CN115088618A

CN115088618A - Method for efficiently regenerating larch and establishing in-vitro cutting orchard

Info

Publication number: CN115088618A
Application number: CN202210812422.3A
Authority: CN
Inventors: 闫学彤; 朱木兰; 齐力旺; 郑珂媛
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2022-07-11
Filing date: 2022-07-11
Publication date: 2022-09-23
Anticipated expiration: 2042-07-11
Also published as: CN115088618B

Abstract

The invention belongs to the technical field of plant tissue culture and rapid propagation, and particularly relates to a method for efficiently regenerating larch and establishing an in-vitro cutting orchard. According to the invention, the larch bud point is taken as an explant to carry out in-vitro regeneration research, the bud and the extension of the fixed bud point are induced, a fast extension plant is obtained, the generation of the fixed bud point is further efficiently induced, and a circulating culture method which is not limited by seasons and is fast in regeneration is provided for the market. The method can be used for establishing an efficient in-vitro cutting orchard system, the single circulation is 8 weeks, the propagation coefficient is 7, the number of the sprouts is increased in a geometric series, and high-activity multiple young branches are produced annually.

Description

Method for efficiently regenerating larch and establishing in-vitro cutting orchard

Technical Field

The invention relates to the technical field of plant tissue culture, namely plant regeneration and rapid propagation, in particular to a method for efficiently regenerating larch and establishing an in-vitro cutting orchard.

Background

Larch leaf (A. falls into)Larix) The deciduous tree is the only deciduous tree in Pinaceae (Pinaceae), contains 18 varieties, is mainly distributed in mountainous areas in the temperate zone of the northern hemisphere, plains in the cold temperate zone and high mountain climatic areas, is the dominant species in the polar frozen soil zone, is extremely cold-resistant and pleasant, and is an important tree species for forest renewal and barren mountain afforestation. Larch has the advantages of fast forest establishment, tall and straight tree posture, strong adaptability and barren resistance, and is valued for forestry production and landscape application. The wood has corrosion resistance, pressure resistance and long length of monomolecular fiber, can be used for obtaining large wood, and is a high-quality buildingBuilding materials are basic raw materials of high-grade printing paper. Taxol and arabinogalactan extracted from Larix Gmelini are widely used in medicine, food and other industries.

The current tending of the artificial forest of the larch is mainly carried out in a cutting mode, but the vitality of cutting seedlings is reduced along with the age of a stock plant of the scion, and the cutting seedlings need to be periodically dried and rejuvenated to keep the vitality of the scion; partial rejuvenation approaches are grafted to sapling stocks through big tree new branches to achieve the effect of rejuvenating good seedlings. The operation is complex, time-consuming and labor-consuming, the propagation coefficient is low, and the method is limited by seasons.

The larch has high heterozygosity, the variation of sexual propagation offspring is large, the breeding period is long, the effect is slow, and the offspring characters can not be directionally improved. Because the in vitro regeneration has the characteristics of high speed, short period, high efficiency, stability and the like, the genetic improvement and clonal propagation of the larch are always paid more attention by forestry decision departments of various countries and are particularly concerned with forest breeding workers and forest operators (Wulijun. conifer in vitro culture research progress. Fujian agricultural bulletin, 2006, 21(4): 415-. However, there has been no major breakthrough in tissue culture of larch, and researchers have searched for it in recent years, for example, yanli uses mature zygote embryo as material, and studies on in vitro regeneration and endogenous hormone content change of hybrid larch and long white larch (yanli. establishment of long white larch in vitro regeneration system, shandong forestry science and technology, 2019(02): 17-22.; yanli. establishment of high efficiency in vitro regeneration system of larch and endogenous hormone change study, northeast forestry university, 2016). Based on comparative study on the regeneration performance of mature zygotic embryo and hypocotyl of Pinus massoniana (Pinus massoniana Lamb.) on Zhengwulin, the hypocotyl of Pinus massoniana is taken as an explant, various factors influencing the isolated regeneration of the hypocotyl of Pinus massoniana are studied, and an isolated regeneration system of the hypocotyl of Pinus massoniana is established (Zhengwulin, establishment of a high-efficiency isolated regeneration system of Pinus massoniana and larch, Jiangxi agriculture university, 2014.); the results of extensive subculture conducted on the effect of various factors on the induction of axillary bud differentiation, and investigated in vitro culture of the young stem of Japanese Larix Gmelini, etc., revealed that the basic culture medium was optimized for ZN and SH, and that the cytokinin was optimized for KT and then 2ip (Pentagon Gmelini, Chengning, Zhuihua, Ropaxia, Sun Xiaomei, isolated culture and plant regeneration of Japanese Larix Gmelini, 2002(01): 21-23).

At present, the zygotic embryo or hypocotyl is mainly used as an explant in the research, the source of the explant is easy to be limited, the regeneration effect needs to be improved, the practicability is not strong enough, and the explant cannot be popularized and applied. From this, the applicant has previously found that a large number of regenerated plantlets of larch can be obtained in a short time by using needle leaves of larch which are thick as starting materials and producing a large number of adventitious buds by culturing, the phenotype of the plantlets is highly consistent, and the emergence of the plantlets is not limited by seasons (CN 2019109073388). There is still a need to find more efficient regeneration systems and for the establishment of cutting orchards.

Disclosure of Invention

According to the invention, the larch bud point is taken as an explant to carry out in vitro regeneration research, the germination and the extension of the fixed bud point are induced, a fast extension plant is obtained, the generation of the fixed bud point is further efficiently induced, and a circulating culture method which is not limited by seasons and can be fast regenerated is provided for the market. The method can be used for establishing an efficient in-vitro cutting orchard system, the single circulation is 8 weeks, the propagation coefficient is 7, the number of the sprouts is increased in a geometric series, and high-activity multiple young branches are produced annually.

Therefore, the invention provides a method for efficiently regenerating larch in vitro, which comprises the following steps:

(1) inoculating the aseptic seedling of the larch to DCR +0.4-0.8 mg/L6-Benzylaminopurine (BA) +0.04-0.08 mg/L naphthylacetic acid (NAA), culturing for 1-3 weeks to obtain a semi-lignified elongated seedling of the larch with a small number of bud points, and cutting off the bud points and stem sections of the attached parts of the bud points to be used as an initial explant;

(2) inoculating the stem segment obtained in the step (1) on a DCR culture medium which additionally contains 0.1-3mg/LBA, 0.01-0.3 mg/LNAA and 0-4g/L Activated Carbon (AC), and promoting the sprouting and the primary differentiation of the fixed bud point after culturing for 2 weeks; preferably, 0.5-2 mg/LBA, 0.05-0.2 mg/L NAA, 1-3 g/L AC are added.

(3) Inoculating the material obtained in the step (2) into a DCR culture medium containing 0.5-5mg/L indolebutyric acid (IBA), and culturing for 2-4 weeks, and then, extending the stem of the larch; preferably, the culture conditions are illuminated (60. mu. mol ∙ m) ^-2 ∙s ^-1 ) Culturing for 16 h/d at 25 + -2 deg.C. Preferably, 1-4 mg/L IBA is added to obtain the best elongation effect.

(4) Transferring the young larch elongated seedling obtained in the step (3) to a DCR culture medium containing 0.5-5mg/LIBA and 0.1-0.8mg/L Ethephon (ETH) for culturing for 2-4 weeks, and inducing isolated axillary buds with clear forms; preferably, the cultivation is carried out by light (60. mu. mol ∙ m) ^-2 ∙s ^-1 ) 16 h/d, and the culture temperature is 25 +/-2 ℃. Preferably, DCR +2-4mg/L IBA +0.1-0.5mg/L ETH is added.

(5) Cutting off the bud points obtained in the step (4) and the attached stem sections thereof, and repeating the cyclic culture of the steps (3) to (4) until elongated buds can be obtained;

(6) cutting the elongated bud seedlings obtained in the step (5), transferring the cut elongated bud seedlings into a DCR culture medium which additionally contains 0.5-5mg/L IBA, 25-200mg/L Phloroglucinol (PG) and 0-2g/L AC, and inducing regeneration plant seedlings with adventitious roots and high lignification after culturing for 4-8 weeks; preferably, the cultivation is carried out under light (60. mu. mol ∙ m) ^-2 ∙s ^-1 ) 16 h/d, and the culture temperature is 25 +/-2 ℃. Preferably, the rooting medium formula is DCR +2-4mg/L IBA +50-150 mg/L PG.

Among them, the following should be mentioned: firstly, the early-stage exploration experiment shows that the addition of the activated carbon is beneficial to the germination of bud points and the extension of new germinated leaves, so that the activated carbon is added in the culture in the steps (2) and (3) to obtain better effect. Secondly, ethylene is a rarely applied hormone in tissue culture, and has a well-known function of promoting maturation and aging. A small number of reports mainly focus on the induction of adventitious roots by ethylene, promotion of root absorption, and the like. At present, no report of ethephon applied to in vitro regeneration of larch exists. However, the inventor of the present invention has tried to hope that the rooting can be promoted by adding ethephon (ethephon is a synthetic hormone, and is hydrolyzed in a culture set to generate ethylene) at the beginning, but in subsequent experiments, it has been unexpectedly found that ethephon has little effect on the induction of rooting but has a significant effect on the induction of adventitious bud in the in vitro regeneration of the larch. Thus, ethephon is added to the medium in step (4). Thirdly, the formula of each hormone is obtained by research and grope, and the obtained effect is obvious.

In a specific embodiment, after the bud point obtained in the step (4) is cut off a morphological lower leaf, the steps (2) to (3) are repeated to increase the number of the obtained regenerated seedlings.

Preferably, the method further comprises the following steps:

(7) the rooted seedling obtained in the step (6) is transferred to 28 +/-2 ℃ together with a culture medium, a culture bottle and the like to be irradiated with light (80 mu mol ∙ m) ^-2 ∙s ^-1 ) After carrying out tolerant culture for 1-2 weeks under the culture condition of 16 h/d, opening the cover of a culture bottle, injecting 10-20 ml of sterile water or deionized water, and hardening seedlings for 5-8 days;

more preferably, the method further comprises the following steps:

(8) transplanting the rooted plantlets into a matrix of peat, vegetable garden soil and perlite (3: 6: 1), and growing well in a greenhouse with natural illumination at 28 +/-2 ℃ to obtain larch regeneration plants consistent with the naturally grown plantlets.

Preferably, sucrose 30 g/L, inositol 0.2 g/L (DCR medium refers to salt medium, sucrose and inositol are additionally added), agar powder 5 g/L, and pH value of the medium is adjusted to 5.8. The culture conditions are 25 + -2 deg.C, and light culture (50-70 μmol ∙ m) ^-2 ∙s ^-1 ) 14-18 h/d, preferably culturing at 25 + -2 deg.C under illumination at 60 μmol ∙ m ^-2 ∙s ^- ¹ 16 h/d。

The invention also provides a method for establishing the larch in-vitro cutting orchard, which is characterized in that a regenerated plant obtained by the larch high-efficiency in-vitro regeneration method is utilized; and collecting the branches of the regenerated plants as cuttings to establish a cutting garden for cutting forestation in the next year (equivalent to a seed garden, except that the branches replace seeds).

And the establishment of the cutting orchard system can select the regeneration seedlings which do not need to be subjected to the rooting step. The tissue culture seedling of the larch can not reach a certain lignification degree and is applied to cuttage generally, and the regenerated seedling obtained by the step aggregation before rooting by the method reaches the specification of the cuttage seedling. Certainly, the regenerated seedlings after rooting can be adopted to establish a cutting orchard system.

Through research and exploration, including experimental researches on processing steps, hormone formulas and the like, the method finally and successfully realizes efficient in-vitro regeneration by taking larch bud points as explants, so that the method can be used for establishing an efficient in-vitro cutting orchard system.

Drawings

FIG. 1 Larix Gmelini semi-lignified material is harvested for bud spots.

FIG. 2 shows the sprouting of larch sprouts.

FIG. 3 shows the sprouting of larch's fixed shoots.

FIG. 4 shows rapid elongation of the larch adventitious bud.

FIG. 5 shows growth of larch with ethephon added.

Fig. 6 shows the growth of larch without ethephon.

FIG. 7 shows the growth of the subsequent bud of the larch plant with ethephon added.

FIG. 8 cut the fixed bud to induce the rooting of larch.

FIG. 9 shows a transplanted seedling of Larix Gmelini.

FIG. 10 shows the induction of adventitious buds by addition of ethephon at the same concentration.

Detailed Description

The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.

First, preparation of explant Material

(1) The aseptic seedling of the larch is transferred to DCR +0.6mg/L BA +0.06 mg/L NAA, and cultured for 2 weeks to obtain a semi-lignified larch elongation seedling with a small number of bud points (figure 1), and the bud points and the stem sections of the attached parts of the bud points are cut off to be used as an initial explant.

Wherein, 30 g/L of sucrose, 0.2 g/L of inositol and 5 g/L of agar powder are added into the DCR culture medium, and the pH value of the culture medium is adjusted to 5.8. The culture conditions are 25 + -2 deg.C, and the culture is performed under illumination (60 μmol ∙ m) ^-2 ∙s ^-1 ) 16 h/d. The culture medium and culture conditions of the subsequent steps are the same and are not repeated.

Preparation of larch fast elongation material

(2) Larch bud points promote germination. The stem sections from the excised explants were inoculated into DCR +0.3mg/L BA +0.03 mg/L NAA +2 g/LAC. After 2-3 weeks of culture, the larch fixed bud germination is promoted to promote the fixed bud point germination (figure 2) and the primary differentiation (figure 3).

The early-stage exploration experiment shows that the addition of the active carbon is beneficial to the germination of bud points and the extension of new germinated leaves; compared with the conditions of 1g/L, 2g/L and 4g/L, the 2g/L active carbon has better effect on the growth of larch, and the specific conditions are shown in Table 1.

TABLE 1 Effect of AC addition on larch sprouting

AC	Inducing sprouting of fixed buds	Mean time to sprout
			0g/L	The leaves are dark green after continuous culture, and the bud points are not easy to sprout	Can not be erupted
1g/L	Slow sprouting of bud and small leaf	21.7 days
			2g/L	The bud points sprout faster and the leaves stretch	11.3 days
4g/L	The regenerated seedlings lose green color and grow poorly	29.7 days

(3) And (3) inoculating the bud-fixing seedlings obtained in the step (2) into DCR culture media with different concentrations of plant growth regulating substance combinations. After 2-4 weeks of culture, larch plantlets elongated rapidly (fig. 4). The experimental data are shown in Table 2, in which it can be seen that the elongation effect is best when 2g/L AC is added with 3mg/L IBA.

TABLE 2 Effect of different concentrations of plant growth regulating substance combinations on shoot apical axillary buds

Serial number	Plant growth regulating substance combination (mg/L)	Axillary bud germination rate (%)	Axillary bud elongation (%)	Average adventitious bud elongation (cm)
					1	6-BA 0.5+NAA 0.05	62.7±0.88 ^e	20.7±0.79 ^d	2.5±0.06 ^f
2	BA 0.5+NAA0.05+AC 2000	87.3±1.15 ^b	75.0±0.13 ^b	3.9±0.03 ^e
					3	BA 0.3+NAA 0.03+AC 2000	92.8±0.44 ^a	86.1±0.43 ^ab	4.3±0.06 ^d
4	6-BA 0.1+NAA 0.01	75.47±0.30 ^d	60.1±10.61 ^c	3.7±0.03 ^e
					5	6-BA 3	0.0	0	0
6	IBA 3	92.6±0.87 ^a	85.6±0.75 ^ab	7.2±0.03 ^a
					7	NAA 3	82.1±1.24 ^c	74.4±0.64 ^b	5.0±0.07 ^c
8	IBA 3+AC 2000	93.8±0.6 ^a	90.5±0.92 ^a	7.0±0.1 ^b

Third, the bud point of larch is induced and cultured

(4) And (3) transferring the young larch elongated seedlings obtained in the step (3) into DCR +3mg/L IBA +0.2mg/L ETH to culture for 3-4 weeks, inducing in-vitro axillary buds with clear shapes (figure 6), and increasing more than 7 fixed bud points (the specific proliferation effect is shown in figure 10). No obvious multiple shoots were produced under culture conditions without ethephon addition (fig. 5).

In the previous experiment, ETH content of 0mg/L, 0.05mg/L, 0.1mg/L, 0.2mg/L, 0.4mg/L, 0.6mg/L and 0.8mg/L are selected, and the effect of 0.2mg/L is best in combination (see figure 10).

(5) And (5) repeating the step (4) to transfer the sterile seedlings obtained in the step (4), namely performing subculture to obtain thickened seedlings (figure 6).

In the experiment, after morphological lower leaves are cut off from the bud point obtained in the step (4), the steps (2) to (3) are repeated, so that semi-lignified elongation seedlings can be further obtained, and the number of regeneration seedlings can be increased.

Fourth, transplanting of roots

(7) Transferring the elongated seedlings obtained in the step (5) into a DCR culture medium or 1/2DCR culture medium attached with IBA, NAA, AC and PG for 4-8 weeks, and inducing roots. The hormone formulations and the corresponding results are shown in Table 2, where DCR +0.5-5 mg/L IBA +25-200mg/LPG +0-2g/L AC are known to be suitable, while the preferred formulation is DCR +3mg/L IBA +100mg/L PG (FIG. 8).

For larch, a high adventitious bud induction rate is important than the onset time.

TABLE 2 minimal medium Effect of different concentrations of NAA, IBA, AC, PG combinations on rooting

(8) Transplanting the rooted plantlets in the step (7) into a matrix of peat, vegetable garden soil and perlite (3: 6: 1), and growing well in a greenhouse with natural illumination at 28 +/-2 ℃ to form larch regeneration plants consistent with the naturally grown plantlets (figure 9).

Claims

1. A method for obtaining high-efficiency in-vitro regenerated seedlings of larch is characterized by comprising the following steps:

(1) culturing aseptic seedling of larch to obtain semi-lignified elongated larch seedling with few bud points, and cutting off the bud points and stem segments of the attached parts of the bud points as initial explants;

(2) inoculating the stem segment obtained in the step (1) on a DCR culture medium which additionally contains 0.1-3mg/LBA, 0.01-0.3 mg/LNAA and 0-4g/L of active carbon, promoting the sprouting and the primary differentiation of the bud points of the fixed buds, and culturing for 1-3 weeks by illumination;

(3) inoculating the material obtained in the step (2) into a DCR culture medium with 0.5-5mg/LIBA, and after 2-4 weeks of illumination culture, extending the stem section of the larch;

(4) transferring the young larch elongated seedlings obtained in the step (3) to a DCR culture medium containing 0.5-5mg/LIBA and 0.1-0.8mg/L ethephon, and culturing for 2-4 weeks by illumination to induce isolated axillary buds with clear shapes;

(5) and (5) cutting off the bud points obtained in the step (4) and the attached stem sections, and repeating the cyclic culture of the steps (3) to (4) until elongated buds are obtained, so as to obtain regenerated seedlings.

2. The method for regenerating larch in vitro with high efficiency as claimed in claim 1, further comprising the steps of:

(6) and (3) cutting the elongated bud seedlings obtained in the step (5), transferring the cut elongated bud seedlings into a DCR culture medium which additionally contains 0.5-5mg/L IBA, 25-200mg/L phloroglucinol and 0-2g/L activated carbon, and inducing regeneration plant seedlings with adventitious roots and high lignification through illumination culture.

3. The method for regenerating larch in vitro with high efficiency as claimed in claim 1, wherein the specific operation of the step (1) is: transferring the aseptic seedling of the larch to a DCR culture medium added with 0.4-0.8mg/LBA and 0.04-0.08 mg/LNAA, and culturing to obtain the semi-lignified larch elongated seedling with a small number of buds.

4. The method for regenerating larix dahuricae in vitro efficiently as claimed in claim 1, wherein in the step (2), 0.5-2 mg/LBA, 0.05-0.2 mg/L NAA, 2g/L activated carbon are added to DCR medium; in the step (3), 1-4 mg/L IBA is added into a DCR culture medium; in the step (4), 2-4mg/L IBA +0.1-0.5mg/L ethephon is added into the DCR culture medium; in the step (6), 3mg/L IBA and 100mg/L phloroglucinol are added into the DCR culture medium.

5. The method for regenerating larch in vitro with high efficiency as claimed in any one of claims 1 to 4, further comprising the steps of:

(7) the rooted seedling obtained in the step (6) is transferred to a culture medium and a culture bottle together with the culture medium and the culture bottle at the temperature of 28 +/-2 ℃ and the illumination of 80 mu mol ∙ m ^-2 ∙s ^-1 And after carrying out tolerance culture for 1-2 weeks under the culture condition of 16 h/d, opening the cover of a culture bottle, injecting 10-20 ml of sterile water or deionized water, and hardening seedlings for 5-8 days for transplanting.

6. The method for regenerating larix gmelinii in vitro according to claim 5, further comprising the steps of:

(8) transplanting the rooted plantlets refined in the step (7) into a medium with the mass ratio of peat to vegetable garden soil to perlite being 3: 6: 1, and growing in a greenhouse with the natural illumination at the temperature of 28 +/-2 ℃.

7. The method for regenerating larix gmelinii in vitro according to claim 5, further comprising the steps of:

and (3) cutting off morphological lower-end leaves of the bud points obtained in the step (4), and repeating the steps (2) to (3) to increase the number of the obtained regenerated seedlings.

8. The method for regenerating larix dahurica in vitro according to claim 5, wherein the light culture condition is 50-70 μmol ∙ m ^-2 ∙s ^-1 14 to 18 h/d, preferably 60. mu. mol ∙ m ^-2 ∙s ^-1 16 h/d。

9. The method for regenerating larch in vitro with high efficiency as claimed in claim 5, wherein the culture temperature of each step is 25 ± 2 ℃.

10. A method for establishing an in vitro larch cutting orchard, which is characterized in that a regenerated seedling obtained by the method for obtaining the larch high-efficiency in vitro regeneration bacteria according to any one of claims 1 to 9 is utilized; and collecting the branches of the regenerated seedlings as cuttings to establish a cutting orchard.