CN112352678A

CN112352678A - Tissue culture rapid propagation technology for slash pine seedlings

Info

Publication number: CN112352678A
Application number: CN202011268576.8A
Authority: CN
Inventors: 张付远; 李丹丹; 宋墩福; 景维杰
Original assignee: Jiangxi Environmental Engineering Vocational College
Current assignee: Jiangxi Environmental Engineering Vocational College
Priority date: 2020-11-13
Filing date: 2020-11-13
Publication date: 2021-02-12
Anticipated expiration: 2040-11-13
Also published as: CN112352678B

Abstract

The invention provides a tissue culture and rapid propagation technology of slash pine seedlings, which belongs to the technical field of plant nutrition and cultivation and comprises the following steps: pretreating a stock plant, taking an explant from the stock plant, and disinfecting the explant; tender stem induction; proliferating cluster lateral buds; rejuvenating and culturing rootless buds; rooting culture; domesticating and culturing; and (5) transplanting the tissue culture seedlings. The tissue culture and rapid propagation technology of the slash pine seedlings provided by the invention obtains a large number of rootless buds through the cluster bud proliferation way after acquiring the germinated seedlings by inducing and culturing the tender stems of the slash pine. Then the rootless bud seedlings are subjected to tissue culture rejuvenation, induced rooting and natural domestication in a greenhouse, and then the seedlings can be transplanted out of bottles. The technique has the advantages of stable inheritance of the bred offspring seedlings, comprehensive character expression and excellent ornamental characters. The technical system has low production cost, environmental protection, no environmental pollution and high mass production speed, and completely meets the technical requirement of large-scale mass production.

Description

Tissue culture rapid propagation technology for slash pine seedlings

Technical Field

The invention belongs to the technical field of plant nutrition and cultivation, and particularly relates to a tissue culture rapid propagation technology of slash pine seedlings.

Background

The slash pine is a good broad-spectrum landscaping tree species and is suitable for subtropical climate areas with dry winter, rain and summer. Has strong adaptability to air temperature, and can endure absolute high temperature of 40 ℃ and absolute low temperature of-20 ℃. The fertilizer grows well on neutral or strongly acidic red soil hilly lands, iron nodule layers and sand clay lands with the surface soil being less than 50-60 cm, and is particularly good at the edges of low-lying marsh lands, so that the fertilizer is famous but relatively drought-resistant, and can grow vigorously on the low hilly lands which are arid and barren. The wind resistance is strong, and the damage is less under the attack of typhoon of 11-12 grades. The root system of the plant can resist seawater irrigation, has drought resistance, fatigue resistance and barren resistance, has good adaptability and stress resistance, is widely distributed in the world, and can be cultivated in plains at the north latitude of China to the south and in low mountains facing the sun. The large pieces of soil in the south of Shandong are suitable for planting.

The wet field pine is vigorous and fast-growing, has a growth speed far faster than the masson pine, is a fast-growing tree species with a short rotation cutting period, has strong adaptability, good material quality and high rosin yield, is also a good economic tree species, and has high yield of rosin and wood. Transplanting seedlings with the average annual tree height of 1m, the average biennial breast diameter of 5 cm and the tree height of 2.5 m; the average breast diameter of 4 years can reach 11 cm, the breast diameter of 7 years can reach 20 cm, the average breast diameter growth of the year can reach 2.8cm, and the average growth amount of wood per mu can reach 12m 3; can be used for afforestation for 8-12 years. If the plants are felled for 15 years, the wood growth amount reaches 22m3 per mu. Wood benefit: pine resin is harvested in the 10 th year, and the pine resin is felled in the 15 th year, so that 22m 3/mu of wood can be produced, the yield value is 19800 yuan/mu according to the calculation of the price of 900 yuan/m 3 (the market price of the hybrid pine wood is higher than that of eucalyptus with the same specification in the same period), and the average benefit is as follows: the yield per mu per year can reach 1320 yuan; rosin benefit: and (3) starting to collect the resin at the 10 th year, and producing 3 kilograms of resin per mu per year according to 90 plants per mu, wherein the price of the rosin is 4 yuan/kilogram (the price is 4.4 to 5.4 yuan/kilogram since 2006), and the value of producing the rosin per mu per year is about 1000 yuan. This additional revenue is incomparable with other tree species. Moreover, the price of rosin in the international market is on the rise.

At present, the seedling propagation technology of the slash pine still depends on the seed sowing and seedling raising technology and becomes a bottleneck link in the industrial development. The mismatching between the current wet land pine seedling expanding propagation technology and the afforestation demand seedling quantity, the seedling production cost and the seedling production efficiency is the biggest problem in the current wet land pine industry development. Firstly, the prior slash pine seeds are generally imported seeds, and the slash pine seeds are expensive and high in cost; secondly, the seeding procedure is many. Before sowing, the seeds are soaked in 1.5 to 2 percent formalin solution or 2 percent Bordeaux mixture for 20 minutes or soaked in 0.5 percent potassium permanganate solution for 3 to 5 minutes and washed by clear water. Soaking the disinfected seeds in warm water of 40-60 degrees, naturally cooling for 18-24 hours, taking out and sowing; thirdly, the requirement of soil preparation is high, and if the land is newly opened in the current year, forest soil and fungus compost are dug from the old forest of the wet land and mixed in the soil of the growing bed for small inoculation of mycorrhiza. From the above, the current state of the art of producing slash pine seedlings needs to be improved, and a new technology is used to assist the development of afforestation industry.

Disclosure of Invention

The invention aims to provide a tissue culture and rapid propagation technology of slash pine seedlings, and aims to solve the technical problems that the existing slash pine seedling propagation technology depends on a seed sowing and seedling raising technology, the cost is high, the sowing procedures are multiple, and the land preparation requirement is high.

In order to achieve the purpose, the invention adopts the technical scheme that: provides a tissue culture and rapid propagation technology of slash pine seedlings, which comprises the following steps:

step 1, pretreating a stock plant, taking an explant from the stock plant, and disinfecting the explant;

step 2, tender stem induction: inoculating the explant into an induction culture medium, and then placing the explant into a culture chamber for culture to obtain a sterile bud;

step 3, proliferating cluster lateral buds: reserving the stem at the physiological upper part of the sterile bud body obtained in the step two, and transferring the sterile bud body to a multiplication culture medium for culturing to obtain cluster buds;

step 4, rejuvenation culture of rootless buds: dividing the cluster buds obtained in the step three into single rootless seedling buds, and transferring the single rootless seedling buds into a rejuvenation culture medium for culture so as to restore the rootless seedling buds to be strong;

step 5, rooting culture: transferring the single-plant rootless seedling bud which reaches the rooting standard and is obtained in the step four to a rooting induction culture medium to promote rooting;

step 6, domestication and culture: transferring the obtained seedling buds growing the adventitious roots or the root primordium to a greenhouse for acclimation under natural light and temperature;

step 7, transplanting tissue culture seedlings: and (5) cleaning the root system of the domesticated tissue culture seedling obtained in the sixth step, and then planting the tissue culture seedling.

As another example of the present application, the inducing medium comprises modified MS medium, 6-benzylamino adenine 0.5-1 mg/L, naphthalene acetic acid 0.1mg/L, sucrose 25g/L, and agar 5g/L, wherein the modified MS medium comprises: 2.2 g/L of potassium nitrate, 1.3 g/L of ammonium nitrate, 0.17 g/L of monopotassium phosphate, 0.17 g/L of sodium dihydrogen phosphate, 0.1mg/L of biotin, 1mg/L of L-cysteine and 3 mg/L of glutamine.

As another example of the present application, the multiplication medium includes the following components: 1/2 modified MS culture medium, 6-benzylamino adenine 0.5-1.5 mg/L, naphthylacetic acid 0.02-0.1 mg/L, indolebutyric acid 0.05-0.1mg/L, kinetin 0.5-1 mg/L, sucrose 30g/L and agar 5g/L, wherein the pH value of the enrichment culture medium is 5.8-6.0; or/and

the rejuvenation culture medium comprises the following components: 1/2 modified MS culture medium, kinetin 1-2 mg/L, naphthylacetic acid 0.1-0.5 mg/L, active carbon 1g/L, sucrose 30g/L and agar 5g/L, wherein the pH value of the rejuvenation culture medium is 5.8-6.0; or/and

the rooting induction culture medium comprises the following components: 1/3MS medium, 0.01-0.1 mg/L naphthylacetic acid, 1-5 mg/L indolebutyric acid, 0.5-1 g/L hydrolyzed casein, 1-3 mg/L riboflavin, 30g/L sucrose and 5g/L agar, wherein the pH value of the rooting induction medium is 5.8-6.0.

As another embodiment of the present application, the step 1 includes: selecting semi-lignified tender stems of the slash pine plants germinated after stumping treatment as mother plants in the middle ten days of 2-4 months; carrying out shading treatment on the stock plant for 2 weeks before material drawing, regularly spraying broad-spectrum bactericide for 7 days/time, and taking the material after spraying for 2 times; wherein the length of the explant is 2-5 cm; washing with running water for 1-2 hours after material drawing, and then airing until no obvious water stain exists on the surface of the explant; cutting off the needle leaves and only keeping the length of the needle leaves between 2 and 3 millimeters; shaking and sterilizing with 75% ethanol solution containing 2-3 drops of hydrochloric acid (1 mol/L) per liter for 45 s, washing with sterile water, air drying, and sterilizing with bleaching water solution containing 1.5% of available chlorine and TWEEN-20(0.15 ml/L) for 18-25 min to allow the disinfectant to permeate into explant; rinsing with sterile water for 2 min 3-5 times, and then blotting the explant with sterile filter paper to remove water.

As another embodiment of the present application, the step 2 includes: inoculating the explant into the induction culture medium, then placing the explant in a culture chamber for culture, performing dark culture for 2 days, and then transferring the explant into illumination culture, wherein the photoperiod is 12-16 hours/day and the illumination is 1500-ion 3000 lux during the illumination culture; controlling the indoor temperature to be 25 +/-2 ℃; periodically observing the pollution and the induced growth condition during the induction culture period; and transferring the explants into the proliferation culture medium for propagation after 25-30 days.

As another example of the present application, the sterile sprouts were cut 1-2 mm off the base and then transferred to the multiplication medium; or/and

the culture conditions include: the light period is 12-16 hours/day, the illuminance is 1500-; or/and

after culturing for 30-35 days, obtaining cluster bud clusters, cutting the cluster buds into individual buds or cluster bud clusters, and continuously transferring the individual buds or cluster bud clusters to the same proliferation culture medium for multiple subcultures, wherein the transfer period is one generation within 30-35 days.

As another example of the present application, after the number of subcultured proliferations reached the desired number, the multiple shoots were divided into single rootless shoots, which were transferred to a rejuvenation medium at a culture temperature of 25. + -. 2 ℃ with a light cycle of 16 hours/day and a light illumination of 1500-.

As another embodiment of the application, the seedlings with the height of 3 cm are transferred to a rooting induction culture medium to promote rooting; repeatedly rejuvenating the weak and small buds with the height less than 3 cm until the height requirement is met, and then regenerating roots for culturing; after rooting inoculation, culturing for 24-48 hours, and then transferring to illumination culture; wherein, the illumination period of the rooting culture is 16 hours/day, and the illumination intensity is 1500-; culturing at 25 + -2 deg.C for 30-35 days.

As another example of the present application, rooting culture is carried out for 10-20 days, adventitious roots grow out and subsequent root primordium germination; transferring the bottle seedlings into a greenhouse facility to be acclimated by natural light and temperature, gradually increasing the temperature amplitude to 25-28 ℃ and the illuminance to 3000 and 8000 luxes during the acclimation, and transplanting the rooted seedlings out of the bottles after the acclimation is carried out for 40-50 days.

As another embodiment of the application, the domesticated rooting tissue culture seedling is taken out from a bottle, the culture medium adhered to the root system is cleaned, and 0.1 per mill of potassium permanganate solution is soaked for disinfection. The medium is mixed by 72-hole sieve trays, turfy soil and vermiculite (2:1= V/V). The substrate is thoroughly irrigated and disinfected by carbendazim solution of 75% wettable powder with the concentration of 2.5g/L, the relative humidity in a greenhouse of a facility after planting is 80-90%, and the temperature is kept at 25-28 ℃; transplanting to field nursery after 3-6 months.

The tissue culture rapid propagation technology of the slash pine seedlings provided by the invention has the beneficial effects that: compared with the prior art, the tissue culture rapid propagation technology of the slash pine seedlings is beneficial to improving the efficiency of slash pine seedling propagation, and promotes the development of economic and ecological benefits of forestry industry. The invention overcomes the key point in the production of the slash pine seedling, namely the regeneration of the rootless seedling. Compared with the conventional cuttage means at present, the technology not only greatly promotes the production efficiency of the slash pine seedlings and reduces the cost, but also effectively solves the problems in the tissue culture production at present. Not only is beneficial to improving the precision of the production process, but also improves the consistency of the quality of the commercial seedlings, and is convenient for production management. The method provided by the invention can greatly improve the planning accuracy of production management and mass production, and can effectively reduce the cost and improve the benefit. The method has the advantages of easy operation, low production cost, fast multiplication of the number of the slash pine seedlings and realization of large-scale production. The slash pine seedling cultivated by the method provided by the invention has stable genetic character, maintains the characteristics of parents, and has the advantages of less investment, high yield, short period and the like. Has obvious advantages in effectively improving the production efficiency of the slash pine seedlings and improving the proportion of effective commercial seedlings of the tissue culture seedlings.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed for the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

Fig. 1 is a flow chart of a tissue culture rapid propagation technique of slash pine seedlings provided by an embodiment of the invention.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Referring to fig. 1, a tissue culture and rapid propagation technique for slash pine seedlings provided by the present invention will now be described. The tissue culture and rapid propagation technology of the slash pine seedlings comprises the following steps:

(1) pretreating a stock plant, taking an explant from the stock plant, and disinfecting the explant;

selecting semi-lignified tender stems of the slash pine plants which germinate after stumping treatment and grow robustly as parent plants in the slash pine cutting orchard from 2 months to 4 months. Shading the stock plant 2 weeks before material selection, periodically spraying broad-spectrum bactericide for 7 days/time, and taking the material after spraying for 2 times. The length of the explant is 2-5 cm. After the materials are taken, the materials are washed for 1 to 2 hours by running water and then dried until no obvious water stain exists on the surface of the explant. The needle leaves are cut off with surgical scissors and only 2-3 mm length is left. Sterilizing by shaking with 75% ethanol solution containing 2-3 drops of diluted hydrochloric acid (1 mol/L) per liter for 45-60 s, washing with sterile water for 3-5 times (each for 1-2 min), and air drying. The cells were sterilized on a clean bench with 1.5% available chlorine in aqueous bleach + TWEEN-20(0.15 mL/L aqueous bleach) for 18-25 minutes. The container is frequently shaken during the sterilization process to allow the sterilant to fully penetrate the explant. Then rinsed 3-5 times with sterile water for 2 minutes each, after which the explant is blotted dry with sterile filter paper. Explants were inoculated into induction medium with sterilized forceps and scalpel.

The volume of 1 drop of liquid is 0.04-0.05 ml.

(2) Tender stem induction: inoculating the explant into an induction culture medium, and then placing the explant into a culture chamber for culture to obtain a sterile bud;

the explant is inoculated into an induction culture medium and then placed in a culture chamber, dark culture is carried out for 2 days, and then illumination culture is carried out, wherein the illumination culture is carried out with the photoperiod of 12-16 hours/day and the illumination intensity of 1500-3000 lux. The indoor temperature is controlled to be 25 +/-2 ℃. Obvious needle growth can be seen in about 7-10 days. Contamination and induced growth were observed periodically during induction culture. Transferring the explant into a proliferation culture medium for propagation in 25-30 days.

The formula of the induction culture medium is as follows:

an improved MS culture medium, 6-benzylamino adenine (0.5-1 mg/L), naphthylacetic acid (0.1 mg/L), sucrose (25 g/L) and agar (5 g/L); the pH value of the induction medium is 5.8-6.0.

Wherein, the components of the improved MS culture medium are as follows: potassium nitrate 2.2 g/l, ammonium nitrate 1.3 g/l, potassium dihydrogen phosphate 0.17 g/l, sodium dihydrogen phosphate 0.17 g/l, magnesium sulfate heptahydrate 0.37 g/l, calcium chloride dihydrate 0.44 g/l, manganese sulfate tetrahydrate 22 mg/l, zinc sulfate heptahydrate 8.6 mg/l, copper sulfate pentahydrate 0.025 mg/l, boric acid 6.2 mg/l, sodium molybdate 0.25 mg/l, cobalt chloride hexahydrate 0.25 mg/l, potassium iodide 0.83 mg/l, ferrous sulfate heptahydrate 22.8 mg/l, disodium ethylenediaminetetraacetate 37.3 mg/l, thiamine hydrochloride 1mg/l, pyridoxine hydrochloride 0.5 mg/l, hydrochloric acid 0.5 mg/l, inositol 200 mg/l, glycine 2 mg/l, biotin 0.1mg/l, magnesium sulfate, sodium dihydrogen phosphate, magnesium sulfate heptahydrate 0.025 mg/l, magnesium sulfate, 1mg/L of L-cysteine and 3 mg/L of glutamine.

(3) And (3) cluster lateral bud multiplication: reserving a physiologically upper stem of the sterile bud body obtained in the step (2), and transferring the sterile bud body to a multiplication culture medium for culture to obtain cluster buds;

the sterile sprouts obtained by induction were cut 1-2 mm from their bases, the physiologically upper stems were retained and transferred to a multiplication medium. The culture conditions include: the light period is 12-16 hours/day, and the illuminance is 1500-3000 lux. The indoor temperature is controlled to be 25 +/-2 ℃. After culturing for 30-35 days, the clump bud with the multiplication coefficient of 2-3 can be obtained. Cutting the cluster buds into individual buds or cluster buds, and continuously transferring the buds or cluster buds to the same proliferation culture medium for multiple subcultures in a transfer period of 30-35 days. Wherein the culture temperature is 25 + -2 deg.C, the illumination is 1500-3000 lux, and the light period is 12-16 h/day, so as to obtain the required quantity.

The formula of the proliferation culture medium is as follows:

1/2 modified MS culture medium, 6-BA (6-benzylamino adenine) 0.5-1.5 mg/L, naphthylacetic acid (NAA) 0.02-0.1 mg/L, indolebutyric acid (IBA) 0.05-0.1mg/L, Kinetin (KT) 0.5-1 mg/L, sucrose 30g/L and agar 5g/L, wherein the pH value of the proliferation culture medium is 5.8-6.0.

(4) Rejuvenation culture of rootless buds: dividing the cluster buds obtained in the step (3) into single rootless seedling buds, and transferring the single rootless seedling buds into a rejuvenation culture medium for culture so as to restore the rootless seedling buds to be strong.

After the subculture multiplication number reaches the expected number, dividing the cluster buds into single rootless seedling buds, transferring the single rootless seedling buds into a rejuvenation culture medium, culturing at the temperature of 25 +/-2 ℃, with the illumination period of 16 hours/day and the illumination intensity of 1500-.

The rejuvenation culture medium comprises the following components:

1/2 modified MS culture medium, Kinetin (KT) 1-2 mg/L, naphthylacetic acid 0.1-0.5 mg/L, active carbon 1g/L, sucrose 30g/L and agar 5g/L, wherein the pH value of the rejuvenation culture medium is 5.8-6.0.

(5) Rooting culture: transferring the single-plant rootless seedling bud which reaches the rooting standard and is obtained in the step (4) to a rooting induction culture medium to promote rooting;

after rejuvenation culture, the single-plant rootless seedling bud recovers to be strong, the individual grows up, and the seedling with the height of 3 cm is transferred to a rooting induction culture medium to promote rooting; repeatedly rejuvenating the weak and small buds with the height less than 3 cm until the height requirement is met, and then regenerating roots for culturing; after rooting inoculation, culturing for 24-48 hours, and then transferring to illumination culture; wherein, the illumination period of the rooting culture is 16 hours/day, and the illumination intensity is 1500-; the culture temperature is 25 +/-2 ℃, and 2-3 roots can be seen after 30-35 days of culture. After rooting, the domestication culture can be started.

The formula of the rooting induction culture medium is as follows:

1/3MS culture medium (the MS culture medium is original MS culture medium), 0.01-0.1 mg/L Naphthalene Acetic Acid (NAA), 1-5 mg/L indolebutyric acid (IBA), 0.5-1 g/L hydrolyzed Casein (CH), 1-3 mg/L riboflavin, 30g/L sucrose and 5g/L agar, wherein the pH value of the rooting induction culture medium is 5.8-6.0.

(6) Domestication and culture: transferring the seedling buds obtained in the step (5) and growing adventitious roots or the germination of the root primordium to a greenhouse for acclimation under natural light and temperature;

rooting culture for 10-20 days (such as 2 weeks), and observing adventitious root growth and subsequent root primordium germination; transferring the bottle seedlings into a greenhouse facility to be acclimated by natural light and temperature, gradually increasing the temperature amplitude to 25-28 ℃ during the acclimation period and the illuminance to 3000-8000 luxes, and transplanting the rooted seedlings out of the bottles after acclimation for 40-50 days (such as 45 days).

(7) Transplanting tissue culture seedlings: and (4) cleaning the root system of the domesticated tissue culture seedling obtained in the step (6), and then planting the tissue culture seedling.

Taking out the domesticated rooting tissue culture seedling from the bottle, cleaning the culture medium adhered to the root system, and soaking and sterilizing by using 0.1 per mill potassium permanganate solution. A 72-hole sieve tray is used for sieving the mixed matrix of the matrix turfy soil and vermiculite (2:1= V/V). The matrix is thoroughly irrigated and disinfected by carbendazim solution of 75% wettable powder with the concentration of 2.5g/L, the relative humidity in a greenhouse of a facility after planting is 80-90%, and the temperature is kept at 25-28 degrees. Transplanting to field nursery after 3-6 months. After transplanting, the Huabao No. 1 water-soluble fertilizer with the N-P-K ratio of 7-6-19 is used regularly for 10 days per time.

Compared with the prior art, the tissue culture rapid propagation technology of the slash pine seedlings provided by the invention is beneficial to improving the efficiency of slash pine seedling propagation and promoting the development of economic and ecological benefits of forestry industry. The invention overcomes the key point in the production of the slash pine seedling, namely the regeneration of the rootless seedling. Compared with the conventional cuttage means at present, the technology not only greatly promotes the production efficiency of the slash pine seedlings and reduces the cost, but also effectively solves the problems in the tissue culture production at present. Not only is beneficial to improving the precision of the production process, but also improves the consistency of the quality of the commercial seedlings, and is convenient for production management. The method provided by the invention can greatly improve the planning accuracy of production management and mass production, and can effectively reduce the cost and improve the benefit. The method has the advantages of easy operation, low production cost, fast multiplication of the number of the slash pine seedlings and realization of large-scale production. The slash pine seedling cultivated by the method provided by the invention has stable genetic character, maintains the characteristics of parents, and has the advantages of less investment, high yield, short period and the like. Has obvious advantages in effectively improving the production efficiency of the slash pine seedlings and improving the proportion of effective commercial seedlings of the tissue culture seedlings.

The invention obtains a large amount of rootless buds through the cluster bud multiplication way after the germinated seedlings are obtained by the induction culture of the tender stems of the slash pine. Then the rootless bud seedlings are subjected to tissue culture rejuvenation, induced rooting and natural domestication in a greenhouse, and then the seedlings can be transplanted out of bottles. The technique has the advantages of stable inheritance of the bred offspring seedlings, comprehensive character expression and excellent ornamental characters. The technical system has low production cost, environmental protection, no environmental pollution and high mass production speed, and completely meets the technical requirement of large-scale mass production.

The technology also provides valuable reference for the development of seedling production technology of other conifer species.

In order that the invention may be more readily understood, specific embodiments thereof will be described further below.

Example 1, regeneration of slash pine shoot tissue culture:

in this example, tender stem explants of socially grown plants were selected from Wugong mountain forest farm of Ji' an, Jiangxi from Jun for several years, and cultured for propagation test production. Shading pretreatment is carried out before material taking, carbendazim is periodically sprayed for 7 days/time and material taking is carried out after 2 times of spraying. Cutting off needles of tender stems of the explants, flushing the tender stems for 1.5 hours by using running water of a tap, taking out the tender stems and drying the tender stems in the air. Sterilized with 2 drops per liter of a 75% ethanol solution with 1M hydrochloric acid added for 60 seconds and rinsed once with sterile water. After the surface moisture was blotted with absorbent paper, the surface was sterilized with an aqueous bleaching solution having an available chlorine content of 1.5% and TWEEN-20 (3 drops/liter aqueous bleaching solution) on a clean bench for 23 minutes. The explants are washed by sterile water for 3 times, then the surface water is absorbed by absorbent paper and inoculated to an induction culture medium, and the explants are placed in a culture room for culture, observation and recording. The new needles can grow fully in the induction culture medium for 30 days, and are transferred to the propagation culture medium for propagation. The proliferation coefficient of 2-3 times can be obtained in the proliferation culture medium for 25-35 days. After 3-5 generations of multiplication, the single bud is cut and transferred into a rejuvenation culture medium, the height and the robustness of the bud are obviously improved about 30 days, and the bud can be transferred into a rooting culture medium. After the transfer, the cells are cultured for 24 hours and then are transferred to illumination culture, and the occurrence of root systems can be seen after about 2 weeks. The rooting and transplanting can be carried out for about 25 days, the seedlings are transferred from a culture room to a greenhouse for acclimatization and culture, after 45 days, the statistical rooting rate can reach more than 85, the seedlings can be transplanted out of bottles, and the survival rate after the transplanting is more than 90%.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the invention, but the present invention also provides the technology for developing the seedling production technology of other conifer species, and all modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A tissue culture and rapid propagation technology of slash pine seedlings is characterized by comprising the following steps:

2. The tissue culture and rapid propagation technology of slash pine seedlings according to claim 1, wherein the components of the induction medium comprise modified MS medium, 6-benzylamino adenine 0.5-1 mg/L, naphthalene acetic acid 0.1mg/L, sucrose 25g/L and agar 5g/L, wherein the components of the modified MS medium comprise: 2.2 g/L of potassium nitrate, 1.3 g/L of ammonium nitrate, 0.17 g/L of monopotassium phosphate, 0.17 g/L of sodium dihydrogen phosphate, 0.1mg/L of biotin, 1mg/L of L-cysteine and 3 mg/L of glutamine.

3. The tissue culture rapid propagation technology of slash pine seedlings as claimed in claim 2, wherein the propagation medium comprises the following components: 1/2 modified MS culture medium, 6-benzylamino adenine 0.5-1.5 mg/L, naphthylacetic acid 0.02-0.1 mg/L, indolebutyric acid 0.05-0.1mg/L, kinetin 0.5-1 mg/L, sucrose 30g/L and agar 5g/L, wherein the pH value of the enrichment culture medium is 5.8-6.0; or/and

4. The tissue culture rapid propagation technology of slash pine seedlings as claimed in any one of claims 1-3, wherein the step 1 comprises: selecting semi-lignified tender stems of the slash pine plants germinated after stumping treatment as mother plants in the middle ten days of 2-4 months; carrying out shading treatment on the stock plant for 2 weeks before material drawing, regularly spraying broad-spectrum bactericide for 7 days/time, and taking the material after spraying for 2 times; wherein the length of the explant is 2-5 cm; washing with running water for 1-2 hours after material drawing, and then airing until no obvious water stain exists on the surface of the explant; cutting off the needle leaves and only keeping the length of the needle leaves between 2 and 3 millimeters; shaking and sterilizing with 75% ethanol solution containing 2-3 drops of hydrochloric acid (1 mol/L) per liter for 45 s, washing with sterile water, air drying, and sterilizing with bleaching water solution containing 1.5% of available chlorine and TWEEN-20(0.15 ml/L) for 18-25 min to allow the disinfectant to permeate into explant; rinsing with sterile water for 2 min 3-5 times, and then blotting the explant with sterile filter paper to remove water.

5. The tissue culture rapid propagation technology of slash pine seedlings as claimed in any one of claims 1-3, wherein the step 2 comprises: inoculating the explant into the induction culture medium, then placing the explant in a culture chamber for culture, performing dark culture for 2 days, and then transferring the explant into illumination culture, wherein the photoperiod is 12-16 hours/day and the illumination is 1500-ion 3000 lux during the illumination culture; controlling the indoor temperature to be 25 +/-2 ℃; periodically observing the pollution and the induced growth condition during the induction culture period; and transferring the explants into the proliferation culture medium for propagation after 25-30 days.

6. The tissue culture rapid propagation technology of slash pine seedlings as claimed in any one of claims 1-3, characterized in that: cutting off the base of the sterile bud by 1-2 mm, and then transferring the sterile bud to the enrichment medium; or/and

7. The tissue culture rapid propagation technology of slash pine seedlings as claimed in any one of claim 6, characterized in that: after the subculture multiplication number reaches the expected number, dividing the cluster buds into single rootless seedling buds, transferring the single rootless seedling buds into a rejuvenation culture medium, culturing at the temperature of 25 +/-2 ℃, with the illumination period of 16 hours/day and the illumination intensity of 1500-.

8. The tissue culture rapid propagation technology of slash pine seedlings as claimed in claim 7, characterized in that: transferring the seedlings with the height of 3 cm to a rooting induction culture medium to promote rooting; repeatedly rejuvenating the weak and small buds with the height less than 3 cm until the height requirement is met, and then regenerating roots for culturing; after rooting inoculation, culturing for 24-48 hours, and then transferring to illumination culture; wherein, the illumination period of the rooting culture is 16 hours/day, and the illumination intensity is 1500-; culturing at 25 + -2 deg.C for 30-35 days.

9. The tissue culture rapid propagation technology of slash pine seedlings as claimed in claim 8, characterized in that: rooting culture for 10-20 days, allowing adventitious roots to grow out and allowing subsequent root primordium to germinate; transferring the bottle seedlings into a greenhouse facility to be acclimated by natural light and temperature, gradually increasing the temperature amplitude to 25-28 ℃ and the illuminance to 3000 and 8000 luxes during the acclimation, and transplanting the rooted seedlings out of the bottles after the acclimation is carried out for 40-50 days.

10. The tissue culture rapid propagation technology of slash pine seedlings as claimed in claim 9, characterized in that: taking out the domesticated rooting tissue culture seedling from the bottle, cleaning the culture medium adhered to the root system, and soaking and sterilizing by 0.1 per mill potassium permanganate solution. Using 72-hole sieve tray, mixing the matrix peat soil and vermiculite (2:1 ═ V/V). The substrate is thoroughly irrigated and disinfected by carbendazim solution of 75% wettable powder with the concentration of 2.5g/L, the relative humidity in a greenhouse of a facility after planting is 80-90%, and the temperature is kept at 25-28 ℃; transplanting to field nursery after 3-6 months.