CN101015279B - Tissue culture method for fast propagation of primula poissonii - Google Patents

Abstract

The invention provides a method for tissue culture and rapid propagation of Primula chinensis, which comprises explant selection, surface disinfection, adventitious bud induction, subculture, rooting culture and test-tube seedling transplantation. According to the tissue culture and rapid propagation method of Primula poisonii of the present invention, one axillary bud can be induced to produce about 40 to 50 new buds, and after about 30 to 40 days of subculture, the multiplication coefficient is 3 to 4, and the rooting rate can reach 95 % or more, the transplanting survival rate is almost 100%, which realizes the preservation of the excellent Primula poisonii (Primula poisonii) single plant through rapid propagation technology, and greatly improves the seedling production capacity of Primula poisonii at the same time, shortening the seedling time It can also reduce the cost, and provides technical support for the large-scale popularization and application of the primrose.

Description

Tissue Culture and Rapid Propagation Method of Primula Haixian

technical field

The invention relates to plant tissue culture, in particular to a tissue culture rapid propagation method of Primulapoisonii.

Background technique

Primula poisonii (Primula poisonii) is a plant of the genus Primula in the Primulaceae family. It is a very beautiful garden ornamental flower. Haixian Primula (Primula poisonii) is a perennial herbaceous flower. The whole plant is in the shape of a rosette, with well-developed rhizomes and 5 or more leaf clumps growing upwards. It is mainly propagated by seeding. After sowing in spring, it grows vegetatively in the next stage. It begins to flower in May, grows slowly, and has a low reproduction coefficient. In addition to seed propagation, the available methods for asexual propagation of Primula are: branch (leaf cluster) propagation, tissue culture propagation, and a small number of perennial species can be propagated by cuttings, but the method of branch propagation is far from meeting production needs.

Tissue culture is an important way of rapidly propagating good plant varieties. Many species of Primula have been reported on tissue culture, but there is no relevant research and report on tissue culture of Primula poisonii, so it needs to be studied. Tissue culture of Primula poisonii (Primula poisonii) and a set of specialized rapid propagation technology system.

Contents of the invention

The object of the present invention is to provide a kind of tissue culture rapid propagation method of Primula poisonii (Primula poisonii), improve the propagation coefficient, rooting rate and transplanting survival rate of Primula poisonii (Primula poisonii), and combine other related technologies Measures, so that the seedling production and germplasm preservation of Primulapoisonii can be better guaranteed.

In order to realize the object of the present invention, a kind of tissue culture rapid propagation method of Primula poisonii (Primula poisonii) of the present invention, explant selection, surface disinfection, adventitious bud induction, subculture, rooting culture and test-tube seedling transplanting.

The explant is the axillary bud of Primula poisonii.

The medium for adventitious bud induction and subculture is MS+2.0mg/l 6-BA+0.1mg/lNAA, and the pH is 5.8-5.9. The lighting conditions are 2500-3000Lux of natural scattered light plus 1500-2000Lux of artificial auxiliary light. Lux (lux) is the unit of illuminance.

The rooting medium is MS or MS+0.2mg/l NAA, preferably MS+0.2mg/lNAA, and the pH is 5.8-5.9.

The lighting conditions are 2500-3000Lux of natural scattered light plus 2500-3000Lux of artificial auxiliary light.

After being cultured in the rooting medium for 2 to 3 weeks, the hardened seedlings are transplanted to fine peat soil in the greenhouse for 3 to 4 days.

The explants were sterilized with 0.1% mercuric chloride solution for 3-4 minutes.

The suitable culture temperature for the tissue cultured seedlings is between 20°C and 23°C, when the temperature exceeds 25°C, the tissue cultured seedlings turn yellow or even gradually die.

The suitable temperature for transplanting tissue culture seedlings is between 18 and 25°C, too high or too low is not conducive to the survival of test tube seedlings.

Specifically, the tissue culture rapid propagation method is as follows: select the axillary buds of Primula poisonii as explants, wash them with water, sterilize them with 0.1% mercuric chloride solution for 3-4 minutes, and wash them repeatedly with distilled water under aseptic conditions. , inoculated into the induction medium MS+2.0mg/l6-BA+0.1mg/l NAA, the pH is 5.8, the light conditions are natural scattered light within 2500-3000Lux plus artificial auxiliary light 1500-2000Lux; then move to the same medium Proliferate, subculture 2-3 times, rooting medium is MS+0.2mg/l NAA, pH is 5.8, light conditions are natural scattered light 2500-3000Lux plus artificial auxiliary light 2500-3000Lux; temperature is kept between 20-23°C Between; rooting culture for 2 to 3 weeks, after 3 to 4 days of seedling hardening, move to fine peat soil for cultivation, keep the humidity higher than 80%, and spray water 2 to 3 times a day. After 2 weeks, the covering film was gradually removed for normal culture.

Generally speaking, using axillary buds as explants is more destructive to plants, but for some materials that are more precious and urgently need to be multiplied, using axillary buds as explants for multiplication can obtain a large amount of regeneration in a short time Plant: the Primulapoisonii test-tube plantlet regenerated by axillary buds grows vigorously, and the observation of the growth in the later stage shows that it can basically keep its excellent seed quality unchanged. Tissue-cultured and fast-propagated test-tube seedlings are not easily infected by diseases and insect pests during the growth process, but in order to further prevent the occurrence of diseases and insect pests, it is better to spray carbendazim, omethoate, etc. 3 to 4 times during the growing season; The temperature when transplanting test-tube seedlings should be controlled between 18 and 25°C, too high or too low is not conducive to the survival of test-tube seedlings, and it is not suitable for transplanting test-tube seedlings in summer.

The tissue culture rapid propagation method of the present invention breeds Primula poisonii (Primula poisonii), and within 2 months, the multiplication coefficient reaches 3 to 4, the rooting rate reaches more than 95%, and the transplanting survival rate is almost 100%, which greatly improves the quality of Primula poisonii. The propagation coefficient of Primula poisonii shortens the seedling period and reduces the cost, which provides a very effective method for large-scale garden application and industrial seedling cultivation in the future; at the same time, the axillary buds are used as explants for multiplication and rapid growth. It can keep the species of Primula poisonii unchanged, and provide guarantee for the preservation of the more precious and rare varieties that appear in the cultivation of new varieties.

Compared with the prior art, the advantage of the tissue culture rapid propagation method of Primula poisonii (Primula poisonii) of the present invention is:

1. Established a kind of tissue culture rapid propagation method of Primula poisonii (Primula poisonii), compared with the traditional method of breeding seedlings of Primula plants, such as seeding propagation and division propagation, greatly improved Primula poisonii (Primula poisonii) The reproduction coefficient of Primula poisonii) provides technical support for scientific research and production cultivation;

2. The seedlings obtained through tissue culture and rapid propagation have strong growth, dark green leaves, strong consistency, easy seedling growth, strong adaptability, less pests and diseases, and easy management;

3. Breeding Primula poisonii by using tissue culture and rapid propagation method, which removes the restrictions of season and climate, thereby shortening the seedling breeding cycle and increasing the reproduction quantity;

4. Propagation of Primula poisonii by tissue culture and rapid propagation can keep the characteristics of elite species or varieties unchanged, thus providing an effective method for variation preservation in new variety breeding research.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1

The optimal medium for adventitious bud induction was explored using the leaves of Primula poisonii as explants: the optimal medium formula for adventitious bud induction was explored by using the leaves of well-growing seedlings as explants. Wash young leaves of Primula poisonii with tap water for 2-3 hours, drop a drop of detergent in the water and shake for 15 minutes to remove surface impurities, then rinse with tap water repeatedly; surface disinfection: 70% alcohol disinfection for 10-15 seconds, sterile water Rinse 4 to 5 times, disinfect with 0.1% mercury chloride for 3 to 4 minutes, and then rinse with sterile water 4 to 5 times. Then the sterilized leaves were cut into small pieces of ¹ cm (with part of the veins) and inoculated on the induction medium for clustering buds, 5-6 pieces were inoculated in each bottle, and 10 bottles were connected to each medium, and cultured in the dark. Among them, the concentration gradient of 6-BA: 0.5mg/l, 1.0mg/l and 2.0mg/l; the concentration gradient of NAA: 0.05mg/l, 0.1mg/l and 0.5mg/l, the pH of the medium is 5.8, A two-factor completely randomized experimental design was used.

Different concentrations of 6-BA and NAA affect the leaf differentiation of Primula poisonii. After 4-5 days of inoculation, the leaves began to swell and shrink, and the incisions became fat and thickened about 15 days later. After 30 days, the leaves of the medium species began to produce calluses and a small amount of adventitious buds. The optimal concentration of adventitious bud differentiation is 6-BA 2.0mg/l and NAA 0.1mg/l, the induction rate of Primula poisonii in this medium reaches 22.5%, and some of them differentiate into buds.

Example 2

The axillary buds of Primula poisonii (Primula poisonii) were used as explants for primary culture and subculture: select axillary buds from robust plants, rinse them with tap water for 2-3 hours, disinfect and sterilize the leaves, and inoculate them on MS+6- BA 2.0mg/l+NAA 0.1mg/l cluster bud induction medium, inoculate 2 to 3 buds per bottle; the lighting conditions are natural scattered light 3000 Lux plus artificial auxiliary light source 1800±200 Lux, and the lighting time is 14 hours.

When the axillary buds are inoculated on the differentiation medium, new leaves are continuously pulled out. From 10 days on, the petioles of the new leaves are thick and the base turns red, and light green callus is gradually formed. After 2 weeks, adventitious buds begin to differentiate; 40 days after inoculation , A large number of adventitious buds are differentiated on the newly drawn petioles and leaves of the axillary buds.

The statistical results show that after 2 months of inoculation on the medium of MS+6-BA 2mg/l+NAA 0.1mg/l, the average number of adventitious buds that can be differentiated from each axillary bud is 40-50, which is significantly more than that induced by leaves. The number of adventitious buds. At the same time, temperature plays a key role in the induction of adventitious buds and the growth of tissue cultured seedlings. Between 20 and 23°C, the leaf color of tissue cultured seedlings is dark green and grows vigorously; Gradually die. Under such culture conditions, the value-added coefficient of the test-tube plantlets reached 3-4 within 2 months, which greatly improved the reproduction coefficient of Primula poisonii.

Example 3

Rooting culture of primula poisonii (Primula poisonii) tissue culture seedlings: when the adventitious buds grow 2 to 3 leaves on the differentiation medium, they are excised from the callus and transferred to the rooting medium, formula: MS , MS+NAA (0.1mg/l, 0.2mg/l, 0.5mg/l), the pH is 5.8; the lighting conditions are 3000Lux of natural scattered light plus 3000Lux of artificial auxiliary light source, and the lighting time is 14h.

From the perspective of the time required for rooting, the time required for rooting in medium MS and MS+0.2mg/l NAA is the shortest, 8-10 days, but in MS+0.2mg/l NAA, the rooting amount of tissue culture seedlings And growth is obviously better than MS basic medium, the average height of tissue culture seedlings can reach 4.8cm, and the rooting rate is over 95%.

Example 4

Transplanting of tissue-cultured seedlings of Primula poisonii: After two weeks of rooting and cultivation, when the tissue-cultured seedlings take root on average 4 to 5 per plant, and the root length reaches 1 cm, the preparations for transplanting can begin. Seedling hardening is the transition before transplanting the test-tube seedlings, which can help the test-tube seedlings gradually adapt to the external environment, making the test-tube seedlings stronger and darker green in leaf color, thereby increasing the survival rate.

After 3 to 4 days of hardening the test-tube seedlings of Primula poisonii, transplant them into the fine peat soil in the greenhouse. The peat soil is covered with the hole trays in advance and soaked with clean water; And spray water 2 to 3 times a day, and normal management can be carried out gradually after 2 weeks, and the transplanting survival rate is almost 100%.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (1)

1. a method for rapid propagation of tissue culture of Primula Haixian, comprising explant selection, surface disinfection, adventitious bud induction, subculture, rooting culture and transplanting of test-tube seedlings, characterized in that, the selection of Primula Haixian Axillary buds are explants, rinsed with water, sterilized with 0.1% mercuric chloride solution for 3 to 4 minutes, washed repeatedly with distilled water under sterile conditions, and inoculated into induction medium MS+2.0mg/l 6-BA+0.1mg/ l In NAA, the pH is 5.8, the light conditions are natural scattered light 2500-3000Lux plus artificial auxiliary light 1500-2000Lux; then move to the same medium for proliferation, subculture 2-3 times, rooting medium is MS+0.2mg/l NAA, pH 5.8, light conditions: natural scattered light 2500-3000Lux plus artificial auxiliary light 2500-3000Lux; temperature kept between 20-23°C; rooting culture for 2-3 weeks, seedling hardening 3-4 days later, move to fine Cultivate in peat soil, transplanting temperature is 18-25°C, keep humidity higher than 80%, spray water 2-3 times a day.