CN106035087B - A kind of method of Changshan grapefruit fast asexual propagation - Google Patents

Abstract

The invention discloses a method for rapid asexual reproduction of Changshan Huyou. In the method, the sterilized explants are inoculated into a cluster bud induction medium, and cultivated at 20-24°C under the conditions of 1000-1200 lux of light, and every 25 Transplant once in ~45 days, transfer and culture 2-3 times to obtain clustered buds; cut the clustered buds into individuals with terminal buds, inoculate them into rooting medium, and cultivate them at 24-26°C and light of 1000-1200lux 30‑60 days to form a complete plant; using this method, an immature embryo can be cultivated, and at least 6 plants can be obtained in about 3 months, which has high efficiency and can reproduce and obtain a large amount of Changshan in a short time. Huyou seedlings are used in production practice.

Description

A method for rapid asexual reproduction of Changshan Huyou

(1) Technical field

The invention relates to a method for rapid asexual reproduction of Changshan Huyou.

(2) Background technology

Changshan Huyou is a new species of Citrus in China. Its fruit is plump and bright yellow in appearance; the fruit is golden yellow and full of juice; it has a strong fragrance, moderately sweet and sour taste, and slightly sweet; it is popular among the masses and reflects good economic benefits. In recent years, the plantation area has stepped up the construction of Changshan pomelo orchards, and the seed area has continued to expand; moreover, more attention has been paid to the yield and fruit quality of pomelo. The fruit trees of Changshan pomelo after multi-generation propagation are not easy to preserve some of the excellent traits of the female parent, and the resulting fruit tastes bad. Some fruit farmers often use cuttings for propagation, but the propagation speed of cuttings is slow and the survival rate is not high. , It is difficult to adapt to the needs of the production and development of Changshan Huyou.

(3) Contents of the invention

The purpose of the present invention is to provide a method for rapid propagation of Changshan pomelo, which is efficient and convenient, and can obtain a large number of excellent seedlings in a relatively short period of time, thereby establishing a fast, efficient, and high-quality industrialized production of seedlings system. This is not only the urgent need of fruit farmers, but also the demand of today's market.

The technical scheme adopted in the present invention is:

The present invention provides a method for rapidly propagating Changshan Huyou, which comprises: (1) acquisition and induced cultivation of explants: select Changshan Huyou explants with good growth and no pests and diseases for disinfection, and obtain sterilized explants Implant: Inoculate the sterilized explant into the cluster bud induction medium, place it in a culture room, cultivate it at 22～24°C, under the conditions of light 1000～1200lux, and transfer once every 25～45 days (preferably 25 days), transfer and culture 2-3 times to obtain clustered buds; the clustered bud induction medium contains 6-benzylaminopurine (6-BA) 0.25～2.5mg/L+brassinolide (BRs) 0.1mg /L+AgNO ₃ 1mg/L MS improved medium, the MS improved medium is to add polyvinylpyrrolidone (PVP) 0.5g/L+arginine (Arg) 0.1g/L+glutamine (Gln) 0.5g /L of MS basic medium; (2) rooting culture: after cutting the clustered buds obtained in step (1) into individuals with terminal buds, inoculate them on the rooting medium, at 22～24°C, under the condition of 1000～1200lux of light Under culture for 30-60 days, a complete plant is formed; the rooting medium is the MS improved medium containing 0.1-0.2mg/L indole-3-acetic acid (IAA); the MS improved medium is the addition of PVP 0.5g MS basal medium of /L+Arg 0.1g/L+Gln 0.5g/L.

Further, the Changshan Huyou explant in step (1) is a young stem, young leaf or young fruit with young buds, more preferably the Changshan Huyou explant is a young fruit 3 months after flowering, and the After placing the young fruit at 4°C for 3 days, take it out and wrap it with plastic wrap and place it at 37°C for 60 minutes, wash it with detergent and soak it for 5 minutes, then rinse it with distilled water to obtain the young fruit after cleaning, and peel off the young embryo. That is the explant.

Further, the explant disinfection method in step (1) is as follows: soak in 20-28g/L sodium hypochlorite aqueous solution for 10 minutes, rinse with sterile water for 3-5 times, and dry the surface moisture with filter paper to obtain the sterilized explant.

Further, the cluster bud induction medium in step (1) is MS modified medium containing 6-BA 0.25-0.75 mg/L+BRs 0.1 mg/L+AgNO ₃ 1 mg/L.

Further, the rooting medium in step (2) is MS modified medium containing 0.1-0.2 mg/L IAA or MS modified medium containing 0.1-0.2 mg/L IAA+0.5 mg/L 6-BA.

Further, the final concentration of the MS basic medium is composed of: NH ₄ NO ₃ 1.65g/L, KNO ₃ 1.9g/L, CaCl ₂ 2H ₂ O 0.44g/L, MgSO ₄ 7H ₂ O 0.37g/L , KH ₂ PO ₄ 0.17g/L, KI 0.83mg/L, H ₃ BO ₃ 5.2mg/L, MgSO ₄ 7H ₂ O 22.3mg/L, ZnSO ₄ 7H ₂ O 3.6mg/L, Na ₂ MoO ₄ 2H ₂ O 0.25mg/L, CuSO ₄ 5H ₂ O 0.025mg/L, CoCl ₂ 6H ₂ O 0.025mg/L, Na ₂ EDTA 37.3mg/L, FeSO ₄ 7H ₂ O 27.8mg/L , inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, niacin 0.5mg/L, sucrose 20～40g/L, agar powder 3～12g/L L, the solvent is water, and the pH is 5.6-6.0.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: up to now, no research reports on the regeneration of plants of Huyou Changshan using explants have been seen, and the present invention is the first report; After cultivation, at least 6 or more plants can be obtained in about 3 months, which has high efficiency and can reproduce and obtain a large number of Changshan Huyou seedlings in a short period of time, which can be used in production practice. The method is efficient and convenient, and can obtain a large number of excellent seedlings in a relatively short period of time, thereby establishing a rapid, efficient, and high-quality industrialized production system for seedlings. This is not only the urgent need of fruit farmers, but also the demand of today's market.

(4) Description of drawings

Fig. 1 is a photo of curling and callus of young leaves after culture, A and B are callus young leaves.

Fig. 2 Photos of Changshan Huyou young stems cultured and expanded.

Fig. 3 Callus and clustered shoots formed by cultured immature embryos; A: callus; B: clustered shoots regenerated from callus; C: clustered shoots directly regenerated from immature embryos.

Fig. 4 The complete plant regenerated from immature embryo culture; A: the regenerated complete plant; B: the transplanted and survived plant.

(5) Specific implementation methods

The present invention will be further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto.

Example 1:

(1) Selection and processing of explants:

The young stems, young leaves and young fruits with young buds of Huyou Changshan were selected as explants. Specifically, Changshan Huyou was planted in an experimental greenhouse, and watered at the right time to keep its growth in good condition. During the germination period of Huyou, the young stems and young leaves with buds of Huyou are obtained by clipping. If there is no new shoot, carry out topping treatment to Huyou, new shoots will grow after 2 weeks, and young stems and young leaves suitable for the experiment can be obtained after 3-4 weeks. Young fruit about 3 months after flowering, young fruit with a diameter of 2-3cm, put the young fruit at 4°C for 3 days, take it out, wrap it with plastic wrap and put it at 37°C for 60 minutes, wash it with detergent and soak After 5 minutes, rinse with distilled water to obtain the pretreated young fruit, and peel off the immature embryo with a size of 1-3mm on the sterile operating table. Young fruits without pretreatment were used as controls.

(2) Disinfection process of explant:

Wash and soak the young pomelo leaves, young stems with buds, and young embryos with detergent for 5 minutes, rinse them with distilled water, and then soak them in 20-28g/L sodium hypochlorite aqueous solution for 10 minutes. Then the treated material was washed 3-5 times with sterile water, and excess water on the surface was blotted with filter paper to obtain sterilized explants.

(3) Induction culture of explants:

On the ultra-clean workbench, inoculate the above-mentioned sterilized explants into the cluster bud induction medium (the cluster bud induction medium is 6-BA0.25～2.5mg/L+BRs 0.1mg/L+AgNO ₃ 1mg/ The MS modified medium of L+, wherein the specific content of 6-BA is shown in Table 1, Table 2 and Table 3), and 20 explants were inoculated with each cluster bud induction medium. Cultivate at a temperature of 22±2°C and a light of 1000-1200lux. The explants were transferred to new cluster bud induction medium every 25 days, and the growth changes were observed in time and photographed and recorded. After one transfer, cluster shoots were obtained. Under the same conditions, 6-BA 0.25-2.5mg/L+BRs 0.1mg/L+AgNO ₃ 1mg/L MS basic medium (see Table 1-Table 3 for 6-BA content) was used as a control.

(4) Rooting cultivation of buds in clusters:

After the clustered buds are cut into individuals with terminal buds, they are inoculated into the rooting medium, and cultivated for about 30 days at 24-26°C and under the condition of light of 1000-1200 lux to form a complete plant.

(5) Experimental results

When young leaves are inoculated into Table 1, young stems are inoculated into Table 2, and young embryos are inoculated into Table 3, the different hormone ratios listed in Table 3 are 6-BA0.25～2.5mg/L+BRs 0.1mg/L+AgNO ₃ 1mg/ On the MS basic medium of L (MS medium without adding PVP 0.5g/L+Arg0.1g/L+Gln 0.5g/L), browning phenomenon occurred in about 5 days of cultivation, and finally stopped growing and died. When the young leaves are inoculated into Table 1, the young stems are inoculated into Table 2, and the young embryos are inoculated into the clustering bud induction medium (6-BA 0.25～2.5mg/L+BRs 0.1mg/L) listed in Table 3 +AgNO ₃ 1mg/L MS medium (MS medium supplemented with PVP 0.5g/L+Arg 0.1g/L+Gln 0.5g/L), cultured for about 5 days, still kept fresh green state.

Picked young fruits have not been subjected to 4°C low temperature and 37°C heat shock treatment. After disinfection in the same way as step (2), the immature embryos are stripped and inoculated into cluster bud induction medium. Even if they are cultivated for more than 60 days, they still do not germinate, and the germination is extremely high. slow. After the young fruit was placed at 4°C for 3 days, it was taken out and wrapped in plastic wrap and placed in a heat shock treatment at 37°C for 60 minutes. After being sterilized by the method of step (2), the immature embryos were stripped and inoculated into the cluster bud induction medium for 2 weeks. Germination can be seen from the left and right.

Therefore, choose 6-BA 0.25～2.5mg/L+BRs 0.1mg/L+AgNO ₃ 1mg/L MS modified medium (add PVP 0.5g/L+Arg 0.1g/L+Gln 0.5g/L MS medium) as the medium for young leaves, young stems and immature embryos to induce clustered buds; after the young fruits were placed at 4°C for 3 days, they were taken out, wrapped in plastic wrap and placed in 37°C heat shock treatment for 60 minutes as the medium for the cultivation of immature embryos. preconditioning conditions.

After the inoculated young leaves were cultured on the cluster bud induction medium, about 15 days later, yellow flowers died on a few leaves, and some leaves curled up, further forming callus. The culture results are shown in Table 1 and Figure 1.

Table 1 Culture results of Changshan Huyou young leaves

After inoculating the young stems on the cluster bud induction medium, the stems swelled 20-30 days after inoculation, but no obvious callus was formed. The specific results are shown in Table 2 and Figure 2.

Table 2 The results of young stem culture of Changshan Huyou

After the young fruit was placed at 4°C for 3 days, it was taken out and wrapped in plastic wrap and placed in a heat shock treatment at 37°C for 60 minutes. Germination begins in about 2 weeks. In the process of immature embryo culture, cluster buds can be directly differentiated under the condition of lower concentration 6-BA (0.25-0.75mg/L); while under the condition of higher concentration 6-BA (1.0-2.5mg/L), then Only dedifferentiated into callus. Moreover, when the callus is transferred to a medium containing a lower concentration of 6-BA (0.25-0.75mg/L), it can also regenerate and sprout. The results are shown in Table 3 and Figure 3.

Table 3 Culture results of Changshan Huyou immature embryos

In addition, in order to verify whether BRs 0.1mg/L+AgNO ₃ 1mg/L can promote the regeneration of immature embryos, the immature embryos were inoculated on the medium with or without BRs 0.1mg/L and AgNO ₃ 1mg/L for experiments. Conditions and results are shown in Table 4. The results showed that BRs 0.1mg/L+AgNO ₃ 1mg/L could significantly promote the regeneration of immature embryos and cluster buds.

Table 4 Effects of BRs and AgNO ₃ on immature embryo culture

Example 2

The immature embryos separated from the pretreated young fruit in Example 1 were inoculated into cluster bud induction medium (6-BA 0.5mg/L+BRs 0.1mg/L+AgNO ₃ 1mg/L MS improved medium), in Cultivate at 22±2°C for about 30 days under the condition of 1000-1200lux light to obtain clustered buds. After cutting into individuals with terminal buds, transfer them to rooting medium (0.1-0.2mg/L IAA MS modified medium or 0.1-0.2mg/L IAA+0.5mg/L 6-BA MS modified medium (see Table 5), cultured for about 30 days at a temperature of 22±2°C and a light of 1000-1200lux, and finally formed a complete plant. On average, each cluster bud can form more than 6 plants after cutting. Roots can be grown on MS modified medium + 0.1-0.2 mg/L IAA, and MS modified medium + 0.1-0.2 mg/LIAA + 0.5 mg/L 6-BA. In about 30 days, the root length can reach 2-3 cm, further forming a complete plant. Therefore, after the above-mentioned cultivation, an immature embryo can obtain clustered buds within 60 days, and at least 6 or more complete plants can be obtained after about 30 days, that is, an immature embryo can obtain at least 6 or more complete plants in about 3 months. The complete plant has the characteristics of short time and high efficiency. The specific results are shown in Table 5 and Figure 4.

The results of clustered shoots taking root and forming complete plants under different culture conditions in table 5

MS modified medium + different plant hormone concentrations (mg/L) Can take root 0.1mg/L IAA+0.5mg/L 6-BA can 0.1mg/L IAA can 0.2mg/L IAA+0.5mg/L 6-BA can 0.2mg/L IAA can 0.1mg/L NAA No, callus formed at the base 0.1mg/L NAA+0.5mg/L 6-BA No, callus formed at the base 0.2mg/L NAA No, callus formed at the base 0.2mg/L NAA+0.5mg/L 6-BA No, callus formed at the base

Claims (5)

1. A kind of 1. method of Changshan grapefruit fast asexual propagation, it is characterised in that methods described is：(1) acquisition of explant and lure Lead culture：Selection is grown fine, the sterilization of the Changshan grapefruit explant of no disease and pests harm, the explant after being sterilized；After sterilizing Explant be seeded in inducing clumping bud culture medium, cultivated under the conditions of 20~24 DEG C, 1000~1200lux of illumination, every Switching in 25~45 days once, switching culture 2-3 times, obtains Multiple Buds；The inducing clumping bud culture medium is that the amino of benzyl containing 6- is fast Purine 0.25~2.5mg/L+ brassinosteroids 0.1mg/L+AgNO₃1mg/L MS improved culture mediums, the MS improved culture mediums To add polyvinylpyrrolidone 0.5g/L+ arginine 0.1g/L+ glutamine 0.5g/L MS minimal mediums；The Changshan Hu shaddock explant is spire, rataria or the young stem with young shoot, and the rataria is to place the Post flowering young fruit of 3 months 3 days at 4 DEG C Afterwards, taking-up is wrapped up with preservative film and placed 60 minutes at 37 DEG C, after cleaning immersion 5 minutes with liquid detergent, is done with distilled water flushing Only, the young fruit after being cleaned, strips rataria；(2) culture of rootage：The Multiple Buds that step (1) obtains are cut into terminal bud After individual, root media is seeded to, cultivates 30-60 days, has been formed under the conditions of 24~26 DEG C, 1000~1200lux of illumination Whole plant；The root media is the MS improved culture mediums of the indole-3-acetic acid containing 0.1-0.2mg/L, the MS improvement culture Base is addition polyvinylpyrrolidone 0.5g/L+ arginine 0.1g/L+ glutamine 0.5g/L MS minimal mediums.
2. 2. the method for Changshan grapefruit fast asexual propagation as claimed in claim 1, it is characterised in that step (1) described explant disappears Malicious method is：10min is soaked with 20-28g/L aqueous sodium hypochlorite solutions, aseptic water washing 3-5 times, surface water is blotted with filter paper Point, the explant after being sterilized.
3. 3. the method for Changshan grapefruit fast asexual propagation as claimed in claim 1, it is characterised in that step (1) described Multiple Buds lure It is containing 6-benzyl aminopurine 0.25~0.75mg/L+ brassinosteroids 0.1mg/L+AgNO to lead culture medium₃1mg/L MS improvement Culture medium.
4. 4. the method for Changshan grapefruit fast asexual propagation as claimed in claim 1, it is characterised in that step (2) described culture of rootage Base is the MS improved culture mediums of+0.5mg/L 6-benzyl aminopurines of indole-3-acetic acid containing 0.1-0.2mg/L.
5. 5. the method for Changshan grapefruit fast asexual propagation as claimed in claim 1, it is characterised in that the MS minimal mediums are whole Concentration forms：NH₄NO₃ 1.65g/L、KNO₃ 1.9g/L、CaCl₂·2H₂O 0.44g/L、MgSO₄·7H₂O 0.37g/L、 KH₂PO₄ 0.17g/L、KI 0.83mg/L、H₃BO₃ 5.2mg/L、MgSO₄·7H₂O 22.3mg/L、ZnSO₄·7H₂O 3.6mg/ L、Na₂MoO₄·2H₂O 0.25mg/L、CuSO₄·5H₂O 0.025mg/L、CoCl₂·6H₂O 0.025mg/L、Na₂EDTA 37.3mg/L、FeSO₄·7H₂O 27.8mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, hydrochloric acid pyrrole Tremble alcohol 0.5mg/L, nicotinic acid 0.5mg/L, 20~40g/L of sucrose, 3~12g/L of agar powder, and solvent is water, and pH is 5.6~6.0.