CN105941143B - A kind of detoxification of lily bulbs method - Google Patents

Abstract

The invention discloses a detoxification method for lily bulbs, comprising the following steps: (1) placing lily bulbs at 3-8°C for 40-50 days; (2) selecting lily bulbs with a bud length of 4-8cm, Peel off the outer scales, leave 3‑5 bulb discs with inner scales, put the bulb discs into the incubator for pretreatment, the pretreatment conditions are: light intensity 1500‑2000Lux, temperature 35‑40°C, treatment time 2 ‑5 days; (3) Select a bulb disc with normal growth, peel off the shoot tip of 0.4‑0.7cm and move it into the differentiation medium, cultivate it in the dark at 23‑25°C for 1‑2 days, then move it into the light incubator, The light intensity is 1500-2000 Lux, the light time is 12-16 hours/day, and the temperature is raised at a rate of 1-5°C/d. After the temperature rises to 35-45°C, maintain the temperature for 35-45 days. The invention combines heat treatment and chemical treatment to remove lily bulb virus, which not only shortens the induction culture period, but also has the characteristics of high detoxification rate and high value-added coefficient, and provides technology for cultivating lily virus-free plants and popularizing the research of non-toxic cultivation Base.

Description

A kind of lily bulb detoxification method

technical field

The invention relates to the field of plant tissue culture rapid propagation technology and biotechnology, in particular to a detoxification method for lily bulbs.

Background technique

Lily is the general name of the genus Lilium (Lilium) of the monocot class Liliaceae (Liliaceae), which is a perennial herbaceous flower. There are more than 90 species of Lilium in the world, and about 47 species originate in my country, accounting for 51% of the world total. There are many varieties of lilies, with huge flowers, rich colors, and beautiful flowers. They can be used as cut flowers, potted flowers, and in gardens and green spaces. Most lilies are edible, and they are the best nourishing products. Lily has the meaning of "a hundred years of harmony" and is deeply loved by people. Lilies are mainly distributed in temperate regions such as China, Japan, North America and Europe. In recent years, the production of cut lilies in my country has developed rapidly, and three major production bases have been formed in Kunming, Shanghai and Shenzhen. However, its propagation mainly adopts the methods of conventional bulb division, bulbil scale cutting, scale embedding and other propagation methods. Long-term asexual reproduction gradually accumulates a large amount of viruses in the lily, causing the lily to suffer from viral diseases, which affects the yield and quality of the lily.

Among the more than ten reported viruses infecting lilies, three are the most common and the most harmful: cucumber mosaic virus (CMV), lily symptomless virus (LSV) and Lily mottle virus (Lily mottle virus, LMoV), they often manifest as compound infection, causing mottled or deformed stems, leaves or flowers of plants, which seriously cause short stature, degeneration or even death of plants, and with the spread of the virus, often It caused a large area of lily to be susceptible to the disease, which seriously affected the yield and quality of fresh cut lily flowers.

For the industrialized production of Lilium longya, bulb propagation is the key. In the large-scale planting of lily, the propagation of lily bulbs in my country is still in the exploratory stage, and the propagation methods can be divided into sexual propagation (seed propagation) and asexual propagation. Among them, sexual reproduction requires 3-4 years for the reproduction cycle, and because most lily hybrid lines are not easy to bear fruit, and the offspring of lily breeding are prone to variation, and cannot fully maintain the excellent traits of the original variety, so in actual production, lily is seldom used. Reproduce sexually rather than asexually. Among them, the main propagation methods adopted by asexual propagation include cauline bulblet propagation, bulbil propagation, scale cutting propagation and tissue culture. The quality of the bulbs obtained from stem bulblet propagation is good, but the propagation coefficient is low; the bulbil propagation takes a long time to form commercial balls, usually after 2-3 years; the cost of scale cutting propagation is low, easy to operate, and the propagation coefficient is high. An effective method for rapid propagation of lilies. The asexual reproduction methods of small bulbs, bulbils, and scale cuttings all lead to easy accumulation of viruses in offspring, and the phenomenon of variety degradation is very serious.

Plant tissue culture technology is widely used in plants that mainly reproduce asexually, and the technology is relatively mature. By adopting tissue culture and rapid propagation of Lily Longya, a large number of high-quality seedlings that maintain the excellent traits of the original varieties can be obtained in a short period of time. If tissue culture can be implemented in industrialized seedling cultivation, significant economic benefits can also be generated. At present, there is no report on the patent for the rapid propagation of Lilium japonicus tissue culture, and the literature research is mainly focused on obtaining tissue culture in the form of seedlings. There are few studies on tissue culture products in the form of bulbs. The bulbs of Lilium argentina have a lot of bacteria, and the explants are very difficult to sterilize. The problem of high pollution rate has not been well resolved.

Lily virus detection methods mainly include indicator plant method, electron microscope detection method, serological method, etc. Since 1990, foreign countries have gradually carried out research on RT-PCR detection technology for lily virus. The RT-PCR technique used in the detection of lily virus is superior to the above-mentioned methods in terms of convenience, sensitivity and specificity. The multiplex RT-PCR technology developed on the basis of traditional RT-PCR has been widely used in lily in recent years because of its advantages of rapidity, sensitivity, strong specificity, and simple operation (Bertolini et al., 2001; Nie and Singh, 2001). There has been some research on virus detection. Multiplex RT-PCR, also known as multiplex RT-PCR, refers to adding multiple pairs of primers into one reaction system to simultaneously amplify multiple sequences. Applying this technology can detect multiple viruses at one time.

At present, the basic methods for blocking virus transmission and obtaining virus-free seedlings in the world include: shoot tip culture, bulbil culture, chemical treatment, and heat treatment.

Therefore, establishing a virus-free system that is easy to implement and has a high rate of virus-free is one of the key issues to reduce the harm of the virus to lilies and restore the seed character.

Contents of the invention

Aiming at the above-mentioned deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a detoxification method for lily bulbs.

The object of the invention is achieved through the following technical solutions:

A kind of lily bulb detoxification method, comprises the following steps:

(1) Place lily bulbs at 3-8°C for 40-50 days;

(2) Select a lily bulb with a bud length of 4-8cm, peel off the outer scales, leave a bulb disc with 3-5 inner scales, put the bulb discs into an incubator for pretreatment, and the pretreatment conditions are: The light intensity is 1500-2000Lux, the temperature is 35-40℃, and the treatment time is 2-5 days;

(3) Select the bulb discs that grow normally after pretreatment, peel off the shoot tips of 0.4-0.7 cm and transfer them to the differentiation medium, and culture them in the dark at 23-25 °C for 1-2 days, then transfer them to the light incubator, and light them. The intensity is 1500-2000 Lux, the light time is 12-16 hours/day, and the temperature is raised at a rate of 1-5°C/day. After the temperature rises to 35-45°C, maintain the temperature for 35-45 days.

Preferably, the formulation of the differentiation medium is: MS medium + 6-BA 1.2-1.8 mg/L + NAA 0.35-0.65 mg/L + antiviral agent 5-15 mg/L.

Preferably, the antiviral agent is a mixture of one or more of dihydrouracil, 2-thiouracil, and morpholine hydrochloride.

More preferably, the antiviral agent is formed by mixing dihydrouracil, 2-thiouracil, and morpholine hydrochloride, and the mass ratio of the dihydrouracil, 2-thiouracil, and morpholine hydrochloride is is (1-3):(1-3):(1-3).

Specifically, in the present invention:

Components of MS Medium:

₍ 1) Major elements: NH ₄ NO ₃ 1.65g/L, KNO ₃ 1.90g/L, KH ₂ PO ₄ 0.17g/L, MgSO ₄ 7H 2 O 0.37g/L, CaCl ₂ (2H ₂ O) 0.44 g/L;

(2) Trace elements: MgSO ₄ 4H ₂ O 22.3mg/L, N ₃ BO ₃ 6.2mg/L, ZnSO ₄ 7H ₂ O 8.6mg/L, KI0.83mg/L, Na ₂ MoO ₄ 2H ₂ O 0.25g/L, CuSO ₄ 5H ₂ O 0.025mg/L, CoCl 6H ₂ O 0.025mg/L, FeSO ₄ 7H ₂ O 27.8mg/L, Na ₂ -EDTA, 37.3mg/L;

(3) Organic ingredients: inositol 100mg/L, niacin 0.5mg/L, L-glycine 2mg/L, vitamin B6 0.5mg/L, vitamin B1 0.1mg/L;

(4) sucrose 30g/L;

(5) Agar 12g/L.

6-BA, namely 6-benzylaminoadenine, CAS number: 1214-39-7.

NAA, namely 1-naphthylacetic acid, CAS number: 86-87-3.

Dihydrouracil, CAS No.: 504-07-4.

2-thiouracil, CAS No.: 141-90-2.

Morpholine hydrochloride, CAS number: 3160-91-6.

The lily bulb detoxification method of the present invention combines heat treatment and chemical treatment to remove lily bulb virus, which not only shortens the induction culture period, but also has the characteristics of high detoxification rate and high value-added coefficient. The research on poisonous cultivation provides a technical basis.

Detailed ways

The present invention will be further described below in conjunction with the embodiments. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention to other forms. Changes to equivalent embodiments with equivalent changes. Any simple modifications or equivalent changes made to the following embodiments according to the technical essence of the present invention without departing from the solution content of the present invention fall within the protection scope of the present invention.

Example 1

Lilium bulb detoxification method, comprises the following steps:

(1) placing lily bulbs (the variety is Siberia, Siberia) at 4°C for 45 days;

(2) Select a lily bulb with a bud length of 5 cm, peel off the outer scales, leave the bulb discs with 4 inner scales, put the bulb discs into the incubator for pretreatment, the pretreatment conditions are: light intensity 1600Lux, The temperature is 38°C, and the treatment time is 3 days;

(3) Select the bulb discs with normal growth after pretreatment, peel off the 0.6cm shoot tips and transfer them into the differentiation medium, and culture them in the dark at 24°C for 1 day (that is, place them in the dark at 24°C for 1 day) , and then moved into the light incubator, the light intensity is 1500Lux, the light time is 12 hours/day, the temperature is 24°C on the first day after moving into the incubator, and the temperature is raised at a rate of 2°C/day, after the temperature rises to 36°C, Cultured at 36°C for 35 days.

The formulation of the differentiation medium is: MS medium+6-BA 1.5mg/L+NAA 0.5mg/L+antiviral agent 9mg/L.

The antiviral agent is prepared by mixing dihydrouracil, 2-thiouracil and morpholine hydrochloride in a mass ratio of 1:1:1.

Example 2

It is basically the same as Example 1, except that the antiviral agent is prepared by mixing 2-thiouracil and morpholine hydrochloride in a mass ratio of 1:1.

Example 3

It is basically the same as Example 1, except that the antiviral agent is prepared by mixing dihydrouracil and morpholine hydrochloride in a mass ratio of 1:1.

Example 4

It is basically the same as Example 1, except that the antiviral agent is prepared by mixing dihydrouracil and 2-thiouracil at a mass ratio of 1:1.

Example 5

It is basically the same as in Example 1, the only difference is that the step (3) is: select a bulb dish with normal growth, strip off the shoot tip of 0.6 cm and move it into the differentiation medium, place it at 24° C. for 1 day, and then move it into the differentiation medium. In the light incubator, the light intensity is 1500 Lux, the light time is 12 hours/day, and the temperature is kept at 24° C. for 30 days.

Example 6

It is basically the same as in Example 1, the only difference is that the step (3) is: select a bulb dish with normal growth, strip off the shoot tip of 0.6 cm and move it into the differentiation medium, place it at 24° C. for 1 day, and then move it into the differentiation medium. In the light incubator, the light intensity is 1500 Lux, the light time is 12 hours/day, and the temperature is maintained at 30° C. for 30 days.

Example 7

It is basically the same as in Example 1, the only difference is that the step (3) is: select a bulb dish with normal growth, strip off the shoot tip of 0.6 cm and move it into the differentiation medium, place it at 24° C. for 1 day, and then move it into the differentiation medium. In the light incubator, the light intensity is 1500 Lux, the light time is 12 hours/day, and the temperature is kept at 36° C. for 30 days.

test case 1

The survival rate of the shoot tips in step (3) in the differentiation medium in Examples 1-7 was counted. The specific test results are shown in Table 1.

Table 1: Survival rate result table

Survival rate, % Example 1 93 Example 2 86 Example 3 89 Example 4 87 Example 5 88 Example 6 91 Example 7 82

Comparing embodiment 1 and embodiment 2-4, embodiment 1 (dihydrouracil, 2-thiouracil, morpholine guanidine hydrochloride compound) survival rate is obviously higher than embodiment 2-4 (dihydrouracil, 2 - Compounding of any two of thiouracil and morpholinidine hydrochloride). Comparing Example 1 with Examples 5-7, the survival rate of Example 1 (using a temperature increase of 2° C./day) is significantly higher than that of Example 2-4 (maintaining a constant temperature).

test case 2

The virus-free rate of the shoot tips cultivated in Examples 1-7 was tested.

Using multiplex RT-PCR to detect three kinds of lily viruses (lily asymptomatic virus, cucumber mosaic virus, lily mottled virus) specifically refer to the literature "RT- Methods in PCR detection and detoxification technology research. The results of detoxification rate are shown in Table 2.

Table 2: The result table of the detoxification rate of the three viruses

Comparing embodiment 1 and embodiment 2-4, embodiment 1 (dihydrouracil, 2-thiouracil, morpholine guanidine hydrochloride compound) three kinds of virus detoxification rates are obviously higher than embodiment 2-4 (dihydrouracil Any two of uracil, 2-thiouracil, and morpholine hydrochloride are compounded). Comparing Example 1 with Examples 5-7, the detoxification rate of the three viruses in Example 1 (using a temperature increase of 2° C./day) is significantly higher than that in Example 2-4 (maintaining a constant temperature).

Claims (1)

1. a lily bulb detoxification method, is characterized in that, comprises the following steps: (1) Place lily bulbs at 3-8°C for 40-50 days; (2) Select a lily bulb with a bud length of 4-8cm, peel off the outer scales, leave a bulb disc with 3-5 inner scales, put the bulb discs into an incubator for pretreatment, and the pretreatment conditions are: The light intensity is 1500-2000Lux, the temperature is 35-40℃, and the treatment time is 2-5 days; (3) Select the bulb discs that grow normally after pretreatment, peel off the shoot tips of 0.4-0.7 cm and transfer them to the differentiation medium, and culture them in the dark at 23-25 °C for 1-2 days, then transfer them to the light incubator, and light them. The intensity is 1500-2000Lux, the light time is 12-16 hours/day, and the temperature is raised at a rate of 1-5°C/day. After the temperature rises to 35-45°C, keep the temperature for 35-45 days; The formula of the differentiation medium is: MS medium+6-BA 1.2-1.8mg/L+NAA 0.35-0.65mg/L+antiviral agent 5-15mg/L; Described antiviral agent is mixed by dihydrouracil, 2-thiouracil, morpholine hydrochloride, and the mass ratio of described dihydrouracil, 2-thiouracil, morpholine hydrochloride is (1- 3): (1-3): (1-3).