessel whose opening remains opened or a vessel whose opening is covered with a CO 2 gaspermeable membrane as described above.

In the method of the present invention for producing a rooted cutting, there is no particular limitation on other conditions such as temperature and light strength. The conditions suitable for the photosynthesis of the plant from which the cutting is derived can be optionally adopted. In general, the \i following conditions are adopted for the present invention: a temperature of about 20 to 30 0 C; a light strength of about 40 to 100 pmol/m 2 /sec. Furthermore, in the present invention, the culture for producing a rooted cutting can be carried out with repeating the light period where the S cutting is cultured under light irradiation, and the dark S period where it is cultured in darkness, alternatively. In this case, since the photosynthesis is conducted only in the light period, the regulation of the concentration of

CO

2 gas is only necessary in the light period.

".As described above, it is not particularly necessary to work under aseptic conditions in the present 9 invention. However, in order to make assurance double sure for producing a sounder plantlet, it is preferable to subject a culturing vessel, a liquid medium and a rooting bed to a sterilization treatment, such as heat sterilization, autoclave sterilization, or the like, prior to insertion of the cutting.

The rooted cutting produced in the present invention can be taken out of the culturing vessel immediately after rooting, and transplanted into a growing pot to grow it. The soil used for the transplantation of the rooted cutting and the growing conditions, such as temperature, light strength, and the like, can be optionally determined so as to be suitable for the plant from which the cutting is desired. Through such growing process, plantlets usable for the predetermined purposes, such as forestation and the like, can be obtained.

Previously, in the case of production and propagation of plantlets by cutting, it is regarded as a taboo to feed nutrients before rooting of the cutting. The reason is that the cutting before rooting lacks normal ability for absorbing nutrients, and therefore, feeding of nutrients at that stage is considered to only result in the rotting of the cutting (for example, Practice of Grafting, •Cutting, and Layering, page 34, published by Taikodo on July i, 1986) oe e go e 10 However, the present inventors have found that even the cutting before rooting can also utilize elements, such as nitrogen, phosphorus, potassium, and the like, and feeding of these elements as nutrients remarkably increases the rooting rate provided that proliferation of unwanted bacteria is prevented. In this case, the cause of the proliferation of unwanted bacteria is a carbon source. In the present invention, therefore, a cutting is cultured with using a liquid medium which contains nitrogen, tC phosphorus and potassium as essential elements and is free of a carbon source.

On the other hand, an energy source necessary for plants as a substitute of the carbon source is CO 2 gas present in the air. In the case of the present invention, artificial regulation of the concentration of CO 2 gas in the culturing environment accelerates rooting of the cutting cultured in the above-mentioned medium to produce a plantlet. The reason is that the utilization of CO 2 gas present in the air only is insufficient for rooting from :°moee Sthe cutting.

"Furthermore, in the case of producing a plantlet according to a cutting method, a cutting is desirably placed under a condition of high humidity until a sound •plantlet is formed. However, cutting has been hitherto carried out in the open air or in a greenhouse or a frame.

Therefore, it is considerably laborious to maintain the 11 environment for culturing the cutting at high humidity.

However, the present invention uses a rooting bed wetted with a liquid medium and produces a rooted cutting in a culturing vessel. Because of this, the humidity in the culturing environment of the cutting can be maintained at a high level without effort and a sound rooted cutting can be easily produced.

Even in the plants whose cuttings are difficult to root according to any conventional cutting method, the \IC rooting rate is remarkably enhanced according to the present invention.

Also, according to the present invention, the environment until the rooting can be easily maintained at high humidity.

Therefore, in the present invention, it becomes possible to produce rooted cuttings from plants whose rooted cuttings are difficult to produce according to any conventional method. Furthermore, in the plants from which rooted cuttings can relatively easily be produced, it is possible to enhance productivity thereof. Moreover, the *operations for the production of the rooted cutting are simple and convenient because the advantages of the operations in the conventional methods for producing rooted cuttings are inherited.

In addition, in the present invention, rooted cuttings are formed in a culturing vessel, so that it is 12 easy to regulate not only humidity but also other conditions such as temperature and light as compared with any conventional method for producing rooted cuttings.

Therefore, according to the present invention, rooted cuttings can be easily produced the year round.

Accordingly, the present invention enables a large scale propagation of clone plantlets with commercial advantage.

The present invention will be explained below in detail with reference to examples. However, the present invention is not limited thereto.

Example 1 r ~70 A branch to be a cutting was collected from a current shoot of 40-year-old Prunus yedoensis. The branch was cut into a 3 cm length with remaining only one leaf bud, and one third of the number of leaves attached thereto were cut off to prepare the cutting.

On the other hand, a polycarbonate vessel of 10 cm square where two holes having a diameter of 1 cm each were opened at the upper part thereof and the openings were covered with a CO 2 gas-permeable membrane (manufactured by Nihon Millipore, "Milliseal") was prepared as a culturing vessel. A foamed phenol resin molded product (manufactured by Nippon Soda Co. Ltd., "Oasis") wetted with 100 mL of MS medium diluted 8-fold or 100 mL of "Hyponex liquid 5-10-5 13 (registered trademark)" diluted 500-fold, to each of which 2 mg/L of IBA was added, was used as a rooting bed.

Rooted cuttings were produced by inserting the cuttings prepared as mentioned above into the rooting bed at a rate of 25 cuttings per one culturing vessel, regulating the concentration of CO 2 gas in each vessel to 350, 500, 1000 or 1500 ppm only in the light period, and culturing them at a temperature of 22 to 24 0 C and a light strength of 50 pmol/m 2 /sec, in a light period of 16 hours and in a dark period of 8 hours. The regulation of the concentration of CO 2 gas in the culturing vessel was carried out by regulating the concentration of CO 2 gas in the environment where the culturing vessel was placed.

After 3 weeks, the rooting was investigated upon 100 cuttings per each test plot. The results are shown in Table 1.

Comparative Example 1 With the exception that water mixed with 2 mg/L of IBA was used as a liquid medium for wetting the rooting bed, the cuttings collected and prepared from Prunus yedoensis were cultured in the same manner as in Example 1 to produce rooted cuttings. This experiment was carried out only on e the case that the concentration of CO 2 gas in the culturing vessel was regulated to 1000 ppm (only in the light period) weeclue nte aemne si Eape1t rdc 14 Three weeks after the insertion of the cuttings into the rooting bed, the rooting was investigated upon 100 cuttings. The results are shown in Table 1.

Comparative Example 2 SExcept that the concentration of CO 2 gas in the culturing vessel was not regulated at all, the cuttings collected and prepared from Prunus yedoensis were cultured in the same manner as in Example 1 to produce rooted cuttings. This experiment was carried out only on the case that MS medium diluted 8-fold and mixed with 2 mg/L of IBA was used as the liquid medium for wetting the rooting bed.

Three weeks after the insertion of the cuttings into the rooting bed, the rooting was investigated upon 100 cuttings. The results are shown in Table 1. In this case, the concentration of CO 2 gas in the culturing vessel was already decreased to 100 ppm or less before the end of the ooeo first light period.

go Comparative Example 3 The production of rooted cuttings was attempted according to a conventional method.

To a base part of each of the cuttings of Prunus o o S• yedoensis collected and prepared in the same manner as in Example 1 was applied talc powder mixed with 1% by weight of IBA, followed by inserting the treated cuttings into a 15 foamed phenol resin molded product (similar to that of Example 1) wetted with tap water. The phenol resin molded product into which the cuttings had been inserted was placed without special operation in a greenhouse where of light was shielded by means of a cheesecloth, and thus the cuttings were cultured.

Three weeks after the insertion of the cuttings into the rooting bed, the rooting was investigated upon 100 cuttings. The results are shown in Table 1.

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Table 1 Investigation of rooting rate on Prunus yedoensis Medium* CO, gas conc. (ppm) Rooting rate MS 8x 350 53 Hyponex 500x 350 43 MS 8x 500 89 Hyponex 500x 500 Ex. 1 MS 8x 1000 81 Hyponex 500x 1000 86 MS 8x 1500 82 Hyponex 500x 1500 83 Comp. Ex. 1 Water 1000 1 Comp. Ex. 2 MS 8x not regulated 0 Comp. Ex. 3 conventional cutting method 1 8x and respectively.) 500x mean 8-fold dilution and 500-fold dilution, As is apparent from Table 1, in the test plots where the concentration of CO 2 gas in the culturing vessel 16 was regulated to 350 ppm or more and a medium for plant tissue culture or a fertilizer for plants was supplied as the liquid medium, extremely high rooting rate was observed in every case as compared with the case where water was supplied instead of the liquid medium, the case where the concentration of CO, gas was not regulated, or the case where the culture was carried out according to a conventional cutting method.

Moreover, in the case that a medium for plant tissue culture was used as the liquid medium, the highest rooting rate was achieved in the test plot where the concentration of CO 2 gas in the culturing vessel was regulated to 500 ppm, while in the case that a fertilizer for plants was used as the liquid medium, it was achieved in the test plot where the concentration of CO 2 gas in the culturing vessel was regulated to 1000 ppm.

Example 2 An apical bud of one-year-old Eucalyptus globulus (hereinafter referred to as globulus") was cut off to grow branches from the axillary buds. After 3 weeks, the branches to the second node from the tip were collected to prepare cuttings.

0*0000 The cuttings were cultured in the same manner as in Example 1 to produce rooted cuttings. On the other hand, in the test plot where the concentration of CO 2 gas in the 17 culturing vessel was regulated to 350 ppm or 500 ppm, rooted cuttings were produced using also MS medium diluted 4-fold other than MS medium diluted 8-fold as a medium for plant tissue culture (in every case, 2 mg/L of IBA was Three weeks after the insertion of the cuttings into the rooting bed, the rooting was investigated upon 100 cuttings. The results are shown in Table 2.

Comparative Example 4 Except that water mixed with 2 mg/L of IBA was used as a liquid medium for wetting the rooting bed, the cuttings collected from E. globulus were cultured in the same manner as in Example 2 to produce rooted cuttings.

This experiment was carried out only on the case that the N\ concentration of CO 2 gas in the culturing vessel is regulated to 500 ppm (only in the light period).

Three weeks after the insertion of the cuttings o into the rooting bed, the rooting was investigated upon 100 cuttings. The results are shown in Table 2.

Comparative Example Except that the concentration of CO, gas in the culturing vessel is not regulated at all, the cuttings collected from E. globulus were cultured in the same manner as in Example 2 to produce rooted cuttings. This 18

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experiment was carried out only on the case that MS medium diluted 8-fold and mixed with 2 mg/L of IBA is used as the liquid medium for wetting the rooting bed.

Three weeks after the insertion of the cuttings into the rooting bed, the rooting was investigated upon 100 cuttings. The results are shown in Table 2.

Comparative Example 6 The production of rooted cuttings was attempted according to a conventional method.

To a base part of each of the cuttings of E.

globulus collected in the same manner as in Example 2 was applied talc powder mixed with 1% by weight of IBA, followed by inserting the treated cuttings into a foamed phenol resin molded product (similar to that of Example 2) wetted with tap water. The phenol resin molded product to which the cuttings had been inserted was placed without special operation in a greenhouse where 70% of light was shielded by means of a cheesecloth, and thus the cuttings were cultured.

Three weeks after the insertion of the cuttings into the rooting bed, the rooting was investigated upon 100 cuttings. The results are shown in Table 2.

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19 Table 2 Investigation of rooting rate on E. globulus Medium* CO, gas cone. (ppm) Rooting rate MS 8x 350 98 MS 4x 350 Hyponex 500x 350 MS 8x 500 97 MS 4x 500 94 Ex. 2 Hyponex 500x 500 MS 8x 1000 88 Hyponex 500x 1000 89 MS 8x 1500 97 Hyponex 500x 1500 97 Comp. Ex. 4 Water 500 12 Comp. Ex. 5 MS 8x not regulated 0 Comp. Ex. 6 conventional cutting method 0 8x, 4x and 500x mean 8-fold dilution, 4-fold dilution and 500-fold dilution, respectively.) As is apparent from Table 2, in the test plots where the concentration of CO 2 gas in the culturing vessel was regulated to 350 ppm or more and a medium for plant tissue culture or a fertilizer for plants was supplied as the liquid medium, extremely high rooting rate was observed in every plot as compared with the plot where water was supplied instead of the liquid medium, the plot where the concentration of CO 2 gas was not regulated, or the plot where the culture was carried out according to a conventional cutting method. Namely, in both cases where a 9 **3 20 medium for plant tissue culture or a fertilizer for plants were used as the liquid medium, it was observed that approximately 90% or more of the cuttings were rooted when the concentration of CO 2 gas in the culturing vessel was regulated to 350 ppm or more.

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Claims (9)

1. A method for producing a rooted cutting comprising: preparing a rooting bed wetted with a carbon source-free liquid medium which comprises nitrogen, phosphorus and potassium as essential elements in a culturing vessel, inserting a cutting therein to culture it, and rooting the cutting while regulating the concentration of CO, gas in the culturing vessel.

2. The method according to claim 1, wherein the liquid medium for wetting the rooting bed comprises a known fertilizer for plants as it is or with dilution. 0. 1 **Se

3. The method according to claim 1, wherein the liquid medium for wetting the rooting bed comprises a known S medium for plant tissue culture as it is or with dilution.

The method according to any one of claims 1 to 3, wherein the liquid medium for wetting the rooting bed .comprises at least one plant growth regulator.

5. The method according to claim 4, wherein the .plant growth regulator is an auxin. plant growth regulator is an auxin. 22 0 23

6. The method according to any one of claims 1 to 5, wherein the concentration of CO 2 gas in the culturing vessel is regulated to a range of from 300 to 1500 ppm.

7. The method according to any one of claims 1 to 6, wherein the cutting is derived from a woody plant.

8. A method for producing a rooted cutting, substantially as hereinbefore described with reference to any one of the examples, but excluding comparative examples.

9. A rooted cutting produced by the method according to any one of claims 1 to 8. Dated 28 December, 2000 Nippon Paper Industries Co., Ltd. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0 0 J (R \LIBVV]02275speci doc njc