JPH11275995A - Production of arboreous plant by photoautotrophic culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently produce small plant bodies, having a genetically homogeneous state, of an arboreous plant by culturing a tissue of the arboreous plant in a specific medium under specific conditions. SOLUTION: These small plant bodies are produced by transplanting a tissue of an arboreous plant preferably of Acasia, Coffea or Garcinia into a sugar free medium, and culturing by using carbon dioxide under illumination. Here, the culturing is carried out preferably at the concentration of carbon dioxide of 350-500 μmol/mol in the culturing vessel, and under the illumination at a photon flux density effective for photosynthesis of 150-300 μmol/m<2> /s.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a woody plant, particularly a plantlet belonging to the genus Acacia, the genus Coffee and the genus Garcinia by photoautotrophic culture.

[0002]

2. Description of the Related Art Conventionally, as a method for producing woody plants, a sexual reproduction method using seed propagation and an asexual reproduction method using cuttings, grafts, cuttings, and plant divisions have been used. However, among these methods, the sexual reproduction method requires genetically heterogeneous in the case of cross-pollinated plants, resulting in variability in the traits and growth of the produced seedlings and stable harvesting. The problem is that the seed germination rate is low and the yield is low, the growth is slow and the seeds are highly hybridized, so it takes time to determine the traits of the seedlings, and the seedling production requires at least two years. There was a point. In addition, in the method of asexual propagation of cuttings, etc., the number of trees that can be collected from one harvesting tree (parent strain) is limited.
Many tree species are difficult for vegetative propagation itself by cuttings,
There was a problem that seedling production required at least two years.

For example, in the case of Acacia mangium, which is a typical tree planting species in Southeast Asia, seed propagation has conventionally been generally used for its propagation, but seed seedlings have a problem of large genetic variation. Had. For this reason, it is desired to establish an effective method for growing a large number of clones having genetically excellent traits.

In coffee, Arabica coffee (Coffea arabica) accounts for 70% of coffee bean production.
, Seed propagation is used because Arabica species are genetically stable by self-pollination. However, the remaining 30% of coffee bean production consists of many species, and most of these species are cross-pollinated and their hybrids are genetically unstable, even though they can breed seeds. Therefore, propagation by cuttings is mainly performed, but also in propagation by cuttings, as with the propagation of cuttings of many tree species, there are problems such as difficulty in rooting and low growth rate. I have.

[0005] In addition, in mangosteen (Garcinia mangostana L.), which is a tropical fruit belonging to the genus Garcinia, seed multiplication has been carried out for parthenogenetic reproduction. However, because the number of seeds is limited,
It has been desired to establish a method for producing seedlings instead of seed propagation.

Therefore, in recent years, clone seedling production by tissue culture has been carried out for seeds that are difficult to propagate vegetatively by seed propagation or cuttings, or those that are not practical.
The production of cloned seedlings using tissue culture technology is large, and the stage of selecting and preparing plants for propagation (stage 0),
It consists of a stage for establishing aseptic culture (stage 1), a stage for propagation (stage 2), a stage for rooting and conditioning in vitro (stage 3), and a stage for acclimating in an ex vitro environment (stage 4) (Hartmann, HTet al., 1990.Principles of tissue
culture for micropropagation.pp459-495.Plant propa
gation: principles and practices (5thed.)). This method is particularly suitable for mass-producing genetically uniform seedlings in a short period of time because the tissue obtained by culturing each organ of the plant is subcultured and propagated, and the plant is regenerated. It can be said that it is a method. In fact, this technique has been used to successfully produce cloned seedlings by tissue culture in many herbaceous plants such as carnations and orchids.

However, there are very few examples of practical use of woody plants compared to herbaceous plants due to the difficulty of establishing a technology such as slow growth, low rooting rate and low growth rate. Is the current situation. Acacia, coffee,
Despite its industrial value, mangosteen has not succeeded in commercializing cloned seedling production by a conventional tissue culture method (a photomixotrophic culture method using a gel medium). Therefore, it has been desired to establish a method capable of producing a large amount of genetically homogeneous cloned seedlings in woody plants such as acacia, coffee, and mangosteen.

[0008]

SUMMARY OF THE INVENTION The present invention provides a method for efficiently producing plantlets of genetically homogeneous woody plants, particularly plants belonging to the genera Acacia, Coffee and Garcinia. As an issue.

[0009]

One of the problems with the conventional plant tissue culture technology (photomixotrophic culture method) is that the use of a medium containing sugar causes the contamination of various bacteria and the growth in an incubator. There is insufficient development of leaves and roots of the plantlets. In particular, root elongation is often difficult in gelled agar,
Roots grown in gelled agar tend to die during acclimation to the new environment (Smith and Spomer, 1995, Vessels, ge)
ls, liquid media, and support systems, p371-404, Auto
mation and Environmental Control in Plant Tissue C
ulture (ed .: Kozai and Smith), Kluwer Academic Publis
hers)).

[0010] The present inventors have found that the above-mentioned problems exist when producing plantlets of woody plants such as acacia, coffee, and mangosteen by conventional photomixotrophic cultivation. Due to the difficulty of production, we considered producing these plantlets by photoautotrophic cultivation in which the plants were cultured in a light-free medium in a light place. Therefore, the present inventors selected acacia, coffee, and mangosteen having high industrial value as woody plants, and studied production of plantlets of these plants by photoautotrophic culture. As a result, the explants obtained from these plants were transplanted to a sugar-free medium, and the culture was performed under light irradiation while maintaining the carbon dioxide concentration in the incubator at a high level. It has been found that plantlets can be rooted and grown very well as compared with culture. In addition, it has been found that the growth of plantlets can be further enhanced by using a support having a high porosity in the culture medium and increasing the ventilation in the incubator during the culture.

That is, the present invention relates to a method for producing a woody plant by photoautotrophic culture, particularly a plant belonging to the genus Acacia, the genus Coffee, and the genus Garcinia, and more specifically, (1) the tissue of the woody plant A method for producing a plantlet of a woody plant, wherein the plant is planted in a medium containing no sugar, and culturing is performed by applying carbon dioxide gas under illumination. (3) The method according to (1), wherein the woody plant is a plant belonging to the genus Coffee.
(4) the method according to (1), wherein the woody plant is a plant belonging to the genus Garcinia; (5) culturing while maintaining the carbon dioxide gas concentration in the incubator at 350 to 500 μmol / mol; ) To (4), (6)
The method according to any one of (1) to (4), wherein the culture is performed under illumination at a photosynthetic effective photon flux density of 150 to 300 μmol / m ² / s, (7) a fibrous support, a porous medium (1) to (6) using a support or a mixture thereof
(8) the support of the medium comprises a mixture of cellulosic fibers and vermiculite;
(9) The method according to any one of (1) to (8), wherein the culturing is performed in an incubator having high gas permeability.

In the present invention, the term "woody plant" refers to a plant that forms a xylem on a stem or a root and has a large number of cell walls that lignify and become strong. It is intended for grassy or fleshy herbaceous plants with poorly developed xylem.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a method for producing plantlets of woody plants by photoautotrophic culture. The production method of the present invention is characterized in that the tissue of a woody plant is transplanted into a medium containing no sugar, and culturing is performed by applying carbon dioxide gas under illumination.

As a material tissue (explant) of the plant used for the culture, for example, in the case of a plant belonging to the genus Acacia, a shoot having leaves can be used. The shoot used is usually a shoot consisting of a plurality of leaves and one stem. Shoots with leaves can be obtained from multiple shoots formed on a medium containing a plant growth regulator. In the case of a plant belonging to the genus Coffee, a single node having leaves can be used as an explant. The number of leaves in a single node is usually one or two. A single node having leaves can be collected by cutting a shoot elongated in a culture vessel between nodes. In plants belonging to the genus Garcinia, for example, shoots can be excised from aseptically germinated seedlings and used as explants.

[0015] In the production method of the present invention, the explant thus obtained is transplanted to a sugar-free medium, and cultivated by applying carbon dioxide gas under illumination. Unlike intact plants, plant tissue cultures do not have the ability to produce sugars by photosynthesis, so it was thought that it was essential to add sugars to the culture medium. No sugar is added as a carbon source to the culture medium to allow the culture to photosynthesize by applying carbon dioxide gas underneath. Addition of a sugar easily causes contamination of various bacteria, which adversely affects the plant body. Therefore, it is not preferable that the sugar is contained. For cultivation, a medium containing no sugar, but containing components such as macroelements such as nitrogen, phosphorus, potassium, magnesium and calcium, and inorganic nutrients such as trace elements such as iron, manganese, copper and zinc is used. Furthermore, vitamins such as nicotinic acid and thiamine hydrochloride, organic nutrients such as amino acids, and growth regulators may be contained, but these are not necessarily essential. As a specific medium, for example, a 1/2 concentration MS medium is suitable.

In the cultivation, it is particularly preferable to use a fibrous, porous, or a mixture thereof in a medium in order to promote rooting and growth of the explant being cultured. The use of a conventionally used medium solidified with agar or the like produces undesirable results in rooting and growth of plantlets. Since the fibrous or porous support has a high porosity, it is considered that the concentration of dissolved oxygen in the culture solution added to the support is increased, thereby promoting rooting and growth. The use of these supports also has the advantage that the pH, EC, etc. of the culture solution can be easily controlled. Specific examples of the support include, but are not limited to, vermiculite, perlite, cellulose fibers, polyester fibers, ceramic fibers, rock wool, and mixtures thereof. However, in the case of polyester fiber, ceramic fiber, and rock wool, there is a problem that the support itself remains without being decomposed when transplanting to the culture soil, and the root is damaged when the support is separated from the root. In addition, when vermiculite or perlite alone is used, a mixture of vermiculite and cellulose fiber (for example, Florialite) which does not have such a problem has problems such as poor fixation of a plant due to being granular. (Manufactured by Nisshinbo) is particularly preferred.

As a culture condition, for example, in a plant belonging to the genus Acacia, the light condition is usually the photosynthetically effective photon flux density (hereinafter sometimes simply referred to as “PPF”).
50 to 300 μmol / m ² / s. If the light conditions are too low, the efficiency of photosynthesis of the plantlets decreases, whereas if the light conditions are too high, plant growth may be inhibited, and costs are increased, which is not preferable. The CO ₂ conditions in the culture chamber are usually 1000 to 2000 μmol / mol. If the CO ₂ concentration is low, photosynthesis is not performed efficiently, which is not preferable. On the other hand, if the CO ₂ concentration is higher than the saturation point, the photosynthetic rate does not increase, which is not preferable. In addition, the temperature condition is usually 25 to 30 ° C., and the humidity condition is usually 50 to 70%. In addition, the ventilation condition in the incubator is usually performed at a ventilation rate of 3 to 6 / h. Among these conditions, the CO ₂ concentration in the incubator is 350-
Particularly preferred is a combination condition of 500 μmol / mol. Since CO ₂ concentration in the incubator, which is also affected by the rate of photosynthesis plantlets in incubator during the culture period, as the culture vessel the CO ₂ concentration is maintained within the preferred range, incubator outside The CO ₂ concentration may be changed appropriately. Even in plants belonging to the coffee genus, it is possible to culture the above conditions, since CO ₂ saturation point of photosynthesis of plants belonging to the coffee genus is higher than in the case of the genus Acacia, cultivation room CO ₂ condition Can be increased to 5000 μmol / mol. Further, in the case of plants belonging to the genus Garcinia, it is possible to culture under the same conditions as in the case of the plants belonging to the genus Acacia, but the PPF under light conditions is 100 to 300.
It is possible to culture at μmol / m ² / s. In the culture period of the plantlets, the light condition is usually such that the light period and the dark period are usually 12 to 16 hours and 12 to 8 hours, respectively. The incubator used for culturing is preferably gas-permeable in order to increase the ventilation rate. For example, the gas permeability can be increased by using a gas permeable film (for example, Missilir (manufactured by Millipore)) for the lid of the incubator. Also,
It is preferable that at least a part of the incubator is light-transmissive in order to cause photosynthesis of the plantlets. For example, an incubator having a transparent lid can be used.

[0018]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Mass Propagation of Acacia Plantlets by Photoautotrophic Culture In order to test whether or not micropropagation by photoautotrophic culture can be applied to Acacia mangium, photoautotrophic culture was performed. Growth and net photosynthetic rates of cultured and conventional photomixotrophically cultured plantlets were compared under different culture conditions.

More specifically, Acacia mangium subcultured by a conventional method (cultured in an MS culture solution supplemented with 20 g / l of sucrose, 1 mg / l of BAP, and 8 g / l of agar) was explanted (50- 100mg
(Body weight / shoot). 60m for 4 explants
Implanted into a 370 ml container containing 1 MS medium. Nine processes were set as shown in Table 1.

[0021]

[Table 1] In the table, “ne” indicates a low CO ₂ concentration (500-600 μmol / mo
l) is shown. "V + CF" indicates a mixture of 10 g of vermiculite and cellulose (Fluorialite, manufactured by Nisshinbo Industries) per container. Agar was used at a concentration of 8 g / l.

The control group was set as a conventional light-mixing culture condition generally used. The temperature in the culture room is 26-27 ° C during 16 hours of light conditions and 8 hours of dark conditions
Met. The relative humidity in the culture room was maintained at 60%. A white fluorescent lamp was used as a light source. The net photosynthetic rate of the plantlets in the incubator under light conditions was measured weekly according to the method of Fujiwara et al. (Agricultural Meteorology, 43 (1): 21-30 (1987)). At the end of the culture period (day 28), the body weight and dry weight were measured and the rooting rate was recorded.

As a result, as shown in Table 2, the biological weight and dry weight of the plantlets were significantly increased by the combination of the highly ventilated container and the CO ₂ concentration in the culture room. On the other hand, the presence or absence of sucrose and growth regulators in the medium had no significant effect. The control-treated plantlets had the lowest biomass and dry weight and did not root. In the table, "NS" and "**" indicate "not significant" and "significant at p <0.01", respectively.

[0024]

[Table 2] Net photosynthetic rate under light conditions, throughout the culture period
High in unsweetened, hyperventilated, and CO ₂ applied plots (S0-
GR-CE section and S0-NR-CE section) (Fig. 1). The above results
Micropropagation by photoautotrophy has been shown to promote growth, photosynthesis, and rooting in Acacia mangium plantlets.

Example 2 Mass Growth of Coffee Plantlets by Photoautotrophic Culture (1) Coffee grown in an incubator (Coffea arabust)
a) Assay of photosynthetic ability of plantlets The photosynthetic ability of coffee (Coffea arabusta) plantlets grown in an incubator was examined in response to different CO ₂ concentrations and PPF levels in the culture vessels. The measurement of photosynthetic rate (hereinafter sometimes simply referred to as “Pn”) is specifically performed by three methods, namely, 1) measuring coffee concentration based on the measurement of CO ₂ concentration inside and outside the culture vessel over 45 days. Measurement of daily changes in photosynthetic rate of plants 2) Niu et al. System (Niu, et al., 1997. J. Japanese Soc. Hort. Sc
i., Suppl.2.300-301), the evaluation of Pn of the plantlets in the incubator on the 10th and 30th days of the culture period,
3) Evaluation of Pn of coffee plantlets in the incubator on day 45 using “Portable photosynthesis (PP) System”
It was examined by.

1) Daily change of net photosynthetic rate (Pn) of coffee plantlets based on CO ₂ concentration measurement Cultivation of coffee plantlets was carried out as follows. First, three single nodes having leaves of coffee plantlets in a culture vessel were cultured in a 480 cm ³ plastic pot-type container. Each container contains 1/2 concentration of MS (Murashige, T
and F.Skoog.1962.Physiol.Plant, 15: 473-497)
75 ml was put, and Florialite (manufactured by Nisshinbo Industries) was used as a support. The pH of the medium was adjusted to 5.70 before autoclaving. A gas permeable membrane having a pore size of 0.5 μm (Milliseal, manufactured by Millipore) was worn on each of the two holes of the transparent lid of the culture vessel. The ventilation rate of the container was determined by the method of the ancient times (Kozai, T. et al., 1986. J. Agr. Meterol., 42
(2): 119-127). Experiments, CO ₂ concentration in the second stage in culture chamber (C _{ou t)} (400-500
(Ambient) and 1400-1500 μmol / mol). Explants were given 50 μmol / m ² / s light for the first 3 days using white fluorescent light (National). For the next 7 days
Increased to 100 μmol / m ² / s, then 150 μmol / m
m ² / s. 150 μmol / m ² / s, 250 μmol / m for the last 25 days
Three levels of light intensity were applied: m ² / s and 350 μmol / m ² / s (Table 3). The temperature of the culture vessel is 28 ° C ± 2 ° C and the relative humidity is 70-80.
%, Performed under light conditions of 16 hours per day.

[0027]

[Table 3] In addition, “L”, “M” and “H” on the left side of the description of each treatment in the table indicate that the PPF from the 21st day is 150, 250 and 350 μm, respectively.
mol / m ² / s, “N” and “E” on the right
Indicates low or high concentration CO ₂ conditions.

The Pn of plantlets in the steady state on days 10, 20, 30, and 45 was determined by Fujiwara et al. (Agricultural Meteorology, 43 (1): 21-30, 1987).
Was calculated by the following equation: The CO ₂ concentration was measured using a gas chromatograph (GC-12A, Shimazu).

[0029]

## EQU1 ## Pn = K ・ EV ・ (C _out -C _in ) (in this equation, “K” is a conversion coefficient from volume to molecular weight (40.5 mol / m ³ ), and “E” is time in a culture vessel. Per hour (h ^-1 ), "V" is the air volume of the culture vessel (c
m ³ ), “C _in ” and “C _out ” indicate the CO ₂ concentration (mol / mol) inside and outside the culture vessel under steady state conditions.) Pn of the plantlets in a 45-day culture of coffee plantlets
FIG. 2 shows the experimental results of the change over time. All ratings are
The average of five measurements is shown. At any level of PPF
C _in is increased with the increase of C _out. The net photosynthetic rate is
There was a slight increase during the first 20 days of culture. During the next 25 days, high CO ₂ concentrations were observed in coffee (coffea arabust
a) It greatly affected the photosynthetic ability of plantlets. At day 45, Pn was three times higher under high CO ₂ conditions (“LE” and “HE”) compared to low CO ₂ conditions (“LN” and “HN”). However, the effect of light intensity on the increase in net photosynthesis rate of coffee plantlets was small. 150μmol
Under the conditions of / m ² / s and 350 μmol / m ² / s, Pn was not significantly different.

Further, the body weight, dry weight, shoot length, and root length on day 45 of the culture were measured (FIG. 3). As a result, high CO ₂ concentrations promoted all of the coffee plantlet biomass, dry weight, shoot length, and root length. In particular, the promoting effect was high under the light condition (LE) of 150 μmol / m ² / s.

2) Evaluation of Pn of coffee plantlets in the incubator In order to evaluate the photosynthetic rate of the coffee plantlets in the incubator without changing the culture environment conditions, Niu et al. Single nodes with leaves were first grown using plastic cups (diameter 25 mm, height 30 mm) containing 10 ml of 1/2 concentration MS medium (sugar (20 g / l) or sugar-free) (FIG. 4). . 3 cups with 2 millipore filters
Place aseptically in a 480 cm ³ container and place the container in a 150 μmo
Transferred to l / m ² / s CO ₂ free room. On days 10 and 30, remove three cups from each media treatment and add C
_In order to measure _in and C _out , the sample was transferred into a polycarbonate magenta-type container having four millipore filters (FIG. 4). The number of ventilations of the container was evaluated as 6.5 / hour. Under each set of conditions (C _out and PPF), C _in and C _out were measured at steady state and Pn was calculated using equation (1) by Fujiwara et al. The temperature of the growth chamber was maintained at 28 ± 1 ° C. In order to measure Pn of the plantlet on the 10th day, the light intensity in two stages (L: 120 μmol / m ²⁾
/ s, H: 247 μmol / m ² / s). On day 30, 320 μmol / m ² / s PPF was applied to evaluate Pn of plantlets. Immediately after the measurement, the dry weight and the leaf area of all the plantlets subjected to each treatment were measured. The CO ₂ concentration was measured using a gas chromatograph (GC-12A, Shimazu).

FIG. 5 shows the results of evaluation of Pn per leaf area using the system by Niu et al. (FIG. 4). The results of this measurement demonstrated that the coffee plantlets have a high ability to develop well under photoautotrophic conditions.
The data obtained in the 10 day and 30 day, CO ₂ concentration in the vessel was increased with increasing CO ₂ concentration in the small growth chamber. On day 10, when light intensity increased, CO ₂ concentration had a significant effect on net photosynthetic rate (FIG. 5a). In the results on day 30, the CO ₂ saturation point of the coffee plantlets in the incubator was very high (about 4500-5000 μmolmo when the light intensity was 320 μmolm ⁻² s ⁻¹ ).
l ^-1 ) (Fig. 5b).

3) "Portable photosynthesis (PP) Syst"
em "and model" CIRAS-1 "(manufactured by PP system) for evaluation of leaf Pn under conditions of PPF 150 μmol / m ² / s
4 using Florialite (manufactured by Nisshinbo) using S medium
The third developed leaf of coffee plantlets cultured for 5 days was used for Pn measurement. Each leaf was placed in a leaf chamber of the PP system (leaf area: 2.5 cm ² ). The temperature in the chamber was set at 28 ° C. Three levels of CO ₂ concentration (46
2, 951, and 1754 μmol / mol) were tested. In each CO ₂ concentration treatment, the light intensity was measured by PPF sensor at 10, 50, 100, 20
It was set to 0, 300, 500, 700 and 1000 μmol / m ² / s. A halogen lamp was used as a light source.

FIG. 6 shows the evaluation of the net photosynthetic rate per leaf area of coffee when the PP system was used. Pn
Increased with increasing CO ₂ concentration and light intensity. 200μ
In PPF of mol / m ² / s, Pn at 1754 μmol / mol CO ₂ concentration (triangle in the figure) is better than that of 462 μmol / mol (square in the figure)
1.8 times higher than at 951 μmol / mol (black circle in the figure)
1.4 times higher. Further, in the case of increasing the PPF to 1000μmol / m ² / s, the Pn of a CO ₂ concentration of 1754μmol / mol, 462
It was 2.1 times higher than that of μmol / mol and 1.7 times higher than that of 951 μmol / mol.

The above results demonstrate that coffee plantlets in vitro have a great ability to grow and grow under photoautotrophic conditions. (2) Coffee with photoautotrophic nutrition (Coffea arabusta)
Sugar and CO ₂ in micropropagation of plantlets
Effect of Concentration and Light Intensity In the first experiment, two single nodes with leaves were placed in a 300 ml polycarbonate column-shaped pot-shaped container (70 mm height, diameter
80mm) with or without sucrose (20g / l)
The cells were cultured on 0 ml of 1/2 concentration MS agar (9 g / l) medium. No growth regulator was used. PH 5.7 before autoclaving
Was adjusted to In the case of a sugar-free medium, a gas permeable membrane (Milliseal, manufactured by Millipore) having a pore size of 0.5 μm was attached to the lid of the container, and was not attached in the case of a sugar-containing medium.
During the 60-day culture period, the plantlets are 70 hours for 10 hours per day.
The cells were grown under light conditions of μmol / m ² / s at 26 ± 2 ° C. and 55-65% relative humidity. Living weight per plantlet, dry weight, shoot length, and leaf area were statistically calculated using analysis of variance. In the second experiment, a single knot with three leaves
Using a 480 ml polycarbonate column-shaped pot-shaped container, the cells were cultured in 75 ml of a sucrose-free 1/2 concentration MS culture solution. Florialite (manufactured by Nisshinbo) was used as a support. Two gas permeable membranes (Milliseal, manufactured by Millipore) having a pore size of 0.5 μm were attached to the lid of the container. The plantlets are exposed to 2 hours of light for 16 hours per day for 45 days.
Step CO ₂ concentration (500 and 1500 μmol / mol), Step PP
Growing at F (150 and 250 μmol / m ² / s). The pH was adjusted to 5.7 before autoclaving. On the 45th day, the data of the increased living weight, dry weight, leaf area, and shoot length of the plantlets were tabulated, and each test group was obtained by repeating three times.
Analysis of variance was performed on the results of the three plants.

The results of measuring the effect of sucrose concentration on the growth of coffee plantlets on day 60 of culture are shown in Table 4. In the test plots in the table, "S20" indicates that the cells were cultured in a medium containing 20 g / l sucrose, and "S0" indicates that the cells were cultured without sugar. “NS” and “*” indicate “not significant” and “significant at p = 0.01”, respectively.

[0037]

[Table 4] The results in Table 4 indicated that the coffee plants grew well in a sugar-free medium. No significant difference was observed in living weight and dry weight between treatments, but shoot length and leaf area of plantlets cultured under sugar-free conditions were significantly increased as compared to sugar medium. did. In a sugar medium,
Callus formed at the base of all shoots. On the other hand, no callus was produced in the sugar-free medium.

Table 5 shows the results of examining the effects of PPF level and CO ₂ concentration on the growth of coffee plantlets in vitro. In addition, "L" in the test plot in the table
And “H” indicate low PPF (150 μmol / m ² / s) and high PPF (250 μmol / m ² / s), respectively, and “N” and “E” indicate low CO ₂ concentration (500 μmol / mol) and high PPF, respectively. CO ₂ concentration (15
00 μmol / mol). Also, "NS", "*" and "*
“*” Indicates “not significant”, “significant at p = 0.05”, and “significant at p = 0.01”, respectively.

[0039]

[Table 5] As shown in Table 5, under the high CO ₂ condition, the plantlets grew remarkably well regardless of the light intensity. Under low CO ₂ conditions, higher PPF promoted growth. However, under high CO ₂ conditions, the plantlets are 250 μmol / m ² / s PP
The growth was better with 150 μmol / m ² / s PPF (LE) than with F (HE). 150 μmol / m ² / s PPF and 1500
In [mu] mol / mol of CO ₂ concentration conditions (LE), biological weight of plantlets, dry weight, shoot length, and an increase in leaf area was the largest as compared to other processes. This indicated that the growth of coffee plantlets was promoted under high CO ₂ conditions. (3) Coffee (Coffea arabu) by photoautotrophic culture
sta) Support and Effect of Ventilation on Micropropagation of Plantlets The coffee (Coffea arabusta) plantlets (single node with a pair of leaves) in an incubator were placed in a 480 ml transparent container in 1/2. 75 ml of MS culture solution (concentration 20 g / l or sugar-free, neither of which has growth regulator) is added, and Florialite (mixture of cellulose fiber and vermiculite, 10 g per container, Nisshinbo Co., Ltd.) is used as a support. ) And Bacto agar (gelled at a concentration of 8 g / l added to the above-mentioned culture solution), and each of the three individuals was implanted and cultured for 40 days. The container lid has no or two holes (diameter 10m
m), and a gas permeable membrane (Milliseal, manufactured by Millipore) having a pore size of 0.5 μm in each of the two holes.
Was attached. The frequency of ventilation of those containers is 1 each
0.24 and 2.34 times per hour. All containers have a temperature of 28
C., humidity 75%, CO ₂ concentration 400-500 μmol / mol, and maintained under a light condition of 16 hours per day by a white fluorescent lamp. PPF was 50, 100, 150, and 200 μmol / m ² / s for the first 3 days of culture, the next 7 days, the next 10 days, and the last 20 days, respectively. The net photosynthesis rate (NPR) is calculated by Fujihara et al.
(1): 21-30, 1987). The CO ₂ concentration inside and outside the container was measured by gas chromatography.
The experiment was performed by repeating the factor experiment of two levels and three factors three times. The obtained data on the increase in the biological weight, dry weight, shoot length, and leaf area were analyzed for variance. To examine the significance of each test interval, an LSD (least significant difference) test at p = 0.05 was performed. Table 6 shows the types of processing performed.
Shown in In the table, "S" and "F" on the left of the test section indicate sugar and sugar-free, respectively, and "A" and "F" in the center indicate agar and cellulose / vermiculite (Florialight), respectively. "L" and "H"
Indicates a low ventilation rate and a high ventilation rate, respectively.

[0040]

[Table 6] Table 7 shows the results of each process. Note that “N” in the table
"S", "*" and "**" are "not significant",
"Significant at p = 0.05" and "significant at p = 0.01" are shown.

[0041]

[Table 7] The presence / absence of sugar in the support did not affect the increase in dry weight and leaf area but the increase in body weight and shoot length after 40 days of culture. The support affected all of the above growth parameters. The number of ventilations is
This affected shoot length and leaf area. These three factors were found to affect shoot length. Increases in body weight, dry weight, shoot length and leaf area are FFH (sugar free, fibrous support, high ventilation rate)
Was the largest. Root growth and development were also greatest in FFH treatment. Root elongation was induced in the fibrous support tests (FFL, SFH and FFH) except for the SFL (sugar containing fibrous support, low ventilation rate) test. In the agar test sections containing sucrose (SAL, SAH), callus was formed at the base of the stem, and root elongation and growth were not observed. The photosynthetic rate was highest in the FFH test plot,
On day 30, day 30, and day 40, they were 0.7, 1.0, 2.1, and 3.9 μmol / h per plantlet per hour, respectively. From the above results, the growth and rooting of the coffee plant in the incubator,
This was shown to be facilitated by using a high porosity support in sugar-free cultures under high ventilation rates.

Example 3 Mass Growth of Mangosteen Plantlets by Photoautotrophy Shoots were excised from aseptically germinated mangosteen seedlings and used as explants (640 to 870 mg / shoot in body weight). Using. Explants were implanted in 370 ml polycarbonate containers containing 100 ml MS medium. Vermiculite (25-35g per incubator)
Was used. Five test plots were set up as shown in Table 8.
In the table, `` Z '' indicates the initial sucrose concentration in the medium, `` Y '' indicates 2-ip of 10 mg / l and IBA of 1 mg / l,
"X" stands for Kozai et al. (Kozai, T. et al., 1986. J. Agr.
l., 42 (2): 119-127).

[0043]

[Table 8] The shoots in the control were cultured under the same culture conditions as the conventional culture conditions except for the support material. The temperature in the incubator was 27.5 ° C under light conditions and 25 ° C in the dark.
The relative humidity in the culture room was maintained at 60%. 110 μmol / m ² / s PPF was irradiated using a fluorescent lamp as a light source. The CO ₂ concentration in the culture chamber was 1300 μmol / mol. In order to obtain a high ventilation rate in the incubator, two gas-permeable membranes (Milliseal, manufactured by Millipore) were mounted on the incubator. On the 30th day, the biological weight and dry weight of six plantlets were measured for each test plot. In addition, the number of leaves and the rooting rate were measured. The net photosynthetic rate in the incubator during the light condition period was determined according to the method of Fujiwara et al. Table 9 shows the results. In the table, “NS”, “*” and “**” are “not significant” and “p =” respectively for the control group (S30-GR-LV).
0.05 is significant "and" p = 0.01 is significant ".
“NS” and “**” in the analysis of variance indicate “not significant” and “significant at p = 0.01”, respectively.

[0044]

[Table 9] As a result, the biological weight and dry weight of the plantlets on the 30th day did not differ significantly between the test groups. Addition of the growth regulator to the medium increased the number of leaves. No rooting was observed in the control group, but when cultivated under high ventilation conditions using vermiculite as a support material, it was 20 to 20 regardless of the presence of sucrose and growth regulators in the medium.
Root induction was observed in 40% of the shoots. The CO ₂ concentration in the incubator in the control plot was the lowest, and the net photosynthetic rate per leaf area in the control plot was 1/12 to 1/7 of the test plot subjected to hyperventilation. Rooting from above, Mangosteen plantlets, vermiculite as a support material, by applying the CO ₂ at high ventilation conditions, photoautotrophically propagate vegetatively, without decreasing the growth rate of the plantlets It was shown that the rate and net photosynthesis rate could be increased.

[0045]

Industrial Applicability According to the present invention, there is provided a method for photoautotrophic production of plantlets of woody plants, particularly plants belonging to the genus Acacia, the genus Coffee, and the genus Garcinia. ADVANTAGE OF THE INVENTION According to the method of this invention, the plantlets of a woody plant genetically homogeneous can be mass-produced in a short period of time.

[Brief description of the drawings]

FIG. 1 shows the daily change in net photosynthetic rate of Acacia plantlets under light conditions. Mean ± SD is shown.

FIG. 2 shows the daily changes in the photosynthetic rate of plantlets in a 45-day culture of coffee plantlets.

FIG. 3 shows the effect of light intensity and CO ₂ concentration on the growth of coffee plantlets in photoautotrophic culture.

FIG. 4 shows a system developed by Niu et al.

FIG. 5 shows the results of evaluating the photosynthetic rate per leaf area of coffee plantlets using the system by Niu et al. (FIG. 4).

FIG. 6 shows the evaluation of net photosynthetic rate per leaf area of coffee plantlets using the PP system.

Claims (9)

[Claims] 1. A method for producing a small plant of a woody plant, comprising transplanting the tissue of a woody plant into a medium containing no sugar, and culturing by applying carbon dioxide gas under illumination. 2. The method according to claim 1, wherein the woody plant is a plant belonging to the genus Acacia. 3. The method according to claim 1, wherein the woody plant is a plant belonging to the genus Coffee. 4. The method according to claim 1, wherein the woody plant is a plant belonging to the genus Garcinia. 5. The cultivation while maintaining the concentration of carbon dioxide gas in the incubator at 350 to 500 μmol / mol.
The method according to any of the above. 6. The photosynthetic effective photon flux density is 150 to 300 μm.
It is cultured under illumination of mol / m ² / s, the method according to any one of claims 1 to 4. 7. The method according to claim 1, wherein a fibrous support, a porous support, or a mixture thereof is used for the medium. 8. The method according to claim 1, wherein the medium support comprises a mixture of cellulosic fibers and vermiculite. 9. The method according to claim 1, wherein the culturing is performed in an incubator having a high gas permeability.