Method for Rooting Seedling of Acacia falcataria Tissue Culture Seedling outside Culture Bottle

Abstract

The present invention discloses a method for rooting and seedling raising of Acacia falcataria tissue culture seedlings outside the bottle, which comprises the steps of: cutting the cluster buds of Acacia falcataria tissue culture seedlings with strong growth and dark green leaves, selecting single buds or cluster buds with apical buds and transferring them to a strong seedling culture medium, performing hardening after strong seedling culture, cutting them into a substrate, and culturing them in the constructed seedling raising microenvironment for 10 d to 15 d, then obtaining the Acacia falcataria seedlings with roots outside the bottle. The method of the present invention realizes the self-culturing and rooting of Acacia falcataria tissue culture seedlings outside the bottle, has a high rooting and seedling formation rate, and has developed roots, rich root hairs and strong plants. At the same time, it shortens the seedling raising cycle, simplifies the transplanting steps and management procedures, and can be widely applied to the rooting of Acacia falcataria tissue culture seedlings with different genotypes outside the bottle.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture. More specifically, the present invention relates to a method for rooting and seedling raising of Acacia falcataria tissue culture seedlings outside the bottle. Background Art

[0002] Looking at the development of micropropagation at home and abroad, plant tissue culture technology not only proves that every living cell of a plant contains all the genetic information of the plant and can develop into a complete plant under certain conditions, but also can change and regulate the development process according to people's wishes within a certain range. This technology has become an important means of factory seedling raising and has been widely used. The plant tissue culture technology system consists of many technical links. Among them, rooting and transplanting are the key links that determine whether large-scale production and application can be carried out.

[0003] Acacia falcataria, as an excellent fast-growing nitrogen-fixing tree species successfully introduced and cultivated in China, has undergone more than 20 years of genetic improvement, and has completed the selection of improved varieties at different levels such as provenance, family, and individual plants, and has successfully obtained regenerated plants through tissue culture technology. At present, under the new situation that the country pays increasing attention to ecological civilization construction and the protection, development and utilization of agricultural germplasm resources, the propagation and application of excellent ecological and economic Acacia falcataria strains and the preservation and protection of important germplasm resources also face higher requirements and challenges. In particular, the key links in the traditional Acacia falcataria tissue culture and rapid propagation technology system, namely rooting and transplanting, urgently need to be innovated and improved.

[0004] The traditional method for rooting and seedling raising of Acacia falcataria tissue culture seedlings needs to go through three stages: in-vitro rooting (6d - 14d), seedling acclimation (10d - 21d), and transplanting and seedling raising (15d - 28d). This method has the following deficiencies:

[0005] 1. The cultivation cycle is relatively long, and it takes 31d - 63d to obtain seedlings with stable growth.

[0006] 2. After rooting and seedling acclimation culture, it is necessary to wash the medium attached to the roots many times before transplanting, and this operation will cause damage and loss to the seedlings.

[0007] 3. The roots are produced in the in-vitro medium, the roots are fleshy and have few root hairs. After transplanting, the roots often cannot absorb water and nutrients normally due to being unaccustomed to the external environment, resulting in unstable transplanting survival rate and a long slow seedling stage.

[0008] 4. At each stage of the occurrence and development of adventitious roots of Acacia falcataria tissue culture seedlings, there will be contradictions in the demand for growth hormones. Auxin plays a key role in the initiation and formation stage of root primordia of Acacia falcataria. After the formation of root primordia, the continued presence of a higher concentration of auxin is not conducive to the growth and development of young roots. Therefore, it is necessary to adjust the concentration of auxin at different stages.

[0009] Therefore, it is very necessary and meaningful to improve the rooting and seedling-raising method of traditional Albizia falcataria tissue culture seedlings. Summary of the Invention

[0010] Based on this, the purpose of the present invention is to provide a method for rooting and seedling-raising of Albizia falcataria tissue culture seedlings outside the bottle. This method realizes the self-rooting of Albizia falcataria tissue culture seedlings outside the bottle, with a high rooting and seedling-forming rate, developed roots, rich root hairs, and strong plants. At the same time, it shortens the seedling-raising cycle and simplifies the transplanting steps and management procedures.

[0011] The specific technical solutions to achieve the above invention purposes are as follows:

[0012] A method for rooting and seedling-raising of Albizia falcataria tissue culture seedlings outside the bottle, comprising the following steps:

[0013] (1) Cut the cluster buds of Albizia falcataria tissue culture seedlings with strong growth and dark green leaves, select single buds or cluster buds with terminal buds and transfer them to a strong seedling culture medium, and culture for 30d - 40d; when the tissue culture seedlings are 4cm - 6cm tall, harden them off.

[0014] (2) Take out the tissue culture seedlings that have completed hardening off, gently hold the rhizome part, cut off the callus head adhering to the culture medium, and retain at least one terminal bud and the first and second lateral branches and leaves for each tissue culture seedling. Cut off all the leaves on the stem where the matrix is to be inserted to obtain the tissue culture seedlings to be cuttaged.

[0015] (3) Fill the matrix into the container for rooting and seedling-raising outside the bottle, then insert the lower end of the stem of the tissue culture seedlings to be cuttaged in step (2) into the matrix, fill the matrix and gently compact it, and pour it thoroughly with pure water.

[0016] (4) Place the container after cutting in a chassis or bottom basin with grooves, add pure water to the grooves so that the water surface in the grooves contacts the bottom of the container; cover the chassis or bottom basin with a transparent cover or plastic film to form a sealed space.

[0017] (5) Spray pure water mist into the sealed space described in step (4) every 2d - 5d, and add water to the grooves in a timely manner to keep the water surface in contact with the bottom of the container; after 10d - 15d, the Albizia falcataria seedlings with roots outside the bottle are obtained.

[0018] In some of these embodiments, the strong seedling culture medium in step (1) comprises the following components: MS medium, 0.1 mg / L to 0.2 mg / L of cytokinin 6-BA, 0.1 mg / L to 0.2 mg / L of naphthaleneacetic acid NAA, 0.1 mg / L to 0.2 mg / L of indolebutyric acid IBA, 25 g / L to 35 g / L of sucrose, 6 g / L to 8 g / L of carrageenan, with a pH of 5.6 to 6; and / or, the strong seedling culture includes: a culture temperature of 20°C to 30°C, a light intensity of 1000 lux to 2000 lux, and a daily light exposure time of 12 h to 16 h.

[0019] In some of these embodiments, the axillary buds of the Acacia falcata tissue culture seedlings in step (1) are axillary buds of tissue culture seedlings that have undergone 3 to 6 subculture cycles.

[0020] In some of these embodiments, the height of the single bud or axillary buds with apical buds in step (1) is 1.5 cm to 2.5 cm.

[0021] In some of these embodiments, the acclimatization of the tissue culture seedlings in step (1) includes the following steps: moving the tissue culture seedlings to the natural light in an outdoor shade shed and placing them for 5 d to 7 d, then injecting pure water covering the strong seedling culture medium by 1 cm to 3 cm, loosening the bottle cap and placing it for 2 d to 5 d.

[0022] In some of these embodiments, the container for rooting and raising seedlings outside the bottle in step (3) is a plug tray, a seedling-raising bag, a seedling-raising pot, or a planting basket.

[0023] In some of these embodiments, in step (3), the lower 1.5 cm to 2.5 cm of the stem of the tissue culture seedlings to be cuttaged is cuttaged into the substrate.

[0024] In some of these embodiments, the substrate in step (3) is vermiculite and quartz sand, vermiculite and perlite, peat soil and quartz sand, or peat soil and perlite with a volume ratio of 0.8 to 1.2:0.8 to 1.2, or a solid substrate with moisture-absorbing properties.

[0025] In some of these embodiments, the depth of the groove in step (4) is 4 mm to 8 mm.

[0026] In some of these embodiments, in step (5), pure water spray is sprayed into the sealed space 1 to 2 times every 2 d to 5 d.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The method for ex vitro rooting and seedling raising of Acacia falcata tissue culture seedlings of the present invention constructs a microenvironment suitable for rooting and seedling raising with appropriate temperature and humidity for the tissue culture seedlings after strong seedling hardening, and cooperates with adding appropriate concentrations of auxin and cytokinin during the strong seedling stage of the tissue culture seedlings, without adding any exogenous auxin during the rooting period. Under this concept, the method of the present invention enables the Acacia falcata tissue culture seedlings to obtain an optimized environmental condition for rooting, fully realizes self-rooting, greatly improves the average rooting and seedling formation rate, reaching more than 87.4%, and has well-developed roots and rich root hairs. Once the seedlings start ex vitro rooting, they can completely form strong plants and grow stably.

[0029] 2. The method for ex vitro rooting and seedling raising of Acacia falcata tissue culture seedlings of the present invention shortens the rooting and transplanting cycle of tissue culture seedlings from 31d - 63d to 17d - 27d, greatly shortens the seedling raising cycle, and does not require washing during the whole process of rooting and seedling raising, reduces the management frequency, avoids the damage and loss of seedlings, greatly simplifies the rooting and seedling raising procedure, and can be widely applied to the ex vitro rooting of tissue culture seedlings of different Acacia falcata genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the cluster buds of Acacia falcata tissue culture seedlings, which are the experimental materials in the present invention.

[0031] Figure 2 It is for strong seedling cultivation of the cluster buds of tissue culture seedlings.

[0032] Figure 3 It is the rootless tissue culture seedlings ready for cutting after hardening.

[0033] Figure 4 It is the growth situation of the seedlings in Example 1 of the present invention.

[0034] Figure 5 It is the root growth situation of the seedlings in Example 1 of the present invention. Among them, A is the root growth situation of the seedlings cut for 15 days; B is the root growth situation of the seedlings cut for 22 days.

[0035] Figure 6 It is the growth and rooting situation of the seedlings cut for 12 days in Example 2 of the present invention.

[0036] Figure 7 It is the growth situation of the seedlings at different stages in Example 2 of the present invention.

[0037] Figure 8 It is the seedling raising microenvironment constructed for the planting basket in Example 3 of the present invention.

[0038] Figure 9 It is the growth situation of the seedlings after cutting for 15 days in Example 3 of the present invention.

[0039] Figure 10For the survival of the transplanted rootless tissue-cultured seedlings on the 5th day and the 15th day in Comparative Example 1 of the present invention.

[0040] Figure 11 For the influence results of adding exogenous hormones during the rooting process on the ex vitro rooting and seedling raising effects of Acacia falcataria tissue-cultured seedlings in Comparative Example 2 of the present invention.

[0041] Figure 12 For the transplanting diagram of the rooted tissue-cultured seedlings by using the existing method of Acacia falcataria clone tissue culture in Comparative Example 3 of the present invention.

[0042] Figure 13 For the survival of the seedlings by using the existing method of Acacia falcataria clone tissue culture in Comparative Example 3 of the present invention. Specific embodiments

[0043] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0044] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0045] A method for ex vitro rooting and seedling raising of Acacia falcataria tissue-cultured seedlings provided by the present invention includes the following steps:

[0046] (1) Selection of bottle seedlings: Select tissue-cultured seedling cluster buds Figure 1 that have gone through 3 to 6 subculture cycles, are vigorous in growth, and have dark green leaves, and conduct strong seedling cultivation;

[0047] The seedlings after multiple subcultures are more juvenile in age, which can expand the occurrence end region of adventitious roots.

[0048] (2) Strong seedling cultivation: Cut off the tissue-cultured seedling cluster buds, select single buds or cluster buds with apical buds and a seedling height of 1.5 cm to 2.5 cm, and transfer them to a strong seedling culture medium for cultivation for 30 d to 40 d Figure 2 ;

[0049] The strong seedling culture medium consists of the following components: MS medium, 0.1 mg / L - 0.2 mg / L of cytokinin 6-BA, 0.1 mg / L - 0.2 mg / L of naphthaleneacetic acid NAA, 0.1 mg / L - 0.2 mg / L of indolebutyric acid IBA, 25 g / L - 35 g / L of sucrose, 6 g / L - 8 g / L of carrageenan; pH 5.6 - 6; the culture temperature is 20°C - 30°C, the light intensity for artificial light source culture is 1000 lux - 2000 lux, and the light duration is 12 h - 16 h per day; providing an appropriate concentration of auxin during strong seedling culture is beneficial to the occurrence of root primordia, while cytokinin can make the bud seedlings strong.

[0050] (3) Hardening off: When the seedling height reaches 4 cm - 6 cm, hardening off is carried out. The bottle seedlings are moved to the outdoor shaded shed and placed in natural light for 5 d - 7 d, then pure water is injected, and the water injection amount is such that it covers 1 cm - 3 cm above the culture medium plane. The bottle cap is loosened (not removed) and placed for 2 d - 5 d;

[0051] During the hardening off of Acacia falcata tissue culture seedlings without roots, the leaves are prone to closing and wilting. The whole process of hardening off requires moisture preservation and calm wind, and air flow should be avoided as much as possible.

[0052] (4) Preparation and treatment of containers and substrates: For the container for rooting seedlings outside the bottle, hole trays, seedling bags, seedling pots, planting baskets, etc. can be selected; for the substrate, vermiculite:quartz sand = 0.8 - 1.2:0.8 - 1.2 (volume ratio), vermiculite:perlite = 0.8 - 1.2:0.8 - 1.2 (volume ratio), peat soil:quartz sand = 0.8 - 1.2:0.8 - 1.2 (volume ratio), peat soil:perlite = 0.8 - 1.2:0.8 - 1.2 (volume ratio) can be selected, or a solid substrate with moisture absorption characteristics can also be used; after the substrate is sterilized at high temperature, it is filled into the container and watered thoroughly with pure water for later use;

[0053] (5) Cuttage: The tissue culture seedlings that have completed hardening off are taken out of the bottle, gently held by the root and stem part, the callus head adhering to the culture medium is cut off, the redundant leaves are appropriately trimmed, at least one terminal bud and the first and second lateral branches and leaves are retained for each plant, and the remaining leaves can be cut by 1 / 2. All the leaves attached to the stem part inserted into the substrate need to be cut off ( Figure 3 ); a bamboo stick is used to make a hole in the center of the substrate, the lower end 1.5 cm - 2.5 cm of the seedling stem is placed in the hole, the substrate is filled and gently compacted, and it is watered thoroughly with pure water;

[0054] The root and stem of Acacia falcata are fragile. Omitting the washing step in the conventional transplanting process of rooted seedlings can avoid damage and loss of the seedlings; cutting off the callus head adhering to the culture medium can avoid pollution and rot caused by attached agar sugars and other nutrients.

[0055] Existing literature proves that auxin plays a key role in the initiation and formation stages of root primordia in tissue-cultured seedlings. Therefore, when rooting tissue-cultured seedlings ex vitro, most plants will add a certain concentration of growth hormone to assist in rooting. However, the inventors of the present invention found that when transplanting rootless tissue-cultured seedlings, no exogenous rooting hormone is added, and the auxin accumulated during the strong seedling cultivation process alone is sufficient to meet the initiation of the root primordia of Albizia falcataria, and at the same time, a matrix environment without growth hormone is more conducive to the growth and development of the roots of Albizia falcataria.

[0056] (6) Construction of the seedling-raising microenvironment: Place the container after cutting in a chassis or bottom basin with grooves (the groove depth is about 5 mm), add pure water to the grooves so that the water surface in the grooves contacts the bottom of the container, cover the chassis or bottom basin with a transparent cover or plastic film for sealing, and the cultivation environment is free from direct sunlight to construct a sealed space;

[0057] The traditional seedling-raising environment is generally directly placed in a seedling-raising greenhouse or on a nursery ground covered with a plastic film for moisture preservation. The inventors found during cultivation that when cultivating Albizia falcataria tissue-cultured seedlings in the traditional seedling-raising environment, due to the loose stem tissue of Albizia falcataria and the unified characteristics of plants in the Mimosoideae family, it is relatively easy to wilt. Although the plastic film is covered for moisture preservation, the bud seedlings of Albizia falcataria are still very easy to dry out. Further discovery shows that rootless tissue-cultured seedlings of Albizia falcataria have higher requirements for the seedling-raising microenvironment. Since the ex vitro rooting seedling-raising substrate selected is a light substrate, the moisture in the light substrate is more likely to be lost, which is more likely to cause the rootless tissue-cultured seedlings to lose water and dry out. Therefore, in the present invention, a seedling-raising and rooting microenvironment is constructed. Through this simple method, it can ensure continuous water supply and ensure that the tissue-cultured seedlings are always in a moist and loose environment before rooting, which can solve the problem that the rootless tissue-cultured seedlings of Albizia falcataria are easy to dry out, greatly promote the self-rooting of Albizia falcataria, and improve the average rooting and seedling formation rate.

[0058] (7) Management: Spray pure water mist into the sealed space 1 to 2 times every 2d to 5d to increase the humidity of the substrate, leaf surface and air. Add pure water to the chassis or bottom basin according to the evaporation situation to keep the water surface in contact with the bottom of the container. The seedling-raising and rooting environment has a significant effect on heat preservation and moisture preservation, which can reduce the watering frequency and simplify the management process. After 10d to 15d, the seedlings completely root by themselves, with strong roots, rich root hairs, strong absorption and adaptation abilities, and the average rooting and seedling formation rate is 87.4%. The rooted seedlings can be gradually ventilated and exposed to sunlight to adapt to the natural environment.

[0059] The reagents, ex vitro rooting seedling-raising containers, substrates, etc. used in the following examples can be commercially available without special instructions.

[0060] The following specific examples are used to illustrate the present invention in detail.

[0061] Example 1 Method for Ex Vitro Rooting and Seedling Raising of Albizia falcataria Tissue-Cultured Seedlings

[0062] In this embodiment, for the clone pf21 (a Acacia falcata clone independently selected and bred by the applicant) with a rooting rate in vitro of less than 30%, rooting and seedling cultivation ex vitro were carried out, which specifically included the following steps:

[0063] (1) Selection of in vitro seedlings

[0064] Select the cluster buds of the clone pf21 tissue culture seedlings that have gone through 5 subculture cycles and are vigorous in growth and dark green in leaf color for strong seedling cultivation;

[0065] (2) Strong seedling cultivation

[0066] Cut off the cluster buds of the tissue culture seedlings, select single buds or cluster buds with apical buds and a seedling height of about 2 cm and transfer them to the strong seedling medium, and culture them at 26°C with an artificial light source illumination intensity of 1500 lux, 16 hours of light per day for 35 days; the strong seedling medium consists of the following components: MS + 0.2 mg / L 6-BA + 0.1 mg / L NAA + 0.1 mg / L IBA + 30 g / L sucrose + 7 g / L carrageenan, pH 6;

[0067] (3) Hardening off

[0068] When the seedling height reaches 4 cm to 6 cm, carry out hardening off. Move the in vitro seedlings to the outdoor shaded shed and place them in natural light for 7 days, then inject pure water, with the injection volume covering 2 cm above the medium plane, and loosen the bottle cap (without removing it) and place it for 3 days;

[0069] (4) Preparation and treatment of containers and substrates

[0070] The ex vitro rooting and seedling cultivation container uses a cell tray; the substrate is peat soil: quartz sand = 1:1 (volume ratio). After the substrate is sterilized at high temperature, it is filled into a 3×4 cell tray and watered thoroughly with pure water for later use;

[0071] (5) Cuttage

[0072] Take out the tissue culture seedlings that have completed hardening off from the bottle, gently hold the rhizome part, cut off the callus head adhering to the medium, appropriately trim the excess leaves, retain at least one apical bud and the first and second lateral branches and leaves for each plant, and the remaining leaves can be cut by 1 / 2. All the leaves on the stem part inserted into the substrate need to be cut off; use a bamboo stick to make a hole in the center of each cell, place the lower end 2 cm of the seedling stem in the hole, fill the substrate and gently compact it, and water it thoroughly with pure water;

[0073] (6) Construction of the seedling cultivation microenvironment

[0074] Place the cell tray after cutting into a bottom basin with grooves (the groove depth is about 5 mm), add pure water to the grooves so that the water surface in the grooves contacts the bottom of the container, cover it with a transparent cover and seal it, and place it in a shaded shed. The cultivation environment has no direct sunlight to construct a sealed space;

[0075] (7), Management

[0076] Spray pure water mist into the sealed space once every 5 days to increase the humidity of the substrate, leaf surface and air. Add pure water to the bottom basin according to the evaporation situation to keep the water surface in contact with the bottom of the container. After 15 days, the growth of the seedlings is stable, and the roots are growing increasingly perfect and developed.

[0077] In this example, a total of 10 trays of these cloned seedlings were transplanted, with a total of 120 plants. After 15 days, the growth of the seedlings was counted. 110 seedlings had their apical buds unfolded and the number of compound leaves increased, indicating that the seedlings had taken root and started normal growth. The average rooting and seedling formation rate was 91.7% (as Figures 4 - 5 shown).

[0078] Method for Rooting and Seedling Raising of Acacia falcata Tissue Culture Seedlings outside the Bottle in Example 2

[0079] The excellent clone pf 160 (an Acacia falcata clone independently selected and bred by the applicant) screened through afforestation measurement has an average tree height of 6.44 m, a breast diameter of 7.08 cm, and a volume of 0.0145 m 3 at 1.5 years old. It is a fast-growing clone with popularization value. The traditional in-vitro rooting of this clone is better, reaching 90%, but the seedling formation rate of the rooted seedlings after transplantation is relatively low, less than 50%. In this example, the clone pf 160 was subjected to rooting and seedling raising culture outside the bottle, which specifically includes the following steps:

[0080] (1), Selection of Bottle Seedlings

[0081] Select the cluster buds of the clone pf160 tissue culture seedlings that have gone through 6 subculture cycles, with strong growth and dark green leaves, and conduct strong seedling culture;

[0082] (2), Strong Seedling Culture

[0083] Cut off the cluster buds of the tissue culture seedlings, select single buds or cluster buds with apical buds and a seedling height of about 2 cm, transfer them to the strong seedling culture medium, and culture them at 26 °C. The light intensity of the artificial light source is 1500 lux, with 16 hours of light per day for 30 days; the strong seedling culture medium consists of the following components: MS + 0.15 mg / L 6-BA + 0.1 mg / L NAA + 0.1 mg / L IBA + 30 g / L sucrose + 7 g / L carrageenan, pH 6;

[0084] (3), Hardening off

[0085] When the seedling height reaches 4 cm - 6 cm, conduct hardening off. Move the bottle seedlings to the natural light in the outdoor shade shed and place them for 7 days, then inject pure water. The water injection volume should cover 2 cm above the medium plane, and loosen the bottle cap (do not uncover) and place it for 5 days;

[0086] (4), Preparation and Treatment of Containers and Substrates

[0087] The rooting and seedling-raising container outside the bottle uses a seedling-raising pot and a seedling-raising bag with specifications of 8 cm (upper diameter) × 8 cm (height). The substrates are peat soil:perlite = 1:1 (volume ratio) and vermiculite:quartz sand = 1:1 (volume ratio) respectively. After the substrates are sterilized at high temperature, they are filled into the containers and watered thoroughly with pure water for standby;

[0088] (5), Cutting

[0089] Take out the tissue-cultured seedlings that have completed acclimatization from the bottle, gently hold the root and stem part, cut off the callus head adhering to the culture medium, appropriately trim the excess leaves, retain at least one apical bud and the first and second lateral branches and leaves for each plant, and the remaining leaves can be cut by 1 / 2. All the leaves attached to the stem inserted into the substrate need to be cut off; use a bamboo stick to make a hole in the center of the container, place the lower end 2 cm of the seedling stem into the hole, fill the substrate and gently compact it, and water it thoroughly with pure water;

[0090] (6), Construction of seedling-raising microenvironment

[0091] Place the container after cutting in a chassis with grooves (the depth of the grooves is about 5 mm), add pure water to the grooves so that the water surface in the grooves contacts the bottom of the container, cover it with a transparent cover and seal it, and place it in a shade shed. The cultivation environment has no direct sunlight to construct a sealed space;

[0092] (7), Management

[0093] Spray pure water mist into the sealed space once every 5 days to increase the humidity of the substrate, leaf surface and air, and add pure water to the chassis according to the evaporation situation to keep the water surface in contact with the bottom of the container. After 12 days, the seedlings grow vigorously and have well-developed roots.

[0094] A total of 3 batches of tests were carried out in this example, with 72 plants per batch, totaling 216 plants. The seedlings in each batch grew stably. After 12 days, the growth of the seedlings was counted. 68 seedlings in each batch had their apical buds unfolded and the number of compound leaves increased. After 30 days, the seedlings were strong, with lush branches and leaves and well-developed roots, and the average rooting and seedling formation rate was 94.4% (as Figure 6 , Figure 7 shown).

[0095] Method for rooting and seedling-raising of Acacia falcata tissue-cultured seedlings outside the bottle in Example 3

[0096] In the 1990s, the Guangdong Academy of Forestry introduced a batch of excellent germplasm resources of Acacia falcata from countries such as Malaysia, the Philippines, and Indonesia. Among them, Y is the seed of an excellent family from Java Island, Indonesia. The preservation period has exceeded 20 years, and the seeds have almost lost their germination ability. Currently, only 1 aseptic seedling (Y1) of the seeds is saved for rescue.

[0097] In this example, the tissue-cultured bud seedlings propagated from Y1 were subjected to rooting and seedling-raising culture outside the bottle, which specifically included the following steps:

[0098] (1) Selection of tissue-cultured seedlings

[0099] Select the cluster buds of asexual clone Y1 tissue-cultured seedlings that have gone through 3 subculture cycles, are growing vigorously, and have dark green leaves for strong seedling cultivation;

[0100] (2) Strong seedling cultivation

[0101] Cut off the cluster buds of the tissue-cultured seedlings, select single buds or cluster buds with apical buds and a seedling height of about 2 cm, transfer them to the strong seedling medium, and culture them at 26°C. The light intensity of the artificial light source is 1500 lux, with 16 hours of light per day for 35 days; the strong seedling medium consists of the following components: MS + 0.1 mg / L 6-BA + 0.1 mg / L NAA + 0.1 mg / L IBA + 30 g / L sucrose + 7 g / L carrageenan, pH 6;

[0102] (3) Hardening-off

[0103] When the seedling height reaches 4 cm - 6 cm, carry out hardening-off. Move the tissue-cultured seedlings in the bottle to the natural light in the outdoor shade shed and place them for 7 days. Then inject pure water, with the water injection volume covering 2 cm above the medium plane, and loosen the bottle cap (do not remove it) and place it for 5 days;

[0104] (4) Preparation and treatment of containers and substrates

[0105] The container for rooting seedlings outside the bottle uses a planting basket with a specification of 2.5 cm (upper diameter) × 3.5 cm (height). The substrates are commercial black cotton soil planting blocks. After the substrates are sterilized at high temperature, soak them in sterile water for 20 - 30 minutes and fully absorb and swell for later use;

[0106] (5) Cutting

[0107] Take out the tissue-cultured seedlings that have completed hardening-off from the bottle, gently hold the root and stem part, cut off the callus head adhering to the medium, appropriately trim the redundant leaves, retain at least one apical bud and the first and second lateral branches and leaves for each plant, and the remaining leaves can be cut by 1 / 2. All the leaves attached to the stem inserted into the substrate need to be cut off; use a tissue-culture knife to make a longitudinal cut on the planting block, place the lower 2 cm of the seedling stem in the center of the planting block and seal it, wrap it with a rubber band around the outside, and then put it into the planting basket;

[0108] (6) Construction of the microenvironment for seedling raising

[0109] Place the planting basket in a chassis with grooves (the groove depth is about 5 mm), add pure water to the grooves so that the water surface in the grooves contacts the bottom of the container, cover it with a transparent lid and seal it, and place it in the shade shed. The cultivation environment has no direct sunlight, and a sealed space is constructed ( Figure 8 );

[0110] (7) Management

[0111] Spray pure water mist into the sealed space once every 2 days to increase the humidity of the substrate, leaf surface and air. Add pure water to the chassis according to the evaporation situation to keep the water surface in contact with the bottom of the container. After 15 days, the seedlings take root.

[0112] In this example, a total of 50 seedlings of this clone were transplanted, and 38 seedlings took root, with an average rooting and seedling formation rate of 76.0% (as Figure 9 shown).

[0113] Effect of the seedling-raising microenvironment in Comparative Example 1 on the ex vitro rooting and seedling-raising of Acacia mangium tissue-cultured seedlings

[0114] This comparative example compared the effects of different seedling-raising microenvironments on the ex vitro rooting and seedling-raising of Acacia mangium tissue-cultured seedlings.

[0115] Steps (1)-(5): The same as in Example 1;

[0116] Step (6): Change the seedling-raising microenvironment to a traditional seedling-raising environment - directly place the plugged hole tray in the shade shed, with no direct sunlight in the cultivation environment, and construct a sealed space.

[0117] Step (7): Spray pure water mist into the sealed space every morning and evening to increase the humidity of the substrate, leaf surface and air.

[0118] In this comparative example, a total of 500 seedlings of the clone were transplanted. After 15 days of cultivation, only 50 seedlings survived, with a survival rate of less than 10% (as Figure 10 shown). It shows that the traditional seedling-raising environment is not suitable for the ex vitro rooting and seedling-raising of Acacia mangium tissue-cultured seedlings.

[0119] Effect of adding exogenous hormones during the rooting process in Comparative Example 2 on the ex vitro rooting and seedling-raising of Acacia mangium tissue-cultured seedlings

[0120] This comparative example compared the effects of adding or not adding exogenous hormones during the rooting process on the ex vitro rooting and seedling-raising of Acacia mangium tissue-cultured seedlings.

[0121] Steps (1)-(4): The same as in Example 3;

[0122] Step (5): Cuttage

[0123] Take out the tissue-cultured seedlings that have completed acclimatization from the bottle, gently hold the rhizome part, cut off the callus head adhering to the culture medium, appropriately trim the excess leaves, retain at least one apical bud and the first and second lateral branches and leaves for each plant, and the remaining leaves can be cut by 1 / 2; use a tissue-culture knife to longitudinally cut a slit on the planting block, dip the lower 2 cm of the stem in ABT rooting powder and place it in the center of the planting block and then seal it, wrap it with a rubber band on the periphery, and then put it into the planting basket;

[0124] Steps (6)-(7) are the same as in Example 3.

[0125] In this comparative example, 50 clonal seedlings were transplanted. After 15 days of cultivation, it was found that most of them showed yellowing and wilting of the plants. Only 15 apical buds gradually unfolded and the leaves remained green, but no roots extended out of the planting blocks. The seedling survival rate was about 30% (as shown in Figure 11 ). It shows that the auxin accumulated during the strong seedling cultivation process relying on the strong seedling culture medium provided by the present invention is sufficient to initiate the root primordium of Albizia falcataria, and there is no need to add exogenous hormones during the rooting process. Adding exogenous hormones during the rooting process will instead affect the ex vitro rooting and seedling raising effect of the tissue-cultured seedlings of Albizia falcataria.

[0126] Comparative Example 3 Comparison of the effects of the ex vitro rooting and seedling raising method of the tissue-cultured seedlings of Albizia falcataria of the present invention and the traditional rooting and seedling raising method of the tissue-cultured seedlings of Albizia falcataria

[0127] This comparative example compared the effects of the ex vitro rooting and seedling raising method of the tissue-cultured seedlings of Albizia falcataria in Example 2 of the present invention and the traditional rooting and seedling raising method of the tissue-cultured seedlings of Albizia falcataria.

[0128] In December 2020, reference was made to ZL 201610012823.5 (Method for tissue culture of Albizia falcataria clones) for rooting and transplanting culture of clone pf 160. The specific method is as follows:

[0129] 1. Rooting culture: When the adventitious buds grew to 3 - 5 cm, they were cut and transferred to the rooting medium for 10 days of culture. The composition of the rooting medium was: 1 / 2 MS medium + NAA (0.2 mg / L) + indolebutyric acid (IBA, 0.2 mg / L) + sucrose (25 g / L) + carrageenan (7 g / L), pH 5.8. The culture temperature was about 28°C.

[0130] 2. Hardening off: The culture bottles were moved to the outdoor shaded shed for 12 days of closed-bottle hardening off while rooting. Then the bottle caps were removed and clear water was injected (the injection volume was 1 cm above the medium surface), and hardening off was carried out for 3 days.

[0131] 3. Plant transplantation: After cleaning the medium on the roots with 5% carbendazim, the plants were transplanted into the seedling-raising substrate at 4:30 pm on sunny days. The seedling-raising substrate was yellow heart soil and peat soil with a volume ratio of 3:1, covered with a transparent plastic film (as shown in Figure 12 ). Sprayers were used to spray water every morning and evening to keep the humidity at 85%. Shading nets needed to be covered on sunny days. After 30 days, the plastic film was removed and cultured at room temperature.

[0132] In this comparative example, a total of 9000 rooted seedlings of clone pf 160 were transplanted. After removing the plastic film after 30 days, the number of surviving seedlings was counted as 3980, and the seedling survival rate was 44.2% (as shown in Figure 13As shown). It shows that compared with the traditional rooting and seedling-raising method of Acacia falcata tissue culture seedlings, the method of rooting and seedling-raising of Acacia falcata tissue culture seedlings outside the bottle of the present invention can significantly improve the seedling survival rate.

[0133] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered that the scope described in this specification.

[0134] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A method for rooting tissue culture seedlings of Falcataria moluccana outside the bottle, Characterized in that, It includes the following steps: (1) Cut the cluster buds of the tissue culture seedlings of Falcataria moluccana with strong growth and dark green leaves, select single buds or cluster buds with apical buds and transfer them to the strong seedling medium, and culture the strong seedlings for 30 d to 40 d; when the tissue culture seedlings are 4 cm to 6 cm high, harden them off; the strong seedling medium consists of the following components: MS medium, 0.1 mg / L to 0.2 mg / L cytokinin 6-BA, 0.1 mg / L to 0.2 mg / L naphthaleneacetic acid NAA, 0.1 mg / L to 0.2 mg / L indolebutyric acid IBA, 25 g / L to 35 g / L sucrose, 6 g / L to 8 g / L carrageenan, pH 5.6 to 6; (2) Take out the tissue culture seedlings that have completed hardening off, gently hold the root and stem part, cut off the callus head adhering to the medium, and retain at least one apical bud and the first and second lateral branches and leaves for each tissue culture seedling. Cut off all the leaves on the stem where the matrix is to be inserted to obtain the tissue culture seedlings to be cuttaged; (3) Fill the matrix into the container for rooting tissue culture seedlings outside the bottle, and then cuttage the lower end of the stem of the tissue culture seedlings to be cuttaged in step (2) into the matrix, fill the matrix and gently compact it, and pour it thoroughly with pure water; (4) Place the container after cuttage in a chassis or bottom basin with grooves, add pure water to the grooves so that the water surface in the grooves contacts the bottom of the container; cover the chassis or bottom basin with a transparent cover or plastic film to form a sealed space; (5) Spray pure water mist into the sealed space described in step (4) every 2 d to 5 d, and add water to the grooves in a timely manner to keep the water surface in contact with the bottom of the container; after 10 d to 15 d, the rooted seedlings of Falcataria moluccana outside the bottle are obtained.

2. The method for rooting tissue culture seedlings of Falcataria moluccana outside the bottle according to claim 1, Characterized in that, The strong seedling culture in step (1) includes: the culture temperature is 20°C to 30°C, the light intensity is 1000 lux to 2000 lux, and the daily light time is 12 h to 16 h.

3. The method for rooting tissue culture seedlings of Falcataria moluccana outside the bottle according to claim 1, Characterized in that, The cluster buds of the tissue culture seedlings of Falcataria moluccana in step (1) are the cluster buds of the tissue culture seedlings that have gone through 3 to 6 subculture cycles.

4. The method for rooting tissue culture seedlings of Falcataria moluccana outside the bottle according to claim 1, Characterized in that, The hardening off in step (1) includes the following steps: Move the tissue culture seedlings to the natural light in the outdoor shade shed and place them for 5 d to 7 d, then inject pure water covering the strong seedling medium by 1 cm to 3 cm, and loosen the bottle cap and place it for 2 d to 5 d.

5. The method for rooting tissue culture seedlings of Falcataria moluccana outside the bottle according to claim 1, Characterized in that, The height of the single bud or cluster bud with apical bud in step (1) is 1.5 cm to 2.5 cm.

6. The method for rooting tissue culture seedlings of Falcataria moluccana outside the bottle according to claim 1, Characterized in that, The container for rooting tissue culture seedlings outside the bottle in step (3) is a cell tray, a seedling raising bag, a seedling raising pot or a planting basket.

7. The method for ex vitro rooting and seedling raising of Albizia falcataria tissue culture seedlings according to claim 1, characterized in that, the substrate described in step (3) is vermiculite and quartz sand, vermiculite and perlite, peat soil and quartz sand, peat soil and perlite or a solid substrate with moisture absorption characteristics in a volume ratio of 0.8~1.2:0.8~1.

2.

8. The method for ex vitro rooting and seedling raising of Albizia falcataria tissue culture seedlings according to claim 1, characterized in that, the depth of the groove described in step (4) is 4 mm to 8 mm.

9. The method for ex vitro rooting and seedling raising of Albizia falcataria tissue culture seedlings according to claim 1, characterized in that, in step (5), pure water spray is sprayed into the sealed space 1 to 2 times every 2 d to 5 d.

10. The method for ex vitro rooting and seedling raising of Albizia falcataria tissue culture seedlings according to any one of claims 1 to 9, characterized in that, in step (3), the lower 1.5 cm to 2.5 cm of the stem of the tissue culture seedlings to be cuttaged is cuttaged into the substrate.

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