CN111406646B - Culture medium for inducing tetraploid paulownia subculture multiplication and application - Google Patents

Abstract

The invention discloses a formula and application of a culture medium for inducing the subculture propagation of tetraploid paulownia. Cutting the aseptic seedlings into stem segments with buds, and transferring the stem segments to a high-efficiency multiplication culture medium, wherein the high-efficiency multiplication culture medium is formed by adding 0.05-5.0mg/L of indoleacetic acid, 0.5-14.0mg/L of 6-benzylaminopurine, 0.05-5.0mg/L of kinetin, 0.05-5.0mg/L of naphthylacetic acid, 0.05-5.0mg/L of spermidine, 4.0-6.0g/L of agar and 30-50g/L of cane sugar into an MS culture medium, and the culture conditions are that the day temperature is 25 ℃, the night temperature is 18 ℃, the illumination intensity is 2500Lx and the illumination time is 13 h/d. After 20 days of culture, the proliferation times can reach 30-40 times at most. The method obviously promotes the efficient proliferation of the tetraploid paulownia, effectively shortens the cultivation period and improves the seedling raising efficiency. Provides technical support for the industrial production of the tetraploid paulownia fine strain and solves the problem of serious short supply of seedlings of the tetraploid paulownia fine strain.

Description

Culture medium for inducing tetraploid paulownia subculture multiplication and application

Technical Field

The invention relates to a formula and application of a culture medium for inducing tissue culture and efficient proliferation of tetraploid paulownia, which are particularly suitable for subculture proliferation in an industrial seedling raising early stage and belong to the technical field of biology.

Background

Paulownia is a plant of the genus Paulownia of the family scrophulariaceae, is native to China, has no flying cotton, beautiful tree body and long cultivation history, is naturally distributed in 25 provinces, cities and autonomous regions of China, and is an important fast growing material and a garden greening local tree species of China. The leaves, flowers, fruits and barks of paulownia can be used as medicine, and the leaves and flowers of paulownia can be used as feed. The paulownia wood is high-quality and high-grade, corrosion-resistant, acid and alkali-resistant, wear-resistant, easy to process, easy to carve and etch, long in fiber, light in color, easy to dye, fine and smooth in texture, natural in color and luster, attractive in pattern, uniform in structure, good in sound guide, not prone to splitting, not prone to deformation, not prone to burning, not prone to being damaged by worms, not prone to deformation, not prone to burning, and deeply popular with domestic and foreign consumers. It is suitable for making musical instrument, aviation, ship model, plywood, life saving apparatus, toy, gift box, etc. and may be also used in building, furniture, artificial board, paper making, packing box, etc.

According to investigation, the resources of paulownia are about 5 hundred million plants in China, wherein about 1 hundred million plants (Chandelong, 2016) exist in Henan province, and 500 to ten thousand meters of paulownia wood is produced annually³. Due to the fast growth, strong adaptability and high economic value of paulownia, the paulownia is introduced or planted by a plurality of countries of Asia, Europe, Africa, North America, south America and oceania. About 1 hundred million paulownia strains exist in Japan, and the annual output is more than 100 ten thousand meters³Paulownia wood; paulownia is nearly 1 hundred million strains in australia; there are about 7000 million plants in the United states in North America, and the annual production of paulownia wood is nearly 100 ten thousand m³(ii) a The number of Brazil, Paraguay, and Uguay in south America is about 3 hundred million plants in the year300 more than ten thousand meters of tung wood³(Chandelong, 2016).

Tetraploid Paulownia (Tetraploid Paulownia) is obtained by double-breeding the chromosome number of diploid Paulownia. Since 2006, the southern agricultural university successfully induced and cultivated one after another with colchicine into autotetraploid plants of paulownia tomentosa, paulownia elongata, paulownia uniflora, paulownia margarita, paulownia uniflora, 9501, paulownia catalpa and paulownia kawakamii (caoyun spring, 2006; weizhen, 2008; fan nationality, 2009; fan nationality, 2010; zhao libi, 2014; populus queuensis, 2014). Compared with diploid paulownia, the tetraploid paulownia has the advantages of faster growth, better material quality, enhanced stress resistance, high natural extension rod rate, obviously reduced arbuscular diseases and the like. A tetraploid paulownia seed root with the length of 10 cm is buried in the early year of the Town Panmobun in Taian, the average tree height is measured at the bottom of the year and is 5m, the average breast diameter is 5 cm, the maximum tree height can reach 8 m, the maximum breast diameter is 8 cm thick and is called as a fast-growing representative in fast-growing tree seeds, each seedling in the current year can be sold to about 30 yuan, and the seedling is called as a money tree which is lovely by common people and is popular with vast forest farmers.

China is the second large wood consumer country and the first large wood import country in the world, the external dependence of wood is over 50 percent, and the safety of wood supply becomes a major strategic problem to be solved urgently in China. The tetraploid paulownia as the new species of the current fastest growing timber tree species in China can just bear the important mission which is provided by the times and meets the national timber supply, and plays an important role in relieving the condition of wood shortage in China, promoting the adjustment of rural industrial structure and promoting the development of national economy.

However, since the emergence of tetraploid paulownia in 2006, seed roots have been used as propagation materials for production. The seed root is preferably a root obtained by pruning 1-year-old seedling (guoshenhua, 2019). Due to the limited propagation material of the seed root, the tetraploid paulownia seedlings have serious short supply and demand. The tissue culture seedling raising method is not limited by geographical environment and seasons, can achieve rapid and efficient large-scale seedling breeding, can also achieve the effect of detoxification, and fundamentally reduces the morbidity of the tetraploid paulownia witches broom. The Caoyanchun (2006) is prepared from tetraploid paulownia tomentosa,The leaves of the tetraploid paulownia elongata and the tetraploid paulownia elongata aseptic seedlings are taken as test materials, and three tetraploid paulownia elongata in-vitro regeneration systems are established. The optimal culture medium for callus induction is MS +0.3mg/L NAA +14.0mg/L BA, MS +0.3mg/L NAA +8.0mg/L BA, MS +0.1mg/L NAA +10.0mg/L BA, and the optimal culture medium for bud induction is MS +0.9mg/L NAA +16.0mg/L BA, MS +0.7mg/L NAA +14.0mg/L BA, MS +0.1mg/L NAA +12.0mg/L BA. The Weizhenzhen (2008) uses tetraploid Yuza I and tetraploid southern paulownia leaf, petiole and stem section which grow on MS culture medium as explant to make callus induction, after 20 days of culture, the optimum culture medium for callus induction of different explants is determined. Then cutting the callus induced by the leaf blade on the optimal culture medium to about 1.0cm²And (5) carrying out adventitious bud induction on the small pieces, and determining an optimal culture medium for bud induction after culturing for 20 days. Considering the factors of callus shape, texture, bud induction capability and the like, the leaf is considered to be the optimal explant for callus induction of tetraploid paulownia yuza I and tetraploid paulownia south, and MS +0.5mg/L NAA +16.0mg/L BA and MS +0.3mg/L NAA +12.0mg/L BA are respectively the optimal culture medium for callus induction. MS +0.5mg/L NAA +16.0mg/L BA and MS +0.3mg/L NAA +12.0mg/L BA are respectively the most suitable culture medium for callus bud induction. Zhao Zhenli (2011) shows that the leaf of paulownia 9501 is the best explant, and the optimal culture for callus, bud and root induction is MS +0.7mg/LNAA +6.0mg/L BA and MS +0.7mg/L NAA +8.0mg/L BA respectively. The Wang Yang (2014) takes the leaves and petioles of the tetraploid paulownia catalpa and the paulownia taiwanensis as explants, and performs the selection of the optimal explants and the selection of the optimal proliferation and rooting culture medium on the basis of the selection. The research result shows that the best explants of the tetraploid paulownia catalpa bungei and the tetraploid paulownia taiwan are both leaves, the optimal culture mediums for inducing the callus of the two paulownia leaves are MS +0.3mg/L NAA +14.0mg/L BA and MS +0.1mg/L NAA +14.0mg/L BA respectively, and the optimal culture mediums for inducing the bud are MS +0.3mg/L NAA +16.0mg/L BA and MS +0.3mg/L NAA +16.0mg/L BA respectively.

Tissue culture experiments are carried out in the laboratory by using the formula reported in the existing literature, the propagation coefficient is very low, and the requirement of large-scale seedling culture cannot be met, which is probably a key reason why the tissue culture technology is not used for carrying out the industrialized seedling culture of the tetraploid paulownia for more than 10 years. The traditional tissue culture research of tetraploid paulownia adopts a two-step induction method, namely, callus is induced firstly, then adventitious buds are induced from the callus, and at least 2 culture cycles are needed.

Therefore, the development of a tetraploid paulownia high-efficiency propagation medium formula is urgently needed, the propagation coefficient is rapidly improved, the cultivation period is effectively shortened, the seedling raising efficiency is improved, and the bottleneck problem of restricting the industrialized tissue culture seedling raising of the tetraploid paulownia is solved. The method has important ecological, economic and social significance for rapidly expanding the asexual propagation of the excellent tetraploid paulownia strains, solving the problem of serious short supply and short demand of tetraploid paulownia seedlings, relieving the contradiction between supply and demand of wood in China, improving the ecological environment, improving the living standard of people and the like.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the method and the formula of the culture medium for inducing the subculture propagation of the tetraploid paulownia to efficiently proliferate are provided, so that the cultivation period of the tetraploid paulownia cultivated in an industrialized mode is shortened as much as possible, the propagation coefficient is improved, technical support is provided for the industrialized production of the excellent strain of the tetraploid paulownia, and the problem that seedlings of the excellent strain of the tetraploid paulownia are seriously short in supply and short in demand is solved.

The technical scheme of the invention is as follows: the culture medium for inducing the tissue culture of the tetraploid paulownia comprises an MS culture medium added with 0.05-5.0mg/L of indoleacetic acid (IAA), 0.5-14.0mg/L of 6-benzylamino adenine (6-BA), 0.05-5.0mg/L of Kinetin (KT), 0.05-5.0mg/L of naphthylacetic acid (NAA), 0.05-5.0mg/L of spermidine (Spd), 4.0-6.0g/L of agar and 30-50g/L of cane sugar.

The invention discloses application of a culture medium for inducing the subculture propagation of tetraploid paulownia to tissue culture of the tetraploid paulownia.

A method for directly inducing and regenerating adventitious buds from tetraploid paulownia explants comprises the steps of shearing aseptic seedlings into stem segments with buds, and transferring the stem segments to a high-efficiency multiplication culture medium, wherein the high-efficiency multiplication culture medium is formed by adding 0.05-5.0mg/L indoleacetic acid (IAA), 0.5-14.0 mg/L6-benzylamino adenine (6-BA), 0.05-5.0mg/L Kinetin (KT), 0.05-5.0mg/L naphthylacetic acid (NAA), 0.05-5.0mg/L spermidine (Spd), 4.0-6.0g/L agar and 30-50g/L sucrose into an MS culture medium, and the culture conditions comprise 25 ℃ of day temperature, 18 ℃ of night temperature, 2500Lx of illumination intensity and 13h/d of illumination time.

Compared with the prior art, the invention has the following beneficial effects:

the method has the advantages that various plant growth regulators are used in a combined mode, the callus induction link is deleted, the explant stem is directly induced to regenerate a new plant, at least 1 culture period can be saved, manpower, material resources and financial resources are effectively saved, efficient multiplication of tetraploid paulownia is remarkably promoted, the culture period is effectively shortened, and the seedling raising efficiency is improved. Provides technical support for the industrial production of the excellent tetraploid paulownia strains. The tetraploid paulownia subculture propagation efficient propagation culture medium formula prepared by the invention can obviously promote the efficient propagation of the tetraploid paulownia, and the maximum propagation multiple reaches 30-40 times after 20 days of culture.

Drawings

FIG. 1 is a photograph of the day after transfer to a high-efficiency proliferation medium.

FIG. 2 is a diagram showing that adventitious buds are directly regenerated after culturing for 6 days by transferring to a high-efficiency multiplication medium.

FIG. 3 example 1 Cluster buds were efficiently regenerated after 25 days of culture.

FIG. 4 example 2 efficient regeneration of clumped shoots after 25 days of culture.

FIG. 5 example 3 Cluster shoots were efficiently regenerated after 25 days of culture.

FIG. 6 high efficiency regeneration of clumpy buds after 25 days of culture at different hormone concentrations.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.

Example 1

The aseptic seedlings are cut into long-strip bud stem segments of about 2cm on an ultra-clean workbench and are transferred to an efficient multiplication culture medium of MS + IAA 0.05mg/L +6-BA 0.5mg/L + KT 0.05mg/L + NAA 0.05mg/L + Spd 0.05mg/L + agar 4.5g/L + sucrose 30g/L (pH value 5.8), the culture conditions are that the day temperature is 25 ℃, the night temperature is 18 ℃, the illumination intensity is 2500Lx and the illumination time is 13 h/d. After 25 days, cluster buds are induced to form, and the multiplication times are 15-20 times, as shown in figure 3.

Example 2

The aseptic seedlings are cut into long-belt bud stem sections of about 2cm on an ultra-clean workbench and are transferred to an efficient multiplication culture medium of MS + IAA 0.5mg/L +6-BA 4.0mg/L + KT 0.5mg/L + NAA0.5mg/L + Spd 0.5mg/L + agar 4.5g/L + sucrose 40g/L (pH value 5.8), the culture conditions are that the day temperature is 25 ℃, the night temperature is 18 ℃, the illumination intensity is 2500Lx and the illumination time is 13 h/d. After 25 days, cluster buds are induced to form, and the multiplication times are 30-40 times, as shown in figure 4.

Example 3

Cutting the aseptic seedlings into long-belt bud stem sections of about 2cm on an ultraclean workbench, and transferring the long-belt bud stem sections to an efficient multiplication culture medium containing MS, IAA 5.0mg/L, 6-BA14.0mg/L, KT 5.0mg/L, NAA 5.0mg/L, Spd 5.0mg/L, agar 4.5g/L and cane sugar 50g/L (pH value 5.8), wherein the culture conditions comprise a day temperature of 25 ℃, a night temperature of 18 ℃, an illumination intensity of 2500Lx and an illumination time of 13 h/d. After 25 days, cluster buds are induced to form, and the multiplication times are 16-23 times, as shown in figure 5.

FIG. 3, FIG. 4 and FIG. 5 show the multiple of the growth of the regenerated multiple shoots after 25 days of culture using the medium of the present invention, which is 15-40 times.

Claims (3)

1. A culture medium for inducing the subculture multiplication of tetraploid paulownia bud stem segments is characterized in that the culture medium is composed of an MS culture medium added with 0.05-5.0mg/L of indoleacetic acid, 0.5-14.0mg/L of 6-benzylaminopurine, 0.05-5.0mg/L of kinetin, 0.05-5.0mg/L of naphthylacetic acid, 0.05-5.0mg/L of spermidine, 4.0-6.0g/L of agar and 30-50g/L of cane sugar.

2. The use of the subculture propagation medium for inducing the stem segments with buds of the tetraploid paulownia as claimed in claim 1 in tissue culture of the tetraploid paulownia.

3. The method for carrying out the secondary propagation of the tetraploid paulownia by applying the secondary propagation culture medium is characterized in that sterile seedlings are cut into stem sections with buds and transferred to the secondary propagation culture medium, wherein the secondary propagation culture medium is prepared by adding 0.05-5.0mg/L of indoleacetic acid, 0.5-14.0mg/L of 6-benzylamino adenine, 0.05-5.0mg/L of kinetin, 0.05-5.0mg/L of naphthylacetic acid, 0.05-5.0mg/L of spermidine, 4.0-6.0g/L of agar and 30-50g/L of sucrose into an MS culture medium, the pH value is 5.8, the culture conditions are that the day temperature is 25 ℃, the night temperature is 18 ℃, the illumination intensity is 2500Lx and the illumination time is 13 h/d.

Images