A method for efficient genetic transformation of oriental lily

Abstract

The invention discloses a method for efficient genetic transformation of oriental lily, which uses filaments of oriental lily as transformation materials, and after infection with Agrobacterium and co-cultivation with drying, directly induces the filaments to differentiate into small bulbs and take root in an integrated screening culture medium, so as to achieve efficient cultivation integrating induction, swelling and rooting, and establish an efficient genetic transformation system of oriental lily. The invention provides a new technical method for the current problems of low genetic transformation efficiency of lily, difficulty in obtaining receptor materials, and sensitivity to Agrobacterium, which is simple, practical, easy to operate, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture and genetic transformation, and particularly relates to a method for efficient genetic transformation of oriental lily. Background Art

[0002] Lily is a perennial bulbous plant of the genus Lilium in the family Liliaceae. At present, domestic lily cut flower varieties are basically dependent on imports, among which Oriental lily has become the mainstream in the market due to its large flowers, rich colors and fragrant smell. After more than ten years of continuous replacement of new products, Oriental lily still maintains an absolute advantage in the market share, which shows that the market recognizes it. Therefore, variety improvement based on it has become the main breeding direction. With the development of modern molecular biology, the use of transgenic technology to overcome the self-incompatibility in traditional breeding and the targeted modification of target traits has become a new means of lily breeding.

[0003] At present, lily is mainly genetically transformed by Agrobacterium-mediated method. Since monocotyledonous plants are not natural hosts of Agrobacterium, although Agrobacterium-mediated genetic transformation of monocotyledonous plants such as lily has been successful, there are still problems such as low transformation efficiency, difficulty in obtaining materials, and sensitivity to Agrobacterium. Therefore, the key to genetic transformation of lily is to select receptor materials that are easy to infect, strengthen the importability of infection solution, and overcome the cell damage caused by Agrobacterium. At the same time, due to the properties of perennial underground bulbs of lily, its scales are difficult to disinfect and cannot be directly used as receptor materials. The establishment period of the first generation of sterile seedlings is long, endophytes are frequent in the later stage, and chimeras are prone to occur in the differentiation process of induced callus. In addition, the explant takes a long time from induction, expansion to rooting, and the operation is complicated, which further hinders the development of genetic transformation of lily. Therefore, the construction of an efficient genetic transformation system for lily is of great significance to the development of directional breeding and variety improvement technology of oriental lily. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a highly efficient genetic transformation method that can be used in the molecular breeding process of Oriental lily.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A method for efficient genetic transformation of oriental lily, comprising the following steps:

[0007] 1) Take sterile lily filaments and cut them into small pieces of 0.5-1 cm in length with a scalpel as explants. Dry them on a clean bench for 30 minutes.

[0008] 2) Place the explants prepared in step 1) in the Agrobacterium infection solution for 10-15 minutes to allow the filaments to fully contact with the Agrobacterium, then transfer to a dry co-culture medium and culture in the dark at 22-25°C for 2-3 days;

[0009] 3) Soak the filaments cultured in step 2) in a 250 mg / L sodium carbenicillin solution for 6-10 minutes, then wash them three times with sterile water, place them on sterile filter paper and blow dry them, then transfer them to the integrated screening culture medium and culture them in a dark environment at 22-25°C to allow the filaments to directly differentiate into small bulbs and take root.

[0010] Step 1) The sterile lily filaments are obtained by soaking the picked lily buds in an alcohol solution with a mass concentration of 75% for 30-60 seconds, washing them once with sterile water, and then placing them in an aqueous solution containing 2wt% sodium hypochlorite and 0.01vol% Tween for disinfection for 20-30 minutes, shaking them continuously during the process, then washing them with sterile water for 3-5 times, placing them on sterile filter paper and blowing them dry, then cutting the buds with a scalpel and taking out the sterile filaments inside.

[0011] Step 2) The Agrobacterium infection solution is obtained by streaking the Agrobacterium strain on a LB solid medium supplemented with 50 mg / L Rif, culturing at 28°C in the dark for 3 days, picking a single colony, and inoculating it into a LB liquid medium containing 50 mg / L Rif, culturing it at 28°C and 180-220 rpm for 16 hours under dark conditions, and then placing the cultured bacterial solution in a sterilized centrifuge tube, centrifuging at 5000 rpm for 5 minutes to collect the bacterial cells, and resuspending the bacterial cells with a modified resuspension solution to adjust the OD600 value to 0.3-0.5. The modified resuspension solution is 10 mmol / L MES + 100 μmol / L AS + 10 mmol / L MgCl2 + 10 mg / L LA, pH 5.8.

[0012] Step 2) The dry co-culture medium is MS+0.1 mg / L TDZ+1 mg / L NAA+1% inositol mother solution+60 g / L sucrose+100 μmol / L AS+10 mg / L LA+7 g / L agar, pH 5.8, and a layer of high-pressure sterilized filter paper is spread on the surface of the culture medium.

[0013] Step 3) The integrated screening culture medium is MS+0.1 mg / L TDZ+1 mg / L NAA+1% inositol mother solution+75 mg / L Kan+250 mg / L Cef+60 g / L sucrose+7 g / L agar, pH 5.8.

[0014] The above-mentioned inositol mother solution is prepared by dissolving 10g inositol, 0.2g glycine, 0.01g thiamine hydrochloride (VB1), 0.05g pyridoxine hydrochloride (VB6), and 0.05g nicotinic acid (VB3) in water and adjusting the volume to 1L.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects:

[0016] The invention provides an efficient genetic transformation method for oriental lily, which includes obtaining explants, agrobacterium infection and dry co-cultivation, and directly inducing small bulbs and rooting. The invention constructs an efficient genetic transformation system with oriental lily filaments as receptors: firstly, the filaments are easy to disinfect and can be directly used for genetic transformation, which solves the problem that transformation materials are difficult to obtain and have a high contamination rate; secondly, through the optimization of culture materials and formulas, small bulbs are directly regenerated, expanded and rooted through explants, and the induction pathway is single, which greatly simplifies the operation and overcomes the defects of chimeras; in addition, the infection ability is the key to the breakthrough of lily genetic transformation. The invention adopts dry co-cultivation treatment and improved heavy suspension, which significantly improves the permeability and infection power of agrobacterium and controls the excessive growth of agrobacterium in the later stage, wherein the use of LA antioxidant solves the adverse damage caused by agrobacterium infection.

[0017] Through the above-mentioned synergistic effects, the present invention can achieve a stable transformation positive rate of oriental lily of more than 45%, with high transformation efficiency and simple operation, providing new ideas and approaches for the establishment of an efficient genetic transformation system for oriental lily.

[0018] The present invention aims at the difficulties existing in the current genetic transformation process of lily, and establishes a simple, efficient and reliable genetic transformation method for oriental lily plants using oriental lily filaments as explants. Through this method, foreign genes can be efficiently and quickly transferred into lilies to obtain positive transgenic lily plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the process of obtaining the transformed plant using Oriental lily "Siberia" in Example 1; wherein a is the newly grafted cut filament; b is the elongated and swollen filament after 7 days of culture using the integrated screening medium; c is the filament that directly differentiates into buds after 20 days of culture; de ​​is the directly swollen small bulb and the roots produced;

[0020] Figure 2 This is a PCR identification diagram of the transgenic plants obtained in Example 1, wherein M is a DNA Marker, 1 is a plasmid positive control, 2 is a wild-type strain negative control, and 3-8 are resistant strains. DETAILED DESCRIPTION

[0021] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0022] Unless otherwise specified, the raw materials described in the examples are common commercial products. Unless otherwise specified, the experimental methods described in the examples are operated according to conventional molecular biology experimental methods. Reagents: thidiazuron (TDZ), lipoic acid (LA), α-naphthaleneacetic acid (NAA), rifampicin (Rif), 2-morpholineethanesulfonic acid (MES), acetosyringone (AS), cephalosporin (Cef), kanamycin (Kan), Agrobacterium (GV3101, purchased from Shanghai Weidi Biotechnology Co., Ltd.).

[0023] Example 1

[0024] A method for efficient genetic transformation of oriental lily, comprising the following steps:

[0025] 1. Treatment of Oriental Lily "Siberia" explants: Soak the lily buds in an alcohol solution with a mass concentration of 75% for 30-60 seconds, wash them with sterile water once, and then put them in an aqueous solution containing 2wt% sodium hypochlorite and 0.01vol% Tween for disinfection for 25 minutes, shaking them continuously during the process, and then wash them with sterile water for 3-5 times, place them on sterile filter paper and blow dry, then cut the buds with a scalpel, take out the sterile filaments inside, cut them into small sections of 0.5-1cm in length, and blow dry them on a clean bench for 30 minutes;

[0026] 2. Preparation of Agrobacterium infection solution: Streak the Agrobacterium strain on LB solid medium supplemented with 50 mg / L Rif, culture at 28°C in the dark for 3 days, pick a single colony, inoculate it into LB liquid medium containing 50 mg / L Rif, culture it at 28°C and 180-220 rpm for 16 hours in the dark, then place the cultured bacterial solution in a sterilized centrifuge tube, centrifuge at 5000 rpm for 5 minutes to collect the bacteria, resuspend the bacteria in a modified resuspension solution (10 mmol / L MES + 100 μmol / L AS + 10 mmol / L MgCl2 + 10 mg / L LA, pH 5.8), and adjust the OD600 value to 0.3-0.5 to obtain Agrobacterium infection solution;

[0027] 3. Agrobacterium infection of filaments: Place the dried explant filaments into the prepared Agrobacterium infection solution for infection for 10-15 minutes, so that the filaments are fully in contact with Agrobacterium, and then transfer them into dry co-culture medium (MS+0.1mg / L TDZ+1mg / L NAA+1% inositol mother solution+60g / L sucrose+100μmol / L AS+10mg / L LA+7g / L agar, pH 5.8, and a layer of autoclaved filter paper is spread on the surface of the culture medium), and culture in the dark at 25℃ for 2-3 days; the inositol mother solution used is 10g inositol, 0.2g glycine, 0.01g thiamine hydrochloride, 0.05g pyridoxine hydrochloride, and 0.05g nicotinic acid dissolved in water and fixed to 1L;

[0028] 4. Agrobacterium degerming and resistance screening: The filaments after co-cultivation in step 3 were soaked in 250 mg / L sodium carbenicillin solution for 6-10 min, then washed with sterile water for 3 times, placed on sterile filter paper and air-dried, and then transferred to integrated screening medium (MS+0.1 mg / L TDZ+1 mg / L NAA+1% inositol mother solution+75 mg / L Kan+250 mg / L Cef+60 g / L sucrose+7 g / L agar, pH 5.8), and cultured at 22-25°C in the dark. During this period, the changes in the filaments were observed. The results showed that the filaments were elongated and swollen after 7 days of culture. After 20 days of culture, small white buds were directly differentiated without the callus stage, and then swelled into small bulbs.

[0029] The obtained bulblet scales were cut into thin slices, and the lily DNA was extracted by CTAB method for PCR amplification. The results were as follows: Figure 2 As shown, PCR amplification products were detected in 4 seedlings among the 6 randomly selected resistant strains, thereby confirming that the exogenous gene had been integrated into the lily genome and establishing a genetic transformation system for lily.

[0030] Example 2

[0031] The scales, leaves and filaments of the bulbs of Oriental lily "Siberia" that grew well outdoors were taken as explants, and the same steps as in Example 1 were used for treatment. The contamination rate, bulblet induction rate and transformation positive rate of the explants were calculated when the integrated screening medium was used for 30 days. The results are shown in Table 1.

[0032] Table 1 Effects of different explants on lily genetic transformation

[0033]

[0034] Analysis of the above data shows that the contamination rate of scales as explants is very high, reaching 74%, and the endophytes are serious and cannot be controlled in the later stage, and positive plants cannot be obtained; the contamination rate of leaves as explants is 32%, the induction rate of small bulbs is 12%, and the positive rate is only 8%; while filaments as explants can achieve no contamination and a positive rate of 48%. This is because the filament tissue is tender, the structure is simple, and there is a one-dimensional tube bundle running through the center, which is more conducive to the penetration and absorption of the infection liquid, thereby improving the conversion rate. Therefore, using lily filaments as transformation receptors does not require the establishment of a first generation of sterile seedlings, and has the characteristics of being not easy to contaminate and having a high conversion rate.

[0035] Example 3

[0036] Sterile filaments of Oriental lily "Siberia" were used as explants and treated with different concentrations of TDZ (0.01 mg / L, 0.1 mg / L, 0.5 mg / L, 1 mg / L) and NAA (0.5 mg / L, 1 mg / L). The induction of bulblets was observed after 30 days.

[0037] Table 2 Effects of different hormone treatments on the induction of lily filament bulblets

[0038]

[0039] The results showed that among the different concentration treatments, when the TDZ concentration was 0.01 mg / L, it could not effectively induce the occurrence of small bulbs, while when the concentration was 1 mg / L, the filaments turned brown and died, and the increase in NAA concentration was conducive to the occurrence of small bulbs, indicating that Oriental lily was more sensitive to TDZ. In the treatment of MS+0.1 mg / L TDZ+1 mg / L NAA+30 g / L sucrose+7 g / L agar, the induction rate was the highest, reaching 56%.

[0040] Example 4

[0041] Sterile filaments of Oriental lily "Suobang" were taken as explants, and different concentrations of silver nitrate (2 mg / L, 5 mg / L, 10 mg / L), VC (50 mg / L, 75 mg / L, 100 mg / L), and LA (2 mg / L, 10 mg / L, 20 mg / L) were used for co-culture treatment with antioxidants. The browning rate of the explants was calculated at 30 days.

[0042] Table 3 Effects of different concentrations of antioxidants on filament induction and regeneration

[0043]

[0044] The results showed that lily silk was sensitive to Agrobacterium. After infection, the silk was prone to browning without antioxidant treatment, with a browning rate of 46.7%. Among different types of antioxidants, the browning rate of 2 mg / L silver nitrate treatment was 15%, and high concentrations produced certain toxicity and increased the browning rate. VC had a weak effect on silk, with a browning rate of 18.3% when treated with 100 mg / L, while the silk was basically not browned under 10 mg / L LA treatment, with a browning rate of only 1.7%. Therefore, the use of LA can effectively solve the tissue necrosis and browning during silk transformation and overcome the adverse damage during Agrobacterium infection.

[0045] Example 5

[0046] To further investigate the infection process of filaments, different resuspensions (① resuspension: MS+100mg / L AS, ② improved resuspension: 10mmol / L MES +100μmol / L AS+10 mmol / L MgCl2+10 mg / L LA) were used to prepare Agrobacterium infection solution, and explants prepared from sterile filaments of Oriental lily “Suobang” were infected in different ways (① co-culture: sterile filaments were not dried before infection, and were placed in a co-culture medium of MS+0.1mg / L TDZ+1mg / L NAA+1% inositol mother solution+60g / L sucrose+100μmol / L AS+10 mg / L LA+7g / L agar after Agrobacterium infection; ② dry co-culture: sterile filaments were blow-dried on a clean bench for 30min before infection, and were placed in a co-culture medium of MS+0.1mg / L TDZ+1mg / L NAA+1% inositol mother solution+60g / L sucrose+100μmol / L AS+10 mg / L LA+7g / L agar after Agrobacterium infection). The culture medium was NAA+1% inositol stock solution+60 g / L sucrose+100 μmol / L AS+10 mg / L LA+7 g / L agar, and a layer of sterile filter paper was placed on the culture surface of the co-culture medium), and the results are shown in Table 4.

[0047] Table 4 Effects of different infection combinations on lily silk transformation

[0048]

[0049] The resuspension is the key to Agrobacterium infection. From the comparison of treatments 1 and 2 in Table 4, it can be seen that removing MS from the resuspension has no effect on transformation, while adding surfactant MES and ion buffer MgCl2 can significantly increase the adsorption of Agrobacterium on filaments, improve cell membrane permeability, and further enhance the infection ability of Agrobacterium. LA reduces the adverse stress of Agrobacterium, so the positive rate of treatment 2 is increased to 33.3%.

[0050] Compared with co-cultivation, the positive rate of treatment 4 after drying co-cultivation reached 48.3%. This may be because appropriate drying treatment can promote the dehydration of filament cells, cause slight plasmolysis, promote the introduction of Agrobacterium, and thus enhance the efficiency of Agrobacterium. In addition, placing a layer of sterile filter paper on the culture surface can significantly inhibit the excessive growth of Agrobacterium in the later stage, reduce Agrobacterium contamination, and promote the recovery of receptor materials, thereby increasing the stable transformation positive rate of lily filaments.

[0051] Example 6

[0052] Treatment 1: Sterile filaments were taken as explants and first placed in an induction medium (MS+1 mg / L TDZ+0.5 mg / L NAA) for 30 days. When buds began to appear, they were transferred to a primary expansion medium (MS+1 mg / L TDZ+0.5 mg / L NAA+90 g / L sucrose) for 15 days, and then transferred to a rooting medium (MS+1 mg / L NAA) for culturing.

[0053] Treatment 2: Take sterile filaments as explants, first place them in induction medium (MS+0.01mg / L TDZ+0.5mg / LNAA) and culture for 30 days. When buds begin to appear, continue to place them in the original culture medium and subculture every 15 days. After the bulblets grow to 2-3cm, transfer them to rooting medium (1 / 2MS+0.5mg / L IBA) for culture.

[0054] Treatment 3: Sterile filaments were taken as explants and placed in induction medium (MS+0.1mg / L TDZ+1mg / L NAA+60g / L sucrose) for culture.

[0055] Treatment 4: Sterile filaments were taken as explants and placed in an integrated culture medium (MS + 0.1 mg / L TDZ + 1 mg / L NAA + 1% inositol mother solution + 60 g / L sucrose) for culture.

[0056] Table 5 Effects of different culture media on bulblet differentiation

[0057]

[0058] Conventional rooting of lilies generally uses auxins such as NAA or IBA. If a certain proportion of auxins is added during the induction period to promote rooting, it is easy to cause abnormal differentiation and failure to induce buds normally, so staged culture is required. Inositol mother solution is an organic matter and does not replace hormones to exert its effects, but it is beneficial to its development and rooting. Inositol in inositol mother solution promotes cell reproduction and differentiation. Thiamine and other B vitamins, as cofactors of metabolism, are involved in the synthesis of amino acids and the occurrence of in vitro roots, which can promote bud induction and direct rooting in the later stage. In addition, the increase in sucrose concentration can accelerate the expansion of small bulbs and shorten the development cycle. Therefore, compared with the conventional three-stage culture method of induction, expansion, and rooting, the use of an integrated culture medium containing 1% inositol mother solution and 60g / L sucrose for non-segmented culture can achieve direct differentiation of small bulbs and rooting of filaments without the callus stage, which greatly simplifies the operation, and its bud induction rate reaches 90%, and the culture cycle can be shortened by about 25d.

[0059] Finally, it should be noted that the above-mentioned embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. Although the present invention has been described in detail through the above-mentioned examples, those skilled in the art should understand that several improvements and modifications may be made to the present invention in terms of form and details without departing from the scope defined in the claims of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. A method for efficient genetic transformation of Oriental lily, characterized in that: The following steps are involved: 1) Take sterile lily filaments, cut them into small pieces of 0.5-1 cm in length as explants, and dry them on a clean bench for 30 minutes; 2) Place the explants prepared in step 1) in the Agrobacterium infection solution for 10-15 minutes to allow the filaments to fully contact with the Agrobacterium, then transfer them to a dry co-culture medium and culture them in the dark at 22-25°C for 2-3 days; 3) Soak the filaments cultured in step 2) in a 250 mg / L sodium carbenicillin solution for 6-10 minutes, then wash them with sterile water for 3 times, place them on sterile filter paper and blow dry them, then transfer them to the integrated screening culture medium and culture them in a dark environment at 22-25°C to allow the filaments to directly differentiate into small bulbs and take root; Step 2) the Agrobacterium infection solution is prepared by streaking the Agrobacterium strain on an LB solid medium supplemented with 50 mg / L Rif, culturing at 28°C in the dark for 3 days, picking a single colony, inoculating it into an LB liquid medium containing 50 mg / L Rif, and culturing it at 28°C and 180-220 rpm for 16 hours under dark conditions, then placing the cultured bacterial solution in a sterilized centrifuge tube, centrifuging at 5000 rpm for 5 minutes to collect the bacterial cells, and resuspending the bacterial cells with a modified resuspension solution to adjust the OD600 value to 0.3-0.5; the modified resuspension solution is 10 mmol / L MES + 100 μmol / L AS + 10 mmol / L MgCl2 + 10 mg / L LA, pH 5.8; Step 2) the dry co-culture medium is MS+0.1mg / L TDZ+1mg / L NAA+1% inositol mother solution+60g / L sucrose+100μmol / L AS+10mg / L LA+7g / L agar, pH 5.8, and a layer of autoclaved filter paper is spread on the surface of the culture medium; Step 3) The integrated screening medium is MS+0.1mg / L TDZ+1mg / L NAA+1% inositol mother solution+75mg / L Kan+250mg / L Cef+60g / L sucrose+7g / L agar, pH 5.8; The inositol mother solution is prepared by dissolving 10 g of inositol, 0.2 g of glycine, 0.01 g of thiamine hydrochloride, 0.05 g of pyridoxine hydrochloride and 0.05 g of nicotinic acid in water and then fixing the volume to 1 L.

2. The method for efficient genetic transformation of oriental lily according to claim 1, characterized in that: Step 1) The sterile lily filaments are prepared by soaking the lily buds in an alcohol solution with a mass concentration of 75% for 30-60 seconds, washing them once with sterile water, and then placing them in an aqueous solution containing 2wt% sodium hypochlorite and 0.01vol% Tween for disinfection for 20-30 minutes, shaking them continuously, washing them 3-5 times with sterile water, placing them on sterile filter paper and blowing them dry, then cutting the buds with a scalpel and taking out the sterile filaments inside.

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