CN115088618B

CN115088618B - Method for efficiently regenerating larch and establishing in-vitro cutting orchard

Info

Publication number: CN115088618B
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: 2023-03-07
Anticipated expiration: 2042-07-11
Also published as: CN115088618A

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 (Larix gmelinii)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. The fallen She Songcheng forest is fast, tall and straight in tree posture, strong in adaptability and barren-resistant, and is valued for forestry production and landscape application. The wood has corrosion resistance, pressure resistance and long monomolecular fiber length, can be used for obtaining large wood, and is a high-quality building material and a basic raw material 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 the clonal propagation of the larch are always paid more attention by decision departments of forestry of various countries and are particularly concerned by forest breeding workers and forest operators (Wu Lijun. The research progress of the in vitro culture of the conifer, fujian agricultural science, 2006, 21 (4): 415-419; wu Kexian, li Wei, xu Miaozhen, and the like. The research on the tissue culture of the larch in Changbai province, forestry science, 1996, 32 (2): 125-133; zhang Suqing. The research progress of the tissue culture of the larch species, lianning forestry, 2015, 56-58. However, there has been no major breakthrough in tissue culture of larch, and researchers have searched for it in recent years, for example, zhang Li uses mature zygote embryo as material, and research on in vitro regeneration and endogenous hormone content change of hybrid larch and long white larch (Zhang Li. Establishment of long white larch in vitro regeneration system, shandong forestry science and technology, 2019 (02): 17-22.; zhang Li. Establishment of larch high efficiency in vitro regeneration system and endogenous hormone change research, northeast forestry university, 2016). Zheng Wulin on the basis of relatively researching the regeneration performance of mature zygotic embryo and hypocotyl of Pinus massoniana (Pinus massoniana lamb.), takes the hypocotyl of Pinus massoniana as an explant, researches various factors influencing the in vitro regeneration of the hypocotyl of Pinus massoniana, and establishes an in vitro regeneration system of the hypocotyl of Pinus massoniana (Zheng Wulin. Establishment of the in vitro regeneration system of Pinus massoniana and Larix Gmelini, jiangxi agriculture university, 2014.); zhou Pengfang and others use young stems of Japanese larch as explants for in vitro culture, and research on the influence of various factors on the induction of axillary bud differentiation and propagation subculture is carried out, and the results show that the basic culture medium is most suitable for ZN and SH, the cytokinin is most suitable for KT and 2ip (Zhou Pengfang, chen Changqing, zhang Huihua, luo Fengxia and Sun Xiaomei).

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 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 progression, and the 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) Transferring the aseptic seedling of the larch to DCR +0.4-0.8 mg/L6-Benzylaminopurine (BA) +0.04-0.08mg/L naphthylacetic acid (NAA), culturing for 1-3 weeks to obtain a semi-lignified larch elongated seedling with a small number of bud points, and cutting 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 section obtained in the step (1) on a DCR culture medium containing 0.1-3mg/LBA,0.01-0.3 mg/LNAA and 0-4g/L Activated Carbon (AC), and after culturing for 2 weeks, promoting the sprouting and the primary differentiation of a fixed bud point; 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 attached with 0.5-5mg/L indolebutyric acid (IBA), and after culturing for 2-4 weeks, extending the stem of the larch; preferably, the culture conditions are light (60. Mu. Mol ∙ m) ^-2 ∙s ^-1 ) 16h/d, the culture temperature is 25 +/-2 ℃. Preferably, 1-4mg/L IBA is added, and the elongation effect is the best.

(4) Transferring the young larch elongation seedlings obtained in the step (3) to a DCR culture medium containing 0.5-5mg/LIBA and 0.1-0.8mg/L Ethephon (ETH) for cultureAfter 2-4 weeks of cultivation, inducing in vitro axillary buds with clear shapes; preferably, the culture is performed by light (60. Mu. Mol ∙ m) ^-2 ∙s ^-1 ) 16h/d, culture temperature 25 + -2 deg.C. 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 culture is performed by light (60. Mu. Mol ∙ m) ^-2 ∙s ^-1 ) 16h/d, and the culture temperature is 25 +/-2 ℃. Preferably, the formulation of the rooting medium 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 little applied hormone in tissue culture, and the well-known function is to promote maturation and aging. A small number of reports mainly focus on inducing the generation of adventitious roots by ethylene, promoting root absorption and the like. At present, no report of ethephon applied to in vitro regeneration of larch exists. The inventor initially tries ethephon to hopefully promote rooting by adding ethephon (ethephon is synthetic hormone and is hydrolyzed in a culture set to generate ethylene), but unexpectedly finds that ethephon has little effect on rooting induction but has obvious effect on bud fixing induction in the in vitro regeneration of the larch in subsequent experiments. Therefore, ethephon is added to the medium in the step (4). Thirdly, the formula of each hormone is obtained by research and exploration, and the obtained effect is obvious.

In a specific embodiment, after morphological lower leaves are cut from the bud point obtained in the step (4), 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 for illumination (80 mu mol ∙ m) ^-2 ∙s ^-1 ) Carrying out tolerance culture for 1-2 weeks under the culture condition of 16h/d, opening the cover of the culture bottle, injecting 10-20ml sterile water or deionized water, and hardening the 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 regenerated plants consistent with the naturally grown plantlets.

Preferably, all the culture media are added with sucrose 30 g/L, inositol 0.2 g/L (DCR culture medium refers to salt culture medium, sucrose and inositol are additionally added), agar powder 5 g/L, and the pH value of the culture 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 of 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

Figure 1 larch semi-lignified material obtains bud points.

FIG. 2 shows the sprouting of larch sprouts.

FIG. 3 shows the germination of larch sprouts.

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

Fig. 5 shows larch growth with ethephon added.

Fig. 6 shows the growth of larch without ethephon.

FIG. 7 shows the growth of the subsequent shoot point of the Larix Gmelini 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 the 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.

1. Preparation of explant Material

(1) The aseptic seedling of the larch is transferred to DCR +0.6mg/L BA +0.06 mg/L NAA, cultured for 2 weeks to obtain the semi-lignified larch elongation seedling with a few buds (figure 1), and the buds and the stem section of the attachment part are cut off to be used as an initial explant.

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

2. Preparation of larch rapid elongation material

(2) The larch bud points promote germination. The excised explants were inoculated into stem sections that were inoculated into DCR +0.3mg/L BA +0.03 mg/L NAA +2g/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	The bud is slow to sprout and the leaf is small	21.7 days
			2g/L	Fast sprout and spread leaves	11.3 days
4g/L	The regenerated seedlings lose green and grow badly	29.7 days

(3) And (3) inoculating the final-budding 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, the seedlings of She Songxiao elongated rapidly (FIG. 4). The experimental data are shown in Table 2, wherein the elongation effect of 2g/L AC is best when 3mg/L IBA is added.

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

3. Larch bud point induction culture

(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 isolated axillary buds with clear shapes (shown in figure 5), and increasing over 7 fixed bud points (shown in figure 10 for specific proliferation effect). No obvious multiple shoots were produced under culture conditions without ethephon addition (FIG. 6).

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 7).

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.

4. Rooting and transplanting

(7) Transferring the elongated seedlings obtained in the step (5) into a DCR culture medium attached with IBA, NAA, AC and PG or 1/2DCR to culture 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 is known to be suitable, and the preferred formulation is DCR +3mg/L IBA +100mg/L PG (FIG. 8).

For colony She Songlai, high adventitious bud induction rate is important over the onset time.

TABLE 2 Effect of minimal Medium addition 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 larch in-vitro regenerated seedlings is characterized by comprising the following steps:

(1) Culturing the aseptic seedling of the larch to obtain a semi-lignified larch elongation seedling with a small number of bud points, and cutting off the bud points and stem sections of the attachment parts of the bud points as an initial explant;

(2) Inoculating the stem section obtained in the step (1) on a DCR culture medium which additionally contains 0.1-3mg/L BA,0.01-0.3mg/L NAA and 1-4g/L active carbon, promoting the germination and the primary differentiation of a fixed bud point, 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/L IBA, and after 2-4 weeks of illumination culture, extending the stem segments of the larch;

(4) Transferring the young larch elongated seedlings obtained in the step (3) to a DCR culture medium containing 0.5-5mg/L IBA 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 (4) 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 obtaining larch regenerated seedlings in vitro as set forth in claim 1, further comprising the steps of:

(6) Cutting the elongated bud seedlings obtained in the step (5), transferring the cut elongated bud seedlings into a DCR culture medium attached with 3-5mg/L IBA, 25-200mg/L phloroglucinol and 0-2g/L activated carbon, and inducing regenerated plant seedlings by illumination culture.

3. The method for obtaining larch regenerated seedlings in vitro as claimed in claim 1, wherein the specific operation of the step (1) is as follows: transferring the aseptic seedling of the larch into a DCR culture medium added with 0.4-0.8mg/L BA and 0.04-0.08mg/L NAA, and culturing to obtain the semi-lignified elongated seedling of the larch with a small number of bud points.

4. The method for obtaining the larch regenerated plantlet in vitro according to claim 2, wherein in the step (2), 0.5-2mg/L BA,0.05-0.2mg/L NAA and 2g/L activated carbon are added into the DCR culture medium; in the step (3), 1-4mg/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 obtaining larch regenerated seedlings in vitro as set forth in claim 2, further comprising the steps of:

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

6. The method for obtaining larch regenerated plantlets in vitro as claimed in claim 5, further comprising the steps of:

(8) Transplanting the rooted plantlets hardened 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 natural illumination at 28 +/-2 ℃.

7. The method for obtaining larch regenerated seedlings in vitro as set forth in 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 larch regenerated seedling in vitro as set forth in any one of claims 1 to 4The method for obtaining (1) is characterized in that the culture conditions under light irradiation are 50 to 70. Mu. Mol ∙ m ^-2 ∙s ^-1 ，14-18h/d。

9. The method for obtaining larch regenerated plantlets in vitro as set forth in claim 8, wherein the light culture conditions are 60 μmol ∙ m ^-2 ∙s ^-1 ，16h/d。

10. The method for obtaining an in vitro regenerated plantlet of larch as claimed in claim 5, wherein the culture temperature of each step is 25 ± 2 ℃.

11. A method for establishing an in vitro cutting orchard for larch, characterized in that a regenerated seedling obtained by the method for obtaining an in vitro regenerated seedling for larch according to any one of claims 1 to 10 is used; and collecting the branches of the regenerated seedlings as cuttings to establish a cutting orchard.