JPH078189B2 - How to Propagate Potatoes - Google Patents

Description

[Detailed Description of the Invention] Technical Field The present invention relates to a method for propagating potatoes.

More specifically, the present invention relates to a method for obtaining a large amount of tubers of potatoes large enough to cultivate seed potatoes in a single crop in soil, such as potato tubers, taro, konjac, corms of Pinellia ternata, rhizomes of Aster japonica, and tuberous roots of Chinese yam (Yam), in a short period of time by tissue culture.

BACKGROUND ART Conventionally, various methods have been known for propagating potatoes by tissue culture.

A method for obtaining potato tubers is known in which the roots and leaves of a plant obtained by culturing potato tissue are removed and the stem is cultured in liquid (Cip Circular, v
ol 13, No.4 December. P.1-5 (1985); Plant Cell, Tissue and Organ Culture (Plan
t Cell, Tissue and Organ Culture) 7:3-10 (198
6).

In this method, the culture medium is filled very shallowly into the flask, so potato tubers are formed in the gas phase and are not numerous.

A method for cultivating potato tubers and growing plants by repeatedly dividing the resulting plants is known (American Potato Journal,
l) 55:691-701 (1978)]. However, even the most efficient method only yielded 140 plants in 16 weeks. An efficient method for multiplying small potato tubers is known (Tissue Culture 11(9), 391-395, 1985). The potato tubers formed by this method range in size from adzuki beans (approximately 0.1 g) to peanuts (approximately 0.7 g) after about 100 days of culture, and a single medium and culture conditions are used throughout the culture period. Furthermore, the culture is carried out in a small vessel, making it difficult to scale up.

There is a need to develop a method for efficiently producing potato tubers weighing more than 1 g through mass cultivation.

In addition, in the case of taro, Chinese yam (Japanese yam), and Pinellia ternata, plant bodies have been successfully propagated by tissue culture, but no technology has been developed to efficiently propagate taro, Pinellia ternata corms, or Chinese yam tuberous roots (Vegetable Experiment Station Report A 9:1-46 (1981); Tissue Culture 11:372-376 (1982)).
5).

There is a need for a method to efficiently mass-produce taro, konjac, Pinellia ternata corms, Euonymus chinensis rhizomes, and Chinese yam tubers by tissue culture.

DISCLOSURE OF THE INVENTION According to the method of the present invention, a source plant obtained by culturing a tuber plant is transplanted into a liquid medium and cultured until the plant material grows to an average length of 15 to 30 cm. If necessary, if at least 90% of the obtained plant body is not submerged in the liquid medium, new liquid medium is added to submerge at least 90% of the plant body, and the liquid medium is then reduced over time while further culturing until 50 to 98% of the plant body is exposed to the gas phase, thereby enabling efficient mass proliferation of tubers, corms, rhizomes or tuberous roots.

The tubers to which the method of the present invention can be applied include potatoes,
Examples include taro, konjac, pinecone, holly, and Chinese yam.

The following explanation will be divided into potatoes and other tubers.

(I) In the case of potato (A) Production and propagation of plants by liquid culture The raw material used in the present invention is a plant obtained by aseptically culturing the main stem, stem, leaves, roots, stems, leaves, etc. of a potato plant by a method known per se. The plant material is cultured in a liquid medium at a concentration of 5 to 50 individuals/plant.
The medium used can be either natural or synthetic, as long as it contains an adequate amount of carbon source, chlorine source, inorganic matter, etc.

Sucrose, maltose, fructose, glucose, molasses, etc. are used as carbon sources.

Nitrogen sources include potassium nitrate, sodium nitrate, ammonium nitrate, calcium nitrate, ammonium sulfate, amino acids (glycine, glutamic acid, lysine, aspartic acid, etc.), yeast extract, meat extract, peptone,
Coconut milk is used.

Inorganic substances include potassium chloride, calcium chloride, manganese chloride, nickel chloride, cobalt chloride, aluminum chloride, iron chloride, magnesium sulfate, sodium sulfate, nickel sulfate, iron sulfate, manganese sulfate, zinc sulfate, copper sulfate,
Potassium phosphate, potassium iodide, boric acid, sodium molybdate, etc. are used.

Other ingredients that may be added to the medium as needed include cytokinin, benzyladenine (hereinafter referred to as BA), N-(2-chloro-4-pyridine)-N-phenylurea (hereinafter referred to as 4PU), kinetin, chlorcholine hydrochloride, abscisic acid, vitamin _B1 , inositol, pyridoxine hydrochloride, nicotinic acid,
Thiamine hydrochloride, biotin, etc. may also be added.

Specific media include Murashige-Skoog medium, Ericson medium, White medium, Linsmeyer-Skoog medium, and the like.

The culture is carried out at a sugar concentration of 60 g/L or less, preferably 25-35 g/L, a culture medium depth of 12 cm or less, preferably 5-10 cm, and a temperature of 10-35°C.
Illumination: 200-10,000 lux, pH: 4-8, aeration: 0.01-0.5vvm, preferably 0.05-0.2vvm, and the average length of the plant material is 15 to 3.
Culture until the seedlings reach 0 cm in height, which usually takes 20-60 days.

(B) Tuber induction and thickening: The sugar concentration of the medium used in step A is 60-150 g/L, preferably
After submerging at least 90% of the plant bodies formed in step A in the medium replaced with 80-100g/, the temperature was 10-35°C and the light intensity was 2
500 lux or less, preferably in the dark, at pH 4-8,
The liquid medium is gradually reduced in volume until 0-98% of the culture is exposed to the gas phase, which usually takes 20-90 days.

Various methods for reducing the liquid medium over time include evaporating the liquid medium by aeration and directly discharging the liquid medium, and these methods may be used alone or in combination.

The aeration rate for the method of evaporating the liquid medium by aeration is in the range of 0.8-2.2 vvm, higher than that of Process A. Directly discharging the liquid medium can be achieved by discharging the liquid medium as needed. For example, when potato tuber formation is observed near the boundary between the medium and the gas phase, an appropriate amount (e.g., 10-20% of the total medium volume) is discharged, and this discharging procedure is repeated 3-10 times. In this manner, a large number of potato tubers can be obtained, including many that have grown to a weight of 1 g or more.

(II) In the case of tubers other than potatoes (taro, konjac, pinellia ternata, yam, and Chinese yam) (A) Production and propagation of plants by liquid culture As plant material, the growing points, stems, etc. of taro, konjac, pinellia ternata, yam, and Chinese yam plants are used.
Plants with an average length of 15 to 30 cm can be obtained by using a medium and a culture method similar to those used in step A for potato in (I) above, except that leaves, roots, stems, etc. are aseptically cultured by a method known per se.

(B) Induction and thickening of taro, konjac, Pinellia ternata corm, Euonymus chinensis rhizome, and Chinese yam tuberous root. The plant body formed in step A of (I) above is transplanted into a liquid medium in which the sugar concentration is changed to 60-150 g/l, preferably 80-100 g/l, and then the plant body is grown at a temperature of 10-35°C, an illumination of 200 lux or less, preferably in the dark, and at a pH of 4-8.
The liquid medium is gradually reduced in volume until 100% of the cells are exposed to the gas phase, which usually takes 20-90 days.

A method for reducing the liquid medium over time includes, for example, evaporating the liquid medium by aeration (0.8-2.2 vvm).

BEST MODE FOR CARRYING OUT THE INVENTION Example 1 Potato stems were cultured in Murashige
The potato plants were transplanted into test tubes with a diameter of 25 mm and a length of 125 mm containing 10 ml of Skoog medium, and cultured at 25°C under continuous illumination at 2500 lux for about one month to obtain 20 potato plants that had grown to about 10 cm in height. The plants were then divided into individual nodes, and then cultured in liquid medium 5 shown in Table 1, which had the agar removed.
16-volume jar fermenter containing 90 ml of medium
m) and incubated at 25°C under continuous illumination of 4000 lux at 0.1 vvm
After culturing for 3 weeks at an aeration rate of 1000 kJ/L, plants were grown to an average length of 20 cm. After removing the entire medium, the agar was removed from the medium shown in Table 1, and sucrose was added at 9% (W/
The liquid medium 10 changed to V) was added again to the 16-volume jar fermenter (medium depth: 18 cm) and incubated at 25°C in the dark.
The plants were cultured at an aeration rate of approximately 0.1 vvm. After adding the medium, clear tuber formation was observed primarily near the boundary between the medium and the air space within five days. On the fifth day after adding the medium, the medium was removed twice to lower the liquid level, and further tuber formation was observed near the boundary between the medium and the air space. Removal of approximately 2 vol of medium was repeated four times every five days to allow tuber formation. The formed tubers continued to enlarge thereafter. Culture was terminated 45 days after adding the medium. The remaining medium volume was 0.8 cm (medium depth: 1 cm), and approximately 95% of the plant was exposed to the air space, but did not die. A total of approximately 370 tubers were obtained near the air space boundary and in the air space of the medium. Of these, approximately 80 tubers weighed 1 g or more.

Table 1 Murashige-Skoog medium Ammonium nitrate 1,650 mg Potassium nitrate 1,900 mg Calcium chloride dihydrate 440 mg Magnesium sulfate heptahydrate 370 mg Potassium phosphate monobasic 170 mg _Na2 EDTA dihydrate 37.3 mg Ferrous sulfate heptahydrate 27.8 mg Boric acid 6.2 mg Manganese sulfate tetrahydrate 22.3 mg Zinc sulfate heptahydrate 8.6 mg Potassium iodide 0.83 mg Sodium molybdate dihydrate 0.25 mg Cuprous sulfate 0.025 mg Cobalt chloride 0.025 mg Vitamin _B1 0.40 mg Inositol 100 mg Pyridoxine hydrochloride 0.50 mg Nicotinic acid 0.50 mg Glycine 2.00 mg Sucrose 30.0 g Agar Dissolve 8.0g of the above ingredients in deionized water to make a solution of 1, adjust the pH to 6.2 with 0.1N aqueous sodium hydroxide solution, dispense into culture vessels, and sterilize at 121°C for 20 minutes.

Example 2 Potato stems were soaked in Murashige
The potato plants were transplanted into test tubes with a diameter of 25 mm and a length of 125 mm containing 10 ml of Skoog medium and cultured at 25°C under continuous illumination at 2500 lux to obtain potato plants that had grown to about 10 cm. The plant bodies were cut into 100 pieces at each node and transplanted into 8-volume jar fermenters (medium depth: 8 cm) containing liquid medium 2, which was the same medium shown in Table 1 but with the agar removed, for 25
The plants were cultured at 0.5°C under continuous illumination of 4000 lux and aeration of 0.1 vvm for 4 weeks, and plantlets were obtained which had grown to an average height of 25 cm.

Next, after removing all of the medium, the agar was removed from the medium in Table 1, and liquid medium 6 with a sucrose concentration of 9% (W/V) was added to an 8-volume jar fermenter (medium depth: 23 cm). The obtained plant bodies were transplanted into the medium and cultured in the dark at 25°C with an aeration rate of 0.8 vvm for 4 weeks. The remaining medium volume was approximately 2 (medium depth: approximately 8 cm).
m), and about 70% of the plant body was exposed to the gas phase, but it did not die.
Of these, 110 weighed more than 1g.
On the other hand, the same culture as above was carried out except that the aeration volume was changed to 0.1 vvm.

The remaining medium volume was 5.2 (medium depth: 19 cm), and the obtained potato tubers were approximately 250 (weight approximately 210 g).
Of these, 80 weighed more than 1g.

Example 3: The buds of the corms of taro (cultivar: Ishikawa Wase) were observed under a stereomicroscope and the growing points were collected aseptically. The growing points were transplanted into test tubes 25 mm in diameter and 125 mm in length containing 10 ml of Murashige-Skoog medium shown in Table 1, and cultured at 25°C under continuous illumination at 2500 lux for about 3 months. The obtained plants were then cultured in 10 ml of liquid medium shown in Table 1, with the agar removed.
The plants were then transplanted into a 300 ml flask containing 100 ml of liquid medium and cultured at 25°C under continuous illumination at 2500 lux with rotary shaking (180 rpm). After approximately two months of culture, 250 plantlets were obtained. The plants were aseptically removed from the flask and then aseptically divided into 10 portions, each divided into 300 ml flasks containing 100 ml of liquid medium of the same composition as above. The plants were then cultured at 25°C under continuous illumination at 2500 lux for one month.
The plants were grown by rotary shaking culture (180 rpm). The same procedure was repeated every month with shaking culture (180 rpm). The plants were grown by repeating the same procedure 20 times every month. As a result of growing the plants, the number of plants gradually increased with each subculture, and a densely grown mass of an average of 700 plants per flask was obtained. The plants thus grown (3 flasks) were cultured in the medium shown in Table 1, with the agar removed, and the medium was then cultured in 9% (W/V) sucrose.
The plants were transplanted into 8-volume jar fermenters (medium depth: 23 cm) containing liquid medium 6 modified with the above formula, and cultured for one month at 25°C under continuous illumination at 2500 lux with an aeration rate of 0.1 vvm, yielding grown plants (average length: 23 cm). Next, the aeration rate was increased to 2 vvm without changing other conditions, and the culture was continued for another month. The liquid medium rapidly evaporated due to aeration, and the grown plants were gradually exposed to the gas phase from the liquid medium. After one month, the medium volume reached 1.2 (medium depth: 5 cm), and approximately 80% of the plants were exposed to the gas phase, but they did not die.
At this point, the culture was terminated and the plants were removed. The bases of almost all of the plants had swelled to form corms, and 2,900 taro corms were obtained per jar fermenter. The total weight was 0.8 kg, with an average weight of 0.3 g per corm. At this time, 370 corms weighed over 1 g. Meanwhile, the aeration rate was not changed during the culture, and the culture was continued for two months at 0.
When cultured at an aeration rate of 1vvm, the medium at the end of the culture was
The yield was 430 taro corms per jar fermenter, with a total weight of 130 g and an average weight of one corm of approximately 0.3 g.
Furthermore, only 65 corms weighed more than 1g.

Example 4 Konjac corms were wrapped in absorbent paper soaked in 500 ppm BA, and then wrapped in vinyl to prevent moisture evaporation.
After leaving the corms at 20°C for 7 days, approximately 100 ambush buds grew per corm, each measuring between 3 and 10 mm. These ambush buds were observed under a stereomicroscope and the growing points were aseptically collected to a size of 0.2 mm or less. The growing points were then soaked in Murashige-Skoog medium (see Table 1) with 4 PU for 1 minute.
A 25mm diameter tube containing 10ml of medium added to achieve a concentration of mg/L.
The plants were then transplanted into test tubes measuring 125 mm in length and cultured at 25°C under continuous illumination at 2500 lux for approximately 5 months to obtain plant bodies. The plants were aseptically divided into 10 pieces and further propagated in the same medium. After repeating this subculture five more times, the resulting plants (20 test tubes) were used for the first
Remove the agar from the medium and add 9% sucrose (w/
The plants were then transplanted into an 8-volume jar fermenter (medium depth: 23 cm) containing liquid medium 6 (modified with 2500 lux) and cultured for 60 days at 25°C under continuous illumination of 2500 lux with an aeration volume of 0.2 vvm, yielding grown plants (average length: 18 cm). Next, the aeration volume alone was increased to 1.5 vvm, and the plants were cultured for another month. The liquid medium rapidly evaporated due to aeration, and the grown plants were gradually exposed to the gas phase from the liquid medium. After one month, the medium volume reached 1.7
The culture medium reached a depth of 6 cm, and although approximately 60% of the plant body was exposed to the gas phase, it did not die. When the culture was terminated at this point, 1,800 konjac corms were obtained per jar fermenter. The total weight was 1.2 kg.
The average weight of a corm was 0.7g, and 270 corms weighed more than 1g.

On the other hand, the aeration volume was not changed during the culture period, and the aeration volume was maintained at 0.2vvm for 2 months.
When cultured at an aeration rate of 1000 m/s, only 47 konjac corms were formed.

Example 5 The buds of the corms of Pinellia ternata were observed under a stereomicroscope and the growing points were collected aseptically. The growing points were placed in a 25 mm diameter, 12 mm long container containing 10 ml of Murashige-Skoog medium shown in Table 1.
The plants were then transplanted into 5 mm test tubes and cultured at 25°C under continuous illumination at 2500 lux for approximately 3 months to obtain plant bodies. The plants were then cultured in the same manner as in Example 3 to obtain plant bodies.
The culture medium (main portion) was transferred to an 8-volume Shar fermenter (medium depth: 23 cm) containing liquid medium 6, which was prepared by removing the agar from the medium in Table 1 and changing it to 9% (w/v) sucrose.
After 40 days of cultivation at 5°C under continuous illumination of 2500 lux and an aeration volume of 0.1 vvm, grown plants (average length: 20 cm) were obtained. Next, the aeration volume alone was increased to 1.5 vvm, and cultivation continued for another month. The liquid medium rapidly evaporated due to aeration, and the grown plants were gradually exposed to the gas phase from the liquid medium. After one month, the medium volume reached 2.0 (medium depth: 8 cm), and approximately 80% of the total plants were grown.
0% of the cultures were exposed to the gas phase, but none died. The culture was terminated at this stage, yielding 3,600 Pinellia ternata corms per jar fermentor. The total weight was 1.8 kg, with an average weight of 0.5 g per corm.
Furthermore, 280 corms weighed over 1g. On the other hand, when the aeration rate was not changed during the culture and the culture was continued for two months at 0.1vvm, many plants were formed but few corms were formed, resulting in 430 Pinellia ternata corms per jar fermenter. The total weight was 130g, with an average weight per corm of 0.3g. Furthermore, almost no corms weighing over 1g were obtained.

Example 6 While observing the stem buds of Chinese yam under a stereomicroscope, the growing points were collected aseptically. The growing points were then placed on Murashige-Skoog medium shown in Table 1 containing 0.1 mg/ml naphthaleneacetic acid and 0.1 mg/ml BA.
25mm diameter, 125mm length, containing 10ml of medium supplemented with 1mg/ml
The plants were then transplanted into test tubes containing 100 ml of a liquid medium with the same composition as above, but with the agar removed, for approximately 5 months at 25°C and 2500 lux of continuous light, to obtain plant bodies.
The plants were transplanted into 300 ml flasks and cultured at 25°C under continuous illumination at 2500 lux with rotary shaking (180 rpm). After approximately two months of culture, 120 plants were obtained. The plants were aseptically removed from the flasks and then aseptically divided into 10, which were then transplanted into 300 ml flasks containing 100 ml of liquid medium of the same composition as above, and cultured for 25 minutes.
The plants were grown by rotary shaking culture (180 rotations per minute) at 2500 lux continuous illumination at 50°C for 2 months.
The same procedure was repeated three times every two months to grow the plants. The Chinese yam plants (three flasks) thus grown were cultured in eight different liquid media, including liquid medium 6, with the same composition but with sucrose changed to 9% (w/v).
The plants were then transplanted into a jar fermenter (medium depth: approximately 23 cm) and cultured at 25°C, 2500 lux, under continuous lighting, and with an aeration rate of 0.1 vvm for two months to obtain grown plants (average length: approximately 24 cm).
When the aeration rate was increased to 0.1 vvm and the culture was continued for another two months, the liquid medium rapidly evaporated due to aeration, and the grown plant bodies were gradually exposed to the gas phase from the liquid medium. After one month, the medium volume was 0.7 (medium depth: 3 cm), and about 85% of the plant bodies were exposed to the gas phase, but they did not die. When the culture was terminated at this point, 1,900 Chinese yam tubers were obtained per jar fermenter. The total weight was 0.4 kg, and the average weight of each tuber was 0.2 g. On the other hand, when the aeration rate was not changed during culture and the culture was continued for four months at an aeration rate of 0.1 vvm, the liquid medium volume at the end of culture was 4.7 (medium depth: 18 cm), and 800 tubers were obtained per jar fermenter. The total weight was 140 g, and the average weight of each tuber was about
The weight was 0.18g.

Example 7: Rhizome buds of Acanthus nigricans were aseptically harvested at approximately 3 mm in size while observing them under a stereomicroscope. The harvested buds were transplanted into test tubes measuring 25 mm in diameter and 125 mm in length containing 10 ml of Murashige and Skoog medium shown in Table 1, and cultured at 25°C under continuous illumination at 2500 lux for approximately two months to obtain plant bodies. The plant bodies were then cultured in Murashige and Skoog medium shown in Table 1 with 1 ml of BA.
25mm diameter tube containing 10ml of medium added to a concentration of g/
The plants were transplanted into test tubes measuring 125 mm in diameter and 2500 lux in length and cultured at 25°C for 40 days under continuous illumination at 2500 lux to obtain plantlets. The plants were aseptically divided into 10 parts and subcultured in a medium having the same composition as above, and cultured in the same manner as above, resulting in further plant proliferation. The plantlets (20 pieces) thus grown were removed from the Murashige and Skoog medium shown in Table 1 and cultured in the same manner as above, resulting in further plant proliferation.
BA 1 mg/ and sucrose 9% (w/
The plants were transplanted into 8-volume jar fermentors (medium depth: 23 cm) containing liquid medium 6 (v) and cultured at 25°C under continuous illumination at 2500 lux and an aeration rate of 0.1 vvm for two months, yielding mature plants (average length: 20 cm). Next, the aeration rate was increased to 1 vvm without changing other conditions, and the culture was continued for another 1.5 months. The liquid medium rapidly evaporated due to aeration, and the grown plants were gradually exposed to the gas phase. After 1.5 months, the plants reached medium 2 (medium depth: 8 cm). More than 50% of the plants were exposed to the gas phase, but they did not die. When the culture was terminated and the plants were removed, rhizomes formed at the base of the plants, yielding 670 rhizomes per jar fermentor. In contrast, when the aeration rate was not changed during the culture period and the culture was continued for 3.5 months at 0.1 vvm, only 420 rhizomes were obtained.

Claims (3)

[Claims] [Claim 1] A method for propagating tubers, comprising transplanting a plant body or a slice thereof (hereinafter referred to as plant material) having at least buds obtained by culturing a tuber plant into a liquid medium and culturing the plant material until the plant material grows to an average length of 15 to 30 cm, and then, if necessary, if at least 90% of the obtained plant body is not submerged in the liquid culture, adding new liquid medium to submerge at least 90% of the plant body, and then further culturing while gradually reducing the amount of liquid medium until 50 to 98% of the plant body is exposed to the gas phase, thereby forming tubers, corms, rhizomes or tuberous roots. [Claim 2] A method for propagating potato tubers, comprising transplanting a plant body or a slice thereof having at least buds obtained by culturing potato (hereinafter referred to as plant material) into a liquid medium and culturing the plant material until it grows to an average length of 15 to 30 cm, then adding new liquid medium so that at least 90% of the obtained plant body is submerged in the liquid medium, and then further culturing the plant body while gradually reducing the amount of liquid medium until 50 to 98% of the plant body is exposed to the gas phase, thereby forming potato tubers. Claim 3: Taro, konjac, pinecone,
A method for propagating taro, konjac or ternate pinecone corms, ternate pinecone rhizomes or ternate yam tubers, characterized in that a plant having at least buds obtained by culturing ternate pinecone or ternate yam, or a slice thereof (hereinafter referred to as the plant material), is transplanted into a liquid medium, cultivated until the plant material grows to an average length of 15 to 30 cm, and then the liquid medium is gradually reduced with time while culturing until 50 to 98% of the plant is exposed to the gas phase, thereby forming taro, konjac or ternate pinecone corms, ternate pinecone rhizomes or ternate yam tubers.