KR20130084028A - The method of shoot regeneration from salix spp. and its improvement - Google Patents

Abstract

PURPOSE: A redifferentiation method of Salix koreensis and a method for improving the same are provided to quickly proliferate selected male trees of Salix koreensis with excellent growth and to effectively supply Salix pseudo-lasiogyne and Salix caprea L. CONSTITUTION: A method for improving the redifferentiation ratio of Salix koreensis shoots comprises the steps of: culturing Salix koreensis by low light for two weeks; and culturing Salix koreensis by strong light for four weeks. The method comprises a pre-cultivation of the shoots in a medium containing 2.0 mg/L of 2ip ( (2-isopentenyl) adenine) and 0.1 mg/L of naphthalene acetic acid (NAA) for two weeks. The method further comprises the step of the shoots in a shoot-inducing medium containing 2.0mg/L of NAA-free 2ip.

Description

Willow tree regeneration method and improved method {The method of shoot regeneration from Salix spp. and its improvement}

The present invention relates to the regeneration of willow shoots. One aspect of the present invention in more detail in the in-flight culture of willow (i) after incubation for 2 weeks in low light; (ii) incubation for 4 weeks in strong light; It relates to a technique for improving the regeneration rate of willow shoots (新 草) characterized in that it comprises a step. Another aspect of the present invention is pre-culture for 2 weeks in the medium added 2ip ((2-isopentenyl) adenine) 2.0mg / L and NAA (naphthalene acetic acid) 0.1 mg / L cultivation) and the step of cultivating in a shoot-derived medium containing 2ip 2.0mg / L of NAA removed after pre-cultivation alone to improve the regeneration rate of willow shoots.

Willow is a deciduous broad-leaved arboreous tree that grows over a large area, depending on the species, in grassland, riverside wetlands, and mountainsides (Zomleter 1994). Most willows grow quickly and adapt well under certain circumstances. Because of these two important features, these species are rapidly increasing in economic value as alternative species for bioenergy production and environmental purification.

Therefore, in order to expand the use of willows, that is, rapid growth and rapid breeding of selected clones and introduction of useful traits through genetic manipulation, mature in-plant culture and regeneration system of male tree, which grows well and does not fly, It must be established. However, mature trees generally die gradually within 6 months of culture, making in-flight cultivation difficult, in particular, regeneration of plants from mature trees.

As mentioned above, willows are a very important species for environmental purification and biomass, and are very difficult to re-differentiate. So far, only three international journals have reported regeneration of willows (Lyyra et al, 2006). This can be said to disprove that it is very different from poplars which are similar in form but very easy to redifferentiate.

Until now, no in-flight cultivation and re-differentiation have been reported in the willow and hawthorn species of our willow species, and this technology was developed for the first time at home and abroad.

Hereinafter, the patent document related to this invention is described.

First, Patent No. 10-2011-0009115 exists as a patent document. The present invention relates to a method for plant regeneration from embryogenic callus, more specifically 25-35 g / L sucrose, 700-800 mg / L MgCl 2 · 6H 2 O and 1-3 g / L gelrite, 1 From embryogenic callus using a medium for plant regeneration using a combination of ˜3 mg / L 2,4-D and 1.5 to 2.5 mg / L BA, or MS medium containing 1 to 3 mg / L kinetin alone. The present invention relates to a method for re-differentiating a plant, and there is no mention of light control of the present invention, and the technical configuration of medium control is completely different.

Second, there is a patent document 10-2008-0084824. The present invention relates to a method of inducing regeneration by culturing a callus of candles in MS medium containing kinetin and NAA, wherein casamino acid is additionally added to MS medium containing kinetin and NAA. The present invention relates to a method and a medium for inducing regeneration from a callus of a candle, which is contained, and is different from the present invention.

Third, Patent No. 10-2009-0120924 exists as a patent document. The present invention also relates to a mass production method of a plant regenerated from shrimp orchid leaf slices. The method for mass production of shrimp orchid plants of the present invention comprises the steps of sterilizing the young shrimp orchid, sterilizing, incubating the sterilized young shrimp orchid on a 1/3 MS medium and incubating the cultured young shrimp orchid. Cutting the leaves of the leaves, and inducing the buds by injecting the leaf fragments of the resulting prawn orchids on a 1/2 MS medium to which sucrose, activated carbon, indolebutyric acid, and naphthalene acetic acid were added. Induced and multiplied by inducing polycincho by incubating in 1 / 2MS medium containing sucrose and gibberellic acid, 1 / 2MS to which the propagated polycynic acid is added one selected from sucrose, indolebutyric acid or naphthalene acetic acid Mass production of shrimp orchid plants by transplanting the produced seedlings into soil, purifying them and then growing them in a greenhouse Consisting of a bar, or less does not have any disclosure or suggestion of the present invention the technical features to be described.

The present invention (i) after incubation for 2 weeks in low light (低 光) during in-flight culture of willow; (ii) incubation for 4 weeks in strong light; The aim of this study is to provide a solution that ultimately improves the regeneration rate of willow buds.

However, the technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned may be clearly understood by those skilled in the art from the description of the present invention. There will be.

In the present invention in order to solve the problems of the prior art (i) after incubation for 2 weeks in low light (低 光) during in-flight culture of willow; (ii) incubation for 4 weeks in strong light; It provides a method for improving the regeneration rate of willow shoots (新 草) comprising the step of.

More specifically, the method is pre-cultivation for 2 weeks in the medium added 2ip ((2-isopentenyl) adenine) 2.0 mg / L and 0.1 mg / L naphthalene acetic acid (NAA) pre-cultivation It can be characterized in that).

Furthermore, after the pre-culture, it may further comprise the step of culturing in shoot-derived medium added with 2ip 2.0mg / L alone NAA is removed.

The light control and the control of the medium composition have an effect of improving the regeneration rate of willow shoots, respectively, and when the light control and the control of the medium composition are combined, it is possible to obtain a higher regeneration rate.

Preferably, the low light may be characterized in that 5 ~ 10μMm ^-2 s ^-1 PPFD (photosynthetic photon flux density), the strong light may be characterized in that 40μMm ^-2 s ^-1 PPFD. It was confirmed experimentally that the range of the low light and the numerical value of the strong light have a critical significance in improving the regeneration rate of shoots.

The willow is also called holly ( Salix) caprea ) or Twill weed ( Salix pseudolasiogyne ).

The following advantageous effects are recognized by the method of improving the regeneration rate of the willow tree which is this invention.

First, according to the present invention, it will be possible to rapidly grow a large amount of mature willow tree selected as a male tree having excellent growth, and thus it will be able to easily adapt to growth places where environmental purification is required, such as salinity soils, abandoned mines and waterfronts. And it is an advantageous effect that it can efficiently supply the hover hovers, which are known to have the best absorption of heavy metals.

Secondly, since the horns and twill weeds are excellent in growth ability, there is an advantageous effect that it is possible to supply the material for biomass based on the present invention.

Third, a very advantageous effect is recognized that the re-differentiation technique of the present invention will enable the willow tree transformation technique known only so far, thereby enabling molecular breeding through the introduction of heavy metal adsorption genes, cellulose biosynthesis genes and the like.

Ultimately, it is expected to bring enormous economic and social value in the future through eco-friendly natural purification and biomass production using willows, which are the effects of the present invention.

Figure 1 relates to the treatment for glances in cultured weeping willow mature tree node.
2 is related to shoot regeneration according to light intensity change during shoot regeneration. More specifically, 'Continuous light' of FIG. 2 means that the culture is continued under the same light condition (40 μMm ^-2 s ^-1 PPFD 6 weeks), and 'Altering light' means that the light condition is modified ('5). ~ 10 μMm ^-2 s ^-1 PPFD 2 weeks '→ '40 μMm ^-2 s ^-1 PPFD 4 weeks').
3 relates to the effect of pretreatment on shoot regeneration.
Figure 4 relates to the optimal medium selection basis on shoot regeneration.
FIG. 5 is a photograph taken after shoot regeneration and purified seedlings from leaf slices of tigers according to the present invention.
A and B: Shoot Regeneration from the Willow Leaf Base and Leaf Sections
C: In-flight commute
D: outside purifying

The present invention (i) after incubation for 2 weeks in low light (低 光) during in-flight culture of willow; (ii) incubation for 4 weeks in strong light; It relates to a method of improving the regeneration rate of willow shoots (新 草) comprising the step of doing.

More specifically, the method is pre-cultivation for 2 weeks in the medium added 2ip ((2-isopentenyl) adenine) 2.0 mg / L and 0.1 mg / L naphthalene acetic acid (NAA) pre-cultivation It can be characterized in that).

Furthermore, after the pre-culture, it may further comprise the step of culturing in shoot-derived medium added with 2ip 2.0mg / L alone NAA is removed.

Preferably, the low light may be characterized in that 5 ~ 10μMm ^-2 s ^-1 PPFD (photosynthetic photon flux density), the strong light may be characterized in that 40μMm ^-2 s ^-1 PPFD.

In addition, the willow tree may be characterized by holly ( Salix caprea ) or stalk ( Salix pseudolasiogyne ).

There are about 300 species of willow in the world of willow (Zomleter 1994). Willow is a deciduous broad-leaved arboreous tree that grows over a large area, depending on the species, in grassland, riverside wetlands, and mountainsides (Zomleter 1994). Most willows grow quickly and adapt well under certain circumstances. Because of these two important features, these species have recently gained further economic value as alternatives for bioenergy production and environmental purification (Elowson1999, Graneletal. 2002, Kuzovkina and Quigley 2005, Pushon and Dickinson 1997). Among these, the well-grown weeping willow ( Salix pseudolasiogyne Leveille, Korean weeping willow) and Salix, which grow naturally in abandoned mines and are expected to be candidates for environmental purification. caprea ) is highly available. In-flight cultures and regeneration systems should be established to expand the use of these species, ie, rapid rapid growth and selective breeding of selected clones.

Willow is a distinctive tree, and the female tree (female tree) in the spring season is attached to the fluffy flies cause allergies, etc. For its use, a good growth of the male tree (male tree) should be selected and used. Therefore, there is no choice but to use mature trees that can be divided into male and female during flowering. However, most mature trees have slow growth and seasonal dormancy in the air, and often die within 6 months without being able to settle on the medium during in-flight culture.

Serial grafting (Valdes et al. 2003), hormonal treatment (Beck et al. 1998), in-flight micrografting ( in ) to rejuvenate or reinvigorate mature plants in a variety of plants in vitro micrografting (Perrin et al. 1994) or laser beam treatment (Rodrigues et al. 2001). In many trees, however, these methods are not yet practically available.

Willows are dormant in an environment that is not suitable for growth.Because of these characteristics, the buds are dormant because of their characteristics. ) Is not predictable. In addition, the degree is even worse when using mature wood as the main material.

In-flight propagation may be one method for this kind of plant. However, willows are one of the most recalcitrant species that are still very difficult to re-differentiate due to their genetic characteristics, but most of them (von Aderkas and Bonga 2000, Selby et al., 2005). Cytokinins among endogenous hormones are known to play an important role in bud breaking and regeneration during in-flight cultivation of mature willows (Beck et al. 1998).

Therefore, in the present technology, it was conceived to provide a method of re-differentiating shoots from the in-flight culture of mature willows and the leaves of the plant derived therefrom, which confirmed that the growth is fast and does not fly down.

Hereinafter, the technical contents of the present invention will be described in detail with reference to the accompanying drawings.

Figure 1 shows the bud-breaking and multiplication rate during in-flight culture of mature willows (Hors and Twill) in connection with the present invention.

As a result of experiment, the following three technical means could be conceived as a method to re-differentiate shoots in leaf segments of plants regenerated from mature willows.

1. Technology to increase shoot regeneration rate after two weeks of low light culture in culture

Generally, bright cultures are performed under ⁴⁰ μMm ⁻² s ⁻¹ PPFD for redifferentiation (no treatment). The initial survival rate of tiger willows was more than three times (about 20% → about 70%) when cultured in low light of 5 ~ 10μMm ^-2 s ^-1 PPFD for 2 weeks and then transferred to 40μMm ^-2 s ^-1 PPFD. , Shoot regeneration per fragment increased more than 2 times (about 2) (see FIG. 2).

2. Pretreatment of pre-culture formation for 2 weeks in medium supplemented with specific hormone to increase regeneration rate (Pre-culture)

As shown in FIG. 3, when cultured in 1-12 kinds of shoot-derived medium without pretreatment (flower seed formation step), callus formation rate was about 60%, and the number of replanted shoots per section was 0-2. However, when they were pretreated for 2 weeks in shoot-forming medium and transferred to shoot-derived medium, callus formation rate increased to about 90% and maximum shoot number of 10.

3. Induction medium to increase the number of shoots during regeneration after incubation in pre-fried medium (Induction medium)

As shown in FIG. 4, the maximum shoot regeneration was achieved with 10 total shoots in 2ip 2.0 mg / L monocultured medium with NAArk removed when transferred to 12 shoot-derived media after 2 weeks of pre-treatment in shoot-derived medium.

Through the present invention it was possible to obtain shoots from leaf slices, stem slices, etc. in both the hover and the twill weeds (see FIGS. 5 and 6), the technology of the present invention has not been reported so far at home and abroad. Furthermore, the present invention can be applied to domestic willows such as kings, weeping willows and pussy willows.

The present invention has been described above in connection with specific embodiments of the present invention, but this is only an example and the present invention is not limited thereto. Those skilled in the art can change or modify the described embodiments without departing from the scope of the present invention, and such changes or modifications are within the scope of the present invention. In addition, the materials of each component described herein can be readily selected and substituted for various materials known to those skilled in the art. Those skilled in the art will also appreciate that some of the components described herein can be omitted without degrading performance or adding components to improve performance. In addition, those skilled in the art may change the order of the method steps described herein depending on the process environment or equipment. Therefore, the scope of the present invention should be determined by the appended claims and equivalents thereof, not by the embodiments described.

Claims (6)

In-flight cultivation of willow (i) after incubation for 2 weeks in low light; (ii) incubation for 4 weeks in strong light; Method for improving the regeneration rate of willow shoots (新 草), characterized in that it comprises a step of.
The method according to claim 1, wherein the method comprises pre-culture for 2 weeks in a medium to which 2.0 mg / L of 2ip ((2-isopentenyl) adenine) and 0.1 mg / L of naphthalene acetic acid (NAA) are added. A method of improving the regeneration rate of willow shoots, characterized by pre-cultivation.
The method of claim 2, further comprising culturing in shoot-derived medium containing 2ip 2.0 mg / L of NAA removed after pre-culture alone.
The method of claim 1, wherein the low light is 5-10 μMm ⁻² s ⁻¹ photosynthetic photon flux density (PPFD).
The method of claim 1, wherein the concentrate is 40 μMm ⁻² s ⁻¹ PPFD.
The willow tree of claim 1, wherein the willow is Salix caprea ) or Twill weed ( Salix pseudolasiogyne ) is a method of improving the regeneration rate of willow shoots.

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