KR101341148B1 - Production method of citrus unshiu marcow - Google Patents

Abstract

The present invention relates to a method for producing citrus trees through an in vitro culture, by establishing in vitro culture conditions for the citrus trees to mass produce the citrus trees. The method comprises the steps of: (a) adding sucrose and Phytagel into an MS basic medium to prepare a germination medium, germinating seeds, which are taken from citrus, in the germination medium, and culturing the germinated sprouts to have multiple joints to form young seedlings; (b) adding one or two of additives, BAP, IAA, and NAA, into an MS basic medium to prepare a shoot growth medium, cutting the young seeding of step (a) into joint units, and transferring the cut joint units into the shoot growth medium, and growing shoots on each of the transferred joint units to form joint seedlings; (c) adding 0.1-3 mg of BAP into 1L of an MS basic medium to prepare a joint seeding growth medium, transferring the joint seedlings of step (c) into the joint seeding growth medium, and growing the joint seedlings; and (d) preparing an MS basic medium or preparing a root medium by adding one or two additives of IBA, NAA, IAA, and 2.4-D, transferring the joint seedlings of step (c) into the root medium, and inducing and growing roots.

Description

Production method of citrus trees by in-flight culture {Production method of citrus unshiu Marcow}

The present invention relates to a method for mass production of citrus trees by in-flight culture to establish the in-flight culture conditions of the citrus tree to mass produce.

Citrus tree (Citrus unshiu Marcow) is an evergreen tree with dicotyledonous hawthorn, and is native to Assam in northeastern India, and is widely distributed wildly in tropical and subtropical regions of Southeast Asia, mainly in the southern Himalayas.

In general, citrus trees are 3 ~ 5m high, branches are spread, leaves are alternate, elliptical with flat edges or wavy serrates, and petiole wings are not clear. Flowers blossom in June, with 5 sepals and petals, 1 pistil, and several stamens.

Fruits are 5 ~ 8cm in diameter, small balls, and when ripe, they turn yellowish red. Ripe fruits are hollow in the middle, peel the skin well, and eat mainly raw fruits.

The most cultivated varieties in Korea are about 10 varieties, and classified into crude, middle, and only species.

It is similar to citrus tree (C. unshiu), but the leaf is oval, the fruit is egg-shaped, and the center of the fruit is tight.

According to the historical record of citrus cultivation in Korea, it is believed that citrus has been cultivated in Korea since the history of offering tribute on Jeju Island in the second year of King Baekje Moonju (A.D. 476). Most of the citrus cultivars are Jeju, 22 varieties including kumquat, wild mandarin, blue mandarin orange, orange mandarin orange, citron, sugar orange, red mandarin orange, potato, and soybean persimmon. It is thought to have been improved, and now only a few traditional varieties can be found in Jeju, including bottle tangerine, sugar citron, citron, blue mandarin orange, tangerine tangerine and tangerine.

The classification of citrus fruits can be divided into natural classification classified systematically based on the perspective of softness and artificial classification classified according to practical use. Swingle and tanaka have concentrated natural classification. Tanaka divided citrus fruits into tanza, citrus, clymenia, and gold persimmons.

The artificial classification of citrus fruits is classified into reproductive, cooking, ornamental and large tree depending on the use. For reproduction, the citrus fruits include Wenzhou mandarin, Clementine mandarine, Ponkan, Mandarin mandarin and Hongju mandarin. Tangerine, citrus and golden persimmon, the third genus of citrus fruits, are used a lot for large trees. In Korea, tanza is mainly used, and citron and mandarin are also used.

Citrus fruits are mainly used for reproduction, but the fruits left behind are often used for processing or cooking.In the case of using citrus trees for ornamental purposes, there are many ornamental varieties because there are no ornamental varieties. To be used for ornamental purposes is not used much due to limitations such as cultivation temperature.

An object of the present invention, citrus by in-flight cultivation to be grown on land of land as well as educational materials, tourism products, character props as a mass growth of individuals by establishing in-flight culture conditions of seeds collected from citrus To provide a method for mass production of wood.

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The configuration for the problem solving means of the present invention for achieving the above object, (a) to the MS basic medium Preparing a germination medium by adding sucrose and pita gel, germinating seeds (seeds) collected from citrus fruits in the germination medium, and then culturing the germinated shoots to a plurality of nodes or more to form seedlings;
(b) Adding shoots of BAP, IAA, NAA or two additives to the MS base medium to prepare shoot growth medium, and cut the shoot seedlings of each seedling of (a) seedlings into the prepared shoot growth medium Moving each cleaved node unit to multiply shoots to form the node seedlings;
(c) adding BAP 0.1-3 mg to 1 L of MS basic medium to prepare a seedling seedling growth medium, and transferring the seedling seedling of (b) to the prepared seedling seedling growth medium to grow the seedling seedling;
(d) adding the additive of any one of IBA, NAA, IAA, 2.4-D to the MS base medium or MS base medium to prepare the rooting medium, and transfer the seedling seedlings of (c) to the prepared rooting medium Inducing and growing; .
And Seeds are first sterilized in commercial seed sterilization liquid, then washed with distilled water, and then secondly sterilized with alcohol, and finally sterilized and disinfected with sodium hypochlorite, and used in the MS basic medium. The addition amount of sucrose and pita gel is 20-50 g of sucrose and 2-5 g of pita gel in 1 liter of MS basic medium.

In addition, the seed germination condition of (a) is germinated by irradiation with light at 2000-3000 lux for 15-17 hours at 22-27 ° C for 10-14 days, or dark cancer without light at 22-27 ° C for 10-14 days. Germinate in state.

The amount of the additive of (b) added to the MS basic medium of (b) was 0.1-3 mg of BAP, 0.1-0.5 mg of IAA or NAA, to 1 L of MS basic medium, and the MS of (d) The addition amount of the additive (d) in the base medium is 0.1-0.5 mg in 1 L of the MS base medium.

Citrus tree mass production method by the in-flight culture of the present invention as described above, as a mass growth of individuals through the establishment of in-flight culture conditions of seeds collected from citrus fruits, as well as educational materials, tourism products, character props, as well as transplanted to land land There is an effect that can be grown.

1 is a photograph showing a germination state of citrus seeds according to the present invention,
Figure 2 is a photograph showing the proliferation state after germination of citrus seeds according to the present invention,
Figure 3 is a photograph of a state further growing shoots in the state of Figure 2,
4 is a photograph of a shoot, a height, a diameter in the state of FIG. 3 grown;
5 is a photograph of a state of rooting in the state of FIG.

Hereinafter, the citrus tree mass production method by in-flight culture of the present invention will be described in detail.

(a) Seed germination and culture stage

The present invention first collects seeds for mass production of citrus trees, and the method of collecting seeds is to collect seeds (seeds) from the fruits opened from citrus trees, and the commercial seeds disinfecting commercially available commercially sold seeds. The solution is first disinfected, then washed with distilled water, and then sterilized with alcohol, and finally disinfected with Sodium hypochlorite.

The reason is to increase the germination rate of seeds (seeds) and to achieve healthy culture by disinfecting and sterilizing external contaminants in seeds (seeds) collected from citrus fruits.

Specific methods for disinfecting and sterilizing the external contaminants on the seeds (seeds) include sterilizing the seeds (seeds) by immersion for 2 to 3 hours in a general commercial seed disinfectant solution, followed by distilled water. After five washes, the solution is immersed for 1 to 3 minutes in 70 ± 5% alcohol and then finally sterilized in 2 ± 0.5% sodium hypochlorite.

Seeds obtained as described above should be germinated and cultured in seed germination medium, wherein the method of producing seed germination medium for germinating and culturing the seed is carried out in MS (murashigae and Skoog) basic medium. It is prepared by adding sucrose and phyta gel.

The sucrose (sucrose) is to act as an energy source (growth hormone) required for the germination of the sprouts after the seed germination, Phyta gel (Phyta gel) serves to gel (solidify) the seed germination medium.

On the MS basic medium The amount of sucrose and pita gel added should be 20-50 g of sucrose and 2-5 g of pita gel in 1 liter of MS base medium. The reason is that when 20 g of sucrose is added and used, the germinated shoots of the seeds grow normally. Insufficient energy sources (growth hormones) to grow normally, when used in excess of 50g, the energy source (growth hormones) required to grow the germinated shoots too much occurs, so that the problem is that the MS basic medium on Sucrose is preferably added in an amount of 20-50 g of sucrose in 1 L of MS medium.

In addition to the MS basic medium The amount of pita gel added is 1-5 ml of MS base medium. The reason is that the pytha gel is 2-5 g. The reason is that the MS base medium is liquid, so that the liquid is gelled (solidified) so that seeds are not flowed and are fixed in place and germinated. If less than 2g is used, the liquid MS basic medium will not gel (solidify), and if it is used more than 5g, the liquid MS basic medium will be too hard to interfere with seed germination. It is preferable to use 2-5 g of Pita gel because the problem occurs.

On the other hand, the seed germination medium in the seed germination medium, can be divided into two methods, the first, the method of germinating by irradiation with light for 15-17 hours at 2000-3000lux for 10-14 days at 22-27 ℃ and Secondly, there is a method of germinating in a dark state without light at 22-27 ℃ for 10-14 days.

An example of germinating seeds (seeds) by the two methods is shown in FIG. 1 and Table 1 below.

division Germination rate (%) Pollution rate (%) Dark condition 65 ± 2.0 10 ± 1.5 Light condition 90 ± 5.0 10 ± 1.5

As shown in Table 1, it can be seen that the method of germinating seeds by irradiating light is 20% or more germination rate than that of germinating seeds in a cancer condition.

And the germinated shoots are cultured to be more than a plurality of nodes in the seed germination medium to form seedlings, the plurality of nodes of seedlings are 2-4 nodes.

(b) Shoot growth and node seedling formation

Seedlings grown in plural nodes as described above are cut in each node unit and transferred to the seedling shoot medium to grow the shoots, wherein the method of producing seedling shoot medium for growing the shoots is based on MS. It is prepared by adding any one of BAP (benzylaminopurine), IAA (Indole acetic acid), NAA (1-Naphtalene acetic acid), to the medium.

The BAP mainly serves as a growth regulator required for seedling formation, that is, shoot growth, and the like, and the IAA and NAA also play a role as a growth regulator required for seedling formation, shoot growth, etc., like the BAP. The addition amount is 0.1-3 mg of BAP to 1 L of MS basic medium, and the addition amount of IAA and NAA is 0.1-0.5 mg.

The reason for using the BAP 0.1-3mg is that less than or when used in excess, seedling formation and growth of seedlings is not normal, and the reason for using 0.1-0.5mg of either IAA or NAA is less than that When used or excessively used, the formation and growth of shoots are slow and abnormal growth occurs, which is why the leaves and roots of seedlings do not grow in a balanced state.

In the method for producing seedling seedling medium, seedling seedling medium is prepared by adding only BAP to MS base medium or adding one of BAP + IAA or BAP + NAA to MS base medium.

When the seedling seedling medium is produced in the same manner as described above, each one of the cut pieces of the seedling seedling medium is transferred to the prepared seedling seedling medium, and light is radiated for 15-17 hours at 2000-3000lux for 4-5 weeks at 22-27 ° C. Investigate and multiply.

Examples of growing shoots by the above method are shown in FIGS. 2 and 3 and Table 2 below.

division MS Basic Medium 1L + BAP 0.1-3mg MS basic medium 1L + BAP 0.1-3mg NAA 0.1-0.5mg MS basic medium 1L + BAP 0.1-3mg NAA 0.1-0.5mg
Shoot
multiplication
Count
Symptom
expression
Work 28 4 ± 0.5cm 1 ± 0.5 cm 1 ± 0.5 cm 30 4 ± 1.2 cm 2 ± 1.3 cm 1 ± 1.2 cm 32 4 ± 1.5 cm 3 ± 1.3 cm 3 ± 0.6 cm 35 3 ± 1.0 cm 2 ± 0.5 cm 2 ± 1.1 cm

As shown in Table 2, the seedlings of seedlings were best added with BAP to MS basal medium, and BAP + IAA or BAP + NAA was similar to MS basal medium, and BAP was best added to MS basal medium. Most preferred was 30-32 days when added.

(c) Node seedling growth stage

As described above, the seedlings with the growth of shoots are transferred to the seedling seedling growth medium to grow the height (length) and the diameter of the seedlings with the seedling growth, wherein the method of producing the seedling seedling growth medium, BAP on the MS basic medium It is prepared by adding.

The BAP serves as an energy source necessary for not only seedling growth of seedlings, but also height (length) and diameter of seedlings, and the amount of BAP used is 0.1-3 mg of BAP in 1 L of MS basic medium.

The reason why BAP 0.1-3mg is used when preparing the seedling seedling growth medium is that when the seedlings are used in excess or in excess of the seedling growth, the seedling growth and height (length) and the diameter of the seedlings grow too slowly or grow too large, resulting in healthy breasts. Therefore, the amount of BAP added to 1 L of MS basic medium is preferably 0.1-3 mg.

Examples of growing the height (length) of seedlings in the seedling seedling growth medium are shown in FIG. 4 and Table 3 below.

division BAP (/ L) 0.1 One 2 3 1 week 0.550.2cm 0.540.3 cm 0.550.6 cm 0.540.3 cm 3 weeks 0.950.4 cm 0.970.4 cm 0.850.4 cm 0.780.4 cm 5 weeks 1.430.4cm 1.520.3 cm 1.220.2cm 1.020.5cm

(d) rooting of seedling seedlings

As described above, the seedling seedling growth as well as height (length) and diameter of the seedling seedlings transfer the grown seedlings to the rooting medium to induce rooting. The method of manufacturing the rooting medium is IBA (Indole butyric acid) ), NAA, IAA, 2,4-D (2,4-Dichlorophenoxyacetic aicd) is added by the addition of any one.

The additives used to prepare the rooting medium is any one of IBA, NAA, IAA, 2.4-D, all of which serves to promote and grow rooting of seedling seedlings in the 1L MS base medium IBA, NAA, It is preferable to add 0.1-0.5 mg of either IAA or 2,4-D.

The reason why the amount of the additive used when preparing the rooting medium is 0.1-0.5 mg, is less than or used in excess, so that rooting of seedlings grows too slow or grows too much, which prevents healthy rooting. The amount of any one of IBA, NAA, IAA, and 2,4-D is preferably 0.1-0.5 mg in 1 L of the medium.

An embodiment of the rooting state in the rooting medium is shown in Figure 5 and Table 4 below.

division
MS Base Badge +
IBA (mg / L) MS Base Badge +
NAA (mg / L) MS Base Badge +
IAA (mg / L) MS Base Badge +
2.4-D (mg / L) 0.1 0.5 0.1 0.5 0.1 0.5 0.1 0.5 2 weeks 28.9 cm 20.0 cm 72.2 cm 60.2 cm 40.0 cm 73.7 cm 0.0cm 0.0cm 4 weeks 35.2 cm 33.5 cm 73.6 cm 65.5 cm 42.0 cm 75.0 cm 5.2 cm 0.5cm

Promoting the growth, growth and rooting of the individual through the establishment of in-flight culture conditions of citrus trees according to the present invention as described above in large quantities in the form of products growing in a very small miniature in a narrow glass bottle or a plastic container It can be used to cultivate land on land as well as educational materials, tourism products, and character props.

The results of testing the survival rate and growth rate of the citrus seedlings according to the growth environment in the glass container are shown in Table 5 below.

division Survival rate (%) Number of leaves (long) Length (cm) 1 week 3 weeks 5 weeks 1 week 3 weeks 5 weeks 1 week 3 weeks 5 weeks Culture room 100 100 100 6.1 8.9 11.8 0.55 0.95 1.43 office 100 90 90 5.44 8.11 8.22 0.48 0.98 1.64 Outdoor bench 100 90 90 6.2 7.8 9.1 0.66 1.11 1.7

As described above, the present invention can mass-produce seeds collected from citrus fruits as citrus trees by in-flight cultivation, and has the advantage of being cultivated by implanting them into the ground as well as educational materials, tourism products, and character props.

Claims (8)

(a) on the MS base medium Preparing a germination medium by adding sucrose and pita gel, germinating seeds (seeds) collected from citrus fruits in the germination medium, and then culturing the germinated shoots to a plurality of nodes or more to form seedlings;
(b) Adding shoots of BAP, IAA, NAA or two additives to the MS base medium to prepare shoot growth medium, and cut the shoot seedlings of each seedling of (a) seedlings into the prepared shoot growth medium Moving each cleaved node unit to multiply shoots to form the node seedlings;
(c) adding BAP 0.1-3 mg to 1 L of MS basic medium to prepare a seedling seedling growth medium, and transferring the seedling seedling of (b) to the prepared seedling seedling growth medium to grow the seedling seedling;
(d) adding the additive of any one of IBA, NAA, IAA, 2.4-D to the MS base medium or MS base medium to prepare the rooting medium, and transfer the seedling seedlings of (c) to the prepared rooting medium Inducing and growing; Citrus tree mass production method by in-flight culture. The method of claim 1,
remind Seeds are firstly sterilized in commercial seed sterilization liquid, then washed with distilled water and then secondly sterilized with alcohol, and finally sterilized and disinfected with sodium hypochlorite, mass production of citrus trees by in-flight culture Way. The method of claim 1,
On the MS basic medium The method of mass production of citrus trees by in-flight culture is characterized in that the addition amount of sucrose and pita gel is 20-50 g of sucrose and 2-5 g of pita gel in 1 L of MS basic medium. The method of claim 1,
Seed germination condition of (a) is a mass production method of citrus trees by in-flight culture, characterized in that germination by irradiation with light at 2000-3000lux for 15-17 hours at 22-27 ℃ for 10-14 days. The method of claim 1,
Seed germination conditions of (a), the mass production method of citrus trees by in-flight culture, characterized in that the germination in a dark dark state without light for 10-14 days at 22-27 ℃. The method of claim 1,
Tangerine by in-flight culture, characterized in that the amount of the additive of (b) is added to the MS basic medium of (b) is 0.1-3mg of BAP, 0.1-0.5mg of IAA and NAA in 1L of MS basic medium Wood mass production method. delete The method of claim 1,
The method of mass production of citrus trees by in-flight culture, characterized in that the amount of the additive of (d) added to the MS basic medium of (d) is 0.1-0.5 mg in 1 L of the MS basic medium.

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