阅读说明：本技术 一种落叶松针叶高效离体再生方法 (High-efficiency in-vitro regeneration method for larch needles ) 是由 朱木兰 郑珂媛 王可佳 闫学彤 于 2019-09-24 设计创作，主要内容包括：本发明公开一种落叶松针叶高效离体再生方法,其以落叶松肥厚的针叶为起始材料,经培养产生大量的不定芽,能够在短时间内获得大量落叶松再生植株,植株表型一致性高,而且这种种苗的发生不受季节限制。可以以此再生系统作为转基因的受体系统进一步制备转基因植株,使之既能保持亲本的遗传表型,又具有所转入的外源目的基因的性状。通过建立以落叶松针叶为起始外植体的离体再生方法,对开展以落叶松为代表的针叶树遗传改良、新品种(系)培育和优质种苗繁殖等研究,对林业经济可持续发展和缩小林业基础研究领域与发达国家间的差距,具有深远意义。(The invention discloses a method for regenerating needle leaves of larch in vitro efficiently, which takes the needle leaves with the thick larch as a starting material, generates a large amount of adventitious buds through culture, can obtain a large amount of regeneration plants of the larch in a short time, has high plant phenotype consistency, and is not limited by seasons. The regeneration system can be used as a transgenic receptor system to further prepare transgenic plants, so that the transgenic plants can not only keep the genetic phenotype of parents, but also have the characters of the transferred exogenous target genes. By establishing the in vitro regeneration method taking the larch needles as the initial explant, the method has profound significance for developing researches on genetic improvement, new variety (line) cultivation, high-quality seedling propagation and the like of the needle trees represented by the larch, and for sustainable development of forestry economy and shortening the gap between the basic research field of forestry and developed countries.)

1. A method for regenerating the needle leaf of larch in vitro features that the germinated stem of larch is inoculated to MS culture medium containing 0.5-3mg/l BA, and the needle leaf is cultured to obtain thick needle leaf as initial explant.

2. The method for the efficient ex vivo regeneration of the coniferous needle of larch as claimed in claim 1, wherein the shoot segments with buds are cultured on the medium for 12-30 days, most preferably 14-28 days.

3. The method for regenerating larix dahurica needle in vitro according to claim 1, wherein the induction stage culture medium of callus of explant, the differentiation induction stage culture medium of adventitious bud, and/or the basal culture medium of rooting culture medium is LYS culture medium, which is based on DCR culture medium and added with L-phenylalanine, tryptophan, L-serine.

4. The method for regenerating larix needle in vitro efficiently as claimed in claim 3, wherein the three amino acids L-phenylalanine, tryptophan and L-serine are added in amounts of 0.3-0.8mg/L, 0.2-0.4mg/L and 0.1-0.3mg/L, more preferably 0.4-0.6mg/L, 0.25-0.35mg/L and 0.15-0.25mg/L, and most preferably 0.5mg/L, 0.3mg/L and 0.2 mg/L.

5. The method for regenerating the needle leaf of larix dahurica in vitro according to claim 3, wherein the hormone formulation added to the culture medium for the induction stage of the callus of the explant is 0.5-3mg/l NAA and 0.1-0.5 mg/l BA, and/or the hormone formulation added to the culture medium for the differentiation induction stage of the adventitious bud is 0.5-3mg/l NAA, 0.1-0.5 mg/l BA and 0.001-0.01mg/l TDZ.

6. The method for regenerating the needle of larix dahurica according to any of claims 3 to 5, wherein the hormone added to the culture medium used in the rooting culture stage is 0.5-5 mg/l IBA.

7. The method for regenerating the needle leaf of the larch as claimed in any one of claims 3 to 5, wherein the differentiation induction of the adventitious bud is performed in stages by first inoculating the explant into LYS medium containing 0.5-3mg/l NAA, 0.1-0.5 mg/l BA to induce callus at the needle wound site; transferring the callus into LYS culture medium containing 0.5-3mg/l NAA, 0.1-0.5 mg/l BA, and 0.001-0.01mg/l TDZ, and continuously culturing until adventitious bud with clear shape is induced; then transferring the adventitious bud into LYS culture medium containing 0.5-3mg/l BA, 0.1-0.5 mg/l NAA, 150-350mg/l banana juice to culture to obtain bud cluster; further culturing on LYS medium containing 0.05-0.3 mg/lBA, 0.01-0.05 mg/l NAA, 150-350mg/l banana juice to elongate the bud.

8. The method for regenerating larix dahurica needles in vitro according to any one of claims 3 to 5, wherein the illumination condition is 16h/d, the culture temperature is 25 ± 2 ℃, and the illumination intensity is preferably 50-70 μmol.m-2.s-1, more preferably 60 μmol.m-2.s-1, in the differentiation induction and rooting culture stage of adventitious buds.

9. The method for regenerating larix needle leaves efficiently in vitro according to any one of claims 3 to 5, further comprising the step of transplanting the rooted shoots after the tolerant culture to obtain larix regenerated plants.

10. The method for the efficient in-vitro regeneration of the larix needle as claimed in claim 9, wherein the tolerant culture is carried out by transferring the rooted shoots together with the culture medium and the culture flask into a culture condition of 27-20 ± 2 ℃, 16h/d of illumination and 70-90 μmol.m-2.s-1 of illumination intensity for 1-2 weeks, then opening the cover of the culture flask, injecting 10-20ml of sterile water or deionized water, and hardening the shoots for 5-8 days; transplanting the rooted seedlings to peat, vegetable garden soil and perlite, preferably a substrate with the weight ratio of 2-4: 4-8: 0.5-2, and growing in a greenhouse with the natural illumination at the temperature of 28 +/-2 ℃.

Technical Field

The invention relates to the technical field of plant regeneration and rapidness, in particular to in-vitro regeneration of larch.

Background

Larix gmelinii (Larix Mill) belongs to Larix of Laticaceae (Pinaceae) and is a kind of larch tree. The fertilizer has the advantages of wide distribution, quick forest formation, strong adaptability and less plant diseases and insect pests, and is emphasized and widely applied in forestry production. Meanwhile, the larch wood has the advantages of long fiber, decay resistance, compression resistance, bending resistance and the like, so the larch can be used as a high-quality building material and a basic raw material of high-grade printing paper. The larch belongs to 18 varieties, is mainly distributed in mountain areas in the temperate zone of the northern hemisphere, plains in the cold temperate zone and high mountain climatic zones, has 10 varieties in China, is mainly distributed in the mountainous areas in the northeast, the lesser Khingan mountains, the north China, the northwest and the south west, and is often a large-area pure forest or a mixed forest.

However, 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 particular attention by decision departments of forestry of various countries and forest operators (Wulijun. needle-leaved tree in vitro culture research progress [ J ]. Fujian agribunda, 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 long white larch ex vivo regeneration system [ J/OL ] Shandong forestry science, 2019(02): 17-22.; yanli high efficiency in vitro regeneration system and endogenous hormone change study [ D ] northeast forestry university, 2016). On the basis of researching the regeneration performance of mature zygote embryos and hypocotyls of Pinus massoniana (Pinus massoniana Lamb.) by comparing Zhengwulin, the hypocotyls of Pinus massoniana are taken as explants, various factors influencing the in vitro regeneration of the hypocotyls of Pinus massoniana are researched, and an in vitro regeneration system of the hypocotyls of Pinus massoniana is established (Zhengwulin, Pinus massoniana and Larix Gmelini efficient in vitro regeneration system is established [ D ]. Jiangxi agriculture university, 2014.); the method is characterized in that the entamoeba pentandra and the like are subjected to isolated culture by taking tender stems of Japanese larch as explants, the influence of various factors on the induction of axillary bud differentiation is discussed, the research of propagation and subculture is carried out, and the result shows that the basic culture medium is most suitable for ZN and SH; cytokinins are most suited as KT, followed by 2ip (congratulatory family, Chengning, Zhang Huihua, Lupinxia, Sun Xiaomei. Ex vivo culture and plant regeneration of Larix japonicus [ J ] Liaoning agriculture, 2002(01):21-23.)

At present, the zygotic embryo or hypocotyl is taken 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. Therefore, no effective regeneration system has been found, and the problem to be overcome is always the one. Therefore, it is necessary to establish a rapid propagation system to achieve the purposes of shortening the breeding cycle and directionally improving the traits.

Disclosure of Invention

In order to realize the establishment of a high-efficiency in-vitro regeneration system taking the needle leaves of the larch as the initial explant as soon as possible, the inventor researches the in-vitro regeneration of the larch and establishes a high-efficiency in-vitro regeneration method taking the needle leaves as the explant.

The invention provides a method for regenerating the needle leaf of larch in vitro efficiently, which is to inoculate the stem segment with bud on the MS culture medium which additionally contains 0.5-3mg/l BA, preferably 1-2mg/l BA, and to culture and obtain the needle leaf with the fat thickness, and the needle leaf is used as the initial explant. Further, the stem segments with buds are taken from young larch shoots.

In a preferred mode, the shoot segments are cultured on the medium for 12-30 days, most preferably 14-28 days.

The explants are important to the proposal of regeneration systems of certain plants, particularly woody plants, the inventor tries various forms of explants in the process of developing the in vitro regeneration system of the larch, for example, the needle leaves on the larch are directly taken as the explants, and experiments prove that the activity is not enough, the regeneration effect is very poor, and an effective regeneration system cannot be established. Through groping analysis, a method for enhancing the activity of the needle leaves is finally adopted, and a better regeneration effect is finally obtained. Further, the basic medium for differentiation induction and elongation using adventitious buds of the present invention is a DCR medium to which three amino acids of L-phenylalanine, tryptophan and L-serine (referred to as LYS medium in the present invention) are added. In a preferred embodiment, the three amino acids are added in an amount of 0.3-0.8mg/l, 0.2-0.4mg/l, 0.1-0.3mg/l, more preferably in an amount of 0.4-0.6mg/l, 0.25-0.35mg/l, 0.15-0.25mg/l, and most preferably in an amount of 0.5mg/l, 0.3mg/l, 0.2mg/l, respectively.

Furthermore, the hormone formula added in the culture medium of the callus induction stage of the explant is 0.5-3mg/l NAA and 0.1-0.5 mg/l BA, preferably 0.15-2.5mg/l NAA and 0.2-0.4mg/l BA.

Furthermore, the LYS medium is used for inducing differentiation of adventitious bud, and the hormone formula is 0.5-3mg/l NAA, 0.1-0.5 mg/l BA, 0.001-0.01mg/l TDZ, preferably 0.15-2.5mg/l NAA, 0.2-0.4mg/l BA, 0.004-0.006mg/l TDZ. The LYS culture medium is adopted for obtaining the bud cluster, and the added hormone formula is 0.5-3mg/l BA, 0.1-0.5 mg/l NAA and 350mg/l banana juice of 150-. In addition, the LYS culture medium is adopted in the rooting culture stage, and the hormone formula is added to 0.5-5 mg/l IBA, preferably 2-4 mg/l IBA.

The generation of adventitious buds through induced differentiation is a key link in regeneration, the differentiation conditions including the selection of a culture medium and the hormone formula need to be deeply researched to be successful, and the method is also one link in which the regeneration effect of a plurality of woody plants is poor or can not be successful. The inventor determines to add three amino acids of L-phenylalanine, tryptophan and L-serine by adding various amino acids and different combinations through a large amount of experimental exploration and matching with a hormone proportion, and the callus obtained by induced differentiation without adding amino acids is fragile and not easy to generate adventitious buds.

In a preferred embodiment, the shoot inducing differentiation step is performed in stages by first inoculating the explant into LYS medium containing 0.5-3mg/l NAA, 0.1-0.5 mg/l BA, preferably 0.15-2.5mg/l NAA, 0.3-0.4mg/l BA to induce callus at the site of the needle wound; transferring the callus into LYS culture medium containing 0.5-3mg/l NAA, 0.1-0.5 mg/l BA, 0.001-0.01mg/l TDZ, preferably 0.15-2.5mg/l NAA, 0.3-0.4mg/l BA, 0.004-0.006mg/l TDZ, and continuously culturing until adventitious bud with clear shape is induced; then transferring the adventitious bud into LYS culture medium containing 0.5-3mg/l BA, 0.1-0.5 mg/l NAA, 150-350mg/l banana juice, preferably 0.15-2.5mg/l BA, 0.3-0.4mg/l NAA, 200-300mg/l banana juice to culture to obtain bud cluster; the bud is further extended by culturing on LYS medium containing 0.05-0.3 mg/l BA, 0.01-0.05 mg/l NAA, 150-350mg/l banana juice, preferably 0.1-0.2 mg/l BA, 0.03-0.04 mg/l NAA, 200-300mg/l banana juice. The banana juice is juice squeezed from banana pulp and can be obtained by a conventional method, and the simple method is to smash the banana pulp and squeeze the juice through a filter gauze for later use.

In the differentiation induction and rooting culture process, the illumination culture is carried out for 16h/d, the culture temperature is 25 +/-2 ℃, the illumination intensity is preferably 50-70 mu mol.m-2.s-1, and the illumination intensity is more preferably 60 mu mol.m-2. s-1.

Furthermore, the method also comprises the step of transplanting the rooted seedlings after the rooted seedlings are subjected to tolerance culture to obtain larch regeneration plants. In a preferred embodiment, the tolerant culture is to transfer the rooted seedlings together with a culture medium and a culture bottle into culture conditions of 27-20 +/-2 ℃, 16h/d of illumination and 70-90 mu mol.m-2.s-1 of illumination intensity for tolerant culture for 1-2 weeks, then open the cover of the culture bottle, inject 10-20ml of sterile water or deionized water and refine the seedlings for 5-8 days.

Transplanting the rooted seedlings to peat, vegetable garden soil and perlite, preferably to a substrate in a weight ratio of 2-4: 4-8: 0.5-2, most preferably to a substrate in a weight ratio of 3: 6: 1, and growing in a greenhouse at 28 +/-2 ℃ under natural illumination.

In a more specific embodiment, the invention provides a method for regenerating larch needles in vitro with high efficiency, which is characterized in that: the method comprises the following steps:

(1) material taking: young tender larch branches in the greenhouse;

(2) and (3) disinfection: cleaning tender larch branches, sucking water on the surface of the material, sterilizing with alcohol, sterilizing with mercuric chloride, and cutting into 1-1.5cm stem with bud;

(3) inoculating the stem section obtained in the step (2) on an MS culture medium containing 0.5-3mg/l BA, preferably 1.5mg/l BA, and culturing for 2-4 weeks to obtain a thickened needle leaf which is used as a starting explant;

(4) inoculating the needle leaf obtained in the step (3) into LYS containing 0.5-3mg/l NAA, 0.1-0.5 mg/l BA, preferably 2mg/l NAA, 0.3mg/l BA, culturing for 2-4 weeks, inducing callus at the wound part of the needle leaf, culturing for 4-8 weeks, and culturing under illumination condition of 60 μmol.m-2.s-1 for 16h/d at 25 + -2 deg.C;

(5) transferring the callus obtained in the step (4) to LYS culture medium containing 0.5-3mg/l NAA, 0.1-0.5 mg/l BA, 0.001-0.01mg/l TDZ, preferably 2mg/l NAA, 0.3mg/l BA,0.005mg/l TDZ, and culturing for 3-4 weeks to induce adventitious bud with clear shape, wherein the light culture condition is 60 μmol.m-2.s-1, 16h/d, and the culture temperature is 25 + -2 ℃;

(6) transferring the adventitious bud obtained in the step (5) into an LYS culture medium containing 0.5-3mg/l BA, 0.1-0.5 mg/l NAA, 150 mg/l banana juice, preferably 2mg/l BA, 0.3mg/l NAA and 250mg/l banana juice, and culturing for 2-3 weeks to obtain a bud cluster;

(7) after the bud cluster obtained in the step (6) is transferred to an LYS culture medium containing 0.05-0.3 mg/l BA, 0.01-0.05 mg/l NAA, 150 mg/l banana juice, preferably 0.2mg/l BA, 0.03mg/l NAA and 250mg/l banana juice for culturing for 2-3 weeks, the bud is obviously elongated under the illumination condition of 60 mu mol.m-2.s-1 and 16h/d, and the culture temperature is 25 +/-2 ℃;

(8) after cutting off morphological lower-end leaves of the elongated bud obtained in the step (7), transferring the elongated bud into an LYS culture medium containing 0.5-5 mg/l IBA, preferably 3mg/l IBA, culturing for 2-4 weeks, and inducing rooting, wherein the illumination condition is 60 mu mol.m-2.s-1, 16h/d, and the culture temperature is 25 +/-2 ℃;

(9) transferring the rooted seedling obtained in the step (8), the culture medium and a culture bottle together to a culture condition of 28 +/-2 ℃, the illumination condition of 80 mu mol.m-2.s-1 and 16h/d for tolerance culture for 1-2 weeks, then opening the cover of the culture bottle, injecting sterile water or deionized water, and hardening the seedling for 5-8 days;

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

Wherein the preparation method of the larch culture medium (LYS) comprises the following steps: minimal medium was prepared according to the following table, pH5.8, sterilized at 121 ℃ for 15-25 minutes.

The in vitro regeneration method of the present invention is applicable to all varieties of larch, preferably long white larch, Japanese long white hybrid larch, and the like.

The invention discloses a method for efficiently regenerating the needle leaves of larch in vitro, which takes the needle leaves with thick larch as the starting explant material on one hand, thereby effectively building the whole regeneration system. On the other hand, in the aspect of induced differentiation, a large number of researches and screens are carried out, the problem that callus is fragile is solved by adding three specific amino acids into a culture medium, the callus is induced to generate a high rate, and a reasonable hormone formula is matched to enable the explant to generate a large number of adventitious buds through culture, and on the basis, the differentiation and the adventitious bud elongation are induced by stages, so that the differentiation and the adventitious bud elongation are more effective. The method of the present invention can produce great amount of larch seedling in short time and the seedling is not limited by season and may be produced all the year round. The obtained larch regeneration plants have high phenotype consistency, and the regeneration system can be used as a transgenic receptor system to further prepare transgenic plants, so that the transgenic plants can not only maintain the genetic phenotype of parents, but also have the characters of the transferred exogenous target genes. By establishing an in-vitro regeneration system taking larch needles as an initial explant, the method has profound significance for developing researches on genetic improvement, new variety (line) cultivation, high-quality seedling propagation and the like of the needle trees represented by the larches, and for sustainable development of forestry economy and shortening of the gap between the basic research field of forestry and developed countries. Particularly, the establishment of the high-efficiency regeneration system of the larch is pioneering and has more pioneering significance.

Drawings

FIG. 1 needle wound callus.

FIG. 2 enlarged callus.

FIG. 3 adventitious bud.

FIG. 4 shows multiple shoots.

FIG. 5 elongated slave shoots.

FIG. 6 rooted regenerated plantlets.

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.