NZ501621A - Culturing method for somatic embryos of coniferous trees - Google Patents

Abstract

A method for culturing somatic embryos is disclosed, in which the process for multiplying the spherical cells, and that for causing the maturation of the cells is based on particular cell densities. Early embryonic cell densities of 2.1% to 10% are used for multiplication of spherical cell clusters whereas spherical cell cluster densities of 0.1 to 1.9% are used for maturation from spherical cell clusters to suspensor extensor cells.

Description

NEW ZEALAND PATENTS ACT, 1953 No: Date: COMPLETE SPECIFICATION CULTURING METHOD FOR SOMATIC EMBRYOS OF CONIFEROUS TREES We, FORESTRY AND FOREST PRODUCTS RESEARCH INSTITUTE, FORESTRY AGENCY, MINISTRY OF AGRICULTURE, FORESTRY AND FISHERIES, of 1, Matsunosato, Kukizaki-machi, Inashiki-gun, Ibaraki, Japan, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page - la -) CULTURING METHOD FOR SOMATIC EMBRYOS OF CONIFEROUS TREES BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a method for the culturing of somatic embryos of coniferous trees, and in greater detail, relates to a method for the large-scale culturing of somatic embryos of coniferous trees, wherein embryogenic cells are efficiently caused to multiply and mature.

Prior Art Methods for obtaining plants employing plant tissue culturation technology are broadly divided into methods in which adventitious buds or adventitious roots are formed, and methods in which somatic embryos are formed. Among these methods, plant regeneration techniques employing cultured cells of calluses or embryogenic cells or the like are extremely important techniques in biotechnologies for breeding, which have come to be regarded as important in recent years, that is to say, in the production or growth of plants having useful and novel properties by means of cell fusion methods or gene transfer methods. la Among plants, there are gymnosperms (coniferous trees) and angiosperms (herbaceous plants, broad-leaved trees). Among coniferous trees, there are reports of the formation of adventitious buds from cultured cells (calluses) of the genus Pinus. the genus Picea. the genus Larix. and the like, of the family Pinaceae. which are important trees, particularly in the forestry industry (family Pinaceae. genus Pinus: Washer, et al., (1997) N. Z. J. For. Sci. 7:321-328, Minocha (1980) Can. J. Bot. 58:366-370, genus Picea: Simola and Honkanen (1983) Physiol. Plant. 59:551-561, Lu and Thorpe (1988) In Vitro Cell Dev. Biol. 24:239-245, and genus Larix: Laliberte and Lalonde (1990) Can. J. Bot. 68:979-989). Laliberte and Lalonde drew attention to the difference in color and tissue state produced in calluses in the cultivation of calluses of hybrid European larch (Larix x eurolepis), and discovered that there is a close connection between the ability to form adventitious buds and the color and tissue state of the calluses. The present inventors have to date conducted research into the relationship between the characteristics of the tissue state of the calluses and the ability to form adventitious buds in Japanese larch (Larix leptolepis) and in Picea iezoensis (Ogita, et al., (1996) Mokuzai Gakkaishi Vol. 42, 11:1042-1048; Ogita, et al., (1997) Journal of Forest Research, Vol. 2, 3:141-145). 2 However, it has been pointed out that adventitious buds originating in calluses sometimes exhibit slow growth, and sometimes have difficulties in producing roots. In other words, currently, sufficient success has not been achieved with methods for forming adventitious buds from calluses in coniferous trees.

Furthermore, conventionally, tissue culture (or cell culture) of coniferous trees was conducted using a solid medium such as agar or the like; however, the cell clusters which grew in the solid medium showed a tendency to form heterogeneous cell aggregates having various properties (Laliberte and Lalonde, (1990) Can. J. Bot. 68:979-989).

On the other hand, the first formation of somatic embryos in coniferous trees of genuses Picea and Larix was reported in 1985 (Picea: Hakman and Bonga, (1985) J. Plant Physiol. 121:149-158, Larix: Nagamani and Bonga, (1985) Can. J. For. Res. 15:1088-1091). Since then, a number of successes related to the formation of somatic embryos in coniferous trees have been reported (for example, Dunstan, et al., (1995) In:Thorpe (ed.). In vitro embryogenesis in plants, Kluwer Academic Publishers, Dordrecht, pp 471-538, etc.).

In general, what is meant by embryogenic cells are cells having the ability to divide into somatic embryos; this is defined as a cell cluster containing a typical early somatic 3 embryo which is characterized in terms of morphology(histomorphology) as having a structure which, in embryogenic cells of coniferous trees, an embryonal region and a suspensor region are connected. The embryonal region is a small cellular part rich in cytoplasm, while the suspensor region is a part of the cell which is rich in vacuoles. On the other hand, the case of angiosperms (herbaceous plants and broad-leaved trees) is different, forming small cell clusters rich in cytoplasm having the ability to form somatic embryos (for example, carrots are discussed in Halperin, (1995) In:Thorpe (ed.). In vitro embryogenesis in plants, Kluwer Academic Publisher, Dordrecht, pp. 1-16).

In recent years, there have been reports with respect to the morphology characteristics and ability to mature into somatic embryos of embryogenic cells of coniferous trees, and furthermore, with respect to the relationship with the ability to regenerate plants, in spruce (Jalonen and von Arnold (1991) Plant Cell Reports 10 384-387, Egertsdotter and von Arnold (1993) J. Plant Physiol. 141 222-229). Furthermore, research has also been conducted with respect to the formation of somatic embryos by means of liquid suspension culture methods (for example, Krogstrup (1990) Plant Sci. 72 115-123, Egersdotter and von Arnold (1998) J. Experimental Botany 49 (319) 155-162). In this way, a great deal of knowledge has 4 been gained with respect to embryogenic cells in coniferous trees; however, many unclear points still exist. In practice, it is difficult to obtain large amounts of somatic embryos when culturing embryogenic cells of larch in solid media, furthermore, the efficiency of plant regeneration was low.

The present inventors have previously derived embryogenic cells of the Japanese larch, and have observed and specified the morphological characteristics at an extremely early stage of development (Ogita et al. (1997) Forest Resources Environ. 35 45-51). Furthermore, in comparison with culturing in a solid medium, by means of employing the method of liquid suspension culturing, which is advantageous in the efficient culturing of the specified cells, they discovered that there is a close connection between the morphological characteristics and the maturation and ability to regenerate plants of the somatic embryos (Ogita 1997) Graduate School of Tokyo Agricultural University, Department of General Agricultural Studies, Ph.D.dissertation).

There is a reported method for growing somatic embryos of plants in a liquid medium in which, when suspension culture of the plant is conducted in a liquid medium, the dissolved oxygen and pH are regulated, and high density culturing of the plant culture cells is conducted (Japanese Patent Application, first publication no. Hei 9-220036).

SUMMARY OF THE INVENTION With respect to the culturing of cells of coniferous trees (including calluses and embryogenic cells), there has been insufficient research in the evaluation of the ability of such cells to divide, from the point of view of morphology, and such methods are important in advancing research into the regeneration of plants from cells.

Furthermore, in the case of culturing in solid media, there is a tendency for the characteristics of the cell clusters to be heterogeneous, and this was a reason why it was difficult to regenerate plants from such cultured cells. If it were possible to specify cells having the ability to divide, and to selectively and efficiently culture such cells, it would enable a high rate of plant regeneration from cultured cells of coniferous trees, which is difficult.

Furthermore, if a method of regenerating plants from somatic embryos was established as a stable culturing technology, this would obviate the problems related to slow growth and the treatment of root generation which were apparent in the formation of adventitious buds, and this could be expected to further advance research into the breeding and culturation of coniferous trees. Somatic embryos have a, portion comprising tissue which divides to form an apical cell. 6 501621 that is to say, a bud, and a portion comprising tissue which divides to form a root, so that they are convenient in that they can be handled in the same manner as common seeds.

The present invention was created in light of the above circumstances; it has as an object to go some way towards providing a technique which will allow the efficient culturing of embryogenic cells of coniferous trees, the formation of a large number of somatic embryos of coniferous trees or at least to provide the public with a useful choice.

As a result of diligent research into the questions described above, the present inventors have discovered that, by means of conducting culturing while appropriately regulating the cell density of the cells to be cultured in a liquid medium, it is possible to cause growth of embryogenic cells of coniferous trees in a uniform manner in terms of morphology and in large amounts, and it is also possible to obtain, efficiently and in large amounts, somatic embryos of coniferous trees which possess the ability to germinate, in a liquid culturing medium; the present invention was arrived at on the basis of this discovery.

In other words, the present invention is a culturing method for somatic embryos of coniferous trees, wherein, in a method for culturing somatic embryos from embryogenic cells in 7 intellectual PROPERTY office of n.z. 1 4 SEP 2001 RECEIVED 50162 1 a liquid medium, the following are included: a process for causing the multiplication of spherical cell clusters in a liquid medium, the early embryogenic cell density of which is within a range of 2.1 - 10%, and particularly preferably within a range of 3-5%, and a process for causing the maturation from spherical cell clusters to suspensor extension cell clusters in a liquid medium, the spherical cell cluster cell density of which is preferably within a range of 0.1 - 1.9%, and particularly preferably within a range of 0.1 - 0.5%. The numerical ranges given above reflect the conditions of the initiation of each process (this is also the case for the numerical ranges for the cell densities given hereinbelow).

Furthermore, the present invention is a culturing method for somatic embryos of coniferous trees, wherein, in a method for culturing somatic embryos from embryogenic cells in a liquid medium, the following are included: a process for multiplying spherical cell clusters in a liquid medium in which the cell density of the spherical cell clusters preferably within a range of 2.1 - 10%, and particularly preferably within a range of 3 - 5%, and a process for causing the maturation from spherical cell clusters to suspensor extension cell clusters in a liquid medium in which the cell density of the spherical cell clusters is preferably within a range 0.1 - 1.9%, and particularly preferably within a range of 0.1 - 0.5%.

Furthermore, in the present invention, it is preferable that, after the process for causing maturation to suspensor extension cell clusters, the suspensor extension cell clusters be cultured in a liquid medium containing a cell growth INTELLECTUAL PROPERTY OFFICE OF N.Z. 1 4 SEP 2001 501621 promoting agent for promoting maturation from suspensor extension cell clusters to somatic embryos.

Furthermore, the present invention is a method for causing the multiplication of spherical cell clusters, which are one type of coniferous tree embryogenic cells, in a liquid medium, wherein the spherical cell clusters are caused to multiply in a liquid medium in which the cell density of the early somatic embryogenic cells or the spherical cell clusters is preferably within a range of 2.1 - 10%, and particularly preferably within a range of 3 - 5%.

Furthermore, the present invention is a method for causing the maturation of spherical cell clusters, which are one type of embryogenic cell of coniferous trees, into suspensor extension cell clusters in a liquid medium, wherein the spherical cell clusters are caused to mature into suspensor extension cell clusters in a liquid medium, in which the density of the spherical cell clusters is preferably within a range of 0.1 - 1.9%, and particularly preferably within a range of 0.1 -0.5%.

In the culturing method for somatic embryos of coniferous trees in accordance with the present invention, by regulating 501621 INTELLECTUAL PROPERTY OFFICE OF N.Z. t < SEP 2001 RECEIVED the cell density of the embryogenic cells in the liquid medium, the multiplication and maturation of the embryogenic cells is controlled, and somatic embryos of coniferous trees are obtained.

In the present specification, the terms "early somatic embryogenic cells," "spherical cell cluster," and "suspensor extension cell cluster" are employed to delineate the differences in morphology in embryogenic cells in the process in which the embryogenic cells become somatic embryos.

"Early somatic embryogenic cells" indicate early stage embryogenic cells (used both in the case of single cells and in the case in which a plurality of cells have formed a single cell cluster), and indicate cells developed from so-called calluses, which are non-uniform and have differing degrees of development.

"Spherical cell cluster" indicates the state in which a cluster (from a few cells to a few tens of cells) forms a small spherical cell cluster. In greater detail, such a cell cluster comprises (i) small cells from 20 - 50 micrometers in diameter which are rich in cytoplasm, and (ii) cells containing large vacuoles having a diameter of 50 - 100 micrometers (there are cases in which cells having a diameter of 100 - 150 micrometers are also included). Depending on the type of coniferous tree employed or their cell density during culturing, the diameter. 11 and the proportion of cells of type (i) and cells of type (ii) making up the cell cluster may be changed. In other words, there are cases in which the majority of the cells are of type (i) or type (ii). Using the larch as an example, the diameter of the cell cluster is normally within a range of 70 - 150 micrometers, and comprises from a few to few tens of cells of type (i) and a few cells (ii).

"Suspensor extension cell cluster" indicates the state in which the suspensor region is extended.

In a cultured cells during the actual culturing of "early somatic embryogenic cell clusters", it is possible that there are cells present which do not ultimately form somatic embryos; however, in the present invention, what is meant by "cell density" regulation of course includes cases in which the cell density of aggregations comprising solely early somatic embryogenic cell clusters (cell cluster groups) is regulated, but also includes cases in which the cell density is regulated in the state in which, ultimately, cells are present having either extremely little or no ability to form somatic embryos. Furthermore, the cases of "spherical cell clusters" and "suspensor extension cell clusters" are the same in this regard.

In the culturing method for somatic embryos of the present invention, in order to control the multiplication of the 12 embryogenic cells and the maturation thereof, a process is provided in which the embryogenic cells are cultured at a cell density exhibiting a tendency for the embryogenic cells to become the spherical cell cluster tissue type, and a process is provided in which the embryogenic cells are cultured at a cell density exhibiting a tendency for the embryogenic cells to become the suspensor extension cell cluster tissue type. When the cell density is regulated so that a large number of the embryogenic cells become the spherical cell cluster tissue type, multiplication of the spherical cell clusters is promoted, and a large number of uniform spherical cell clusters can be obtained.

Furthermore, by conducting culturing while regulating the cell density such that it is possible to cause the maturation of the spherical cell clusters obtained to suspensor extension cell clusters at a high rate, it is ultimately possible to obtain a large amount of somatic embryos in an efficient manner.

The regulation of the cell density may be accomplished by regulating the amount of cells in the liquid medium, so that this may be accomplished, for example, by measuring and regulating the packed cell volume (PCV). In the present specification, the cell density numerical ranges indicated were obtained as packed cell volumes (PCV) in the liquid medium 13 containing cells, that is to say, in the culturing liquid. PCV indicates the volume of cells obtained when a standard amount of a liquid medium containing cells (hereinbelow referred to as "culture liquid") is centrifuged under standard conditions (standard centrifugal force, time, and the like), and the cells are precipitated; for example, if one milliliter of packed cells are contained in 10 milliliters of culture liquid, the cell density is 10%. During the measurement of cell density in the present specification, PCV measurement was conducted at 500 rpm and for 3 minutes.

Apart from the conditions described above, it is possible to conduct culturing by following the somatic embryo culturing method commonly employed, and regulating the conditions appropriately; however, a more concrete mode of the method of the present invention will be explained hereinbelow.

Hereinbelow, a more concrete mode of the method of the present invention will be explained.

First, as a first concrete mode of the present invention (hereinbelow referred to simply as "first mode"), a culturing method for somatic embryos of coniferous tree will be explained in which, in a method for culturing somatic embryos from embryogenic cells in a liquid medium, the following are provided: 14 (A) a process for causing the multiplication of the spherical cell clusters in a liquid medium, the cell density of early somatic embryogenic cells of which is regulated, and (B) a process for causing maturation from spherical cell clusters to suspensor extension cell clusters in a liquid medium in which the cell density of the spherical clusters is regulated.

In the first mode, using early somatic embryogenic cells as the starting material, somatic embryos are obtained via spherical cell clusters and suspensor extension cell clusters.

Cell groups containing early somatic embryogenic cells were employed as the early somatic embryogenic cells of coniferous tree employed as the starting material; however, it is preferable that cultured cells containing a high proportion of early somatic embryogenic cells be employed. Depending on the type of tree and the like, differences are produced; however, it is possible to obtain early somatic embryogenic cells by following, for example, Ogita et al. That is to say, following the method of, for example, Ogita et al. (Ogita, et al., (1997) Forest Resources Environ. 35 45-51), the early somatic embryogenic cells of coniferous trees are derived in a solid modified CD medium (modified Campbell and Durzan medium; where not otherwise limited, the mCD medium and the like are liquid media) containing 7 MM (M:mol/liter) 2,4-D, which is an auxin, and 3 micromolar BAP, which is a cytokinin, as plant hormones, and multiplication was conducted in this same solid mCD medium, and the early somatic embryogenic cells were obtained as the cells to be cultured. The mCD medium with the appropriate regulation of plant hormones and the like is effective in the derivation of embryogenic cells not only of larch, but also of other coniferous tree types (for example, Japanese cedar: Ogita et al. (1998), 109th Japan Forestry Society Conference, Collected Presentations, page 189). The present inventors had previously obtained early somatic embryogenic cells of the Japanese larch as a model culturing system. The "early somatic embryogenic cells" of the present specification correspond to the "embryogenic callus" in "Ogita et al. (1997) Forest Resource Environ 35 45-51".

The early somatic embryogenic cells used in process (A) may be cultured and stored on solid media, and these may then be transferred to liquid media, suspended, and cultured. Furthermore, if the initial amount of early somatic embryogenic cells is small, the suspension and culturing of the early somatic embryogenic cells may be conducted prior to the multiplication process of the spherical cell clusters, and the appropriate amount for regulating the cell density may be obtained, and conducting this suspension and culturing as preprocessing to the multiplication process is also preferable 16 in that it facilitates the operation of regulating the cell density. Examples of media which may be employed in this suspension and culturing of the early somatic embryogenic cells included, for example, CD medium, MS (Murashige and Skoog medium) medium, GD medium (Gresshoff and Doy medium), and the like. Furthermore, it is also possible to employ mCD medium, mMS medium, mGD medium, and the like, in which these media are employed as a base and the components thereof are appropriately altered. The suspension may be conducted by means of a standard method such as shaking or the like, and furthermore, after suspension in the liquid medium, stationary culturing is also possible. Depending on the type of coniferous tree employed, the culturing conditions, such as the degree of shaking, the culturing period, the amount of light, and the like may be appropriately regulated.

The process (A) described above signifies that spherical cell clusters are caused to multiply in a liquid medium, the early somatic embryogenic cell density whereof is regulated. When this process (A) is initiated, it is necessary to regulate the cell density of the early somatic embryogenic cells in the liquid medium. In greater detail, the cell density is preferably within a range of 2.1 - 10%, and particularly preferably within a range of 3 - 5%. 17 Within these ranges, it is possible to cause the efficient and large scale multiplication of spherical cell clusters from early somatic embryogenic cell clusters, and furthermore, the culture liquid does not take the form of a gel to as great an extent, so that handling is facilitated. On the other hand, when the density is below the lower limit, there is a tendency for the uniformity and extent of multiplication of the spherical cell clusters to decline. Even if the upper limit of the cell density is exceeded while the culturing continues during the multiplication process, the culturing may be continued without conducting a further regulation of the cell density insofar as this density does not disturb the multiplication tendency. However, there are cases in which the culturing liquid takes on the form of a gel, and later handling becomes difficult.

Examples of the liquid media employed in process (A) include, for example, CD medium, MS medium (Murashige and Skoog medium), GD medium (Gresshoff and Doy medium), and the like. Furthermore, it is also possible to employ mCD medium, mMS medium, mGD medium and the like, in which these media are employed as a base and the components thereof are appropriately adjusted.

Furthermore, the temperature conditions of the culturing in the multiplication process of the spherical cell clusters 18 depend on the type of coniferous tree employed; however, a range of 20 - 27° C is preferable, and a temperature of approximately 25° C is particularly preferable.

The culturing may be conducted by means of shaking culture using an rotatory shaker or the like, or by means of a stationary culture or the like. The degree of shaking (suspension), the culturing period, the amount of light, and similar conditions may be appropriately adjusted in accordance with the type of coniferous tree.

Furthermore, spherical cell clusters are multiplied in the culture liquid by means of process (A), so it is possible that spherical cell clusters are multiplied successively by repeating the regulation of the cell density of spherical cell clusters appropriately. Depending on the type of coniferous tree; however, it is preferable to repeat the regulation approximately every 3-4 weeks.

Furthermore, by means of process (A) described above, it is possible to efficiently obtain a large number of spherical cell clusters which are uniform in terms of morphology from early somatic embryogenic cells which are generally in a nonuniform state in terms of morphology.

Next, with respect to process (B), this process is one in which, after the spherical cell clusters have been obtained in large amounts in process (A), the cell density of the spherical 19 cell clusters is regulated, and maturation is carried out from spherical cell clusters to suspensor extension cell clusters.

In this process (B), the cell density of the spherical cell clusters within the liquid medium is preferably adjusted to within a range of 0.1 - 1.9%, and more preferably within a range of 0.1 - 0.5%. If the density is within such ranges, it is possible to conduct the maturation of spherical cell clusters to suspensor extension cell clusters at a high rate, and it is ultimately possible to obtain a large amount of somatic embryos. On the other hand, when the density is below the lower limit, the efficiency of large scale production of the somatic embryos is low, while when the upper limit is exceeded, it is difficult to sufficiently conduct maturation. When maturation is promoted and PCV increases and the maturation becomes suppressed, the medium may be appropriately exchanged for a fresh medium, and furthermore, it is preferable to repeat the regulation of the cell density; however, insofar as the maturation is not suppressed, the culturing may be continued without again regulating the cell density.

Examples of the liquid medium employed in process (B) include, for example, CD medium, MS medium, GD medium, and the like. Furthermore, it is also possible to employ mCD medium, mMS medium, and mGD medium and the like in which these media are used as a base and the components thereof are appropriately-adjusted.

Furthermore, the temperature conditions of the culturing in process (B) depend on the type of coniferous tree employed; however, they are preferably within a range of 20 - 27°C, and particularly preferably in the vicinity of 25" C.

It is preferable that shaking culture using, for example, an rotatory shaker or the like be employed as the culturing method. Furthermore, the maturation progresses whether the conditions are light or dark; however, dark conditions are preferable.

Furthermore, by continuing the culturing and maturation of the suspensor extension cell clusters obtained in process (B) under the conditions described above, it is possible to obtain somatic embryos. A preferable mode for promoting the maturation after suspensor extension cells are obtained will be separately explained below.

As described above, it is possible to cause the spherical cell clusters to multiply in process (A), and by means of process (B), it is possible to cause the maturation from spherical cell clusters to suspensor extension cells at a high rate, so that by means of the method of the present invention, it is possible to obtain a large amount of somatic embryos in an extremely efficient manner. Conventionally, in the process 21 of producing embryogenic cells, when the embryogenic cells take the tissue form of the spherical cell cluster state, even if culturing in a solid medium or suspension culturing in a liquid medium or the like was continued, there were many cases in which development was suppressed, and the proportion maturing from spherical cell clusters to somatic embryos was not high, so that it was thought to be difficult to form suspensor extension cell clusters from spherical cell clusters; however, the present invention overcomes the difficulties present in the technique and achieves the effects described above.

Furthermore, the spherical cell clusters and suspensor extension cell clusters obtained by means of the processes (A) and (B) are uniform in terms or morphology, and there is little need for operations such as the selection of cells having specified morphologies by means of cell mesh filtering or the like which were conducted in the conventional culturing of other plants such as carrots and the like, so that the present invention is superior in terms of operational efficiency.

Next, a second mode will be explained, concentrating on those parts which are different from the first mode.

In the first mode, by means of conducting culturing while regulating the cell density of the early somatic embryogenic cells, the spherical cell clusters where caused to multiply; however, in the second mode, the cell density of spherical cell 22 clusters is regulated, and the spherical cell clusters are caused to multiply. In other words, after the spherical cell clusters have been obtained, it is not absolutely necessary to initiate culturing from early somatic embryogenic cells, and by repeating the regulation of the cell density of the spherical cell clusters, it is possible to obtain larger numbers of spherical cell clusters. The spherical cell clusters obtained may be used to derive large numbers of somatic embryos via the process (B) described in the first mode. In the process of causing the spherical cell clusters to multiply, the cell density of the spherical cell clusters within the liquid medium is preferably within a range of 2.1 - 10%, and particularly preferably within a range of 3 - 5% as was the case with that of the early somatic embryogenic cell cluster. In the process of causing in the maturation from spherical cell clusters to suspensor extension cell clusters, the cell density of the spherical cell clusters within the liquid medium is preferably within a range of 0.1 - 1.9%, and particularly preferably within a range of 0.1 - 0.5%.

Furthermore, in order to obtain sufficiently developed somatic embryos at a higher rate, in the first or second mode methods, it is preferable that the suspensor extension cell clusters matured from spherical cell clusters via the process (B) be cultured in a liquid medium containing cell growth 23 promoting agents capable of promoting the maturation from suspensor extension cell clusters to somatic embryos.

The cell growth promoting agent may be appropriately selected in accordance with the type of coniferous tree employed; however, concretely, preferable cell growth promoting agents include, in addition to plant hormones such as abscisic acid (ABA), cytokinins (BAP and the like), and auxins (2,4-D, NAA and the like), mannitol, polyethylene glycol, and the like, and particularly preferable among these are ABA and/or mannitol.

To explain in greater detail with references to larch, when ABA is added, the concentration in the liquid medium is preferably within a range of 0.1 - 100 PLM, and particularly preferably within a range of 50 - 100MM, and furthermore, when mannitol is added, the concentration thereof in the liquid medium is preferably within a range 0.1 - 0.6 M, and particularly within a range of 0.1 - 0.3 M. When culturing is carried out in a medium containing ABA, it is preferable that after washing the suspensor extension cell clusters using mCD medium containing no auxins nor cytokinins, culturing be conducted using, as the maturation medium, a liquid medium in which ABA or the like is added to mCD medium containing no auxins or cytokinins. Furthermore, when the suspensor extension cell clusters are transferred to the maturation 24 medium containing ABA, it is preferable that the cell density be readjusted.

By means of conducting culturing in a medium containing cell growth promoting agents as described above, it is possible to promote maturation from suspensor extension cell clusters to somatic embryos.

Furthermore, in the present invention, the method provided is one in which, in a method for causing the multiplication of spherical cell clusters, which are a form of embryogenic cells of coniferous tree, in a liquid medium, the spherical cell clusters are caused to multiply in a liquid medium in which the cell density of the early somatic embryogenic cells or the spherical cell clusters has been regulated. The cell density of the early somatic embryogenic cells or spherical cell clusters in the liquid medium is preferably within a range of 2.1 - 10%, and more preferably within a range of 3 - 5%. The method for the multiplication of spherical cell clusters of the present invention corresponds to the process for causing the multiplication of spherical cell clusters in the culturing method for somatic embryos of coniferous trees described above, and may be conducted in the same manner as this process.

By means of appropriately repeating the regulation of the cell density, it is possible to cause the continuous multiplication of the spherical cell clusters. The spherical cell clusters obtained by means of the method in which the spherical cell clusters are caused to multiply which was described above are thought to enable the successive readjustment of the cell density in fresh medium at intervals of 3 - 4 weeks and subculture, although this depends on the type of coniferous tree employed, so that maintainance of this for one year or more may be possible.

Furthermore, the present invention provides a method for maturing spherical cell clusters, which are a type of embryogenic cell of coniferous trees, to suspensor extension cell clusters in a liquid medium, wherein maturation of the spherical cell clusters to suspensor extension cell clusters takes place in a liquid medium in which the cell density of the spherical cell clusters is regulated. The cell density of the spherical cell clusters within the liquid medium is preferably within a range of 0.1 - 1.9% and particularly preferably within a range of 0.1 - 0.5%. The method for maturing the embryogenic cells or the spherical cell clusters to suspensor extension cell clusters corresponds to the process for maturation to suspensor extension cell clusters in the first and second modes described above and may be conducted in a manner identical to this process.

By means of the method for maturation to suspensor extension cells of the present invention, it is possible to 26 conduct the maturation of spherical cell clusters to somatic embryos, which was conventionally thought to be difficult, at a high rate.

The present invention is optimal for use with coniferous tree types, and concrete examples thereof include, preferably, coniferous trees of the family Pinaceae (the genus Pinus. the genus Larix, and the genus Picea). the Taxodiceae family, the Cypressaceae family, and the like, and more preferable are the larch, the spruce, and the Japanese cedar and the like, and particularly preferable is Larix leptolepis.

In the culturing method of the present invention, it is possible to observe the changes in the morphology of the embryogenic cells in the culturing process of the embryogenic cells in an uninterrupted, clear, and continuous fashion in the liquid medium using an inverted microscope. Accordingly, the method of the present invention facilitates the search for the optimal conditions for the culturing of somatic embryos.

(Brief Description of the Diagrams) [Figure 1] A diagram showing an image of the early somatic embryogenic cells of a larch prior to the setting of the cell density.

[Figure 2] A diagram showing an image of the suspensor extension cell clusters obtained by culturing at a cell density of 0.1%. 27 [Figure 3] A diagram showing an image of spherical cell clusters multiplied at a cell density of 5%.

[Figure 4] A diagram showing an image in the case in which suspensor extension cell clusters were cultured in a medium containing no plant hormones (ABA).

[Figure 5] A diagram showing an image in the case in which the suspensor extension cell clusters were matured in a medium containing 0.1 micromolar ABA.

[Figure 6] A diagram showing an image in the case in which the suspensor extension cell clusters were matured in a medium containing 50 micromolar ABA.

[Figure 7] A diagram showing an image in the case in which the suspensor extension cell clusters were matured in a medium containing 50 micromolar ABA and 0.3 M mannitol.

[Figure 8] A diagram showing an image of spherical cell clusters multiplied while maintaining the morphology characteristics and high ability to form embryos under conditions of 5% cell density.

[Figure 9] A diagram showing an image of somatic embryos developed from spherical cell clusters.

[Figure 10] An outline of the culturing method of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 28 In order to clarify the relationship between the cell density of the embryogenic cells and the morphology, the following experiments were performed.

Experimental Example 1> Relationship between cell density of the embryogenic cells and the morphology (Experimental Example 1-1) Adjustment using a suspension culture of the early somatic embryogenic cells The present inventors have obtained early somatic embryogenic cells of the Japanese larch as a model culturing system (Ogita et al. (1997) Forest Resources Environ 35 45-51), and have employed these as a starting material.

Early somatic embryogenic cells with a fresh weight of 100 mg were placed in 10 ml of liquid mCD medium, and this was placed in a 100 ml conical flask, and covered with a transparent polyester film (trade name "Lumilar", produced by Toray), the culturing temperature was set to 25°C, and setting the light period (4000 - 5000 lux) to 16 hours, and the dark period to 8 hours, culturing was conducted for approximately 3 weeks, and the suspension culture liquid containing the early somatic embryogenic cells in the liquid medium was adjusted (Figure 1). With respect to the shaking culture, using an rotatory shaking culture apparatus, the rotational speed was set to 100 rpm. 29 In the present embodiment (including the experimental examples), where not otherwise limited, the "mCD medium" having the composition shown in Table 1 was employed.

[Table 1] Table 1 Composition of the mCD Medium in the Present Embodiment (pH 5.6) Composition Amount Contained Inorganic Nutrient nh4no3 400 mg/L Components KN03 340 mg/L Ca( N03) 2 • 4H20 980 mg/L kh2po4 170 mg/L KC1 65 mg/L MgS04 • 7H20 370 mg/L FeS04 • 7H20 27.8 mg/L Na2-EDTA 37.3 mg/L MnS04 • 4H20 16.9 mg/L ZnS04 • 7H20 8.6 mg/L CoC12 • 6H20 0.025 mg/L CuS04 • 5H20 0.025 mg/L NaMo04 • 2H20 0.25 mg/L KI 0.83 mg/L h3bo3 6.2 mg/L Organic Nutrient Thiamine • HC1 0.4 mg/L Components Myo-inositol 100 mg/L L-glutamine 600 mg/L D-sucrose 30000 mg/L Plant Hormones 2,4-D 7 MM BAP 3 MM 31 (Experimental Example 1-2) Adjustment of the cell density of the early somatic embryogenic cells in the suspension culture liquid The cell density of the early somatic embryogenic cells in the suspension culture liquid obtained in experimental example 1-1 was set to within a range of 0.1 - 10%. That is to say, the cell density was regulated so that 0.01 - 1 ml of early somatic embryogenic cells, as measured by PCV, were contained in 10 ml of the suspension culture liquid. The PCV measurement for regulating the cell density in the suspension culture liquid was conducted at 500 rpm and for a period of 3 minutes. After the regulation of the cell density, suspension culturing was carried out for a further 4 weeks, and the cell density was again measured. Hereinbelow, the results of the changes in cell density are shown. Furthermore, the state of the early somatic embryogenic cells prior to the setting of the cell density as observed by an inverted microscope is shown in Figure 1, while the state after culturing for 4 weeks at a cell density of 0.1% is shown in Figure 2, and the state after culturing for a period of 4 weeks with a cell density of 5% is shown in Figure 3. The observation of the flasks by the inverted microscope can be facilitated by removing the condenser portion of the inverted microscope. 32 [Table 2] Table 2 Effects of Cell Density in the Multiplication of Early Somatic Embryogenic Cells of the Japanese Larch Cell Density Initially Set Conditions Cell Density (PCV in 10 ml of Suspension Culture Liquid) After 4 Weeks of Suspension Culturing Cell Density (PCV in 10 ml of Suspension Culture Liquid) 0.1% (0.01 ml) .3% (1.03 ml) 0.5% (0.05 ml) .7% (1.57 ml) 1% (0.1 ml) 18.7% (1.87 ml) 2% (0.2 ml) 22.0% (2.20 ml) % (0.5) 43.3% (4.33 ml) % (1 ml) 52.7% (5.27 ml) Note: The numerical values in Table 2 represent average values obtained by conducting the testing three times.

(Experimental Example 1-3) Results of Experimental Example 1 As shown by the results of experimental examples 1-1 and 1-2 (Figures 1-3) and by Table 2, by means of conducting culturing for 4 weeks, the somatic embryogenic cells multiply to approximately 1 - 5.3 ml (a cell density of approximately 10 - 53%) by PCV. In this case, PCV increased by from about 100 times to about 5.3 times. 33 However, with respect to the culturing results, in the present invention, it is necessary to consider the multiplication or maturation of the embryogenic cells from the point of view of morphology.

In other words, the startling increase in PCV (particularly startling at 0.1%) in the cell densities from 0.1 - 1.9% was confirmed to result from the growth of a large number of suspensor extension cell clusters, as shown in Figure 2, by means of observation using an inverted microscope.

On the other hand, a tendency was observed at a cell density of 2% for multiplication with morphologies closely resembling those of the early somatic embryogenic cells prior to density setting (see Figure 1: the morphology of Figure 1 closely resembles that of embryogenic cells multiplied by culturing using solid media).

Furthermore, at a cell density of 5 or 10%, the "spherical cell clusters" shown in Figure 3 actively multiplied, and the number of spherical cell clusters increased.

By means of the experimental example 1 employing the embryogenic cells of larch, the present inventors discovered that there is a "border line between multiplication and maturation" in the embryogenic cells based on changes in morphology. When the early somatic embryogenic cells were at a certain specified cell density, they either maintained the same 34 morphology or exhibited a tendency to be reduced in size and multiply, while when a different cell density was present, the exhibited a tendency to mature in terms of morphology, and that specified cell density is thought to be approximately 2%.

Accordingly, it is thought to be ultimately preferable that, in order to increase the population of embryogenic cells, the cell density be set to a level higher than 2%.

Experimental Example 2> Observation of the morphology during maturation of the embryogenic cells The embryogenic cells (spherical cell clusters, suspensor extension cell clusters) which were caused to multiply or mature in experimental example 1 were cultured for a period of 3 weeks using a specified maturation medium, and the state of development thereof was observed using an inverted microscope. As a result, these developed into somatic embryos which matured as shown in Figure 6 when ABA was added in high concentrations. On the other hand, when the concentration of ABA was low, development such as that seen when ABA concentration was high was not observed. Hereinbelow, this will be explained in detail.

(Experimental Example 2-1) Maturation in media containing ABA In general, abscisic acid (ABA) is effective as a plant hormone which promotes the maturation into somatic embryos.

Based on the morphology, the embryogenic cells obtained in experimental example 1 can be divided into suspensor extension cell clusters (Figure 2) and spherical cell clusters (Figure 3).

Using mCD media containing 0, 0.1, 1, 10, 50, and 100 MM ABA and containing no auxins and cytokinins as maturation media, the spherical cell clusters or suspensor extension cell clusters were transferred to these maturation media and culturing was conducted for a period of 3 weeks, and the state of the development thereof was observed using an inverted microscope.

When the suspensor extension cell clusters at a cell density of 3% (0.3 ml in PCV) were transferred to the maturation medium, there was a clear difference in the reaction with respect to each ABA concentration.

That is to say, when ABA was at a high concentration (in particular at 50 or 100MM), development occurred to somatic embryos having the morphology shown in Figure 6 (in the case of 50MM). On the other hand, in the case in which ABA was not contained (Figure 4) or in the case in with ABA was at a low concentration of 1MM or less (see Figure 5: as an example, the case of a medium to which 0.5MM of ABA was added is shown), development equaling that at 50 or 100MM was not observed. 36 Furthermore, with respect to the spherical cell clusters, as well, when the same type of experiment was conducted as in case of the suspensor extension cell clusters described above, in the spherical cell cluster state, as opposed to the suspensor extension cell clusters, no clear tendency to develop and mature into somatic embryos was observed at any ABA concentration.

From these results, it was confirmed that, in order to obtain a large quantity of somatic embryos, it is desirable to conduct the culturing of embryogenic cells which have developed to suspensor extension cell clusters in an appropriate maturation medium containing ABA or the like.

(Experimental Example 2-2) Readjustment of Cell Density Culturing of suspensor extension cell clusters was conducted in the same manner as in experimental example 2-1, except that the cell density was adjusted to 0.1, 0.3, or 1% in an amount corresponding to PCV, and a maturation medium was employed which contained 50MM ABA, and it was determined that the media containing 0.1 and 0.3% exhibited maturation which was superior to that of the medium containing 1%.

(Experimental Example 2-3) Addition of Mannitol Mannitol was added to a maturation medium containing 50 micromolar ABA in an amount of, respectively, 0, 0.1, 0.2, 0.3, 0.4, 0.5, or 0.6 M, and the maturation of the suspensor 37 extension cell clusters was assayed, and it was determined that by adding 0.3 M of mannitol, the maturation of the somatic embryos was promoted to the greatest extent and somatic embryos were sufficiently obtained (Figure 7). That is to say, by means of culturing in a medium containing ABA and an appropriate amount of mannitol, somatic embryos were obtained which were elaborate in terms of morphology and exhibited white or yellow whitish color.

Embodiments > Hereinbelow, the large scale culturing of somatic embryos by means of the present invention will be explained based on embodiments. An outline of the embodiments described hereinbelow is shown in Figure 10. (1) Suspension Culturing of Early Somatic Embryogenic Cells Early somatic embryogenic cells were suspension cultured in accordance with the method described above (Experimental Example 1-1), and a suspension culture liquid containing early somatic embryogenic cells was prepared. (2) Multiplication of Spherical Cell Clusters from Early Somatic Embryogenic Cells The suspension culture liquid of the early somatic embryogenic cells was adjusted to a cell density of 5%. That 38 is to say, adjustment was conducted so that the early somatic embryogenic cells previously prepared were contained in an amount of 0.5 ml in packed cell volume in 10 ml of the suspension culture liquid. The measurement of the packed cell volume (PCV) for the adjustment of the cell density in the suspension culture liquid containing the early somatic embryogenic cells was conducted at 500 rpm and for a period of 3 minutes.

After adjusting the cell density, culturing was conducted for a period of 4 weeks in mCD medium, and the multiplication of the spherical cell clusters was conducted. After 4 weeks, when the cell density of the spherical cell clusters was measured, it was found to have increased to 43.3%. When an observation was conducted using an inverted microscope in order to assess the morphology characteristics, it was found that a plurality of spherical cell clusters were contained in the culturing liquid, and almost none of these had a morphology other than that of the spherical cell clusters (Figure 8). (3) Maturation from Spherical Cell Clusters to Suspensor Extension Cell Clusters The cell density of the spherical cell clusters in mCD medium was adjusted to 0.1% and the spherical cell clusters obtained by the multiplication process for spherical cell clusters of embodiment (2) were cultured for a period of 3 39 weeks, and caused to mature to suspensor extension cell clusters. (4) Maturation from Suspensor Extension Cell Clusters to Somatic Embryos It was confirmed by inverted microscope that the suspensor regions of the suspensor extension cell clusters were sufficiently extended (see Figure 2).

After washing the suspensor extension cell clusters in an mCD medium having the composition shown in table 1, from which auxins and cytokinins were removed, the cell clusters were allowed to culture for a period of 3 weeks in liquid media in which, respectively, 50 and 100MM ABA was added to the mCD media containing no auxins or cytokinins. After a period of 3 weeks, it was confirmed by observation with an inverted microscope that a large number of somatic embryos were obtained.

Furthermore, 0.3 M of mannitol was further added to the liquid media containing 50MM and 100MM, respectively, ABA, and culturing was conducted. Culturing was conducted for a period of 3 to 4 weeks, and when the state of development was observed using an inverted microscope, it was confirmed that a large number (1.5 - 2xl05) of somatic embryos which had elaborate morphology and exhibited white color or yellow 40 whitish color and were sufficiently matured were obtained (Figure 9, Table 3).

Furthermore, the processes of (2) multiplication of the spherical cell clusters, (3) maturation of the spherical cell clusters to suspensor extension cell clusters, and (4) maturation from suspensor extension cell clusters to somatic embryos, were repeatedly conducted, and a large amount of somatic cell embryos of Japanese larch were obtained in liquid medium. (5) Germination of the Somatic Embryos The somatic embryos obtained were transplanted to a solid mCD medium containing no plant hormones, and allowed to germinate. The mCD medium was solidified using 0.2% gellan gum. <Comparative Examples> The culturing method of the present invention was compared with the culturing method for somatic embryos of the conventional methods employing solid media.

Early somatic embryogenic cells corresponding to 100 mg in fresh weight were cultured for 9 weeks on solid mCD media, and then cultured cells obtained by culturing for a period of 3 weeks were transferred to a solid mCD medium containing 50MM ABA and containing no auxins or cytokinins, and a number of mature somatic embryos in an amount within a range of a few to 41 a few hundred were obtained. The somatic embryos exhibited the ability to germinate.

Table 3 shows a comparison between the method of the present invention and the case of the comparative example. 42 [Table 3] Conventional Method Method of the (Culturing on Solid Present Invention Medium) (Culturing in Liquid Medium with Adjusted Cell Density) Number of Somatic A few - a few 1.5 - 2 x 10s Embryos Formed hundred Reports relating to the promotion of the maturation of somatic embryos in liquid media have been made with respect to herbs (for example, carrots); however, this has been difficult in trees, and in particularly in coniferous trees. However, by appropriately adjusting the cell density and conducting culturing in a liquid medium, it is clear that it is possible to culture a large number of somatic embryos in a liquid medium using coniferous trees.

[Effects of the Invention] By means of the present invention, it is possible to efficiently culture a large number of somatic embryos in liquid media. It is expected that the somatic embryos will find use as artificial seeds, and by means of the present invention, it is expected that it will be possible to arrange for a stable supply of seeds even of useful coniferous trees, which have a 43 wide difference between good and poor crop year. (For example, the Larix leptolepis and cypress, which are Japanese coniferous trees, only produce a harvest once per several years, and a stable supply of seeds thereof is desirable.) Furthermore, by means of the method of the present invention, even if a very small amount of embryogenic cells are present, it is possible to produce a large number of plants there from via somatic embryos.

Furthermore, the method of the present invention is not merely useful in the large scale multiplication of clones having the same morphology, but it is also expected to facilitate the production of trees from embryogenic cells provided with useful novel properties by means of biotechnologies for breeding such as cell fusion or genetic engineering or the like. The provision of novel properties in plants by means of current cell fusion or genetic manipulation methods has an extremely low efficiency of transformation in trees, and in particular in coniferous trees, and the regeneration of plants from cultured cells provided with such morphologies has been difficult, so that these were unstable techniques. By applying the method of the present invention, it is expected that this will lead to the stabilization and increased efficiency of such techniques. 44 Furthermore, by applying the method of the present invention, which allows for the high-rate reproduction of development processes leading to mature somatic embryos from early somatic embryogenic cells or spherical cell clusters, which represent morphology types of embryogenic cells, it is possible to make great advances in research into the cytology, plant physiology, or molecular biology of somatic embryo formation in coniferous trees, in which fields there has been little success to date. 45

Claims (5)

WHAT WE CLAIM IS:

1. A culturing method for somatic embryos of coniferous trees, wherein, in a method for culturing somatic embryos from embryogenic cells in a liquid medium, the following are included: a process for causing the multiplication of spherical cell clusters from early embryogenic cells, in a liquid medium, wherein the early embryogenic cell density is within a range of 2.1 - 10%, and a process for causing the maturation from spherical cell clusters to suspensor extension cell clusters in a liquid medium wherein the spherical cell cluster cell density is within a range of 0.1 - 1.9%.

2. A culturing method for somatic embryos of coniferous trees, wherein, in a method for culturing somatic embryos from embryogenic cells in a liquid medium, the following are included: a process for multiplying spherical cell clusters in a liquid medium in which the cell density of the spherical cell clusters is within a range of 2.1 - 10%, and a process for causing the maturation from spherical cell clusters to suspensor extension cell clusters in a liquid medium in which the cell density of the spherical cell clusters is within a range of 0.1 - 1.9%.

3. A culturing method for somatic embryos of coniferous trees in accordance with one of claim 1 or claim 2, wherein, after the process for causing maturation to suspensor extension cell clusters, the suspensor extension cell clusters are cultured in a liquid medium containing a cell growth promoting agent for promoting maturation from suspensor extension cell clusters to somatic embryos. 46 intellectual property office of n.z. 1 5 OCT 2001 RECEIVED 50162

4. A method for causing the multiplication of spherical cell clusters, which are one type of coniferous tree embryogenic cells, in a liquid medium, wherein the spherical cell clusters are caused to multiply in a liquid medium in which the cell density of early embryogenic cells or the spherical cell clusters within a range of 2.1 - 10%.

5. A method for causing the maturation of spherical cell clusters, which are one type of embryogenic cell of coniferous trees, into suspensor extension cell clusters in a liquid medium, wherein the spherical cell clusters are caused to mature into suspensor extension cell clusters in a liquid medium, the spherical cell cluster cell density is within a range of 0.1 -1.9%. 47 ^' tSncPROPERIY" OFFICE OF N.Z. ' < SEP 2001 I RECEIVED I