KR20220098418A - Methods for mass propagation of tissue culture-derived plants of Vaccinium oldhamii - Google Patents

Abstract

In the present invention, disclosed is a method for mass production of tissue cultured seedlings of Vaccinium oldhamii through an ex vitro rooting process of shoots of the Vaccinium oldhamii. According to the method for mass production of the tissue-cultured seedlings of the Vaccinium oldhamii according to the present invention, instead of usual in vitro rooting, ex vitro rooting is used to remarkably improve a rooting rate (regeneration rate) of the shoots of the Vaccinium oldhamii, and moreover, since a separate environmental purification process is not required to mass-produce the seedlings of the healthy Vaccinium oldhamii. In addition, the tissue cultured seedlings of the Vaccinium oldhamii according to the present invention has the same genetic traits as the parent with excellent genetic traits to be useful for distributing fruit farms.

Description

Methods for mass production of tissue-cultured seedlings of pure gold tree {Methods for mass propagation of tissue culture-derived plants of Vaccinium oldhamii}

The present invention relates to a method for mass production of tissue-cultured saplings of S. oleracea, and more particularly, to a method for mass-producing Tissue-cultured seedlings of S. oleraceae through an extra-extra-root rooting process of new shoots of S.

Vaccinium oldhamii ( Vaccinium oldhamii ) is a deciduous small tree belonging to the genus Rhododendron family, and is native to Korea and grows in low mountains in the central and southern regions. Blueberries are the fruits that grow on trees belonging to the genus Prunus sycamore. Blueberry is one of the superfoods that is good for the body. It is known for its strong antioxidant action, which not only suppresses cellular aging, but also has a high content of anthocyanins and is good for strengthening immunity and preventing adult diseases. In particular, according to a recent study, Korean native pure gold tree showed higher useful ingredients and antioxidant activity than North American blueberry, so the industrialization movement such as the development of health functional food using native pure gold tree fruit is becoming active. Accordingly, the demand for seedlings for cultivation for the production of fruit of the gold tree is increasing recently.

There are two methods of propagation of gold trees: irrigation after sowing seeds collected in autumn into moss, and cuttings in greenery in the summer. However, since the germination rate of the ginseng tree seeds is as low as 10% or less, and the cutting period for green cuttings is also limited in the summer, the efficiency is very low. For the production of saplings for cultivation, studies have been conducted on asexual propagation methods such as seeds and cuttings, but the development of efficient mass production technology is still insignificant.

The yield of seedlings is low due to the low reproductive efficiency of these saplings. As an alternative, a propagation method using tissue culture technology, which is one of the biotechnology techniques, has been partially developed. As an example, an in-flight propagation method of ginseng tree by inducing embryogenic callus from immature seeds or roots through in-flight rooting (formation of plants in the medium) was developed, but the production efficiency was low, so it was not suitable for mass production of tissue-cultured seedlings of ginseng tree ( Registered Patent No. 10-1852799).

Therefore, there is an urgent need for the development of a method for mass production of saplings of pure gold trees.

Accordingly, the present inventors have continuously studied to meet the needs in the prior art, and as a result, when new shoots of ginseng trees are externally rooted, surprisingly, plant redifferentiation through rooting of shoots is remarkably enhanced, resulting in a large amount of cultivar tissue-cultured seedlings. It was confirmed that it can be produced and completed the present invention.

Accordingly, it is an object of the present invention to provide a method for mass production of saplings of cultivar tissue culture.

Another object of the present invention is to provide a sapling of tissue cultured gold tree produced by the above method.

In order to achieve the above object of the present invention, the present invention

i) inducing polyclinic plants by in-flight culturing of spermatozoa explants of the genus chrysanthemum;

ii) re-differentiating into plants through explant rooting by cutting the shoots derived from multi-stemmed plants in top soil; and

It provides a method for mass production of seedlings for tissue culture of ginseng tree comprising: iii) transplanting a plant into the soil and seeding it.

The method according to the invention does not undergo a separate purification step.

In the present invention, 'apical bud' refers to an eye (bud-generating structure) located at the tip of a stem or branch of a plant.

In the present invention, 'shoot' refers to a newly grown branch, and a branch that grows from the in-flight culture of the tissue, a branch that grows from the lateral bud when the tip of the stem is wetted, and the hidden eye or upper part of the branch or on the back Includes strong branches, such as branches growing out of located eyes.

In the present invention, 'multiple shoot' refers to having several shoots.

In the present invention, 'in-flight culture' refers to a method of cultivating an organism using an artificial incubator.

In the present invention, 'rooting' refers to rooting, and includes differentiation (formation) of roots in organs of plants.

In the present invention, 'redifferentiation' refers to restoring an original organ or organism from a dedifferentiated cell to an original organ or organism. Includes phenomena that serve as a starting point.

In the present invention, 'acclimation' means that the plant adapts to the climatic conditions of the land or to the climate change of the same area.

In the present invention, 'top soil (床土)' refers to cultured soil (artificial soil) used for the purpose of seeding crops.

In the present invention, 'seedling (苗木, seedling)' refers to a young tree that has been artificially grown in large quantities, and a young tree grown non-artificially is sometimes distinguished by using terms such as a seedling.

In the present invention, 'raising seeding' refers to the general operation of cultivating a plant for a certain period of time to raise a seedling of good quality most suitable for formal planting.

Step i) In-vitro incubation of spermatozoa of Spermia

Induction of polyclinic plants by in-flight culturing of spermatozoa explants of genus saprum.

In this step, the bud explants are prepared by cutting the tip of the stem containing the buds of the rhizome tree to a length of about 1 cm.

Sperm explants are transplanted into a culture medium and cultured to induce polyclinic sheaths.

In this step, the culture medium is 1.0 mg/L to 2.0 mg/L of 6-(γ,γ-dimethylallyl)aminopurine (6-(γ,γ-Dimethylallyl)aminopurine), 2% to 3% sucrose and A solid phase MS (Murashige and Skoog) medium to which 0.3% to 0.35% of gelite is added is used, and most preferably, an MS medium containing 2.0 mg/L, 3% sucrose and 0.35% gelite is used. .

At this stage, the embryonic explant culture is induced by culturing at 25±1° C. for 15 to 17 hours a day, 30-50 μmol·m ^-2 ·s ^-1 lighting is maintained.

The period of the culture is preferably 6 to 10 weeks, and most preferably 8 weeks.

When culturing the explants of the genus chrysalis under the above culture conditions, it is possible to induce multi-shooting plants with a long shoot length and a large number of shoots while having excellent shoot induction rate (Table 1, FIG. 1).

Step ii) Plant redifferentiation through explant rooting of new shoots

New shoots derived from polysacral plants are cut into the top soil and redifferentiated into plants through extra-vegetative rooting.

In this step, as a new plant, remove the apical part from the multi-stem plant and cut the stem length to 2.5-3 cm so that 4-6 axillas are included.

In this step, external rooting is performed by treating new shoots with auxin for rooting induction, then cutting them into the top soil to induce rooting and redifferentiating into plants.

In this step, auxin treatment for rooting induction is 0.5 ~ 1.0 g/L indole-3-butyric acid (IBA, Indole-3-butyric acid) solution with auxin carrier (facilitating auxin absorption) talc 1 : 1 (v:w) mixed with the cut surface of the shoot and used, and most preferably 1.0 mg/L IBA solution is used.

In this step, the external rooting environment is carried out under the conditions of 15 to 17 hours a day, 30-50 mol·m ^-2 ·s ^-1 lighting, and 80 to 90% humidity are maintained at 25±1°C. Preferably, it is carried out under conditions in which illumination is maintained within the range of 40 μmol·m ⁻² ·s ⁻¹ at 25° C. for 16 hours a day.

The out-of-plant rooting period is preferably 6 to 10 weeks, and most preferably 8 weeks.

Under the above conditions, when rooted outside the ground, the rooting proceeds well, and the rooting rate is high, and the stem length is long, so that the plant is differentiated into a properly grown plant. Accordingly, compared to the in-flight rooting, the production rate (redifferentiation rate) of the chrysanthemum tree tissue-cultured seedlings improved 6.7 to 9.4 times (Table 2, FIG. 2).

In general, it is common knowledge that using in-flight rooting using a culture medium to produce tissue cultured seedlings has a high redifferentiation rate, but in the case of ginseng shoots, surprisingly, when using out-of-plant rooting as described above, the redifferentiation rate is significantly improved, Finally, it significantly improves the production rate of tissue culture seedlings.

In addition, by going through this step through external rooting, the production method of the present invention does not require an additional acclimatization step, so that the production period for seedlings can be significantly shortened.

iii) seedling

Plants are transplanted into soil for seeding.

After transplanting the differentiated plant from step ii) into the soil (pot), it is grown for 4 to 5 months and mass-produced into high quality seedlings most suitable for formal planting.

According to the production method of the present invention, it is possible to mass-produce high-quality saplings of tissue cultured ginseng trees.

According to another object of the present invention, the present invention provides a sapling of the tissue cultured saplings produced by the above production method.

Tissue-cultured seedlings according to the present invention have good growth and are suitable for large-scale fruit tree cultivation.

According to the method for mass production of Tissue-cultured seedlings according to the present invention, the rooting rate (re-differentiation rate) of S. rhododendron shoots is significantly improved up to 80% by using outside rooting instead of the conventional in-flight rooting, and with Since a separate environmental acclimatization process is not required, it is possible to mass-produce healthy ginseng tree tissue cultured seedlings in a short time.

According to the present invention, the production rate (redifferentiation rate) is increased by up to 9.4 times compared to the conventional method by in-flight rooting in the method for mass production of seedlings for cultured seedlings of ginseng tree tissue according to the present invention.

In addition, the saplings of the present invention are useful for supply to orchard farmers because they have the same genetic traits as the parent with excellent genetic traits.

1A is a photograph showing the results of induction of multi-crested plants according to the type and concentration of plant growth hormone in the explants of the genus chrysalis. a is 1.0 mg/L zeatine, b is 2.0 mg/L zeatin, c is 5.0 mg/L zeatin, d is 1.0 mg/L cydiazuron, e is 2.0 mg/L cydiazuron, f is 5.0 mg/L cydiazuron, g is 1.0 mg/L 6-benzylaminopurine, h is 2.0 mg/L 6-benzylaminopurine, I is 5.0 mg/L 6-benzylaminopurine, j is 1.0 mg/L 6 -(γ,γ-dimethylallyl)aminopurine, k is: 2.0 mg/L 6-(γ,γ-dimethylallyl)aminopurine, l is 5.0 mg/L 6-(γ,γ-dimethylallyl)aminopurine It is the result of using plant growth hormone of 1b is an enlarged photograph of k.
Figure 2 is a photograph showing the results of in-flight rooting according to the plant growth hormone of the shoots of ginseng. a is control (no auxin added), b is 0.5 mg/L NAA, c is 1.0 mg/L NAA, d is 2.0 mg/L NAA, e is 5.0 mg/L NAA, f is 0.5 mg/L IBA, g is 1.0 mg/L IBA, h is 2.0 mg/L IBA, and i is the result of using 5.0 mg/L IBA of auxin.
Figure 3 is a photograph showing the process and results of extra-vessel rooting through cuttings of topsoil of new shoots of ginseng.
Figures 4a and 4b are photographs showing the process and results of extra-vessel rooting through the plug cuttings of the new shoots of ginseng.
5 is a photograph showing the seedling process by transplanting the redifferentiated plant (Geum tree) into the soil (pot) according to the present invention.
Figure 6 is a photograph showing the tissue cultured seedlings produced according to the present invention.

Hereinafter, the configuration and effect of the present invention will be described in more detail with reference to specific examples in order to help the understanding of the present invention. However, the following examples are merely illustrative in order to understand the present invention more clearly, and the scope of the present invention is not limited by the following examples.

Example 1: Induction of polycrystals through culturing of sperm explants

The explants of the buds of the genus buds were prepared by cutting the tip of the stem containing the buds to a length of about 1 cm.

25 spermatogonial explants were taken from the plant growth hormone [zeatin, 6-benzylaminopurine (BA), 6-(γ,γ-dimethylallyl) aminopurine (6- (γ,γ-Dimethylallyl)aminopurine), or Thidiazuron (TDZ)], 3% sucrose and 0.35% gelite added to solid MS (Murashige and Skoog) medium (Table 1) after transplantation 8 By weekly culture, polyclinic plants were induced. The medium was adjusted to pH 5.7 and used after sterilization for 20 minutes at 121°C and 15 atm in an autoclave, and 50mL of medium was dispensed into a sterilized glass bottle (6.4×11cm) culture vessel. Five dentitions per bottle.

element name mg/L MS Basic Medium trace elements CoCl ₂ .6H ₂ O 0.025 CuSO ₄ .5H ₂ O 0.025 FeNaEDTA 36.70 H ₃ BO ₃ 6.20 KI 0.83 MnSO ₄ .H ₂ O 16.90 Na ₂ MoO ₄ .2H ₂ O 0.25 ZnSO ₄ .7H ₂ O 8.60 bulk element CaCl ₂ 332.02 KH ₂ PO ₄ 170.00 KNO ₃ 1900.00 MgSO ₄ 180.54 NH ₄ NO ₃ 1650.00 vitamin Glycine 2.00 myo-Inositol 100.00 Nicotinic acid 0.50 Pyridoxine HCl 0.50 Thiamine HCl 0.10 plant growth hormone one of four 1 to 5 carbon source Sucrose 30000 hardener Gelrite 3500 Acidity (pH) 5.7

The culture environment was maintained with illumination (cold white fluorescent lamp, 30-50 μmol·m ^-2 ·s ^-1 ) at a temperature of 25±1° C. for 16 hours a day. A photograph of the culture result is shown in FIG. 1 , and the results are shown in Table 2 by averaging the shoot induction rate, the number of shoots, and the shoot length.

Plant growth hormone type and concentration (mg/L) shoot induction rate
(%) number of shoots
(dog) shoot length
(mm) Zeatin 1.0 (a) 100.0 5.8 ± 2.4 34.1 ± 2.4 2.0 (b) 100.0 7.5 ± 2.8 31.5 ± 2.9 5.0 (c) 100.0 23.5 ± 10.4 4.4 ± 0.4 Cydiazuron (TDZ) 1.0 (d) 84.0 12.2 ± 6.3 5.3 ± 1.4 2.0 (e) 88.0 10.2 ± 5.4 5.3 ± 1.1 5.0 (f) 100.0 8.0 ± 5.3 5.0 ± 1.3 6-benzylaminopurine
(B.A.) 1.0 (g) 0 0 0 2.0 (h) 0 0 0 5.0 (i) 0 0 0 6-(γ,γ-dimethylallyl)aminopurine 1.0 (g) 88.0 5.2 ± 2.9 38.9 ± 2.7 2.0 (k) 100.0 7.4 ± 3.0 51.7 ± 5.5 5.0 (l) 92.0 4.3 ± 1.6 25.5 ± 3.2

As shown in Figure 1a and Table 2, in the culture medium to which 6-benzylaminopurine was added, shoots were not induced at all from the explants of Spermia japonica, regardless of the concentration, and in the culture medium to which zeatin was added, the shoot induction rate was It appeared as 100% and the number of shoots was large, but the length of the shoots was not sufficient. In the culture medium to which Cidiazuron was added, the number of shoots was the highest, but the length of shoots was insufficient.

In contrast, according to the present invention, in the culture medium containing 6-(γ,γ-dimethylallyl)aminopurine at a concentration of 1.0 to 2,0 mg/L according to the present invention, the shoot induction rate was high, the number of shoots was large, and in addition, the shoot length was long enough. It can be seen that the polyspinous plant is well induced from the explants of the genus chrysanthemum. In particular, in the culture medium to which 6-(γ,γ-dimethylallyl)aminopurine was added at a concentration of 2,0 mg/L, the shoot induction rate was 100%, the number of shoots was 7.4 ± 3.0, and the length of the shoots was 51.7 ± 5.5 mm ( Figure 1b). Therefore, it is most effective to add 6-(γ,γ-dimethylallyl)aminopurine among plant growth hormones for inducing multi-headed shoots of ginseng plants, and considering the shoot length (promoting growth), the most desirable concentration is 2.0mg/L It can be confirmed that

Comparative Example 1: Plant redifferentiation and purification through in-flight rooting

<In-flight rooting>

As in Example 1 (group k), shoots were prepared by cutting the stems to 2.5-3 cm so that 4-6 axillas were included from the induced polyclinic plants.

30 each of the prepared shoots were treated with auxin (indole-3-butyric acid (IBA, indole-3-butyric acid) or 1-naphthaleneacetic acid (NAA, 1-Naphthaleneacetic acid)) and 1% of the same type and concentration as in Table 3 below. Roots were induced by transplantation into MS medium supplemented with sucrose and 0.35 gelite and cultured for 8 weeks. The culture environment was illuminated at a temperature of 25° C. for 16 hours a day (cold white fluorescent lamp, 30-50 μmol·m ^-2 ·s). ^-1 ) The rooting rate, number of roots, and root length of each group were averaged and calculated, and the results are shown in Table 3. A photograph of the rooting-induced and redifferentiated plant is shown in FIG.

Auxin (mg/L) Rooting rate
(%) root water
(dog) root length
(mm) Control 0 (a) 3.3 1.0 23.2 IBA 0.5 (f) 30.0 ± 10.0 2.4 ± 2.2 17.6 ± 7.5 1.0 (g) 30.0 ± 10.0 3.7 ±3.5 20.0 ± 6.3 2.0 (h) 43.3 ± 30.0 8.4 ± 7.7 19.3 ± 5.0 5.0 (i) 60.0 ± 10.0 10.1 ± 7.1 14.3 ± 2.8 NAA 0.5 (b) 60.0 ± 26.5 5.2 ± 3.6 12.8 ± 2.6 1.0 (c) 66.7 ± 20.8 8.2 ± 4.2 10.7 ± 1.8 2.0 (d) 86.7 ± 15.3 7.1 ± 4.0 3.7 ± 1.2 5.0 (e) 10.0 ± 10.0 3.3 ± 2.1 1.7 ± 0.5

As shown in Figure 2, it can be confirmed that the rooting is not smooth due to over-formation of the callus during in-flight rooting (b-e) in the culture medium to which NAA is added, and the in-flight rooting in the culture medium to which IBA is added. Trial callus was formed, and rooting was not performed well.

As shown in Table 3, in the culture medium to which IBA was added at a concentration of 0.5 to 5.0 mg/L, the rooting rate of Zinnia shoots was as low as 60% or less during in-flight rooting. The rooting rate of young quinceanium shoots was similarly low, and in the culture medium with high concentration (2.0 ~ 5.0 mg/L) of NAA added, the rooting rate of shoots of quinceae during in-flight rooting was high, but the root length was less than 3.7mm, which was insufficient. did.

<purification>

Thirty young plants redifferentiated through in-flight rooting were transplanted into horticultural media (Farm Hannong) in an acclimatization container and grown for 4 weeks. After transplantation, water was sufficiently irrigated and the humidity in the acclimatization vessel was maintained at 80-90% by covering it with the acclimatization vessel cover. The culture environment in the acclimatization chamber was maintained at a temperature of 25±1° C. for 16 hours a day, 30-50 mol·m ^-2 ·s ^-1 lighting.

After 4 weeks of growth, the survival rate of the pure-flowered seedlings (Geum tree) was calculated, and the results are shown in Table 4.

auxin
(mg/L) survival rate
(%) Control 8.9± 2.1 IBA 0.5 8.3 ± 1.9 1.0 11.8 ± 2.4 2.0 6.7 ± 1.7 5.0 2.3 ± 0.4 NAA 0.5 0 1.0 0 2.0 0 5.0 0

As can be seen in Table 4, in the case of plants rooted in the NAA-added medium at a concentration of 0.5 to 5.0 mg/L, all of them were killed (viability 0%), and even in the case of plants rooted in the IBA-added medium, the maximum survival rate was remarkable as 11.8%. As it appears very low, it can be seen that the pure gold tree redifferentiated by in-flight rooting is not acclimatized properly.

As described above, in the case of in-flight rooting, the reason that the plant acclimatization rate was low at 11.8% or less is because the roots are not formed directly at the lower end of the stem, and the callus is first formed excessively on the cut surface of the lower end and the roots are generated on the surface of the callus. It is judged that the plant did not grow normally and died.

As a result, it was found that the production efficiency of tissue culture seedlings through in-flight rooting of ginseng plants was very low.

Example 2: Plant redifferentiation and seedling through explant rooting

<External rooting>

As in Example 1 (group k), shoots were prepared by cutting the stems to 2.5-3 cm so that 4-6 axillas were included from the induced polyclinic plants.

30 each of the prepared shoots were treated with auxin at a concentration as shown in Table 5 below, and then transplanted with plug (FLEXI-PLPUG) or top soil (Farm Hannong) to induce extra-vegetative rooting and redifferentiate into plants. For auxin treatment, 2.0 g of an auxin carrier (facilitating auxin absorption) Talc (Samjeon Chemical, Japan) was mixed with 2.0 mL of an indole-3-butyric acid (IBA, Indole-3-butyric acid) solution and buried on the cut surface of the shoot.

For explant rooting, the air humidity in the cutting container (54×28×6.5 cm) was maintained at 90%, and the lighting was maintained within the range of 40 μmol·m ^-2 ·s ^-1 at 25°C for 16 hours a day for 8 weeks. Rooting rate, root length, and stem length were measured and calculated, and the results are shown in FIGS. 3 to 4b and Table 5.

cutting method IBA
(g/L) Rooting rate
(%) root length
(mm) stem length
(mm) plug insert 0 20.0 ± 10.0 23.5 ± 2.1 34.6 ± 3.0 0.05 40.0 ± 10.0 24.3 ± 3.7 31.7 ± 4.7 0.1 26.7 ± 15.3 24.8 ± 3.5 34.7 ± 3.9 0.5 50.0 ± 10.0 27.9 ± 4.3 34.1 ± 7.4 1.0 36.7 ± 11.6 32.7 ± 5.2 35.3 ± 6.4 2.0 24.5 ± 10.4 25.3 ± 4.1 32.6 ± 5.9 soil cuttings 0 0 0 0 0.05 33.3 ± 5.8 18.1 ± 4.3 40.3 ± 8.2 0.1 46.7 ± 15.3 27.0 ± 4.7 37.3 ± 8.5 0.5 63.3 ± 15.3 33.8 ± 5.4 42.1 ± 10.2 1.0 80.0 ± 17.3 32.9 ± 5.1 41.7 ± 5.6 2.0 57.8 ± 14.3 28.4 ± 3.8 38.4 ± 7.5

Figure 3 is a photograph of the ex-plant rooting process (above) and the re-differentiated genitalia plant using a plug cuttings. As shown in FIG. 3 , the growth of roots and stems was insufficient throughout the redifferentiated plant.

Figures 4a and 4b are photographs of the re-differentiation process of the rooting process and the redifferentiation of the plant of the plant of the plant of the plant of the plant of the plantation plant using the cuttings of the top soil. As shown in FIGS. 4A and 4B , it can be confirmed that the growth of roots and stems is well performed throughout the redifferentiated plant.

Table 5 shows the effect of induction of induction of external rooting and stem growth of ginseng shoots according to the cutting method and the auxin (IBA) treatment concentration.

When externally rooted by plug cuttings, the rooting rate of the genus root was as low as 50% or less, regardless of the concentration of auxin treatment, and the root length and stem growth were also insufficient.

On the other hand, when rooted out-of-the-field with topsoil cuttings, the rooting rate of the rhododendron shoots treated with 1.0 mg/L IBA was the highest at 80.0%, and the root and stem lengths of the shoots treated with 0.5 mg/L IBA were 33.8, respectively. mm and 42.1 mm. Therefore, for the extra-vegetative rooting of new shoots of ginseng, top soil cuttings and IBA 0.5 to 1.0 mg/L treatment are preferable, and most preferably IBA 1.0 mg/L treatment.

It can be seen that the redifferentiation through in vitro rooting according to the present invention was improved by 6.7 to 9.4 times compared to the redifferentiation through in vitro rooting of Comparative Example 1 (compared to IBA 0.5 to 1.0 mg/L treatment).

When inducing extra-vegetative rooting of gold tree shoots through topsoil cutting, unlike in-flight rooting, rooting takes place without excessive callus formation, and it is judged that re-differentiation is performed smoothly because of good root formation.

<Nursery>

As described above, the redifferentiated ginseng plant was transplanted into the soil in the pot and grown for 4 months in a greenhouse (watering once or twice a week) and mass-produced as the best quality seedlings suitable for formal planting (FIG. 5), as a result The photograph is shown in FIG. 6, and the survival rate after 4 months of seedling was calculated and shown in Table 6.

cutting method IBA
(g/L) survival rate
(%) soil cuttings 0 - 0.05 100.0 0.1 100.0 0.5 100.0 1.0 100.0 2.0 100.0

As shown in Table 6, the survival rate through seedlings of the re-differentiated spruce plants by ex-root rooting using topsoil cuttings was 100%.

In the present invention, since the soil acclimatization of the redifferentiated genus rooted from outside rooting was also performed at the same time, tissue culture seedlings could be grown without a separate additional acclimatization step.

Claims (11)

i) inducing polyclinic plants by in-flight culturing of spermatozoa explants of the genus chrysanthemum;
ii) re-differentiating into plants through explant rooting by cutting the shoots derived from multi-stemmed plants in top soil; and
iii) a step of transplanting a plant into the soil and seeding it: A method for mass production of a sapling of tissue cultured spruce, comprising the.
The method according to claim 1, wherein the culture medium in step i) contains 1.0 mg/L to 2.0 mg/L of 6-(γ,γ-dimethylallyl)aminopurine, 2% to 3% sucrose and 0.3% to 0.35% A method for mass production of tissue-cultured seedlings of pure gold tree, characterized in that it is a solid phase MS (Murashige and Skoog) medium to which gelite is added.
The method of claim 2, wherein the concentration of 6-(γ,γ-dimethylallyl)aminopurine is 2.0 mg/L.
The method according to claim 1, wherein the culture in step i) is carried out at 25±1° C. for 15-17 hours a day, 30-50 μmol·m ^-2 ·s ^-1 Lighting is maintained for 6 to 10 weeks. A method for mass production of tissue-cultured seedlings of pure gold tree.
According to claim 1, wherein in step ii), the shoots remove the apical part from the polycrystals and cut the stems to 2.5-3 cm in length so that 4-6 axillas are included. Way.
According to claim 1, wherein the cut surface of the shoot in step ii) is 0.5 ~ 1.0 g / L indole-3-butyric acid and talc 1:1 (v:w) mixture of talc, characterized in that the treated gold tree A method for mass production of tissue culture seedlings.
The method of claim 6, wherein the concentration of indole-3-butyric acid is 1.0 g/L.
The method according to claim 1, wherein in step ii), explant rooting is carried out at 25±1° C. for 15 to 17 hours a day, 30 to 50 mol·m ^-2 ·s ^-1 lighting, and 6 to in an environment where humidity is maintained at 80 to 90%. A method of mass production of saplings for tissue culture of pure gold tree, characterized in that it is carried out for 10 weeks.
The method of claim 1, wherein the redifferentiation rate is 63 to 80%.
[10] The method of claim 9, wherein the redifferentiation rate is 80%.
[Claim 11] The tissue culture seedlings produced by the method according to any one of claims 1 to 10.

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