阅读说明：本技术 一种适合工厂化生产的彩叶芋的组培快繁方法 (Tissue culture rapid propagation method of colocasia esculenta suitable for industrial production ) 是由 官锦燕 谭嘉娜 罗青文 罗剑飘 文明富 许玉婵 黄海英 于 2020-12-17 设计创作，主要内容包括：本发明属于组织培养技术领域,是基于泰国引进的彩叶芋新品种进行2年的实验研究和生产基础上总结归纳的适合工厂化生产组培快繁研究方法,可操作性强,完全可用于指导生产。本发明首次建立了一套完整的具有高诱导率及增殖倍数的适合工厂化生产的诱导、增殖、生根和炼苗的快繁方法,形成批量快速的繁殖局面,通过控制增殖代数、培养基激素浓度和配比随着代数的变化有所改变,确保种苗的稳定性、活力与增殖系数,提高产量和质量；本发明采用边增殖边生根的生产方式,降低生产成本的同时加快生产进度,保证增殖系数的同时确保出根苗的质量,完全适合大规模工厂化的生产,极大地提高彩叶芋繁育的效率。(The invention belongs to the technical field of tissue culture, and relates to a tissue culture and rapid propagation research method suitable for industrial production, which is summarized and summarized on the basis of 2-year experimental research and production of new variety of colocasia esculenta introduced in Thailand, has strong operability and can be completely used for guiding production. The invention establishes a set of complete rapid propagation methods with high inductivity and multiplication multiple and suitable for industrial production for induction, multiplication, rooting and seedling hardening for the first time, forms a batch rapid propagation situation, ensures the stability, the activity and the multiplication coefficient of the seedlings and improves the yield and the quality by controlling the multiplication algebra, the concentration of culture medium hormone and the proportion to be changed along with the change of the algebra; the invention adopts a production mode of proliferation and rooting, reduces the production cost, accelerates the production progress, ensures the proliferation coefficient and ensures the quality of rooted seedlings, is completely suitable for large-scale industrialized production, and greatly improves the breeding efficiency of the colocasia esculenta.)

1. A tissue culture medium of colocasia esculenta suitable for industrial production is characterized by comprising a callus induction culture medium, a bud induction culture medium, a proliferation culture medium and a rooting culture medium;

the callus induction culture medium is a basal culture medium, 3.0 mg/L6-BA and 0.2mg/L NAA;

the bud induction culture medium is a basic culture medium, 2.0 mg/L6-BA and 0.2mg/L NAA;

the enrichment culture medium comprises an enrichment culture medium A and an enrichment culture medium B, wherein the enrichment culture medium A is a basal culture medium +1.5 mg/L6-BA +0.2mg/L NAA, and the enrichment culture medium B is a basal culture medium +1.0 mg/L6-BA +0.1mg/L NAA;

the rooting culture medium is a nutrient culture medium, 0.4mg/L IBA and 0.2mg/L NAA;

wherein the basic culture medium comprises the following components: adding sucrose and carrageenan into an MS culture medium; the nutrient medium comprises the following components: MS culture medium, adding active carbon, sucrose and agar.

2. A tissue culture and rapid propagation method of colocasia esculenta suitable for industrial production is characterized by comprising the following steps:

s1, callus induction culture: taking the rolling-rod-shaped tender leaves of the taro as explants, and inoculating the disinfected taro into a callus induction culture medium for callus induction;

s2, bud induction culture: inoculating the callus induced by S1 into a bud induction culture medium for culture, and differentiating cluster buds and embryoids by strong light stimulation;

s3, sequentially inoculating the germinated cluster buds and the embryoids to a multiplication culture medium A for culture in a multiplication culture stage I, or sequentially inoculating the germinated cluster buds and the embryoids to a multiplication culture medium B for culture in a multiplication culture stage II to differentiate plants and embryoids;

wherein the proliferation culture stage I refers to the 5 th-9 th generation of the growth of the colocasia esculenta; the proliferation culture stage II refers to the 10 th-13 th generation of the growth of the colocasia esculenta;

s4, rooting culture: cutting the stout plant in S3 into single plants, inoculating the single plants into a rooting culture medium for culturing, transferring the thin and weak cluster buds and embryoids into a propagation culture medium of S3 for propagation culture, and repeating the transfer of S3 and S4 from the 4 th generation of the growth of the colorama taro.

3. The tissue culture and rapid propagation method of colocasia esculenta suitable for industrial production according to claim 2, wherein the sterilization method of the explant of S1 is as follows: processing tender leaf of Colocasiana edulis roll with 75% alcohol solution and mercuric chloride solution, and washing with sterile water.

4. The tissue culture and rapid propagation method of colocasia esculenta suitable for industrial production according to claim 2, wherein S4 is operated as follows: and when the seedlings grow to 2-5 cm and the number of leaves is 3, transferring the seedlings into a rooting culture medium for culture, and transferring the remaining buds and embryoids back into the propagation culture medium of S3.

5. The tissue culture and rapid propagation method of colocasia esculenta suitable for industrial production according to claim 2, wherein S4 rooted seedlings are transplanted to the outside for culture when the length is 5-7 cm and the number of the rooted seedlings is more than 8.

6. The tissue culture and rapid propagation method of colocasia esculenta suitable for industrial production according to claim 5, wherein the transplanting is hardening seedling transplanting, and bag seedlings with successful rooting are placed in a greenhouse for hardening seedlings for 7-10 days, cleaned, treated with 1000 times of carbendazim for 10min, and then cultured in a matrix.

7. The tissue culture and rapid propagation method of colocasia esculenta suitable for industrial production according to claim 5, wherein the matrix is prepared from imported peat soil: perlite in a volume ratio of 3: 1 mixing the components.

Technical Field

The invention relates to the technical field of tissue culture, in particular to a tissue culture rapid propagation method of colocasia esculenta suitable for industrial production.

Background

Taro (caladium bicolor), also known as colocasia esculenta, belongs to the genus taro of the family Araceae, native south America, has gorgeous leaves, is elegant and clear, is durable and ornamental, is a top-grade product in foliage plants, can well grow in indoor weak light, and is a popular indoor foliage plant at present.

At present, the colocasia esculenta is mainly propagated by tuber division, but the tuber propagation is limited, at most 3-4 seedlings can be generated by one good tuber once a year, the propagation rate is low, and the growth is slow. The commercial propagation can also pass through seeds, but the propagation difficulty of the seeds is high because the seeds are very small and have high death rate, the cost of plants obtained by the growth and the propagation of the seeds is too high, and the offspring propagated by the seeds is easy to mutate and degenerate and is difficult to maintain the original characters.

The tissue culture technology is one of the most effective methods for plant rapid propagation at present, has the advantages of high propagation coefficient, short seedling time and convenience for commercialization and industrial production, and can maintain the excellent properties of the variety. At present, a plurality of companies and nurseries for producing the colorama have started to utilize tissue culture technology to carry out mass production, and rapidly propagate the colorama plants which can maintain characters and are free of viruses.

In the prior art, the tissue culture research report of the colocasia esculenta is mainly to carry out tissue culture and rapid propagation research on a single variety. And few systematic researches on tissue culture and rapid propagation of the colocasia esculenta suitable for factory production are carried out.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the technical defects in the prior art and provides the tissue culture and rapid propagation method of the colocasia esculenta suitable for industrial production.

The purpose of the invention is realized by the following technical scheme:

a tissue culture medium for colorfully-grown colocasia esculenta suitable for industrial production comprises a callus induction culture medium, a bud induction culture medium, a proliferation culture medium and a rooting culture medium;

the callus induction culture medium is a basal culture medium, 3.0 mg/L6-BA and 0.2mg/L NAA;

the bud induction culture medium is a basic culture medium, 2.0 mg/L6-BA and 0.2mg/L NAA;

the enrichment culture medium comprises an enrichment culture medium A and an enrichment culture medium B, wherein the enrichment culture medium A is a basal culture medium +1.5 mg/L6-BA +0.2mg/L NAA, and the enrichment culture medium B is a basal culture medium +1.0 mg/L6-BA +0.1mg/L NAA;

the rooting culture medium is a nutrient culture medium, 0.4mg/L IBA and 0.2mg/L NAA;

wherein the basic culture medium comprises the following components: adding sucrose and carrageenan into an MS culture medium; the nutrient medium comprises the following components: MS culture medium, adding active carbon, sucrose and agar.

The invention defines the algebra of the industrial production control of the colocasia esculenta, optimizes the component content and the culture time of the culture medium used in each culture stage, and can completely adopt the culture in the modes of proliferation and rooting at the same time in production. Through a great deal of experimental research, the inventor finds that the hormones used in the whole production process from callus induction to the last generation of multiplication culture are reduced, and the algebraic range of culture in each stage has great influence on the yield and the cost: callus induction (first 2 generations) -shoot induction (3, 4 generations) -proliferation culture stage I (5-9 generations) -proliferation culture stage II (10-13 generations) -rooting culture (beginning of 4 generations).

The invention also provides a tissue culture and rapid propagation method of the colocasia esculenta suitable for industrial production, which comprises the following steps:

s1, callus induction culture: taking the rolling-rod-shaped tender leaves of the taro as explants, and inoculating the disinfected taro into a callus induction culture medium for callus induction;

s2, bud induction culture: inoculating the callus induced by S1 into a bud induction culture medium for culture, and differentiating cluster buds and embryoids by strong light stimulation;

s3, sequentially inoculating the germinated cluster buds and the embryoids to a multiplication culture medium A for culture in a multiplication culture stage I, or sequentially inoculating the germinated cluster buds and the embryoids to a multiplication culture medium B for culture in a multiplication culture stage II to differentiate plants and embryoids;

wherein the proliferation culture stage I refers to the 5 th-9 th generation of the growth of the colocasia esculenta; the proliferation culture stage II refers to the 10 th-13 th generation of the growth of the colocasia esculenta;

s4, rooting culture: cutting the stout plant in S3 into single plants, inoculating the single plants into a rooting culture medium for culturing, transferring the thin and weak cluster buds and embryoids to a propagation culture medium of S3 for propagation culture, and repeating the transfer of S3 and S4 from the 4 th generation of the growth of the colorama.

Preferably, the culture time of the proliferation culture medium A of S3 is 200-225 days, and each generation is 40-45 days; the culture time of the proliferation culture medium B is 160-180 days, and each generation is 40-45 days.

S1 inducing and culturing to induce granular callus from tender leaf of Coleus blume. Generally, the callus induction time is 20-30 days after the callus is inoculated to an induction culture medium, 35-50 days (related to the variety) are also available, and the colocasia esculenta can be inoculated to the bud culture medium S2 after callus blocks are completely induced to proliferate for one generation.

Conditions for S1 induction of calli: under the condition of total darkness, the temperature is 25 ℃; the culture conditions of S2 were: the illumination time is 10-12 h per day, the intensity is 2000lux, and the temperature is 25 ℃.

Preferably, the callus pieces are cultured into embryoid bodies or buds at S2 for 30 days after being inoculated onto the bud induction medium; and S4, performing rooting culture to obtain complete plants with roots. The time for obtaining the complete plant with roots is 20 days after the complete plant is inoculated on a rooting culture medium.

Preferably, the method for disinfecting the explant in S1 comprises the following steps: processing tender leaf of Colocasiana edulis roll with 75% alcohol solution and mercuric chloride solution, and washing with sterile water.

The concentration and time of the disinfectant are strictly controlled to prevent the explant cells from being killed and influence the induction rate. Alcohol has strong permeability, and the alcohol enters into cells of bacteria to denature protein, so that the bacteria are prevented from being killed, tender plant cells are prevented from being damaged, and the alcohol treatment mode is cotton wiping.

Specifically, the mass volume concentration of the mercuric chloride of S1 is 0.1-0.2 g/L, and the treatment time is 5-15 min. More preferably, the mass volume concentration of the mercury bichloride is 0.1g/L, and the soaking time is 7 min. The mercuric chloride denatures proteins of germs and has a stronger disinfection capacity on bacteria than fungi.

Preferably, S4 operates as: and when the seedlings grow to 2-5 cm and the number of leaves is 3, transferring the seedlings to a rooting culture medium for culture, and transferring the remaining buds and embryoids back to S3 for enrichment culture.

Preferably, S4 rooted seedlings are transplanted to outdoor for culture when the seedlings grow to 5-7 cm and the number of the S4 rooted seedlings is more than 8.

Preferably, the transplanting is hardening seedling transplanting, namely, the bagged seedlings which successfully take root are placed in a greenhouse for hardening seedlings for 7-10 days, cleaned, treated with 1000 times of carbendazim for 10min and then cultivated in a matrix.

Preferably, the matrix consists of imported peat soil: perlite in a volume ratio of 3: 1 mixing the components.

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

the tissue culture and rapid propagation research method is suitable for industrial production and is summarized and summarized on the basis of 2 years of experimental research and production of new variety of colocasia esculenta introduced in Thailand, strong in operability and completely applicable to production guidance.

The invention establishes a set of complete rapid propagation methods with high inductivity and multiplication multiple and suitable for industrial production for induction, multiplication, rooting and seedling hardening for the first time, forms a batch rapid propagation situation, ensures the stability, the activity and the multiplication coefficient of the seedlings and improves the yield and the quality by controlling the multiplication algebra, the concentration of culture medium hormone and the proportion to be changed along with the change of the algebra; the method adopts a production mode of proliferating and rooting at the same time, adopts a production mode of proliferating and rooting at the same time from the 4 th generation, has normal seedling shape in the propagation process, has a monthly root growth coefficient of 1.5-2 times, and has excellent seedling quality; the monthly propagation coefficient reaches 3-4 times, the cluster bud propagation activity is strong, and the degradation phenomenon is avoided; ensures the quality of rooted seedlings while ensuring the multiplication coefficient, is completely suitable for large-scale industrial production, and greatly improves the breeding efficiency of the colocasia esculenta.

Drawings

FIG. 1 is a photograph of cultivated Coleus edulis C9;

FIG. 2 is a photograph of different culture generations in the tissue culture process of colocasia esculenta C9;

FIG. 3 is a photograph of different culture generations in the tissue culture process of colocasia esculenta C3;

FIG. 4 is a photograph of different culture generations in the tissue culture process of colocasia esculenta C8;

FIG. 5 is a photograph of different culture generations in the tissue culture process of colocasia esculenta C10;

FIG. 6 is a photograph of different numbers of culture generations in the tissue culture process of colocasia esculenta C13.

Detailed Description

The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1 Effect of different Induction media on the Induction of Doudnera edulis callus

Collecting un-unfolded rolling rod-shaped tender leaves of Colocasiana edulis, wiping with 75% alcohol cotton, and packaging into a clean bag. Sterilizing with 0.1% mercuric chloride on a clean bench for 7min, and washing with sterile water for 4 times. Cut into 1mm slices, inoculated with an induction medium (the specific formula of the induction medium is shown in table 1) and cultured for callus induction. Shading with black cloth, and culturing completely in dark.

The induction culture medium consists of a basic culture medium and a hormone combination, wherein the basic culture medium comprises the following components: MS culture medium, 30g/L of cane sugar, 7g/L of carrageenan and 5.8 of pH value.

10 young leaf slices were inoculated per treatment and the experiment was repeated 3 times. And (4) counting the healing time and the healing rate according to the growth condition.

The average recovery rate is the total number of callus pieces/total number of explants.

TABLE 1

As can be seen from Table 1, different hormone concentration ratios have great influence on the callus induction rate, the callus growth time and the callus state. The 6-BA is less than 2.0, qualified callus is difficult to induce, callus is easy to induce when the 6-BA is between 2.0 and 4.0, the callus-out time is in a descending trend, and the formula of 3.0mg/L of 6-BA and 0.2mg/L of NAA is most suitable for inducing callus of colocasia esculenta in combination with the later callus-induced budding condition.

Example 2 Effect of different shoot Induction Medium on shoot Induction culture of Colocania

The callus induced in example 1 was inoculated into a shoot induction medium (see Table 2 for the formulation of shoot induction medium) for shoot induction culture. The culture light intensity is 2000lux, and the light time is 12 hours/day.

The bud induction culture medium consists of a basic culture medium and a hormone combination, wherein the basic culture medium comprises the following components: MS culture medium, 30g/L of cane sugar, 7g/L of carrageenan and 5.8 of pH value.

20 shoot segments were inoculated per treatment and the experiment was repeated 3 times. And (4) counting the induction time, induction rate and bud state of corresponding embryoids and multiple buds according to the growth condition.

TABLE 2 Effect of different hormone combinations on the Induction of colocasia esculenta bud and embryoid bodies

As shown in Table 2, different hormone combinations have a great influence on the bud induction time, the induction rate and the growth vigor, and the colocasia esculenta buds and embryoids are induced simultaneously, and the embryoids are precursors of multiple buds with each generation. When the amount of 6-BA is 3.0mg/L, the number of clumpy buds is large and strong, but the embryoid body is translucent and easily causes the variation of the next generation seedling. When the 6-BA is 2.0mg/L and the NAA is 0.1mg/L or 0.2mg/L, the cluster buds and the embryoid bodies are normal, the 2.0mg/L + NAA of the 6-BA is 0.2mg/L, the inductivity is the highest, and the cluster buds are more robust and are the better hormone combination. 6-BA is 1.0mg/L, and cluster buds and embryoid bodies are difficult to induce due to too low hormone.

EXAMPLE 3 Effect of different multiplication media on the multiplication culture of colocasia esculenta

The clumpy buds and embryoid bodies induced in example 2 were inoculated into a growth medium (the specific formulation of the growth medium is shown in Table 3) and cultured for growth. The culture illumination intensity is 1500-2000 lux, and the illumination time is 12 hours/day.

The proliferation culture medium consists of a basic culture medium and a hormone combination, wherein the basic culture medium comprises the following components: MS culture medium, 30g/L of cane sugar, 7g/L of carrageenan and 5.8 of pH value.

The data of this stage are the average rooting and proliferation coefficient and the growth vigor of the seedlings obtained by counting the corresponding data in the experiment and mass production.

TABLE 3 Effect of different hormone combinations on Coleus blume bud and embryoid proliferation

As can be seen from Table 3, the algebraic influence of different hormone combinations on the growth of the colocasia esculenta is large, and in the 5 th to 9 th generations, the higher hormone (1.0<6-BA <2.0) is beneficial to the proliferation and rooting of the colocasia esculenta, the seedlings are thicker, and the buds and leaves are more. In the 10 th to 13 th generations, the high hormone is not beneficial to the proliferation of the colocasia esculenta, the budding amount is small, and the size is not uniform; at the moment, the lower hormone combination is beneficial to the proliferation and rooting of high-generation colocasia esculenta. Because endogenous hormones in plants are continuously accumulated along with the increase of the generation number, the required exogenous hormones are reduced when the generation number is higher. According to a large number of experiments and production analysis, 1.5mg/L of 6-BA and 0.2mg/L of NAA are the best hormone combination in the 5 th to 9 th generations, the rooting and proliferation coefficients are the highest, and seedlings are strong, large in quantity and uniform. In the 10 th-13 th generation, 1.0mg/L of 6-BA and 0.1mg/L of NAA are the best hormone combination, the rooting and proliferation coefficients are higher, and the seedlings are robust and have a large number.

Example 4 Effect of different rooting media on rooting of tissue culture seedlings of Colocasianus Koreana

And cutting out a single plant while proliferating and inoculating the single plant to a rooting culture medium (the specific formula of the rooting culture medium is shown in table 4) for rooting culture, wherein the culture illumination intensity is 2000-2500 lux, and the illumination time is 12 hours/day. And (4) counting the average rooting rate, the root condition and the plant growth vigor according to experimental and production data.

The rooting culture medium consists of a basic culture medium and a hormone composition, wherein the basic culture medium comprises the following components: MS culture medium, 0.3g/L of active carbon, 30g/L of cane sugar, 7g/L of agar and 5.8 of pH value.

The rooting rate is that the rooting plant tree/total plant tree is multiplied by 100%.

TABLE 4 influence of different hormone ratios on the rooting of colocasia esculenta tissue culture seedlings

Serial number	Hormone combination (mg/L)	Rooting percentage (%)	Root condition of the root system	Growth vigor of plants
					1	IBA 0.2	100	4 to 6 pieces of root system	The leaf stalk is thin and weak, and the leaf stalk is thin and weak,yellow leaves
2	IBA 0.2+NAA0.1	100	Good root system, 6-8 strips	Strong petiole and green leaf
					3	IBA 0.4+NAA 0.2	100	8-12 developed root systems	Robust, large leaf and emerald green
4	IBA 0.5	100	6-8 developed root systems	Thick petiole and green leaf

As shown in Table 4, colocasia esculenta is easy to root, and different hormone combinations have obvious influence on the root condition and the plant growth. Within a certain range, the higher the hormone, the more favorable the growth of plants, the faster the new leaves come out, the stout leaf stems, the bigger and emerald leaves. IBA 0.4mg/L + NAA 0.2mg/L is the best hormone combination, and the plants are obviously the most robust, tall and big, and have large leaves and emerald green.

Comparative example 1 comparison of the Effect of NAA and IBA on the multiplication culture of Colocania insignis

Based on the optimal shoot induction culture and proliferation medium selected in example 2 and example 3, the basal medium, cytokinin 6-BA concentration and auxin concentration were unchanged, and the type of auxin was changed (i.e., NAA was used instead of IBA) to prepare a proliferation medium.

The culture medium consists of a basic culture medium and a hormone combination, wherein the basic culture medium comprises the following components: MS culture medium, 30g/L of cane sugar, 7g/L of carrageenan and 5.8 of pH value.

TABLE 5 comparison of the effects of NAA and IBA on the induction culture of colocasia esculenta buds

Serial number	Hormone combinations	Induction time (d)	Bud induction ratio (%)	Growth vigor of bud and embryoid
					1	BA 2.0+NAA 0.2	25～30	95	Numerous and strong clumpy buds, and a verdure embryoid
2	BA 2.0+IBA 0.2	30～40	75％	Less cluster buds and pale yellow embryoid

TABLE 6 comparison of the effects of NAA and IBA on the multiplication culture of colocasia esculenta buds

As can be seen from tables 5 and 6: in the bud induction and bud multiplication culture medium, NAA and IBA have obvious difference on the growth of the colocasia esculenta, NAA is more beneficial to the induction of callus, the induction and multiplication of buds and the growth of seedlings of the colocasia esculenta, and compared with IBA, the induction and promotion effects on the colocasia esculenta are poor. Therefore, from the view of the whole production cost and efficiency, the NAA is suitable for being used as the auxin for the industrial tissue culture and rapid propagation of the colocasia esculenta.

FIGS. 3 to 6 are photographs of tissue culture of different varieties of colocasia esculenta in rapid propagation according to the method of the present invention, respectively, and illustrate that the tissue culture medium and the tissue culture method provided by the present invention are suitable for tissue culture and rapid propagation of various varieties of colocasia esculenta.