CN103329807A - Method for rapidly breeding hybrid orchid by root, as well as culture medium - Google Patents

Abstract

The invention discloses a method and a culture medium for rapid propagation of hybrid orchids by using roots in the technical field of plant tissue culture. The method of the present invention comprises the following steps: select the root segment of the hybrid orchid, inoculate it on an induction medium and culture it in dark to induce the protocorm; after the protocorm proliferates in large quantities, transfer to light culture, and the protocorm develops into a clustered seedling; cut the clustered seedling into a The single seedlings are transferred to the rooting medium of strong seedlings, and cultivated to obtain rooted plants. The invention uses MS as the basic medium, the induction medium needs to be added with 6-BA, PIC and hydrolyzed casein, and the strong seedling rooting medium is the MS medium without adding hormones. The invention has the advantages of high propagation efficiency and simple operation, and the protocorm induction rate of the root explant, the plant rooting rate and the transplanting survival rate all reach 100%.

Description

Method and culture medium for rapid propagation of hybrid orchids by using roots

technical field

The invention belongs to the technical field of plant tissue culture, and in particular relates to a method and culture medium for rapid propagation of hybrid orchids by using roots.

Background technique

Hybrid orchid is a new type of orchid selected from the hybridization of Chinese orchid and Cymbidium [Lu Jiliang. A preliminary study on the production and marketing status of Yunnan hybrid orchid [J]. Huamu Bonjing (Flower Gardening), 2013, (2): 36-37; Liang Fang , Cui Bo, Ma Jie, Ye Yongzhong. Proliferation and differentiation of hybrid orchid protocorms [J]. Anhui Agricultural Sciences, 2008, 36(13): 5309-5310, 5353]. Hybrid orchids are medium-sized, colorful, fragrant and elegant, making up for the lack of fragrance of Cymbidium. High ornamental value and broad market prospects [Zhang Xianyun, Yuan Xiuyun, Ma Jie. Research on tissue culture and rapid propagation technology of hybrid orchids [J]. Henan Science, 2009, 27(4): 419-421; Lu Ran. Yunnan hybrid orchids are popular [J]. Chinese Flower Horticulture 2012, (1): 18]. Hybrid orchids have strong adaptability, and some early-flowering varieties can bloom twice a year, which makes up for the lack of concentrated flowering of Cymbidium. Due to the above-mentioned advantages, hybrid orchids are popular among growers and consumers, and have been in a state of booming production and sales in recent years, with rapid development [Lu Jiliang. A Preliminary Study on the Production and Marketing Status of Hybrid Orchids in Yunnan[J]. Flower and Tree Bonsai (Flower Gardening), 2013, (2 ): 36-37; Lu Ran. Yunnan hybrid orchid hot sale [J]. Chinese Flower Horticulture 2012, (1): 18].

Traditional ramets have a long reproductive cycle, slow speed, and low reproductive coefficient, which are far from meeting the requirements of the industrial production of hybrid orchids; therefore, the application of tissue culture to rapidly propagate hybrid orchids is of great significance for the industrial development of hybrid orchids.

Hybrid orchids carry the genetic factors of Chinese orchids. Compared with Cymbidium, Phalaenopsis and other foreign orchids, tissue culture is more difficult. At present, only 3 classic papers on the rapid propagation of hybrid orchids have been found [Liang Fang, Cui Bo, Ma Jie, Ye Yongzhong. Proliferation and differentiation of hybrid orchid protocorms[J]. Anhui Agricultural Sciences, 2008, 36(13): 5309-5310, 5353; Zhang Xianyun, Yuan Xiuyun, Ma Jie. Tissue culture of hybrid orchid and Rapid Propagation Technology [J]. Henan Science, 2009, 27(4): 419-421; Shen Hanguo, Deng Ying, Yang Zhenming, Lan Weiquan, Luo Lixia. Research on tissue culture of hybrid orchid 'Eighteen Gege'. Zhang Qixiang. China Advances in Ornamental Horticulture Research [M]. Beijing: China Forestry Press, 2012: 311-313].

The explants are mainly stem tips and lateral buds, which cause great damage to the plants; the explants are prone to browning after inoculation, and the initial cultivation is difficult; the cultivation process is complicated and the reproduction coefficient is low; it is the rapid propagation of hybrid orchid tissue culture at present. main problem.

Using roots as explants for tissue culture has the unique advantages of large amount of material, easy acquisition, and little damage to plants. However, there are no reports and invention patents of using roots as explants in the current research on tissue culture of hybrid orchids.

Contents of the invention

The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and provide a method and a culture medium for rapidly propagating hybrid orchids using roots. Specifically, the roots are used as explants to induce and proliferate protocorms on the induction medium, and make the protocorms develop into clustered seedlings to rapidly propagate hybrid orchids; at the same time, the specific medium and cultivation techniques required for each cultivation stage are proposed. The method for cultivating regenerated plants with hybrid orchid roots provided by the present invention can complete the protocorm induction, multiplication and seedling formation process in one step; the protocorm induction rate of explants reaches 100%, the plant rooting rate is 100%, and the transplanting survival rate is 100%. 100%; it has the characteristics of high reproduction coefficient, high seedling rate, high transplanting survival rate and low cost.

The purpose of the present invention is achieved through the following technical solutions:

First aspect, the present invention relates to a kind of method utilizing root rapid propagation hybrid orchid, comprises the steps:

Step 1, select the root section of the hybrid orchid, inoculate it on the induction medium and cultivate it in dark, induce and proliferate the protocorm;

Step 2, transferring the protocorms on the induction medium to light culture, and the protocorms develop into clustered seedlings;

Step 3, cutting the clustered seedlings into single seedlings and transferring them to the rooting medium of strong seedlings, and culturing to obtain rooted plants.

Preferably, the induction medium in step one and the medium used for light culture in step two are the same induction medium, and the induction medium is MS+6-BA0.5mg/L+PIC0.6mg/L+ Hydrolyzed casein 200mg/L+ sucrose 30000mg/L+ agar powder 5000mg/L. The cultivation is dark cultivation, and the cultivation temperature is 25±2°C.

Preferably, in step 2, the induction medium is MS+6-BA0.5 mg/L+PIC0.6 mg/L+hydrolyzed casein 200 mg/L+sucrose 30000 mg/L+agar powder 5000 mg/L. The cultivation is light cultivation, and the cultivation temperature is 25±2°C.

Preferably, in step 3, the strong seedling rooting medium is MS+AC100mg/L+hydrolyzed casein 500mg/L+sucrose 30000mg/L+agar powder 5000mg/L. The cultivation is light cultivation, and the cultivation temperature is 25±2°C.

The present invention also relates to an induction medium, which can induce and proliferate protocorms and make the protocorms develop into bushy shoots, and the induction medium is that 6-BA and PIC are simultaneously added to MS basic medium.

Preferably, the induction medium is MS+6-BA0.5 mg/L+PIC0.6 mg/L+hydrolyzed casein 200 mg/L+sucrose 30000 mg/L+agar powder 5000 mg/L.

The MS components in the culture medium used in the present invention are shown in the document "Murashige T, Skoog F.A revised medium for rapid growth and bioassays with tobacco tissue cultures[J].Physiol.Plant.1962,15,473-497", which belongs to the art common knowledge.

The chemical name of PIC used in the culture medium of the present invention is 4-amino-3,5,6-trichloropyridine-2-acid, the molecular formula is C ₆ H ₃ Cl ₃ N ₂ O ₂ , and the common name in Chinese is picloram Or amiloride, the English name is Picloram.

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

1. For the first time, the roots of hybrid orchids were used as explants to obtain regenerated plants, which expanded the source of explants for tissue culture of hybrid orchids. Roots are easy to obtain as explants. Compared with seeds, using roots as explants can maintain the traits of the mother plant. Compared with other vegetative tissues, the amount of material taken is larger and the damage to plants is small. The present invention is useful for the production of hybrid orchid seedlings. important to industrial development.

2. High reproductive efficiency, strong plants, and high survival rate after transplanting. The protocorm induction rate of the explant of the present invention reaches 100%, no browning and vitrification phenomena are observed in the whole cultivation process, the cultivation process is accelerated, and the seedling quality is improved. The obtained regenerated plant has a well-developed root system, many strong roots can be produced without rooting agent, and the plant rooting rate is 100%; the plant grows robustly, and the transplanting survival rate is 100%.

3. Protocorm induction and seedling growth only need two-step cultivation process, and the cultivation process and the medium used are simple. The first step of protocorm induction culture only needs to add 6-BA, PIC and hydrolyzed casein, and does not need to add other complex organic substances such as banana puree and coconut milk, and does not need light to save energy. The second step only needs to turn the original medium to light, and the protocorms can develop into clustered seedlings. The third step is the rooting cultivation of strong seedlings. Without any growth regulator, a complete plant that takes root directly can be obtained, and the cost is low.

4. For the first time, picloram (PIC) was applied to the tissue culture of hybrid orchid roots, and the effect of protocorm induction and differentiation was good.

5. More root explants can be obtained from root regenerated plants, and then more protocorms can be induced in large quantities and continuously to realize cyclic proliferation, which is beneficial to industrial production.

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

Fig. 1 hybrid orchid root section induces the schematic diagram that forms protocorm;

The schematic diagram of Fig. 2 protocorm proliferation and growth under no-light condition;

Fig. 3 is a schematic diagram of the protocorm turning green and differentiating into clustered seedlings after being transferred into the light;

Fig. 4 changes the schematic diagram of the rooted plant that forms after the strong seedling rooting medium;

Fig. 5 is a schematic diagram of transplanted surviving hybrid orchids in the greenhouse.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments and accompanying drawings. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. In the following examples, the various processes and methods that are not described in detail are conventional methods well known in the art, and the reagents used and those who need to list their components are indicated when they appear for the first time, and the same reagents used thereafter are as follows: Unless otherwise specified, the contents are the same as those indicated for the first time.

Example 1

The root of hybrid orchid test tube seedlings is used as material, cut into sections and inoculated, cultured in dark at a temperature of about 25°C (25±2°C), to induce protocorms, and the protocorm induction medium is MS (Murashige and Skoog, 1962)+6 -BA (6-benzylaminoadenine) 0.5mg/L+PIC (4-amino-3,5,6-trichloropyridine-2-acid) 0.6mg/L+hydrolyzed casein 200mg/L+sucrose 30000mg/L+agar Powder 5000mg/L. After 12 days, the root section swelled and began to produce protocorms, as shown in Figure 1, and then the protocorms proliferated in large quantities, and gradually differentiated sprouts, as shown in Figure 2, the protocorm induction rate was 100%.

Transfer the protocorm induction medium that has induced and proliferated a large number of protocorms and sprouts to the light at a temperature of about 25°C (25±2°C). After 3 days, the protocorms and sprouts begin to turn green and gradually develop into clusters Seedlings, while protocorms continued to proliferate, as shown in Figure 3.

Cut the bushy seedlings into single seedlings and transfer them to the strong seedling rooting medium of MS+AC (activated carbon) 100mg/L+hydrolyzed casein 500mg/L+sucrose 30000mg/L+agar powder 5000mg/L, light 16 hours a day, temperature 25℃ Left and right, the rootless seedlings will gradually develop into normal plants and take root simultaneously, as shown in Figure 4, the plant rooting rate is 100%. After about 65 days of cultivation, each root explant can induce 60-80 rooted plants.

After the rooted plant grows 5-6 leaves, open the bottle cap, harden the seedlings at room temperature for 7-10 days, wash off the agar and plant them in the greenhouse. The growth is robust and the survival rate is 100%, as shown in Figure 5.

Example 2

Take the root of the test-tube plantlet obtained in Example 1 as material, inoculate after cutting into sections, culture in dark at a temperature of about 25°C (25±2°C), induce protocorms, and the protocorm induction medium is MS+6-BA0.5mg /L+PIC0.6mg/L+hydrolyzed casein 200mg/L+sucrose 30000mg/L+agar powder 5000mg/L, protocorm induction rate is 100%. After 30 days of culture, select the differentiated sprouts and move them to the new protocorm induction medium MS+6-BA0.5mg/L+PIC0.6mg/L+hydrolyzed casein 200mg/L+sucrose 30000mg/L+agar powder 5000mg/L Light culture, while leaving the undifferentiated protocorms on the original medium to continue to proliferate, differentiate and then transfer to light culture, and then adopt the same culture steps as in Example 1. After about 80 days of culture, each root explant has an average More than 100 plants can be induced to root.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims (8)

1. one kind is utilized root to breed the blue method of hybridization fast, it is characterized in that, comprises the steps:

Step 1 is chosen the blue root segment of hybridization, is seeded on the inducing culture and secretly cultivates, and induces, breeds protocorm;

Step 2 changes the protocorm on the described inducing culture over to light and cultivates, and protocorm develops into the seedling of growing thickly;

Step 3 cuts into single seedling with the described seedling of growing thickly and is transferred on the strengthening seedling and rooting medium, cultivates to obtain the plant that takes root.

2. the root that utilizes as claimed in claim 1 is bred the blue method of hybridization fast, it is characterized in that, it is with a kind of inducing culture that inducing culture in the step 1 and step 2 transfer light inlet are cultivated used medium, and described inducing culture is MS+6-BA0.5mg/L+PIC0.6mg/L+ caseinhydrolysate 200mg/L+ sucrose 30000mg/L+ agar powder 5000mg/L.

3. the root that utilizes as claimed in claim 1 is bred the blue method of hybridization fast, it is characterized in that in the step 3, described strengthening seedling and rooting medium is MS+AC100mg/L+ caseinhydrolysate 500mg/L+ sucrose 30000mg/L+ agar powder 5000mg/L.

4. the root that utilizes as claimed in claim 1 is bred the blue method of hybridization fast, it is characterized in that in the step 1, the temperature of described dark cultivation is 25 ± 2 ℃.

5. the root that utilizes as claimed in claim 1 is bred the blue method of hybridization fast, it is characterized in that in the step 2, the temperature that described light is cultivated is 25 ± 2 ℃.

6. the root that utilizes as claimed in claim 1 is bred the blue method of hybridization fast, it is characterized in that, in the step 3, described cultivation is that light is cultivated, and the temperature of cultivation is 25 ± 2 ℃.

7. one kind is used for utilizing root to breed the inducing culture of the blue method of hybridization fast, it is characterized in that, described inducing culture can be induced, be bred protocorm and make protocorm develop into the seedling of growing thickly, and described inducing culture is in the MS minimal medium, adds 6-BA and PIC simultaneously.

8. inducing culture as claimed in claim 7 is characterized in that, described inducing culture is MS+6-BA0.5mg/L+PIC0.6mg/L+ caseinhydrolysate 200mg/L+ sucrose 30000mg/L+ agar powder 5000mg/L.

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