Taro chemical mutagenesis method

Abstract

The invention discloses a method for chemical mutagenesis of taros. The method comprises the following steps: cutting off roots and leaves on a taro test-tube taro, then putting the treated test-tube taro into a filtered and sterilized Pingyangmycin mutagenic agent containing SilwetL-77, carrying out shake culture for mutagenesis, washing a test-tube plantlet subjected to mutagenesis treatment, carrying out bud induction culture, multiplication culture and rooting culture, and carrying out mutant identification to obtain the taro mutant. According to the method, 0.02% of SilwetL-77 is added into the mutagenic agent, so that the mutagenic agent is accelerated to be absorbed, attached and permeated into the explant, the toxic action of the mutagenic agent on the explant is also avoided, and a very good mutagenic effect can be achieved at a relatively low concentration.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant breeding, and particularly relates to a method for chemical mutagenesis of taro. Background Art

[0002] Taro (Colocasia esculenta (L.) Schott) is a perennial herb of the genus Colocasia in the Araceae family. It is mostly cultivated annually. Under conventional cultivation, it is very difficult for taro to flower and set seeds. In production, asexual reproduction is mostly used. Compared with other vegetable crops, the breeding work of taro is relatively backward. At present, the taro varieties used in production are basically traditional local varieties. Some newly bred varieties are also selected through methods such as field systematic selection or tissue culture bud mutation. The variety replacement is still relatively slow, resulting in the lack of strong guarantee for the yield and quality of taro.

[0003] Chemical mutagenesis has the characteristics of simple and easy operation, high mutation frequency, strong specificity, etc. Combining chemical mutagen with tissue culture technology is an important means for current plant breeding. In the existing plant tissue culture rapid propagation combined with chemical mutagenesis breeding technology, in terms of explant selection, when using single buds as explants, they need to be disinfected first and then treated with mutagen. After multiple treatments in a short time, the damage to the explants is too large and the effect is difficult to guarantee; when using adventitious buds of tissue culture seedlings as explants, the tissue culture seedlings are relatively tender and are easily killed completely. If the concentration is low, the mutation rate is low and the dosage is difficult to screen; when using callus as explants, embryogenic callus needs to be induced first and an efficient plant regeneration system needs to be established. This is not only a cumbersome process, but also the existing technology is difficult to ensure the induction rate of adventitious buds. In terms of the selection of mutagen, colchicine, ethyl methanesulfonate (EMS), etc. are mostly used, but they are highly toxic and can cause lethal damage to plants and pose a greater threat to human health; there are also herbicides used, such as trifluralin and oryzalin for mutagenesis of Hemerocallis fulva, Arnebia euchroma, etc. Although there are good induction effects, they also pose a greater threat to human health and have obvious toxic effects on plants; Bleomycin (PYM) is a unique antibiotic and also a new type of chemical mutagen. Its mutagenic characteristics are similar to those of EMS, but its chemical properties are more stable and the damage to plants is smaller.

[0004] At present, there are few reports on the chemical mutagenesis of taro. Generally, single buds of taro or adventitious buds of tissue culture seedlings are used as explants, and colchicine or herbicides such as oryzalin are used as mutagens. The mutagenesis rate is low and it is harmful to humans. There is an urgent need for a more effective mutagenesis method. Summary of the Invention

[0005] In view of the existing technical problems, the present invention provides a method for chemical mutagenesis of taro, which has certain reference and reference value for taro chemical mutagenesis breeding, germplasm innovation and new variety cultivation.

[0006] The method for chemical mutagenesis of taro in the present invention is to completely remove the roots and leaves on the test-tube taro of taro, and then put the treated test-tube taro into the pingyangmycin mutagen containing Silwett L-77 that has been filtered and sterilized, and perform mutagenesis by shaking culture, wash the test-tube seedlings after mutagenesis treatment, carry out bud induction culture, proliferation culture, rooting culture, and then obtain taro mutants through mutant identification.

[0007] Preferably, for the pingyangmycin mutagen containing Silwett L-77, the concentration of Silwett L-77 is 0.02% by mass fraction.

[0008] Preferably, for the pingyangmycin mutagen containing Silwett L-77, the concentration of pingyangmycin is 50 μg / mL.

[0009] Preferably, for the mutagenesis, it is oscillating immersion culture at 180 rpm and 25 ± 2 °C on a rotary shaker for 48 h.

[0010] Preferably, for the test-tube seedlings, the induction method is: cut a single plant seedling of 2 - 4 cm from the proliferated buds of taro, inoculate it in the test-tube taro induction medium for culture, and the test-tube taro induction medium is GS + 60 g / L sucrose + 6 g / L carrageenan, and the culture conditions are: temperature 25 ± 2 °C, 12 h of daily illumination, and illumination intensity 2000 lux;

[0011] The GS medium contains the following components: 1200 mg / L NH 4 NO 3 、2100 mg / L KNO 3 、220 mg / L CaCl 2 ·2H 2 O、370 mg / L MgSO 4 ·7H 2 O、255 mg / L KH 2 PO 4 、22.3 mg / L MnSO 4 ·4H 2 O、8.6 mg / L ZnSO 4 ·7H 2 O、6.2 mg / L H 3 BO 3 、0.83 mg / L KI、0.25 mg / L Na 2 MoO 4 ·2H 2 O、0.25 mg / LCuSO 4 ·5H 2 O、0.025 mg / LCoCl、37.3 mg / L FeSO 4 ·7H2 O, 27.8 mg / L Na 2 ·EDTA, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L nicotinic acid, 0.5 mg / L pyridoxine hydrochloride, pH 5.8, and the solvent is water.

[0012] Preferably, the washing is to wash the mutagenized tube taro with sterile water 5 times;

[0013] Preferably, for the bud induction culture: inoculate the washed tube taro into the bud induction medium for culture. The bud induction medium is: YS + 30 g / L sucrose + 6 g / L carrageenan, the culture temperature is 25 ± 2 °C, dark culture for the first 7 days, and then 8 h of light per day, with a light intensity of 1500 lux.

[0014] Preferably, for the proliferation culture: cut the adventitious buds germinated on the tube taro and inoculate them into the proliferation medium for culture. The proliferation medium is: YS + 2 mg / L 6 - BA + 0.1 mg / L NAA + 30 g / L sucrose + 6 g / L carrageenan, the culture temperature is 25 ± 2 °C, dark culture for the first 7 days, and then 8 h of light per day, with a light intensity of 2000 lux.

[0015] Preferably, for the rooting culture: cut a single - plant seedling with a length of 2 - 4 cm from the proliferated buds and inoculate it into the rooting medium for rooting culture. The rooting medium is: 1 / 2MS + 0.1 mg / L NAA + 30 g / L sucrose + 6 g / L carrageenan + 0.5 g / L activated carbon, the culture temperature is 25 ± 2 °C, 12 h of light per day, with a light intensity of 2500 lux;

[0016] Preferably, for the mutant identification: first, preliminarily identify from morphological indexes such as plant height, leaf morphology, leaf size, petiole color, etc., and screen out mutant single plants that are different from the tissue - cultured seedlings of the original variety; then conduct SSR molecular marker identification on the screened single plants.

[0017] Preferably, the YS medium contains the following components: 1650 mg / L NH 4 NO 3 、1900 mg / L KNO 3 、220 mg / LCaCl 2 ·2H 2 O、277 mg / L MgSO 4 ·7H 2 O、255 mg / L KH 2 PO 4 、22.3 mg / L MnSO 4 ·4H 2 O、8.6 mg / LZnSO4 ·7H 2 O, 6.2 mg / L H 3 BO 3 , 0.83 mg / L KI, 0.25 mg / L Na 2 MoO 4 ·2H 2 O, 0.25 mg / L CuSO 4 ·5H 2 O, 0.025 mg / L CoCl, 37.13 mg / L FeSO 4 ·7H 2 O, 49.6 mg / L Na 2 ·EDTA, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L niacin, 0.5 mg / L pyridoxine hydrochloride, pH 5.8, solvent is water.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) The present invention uses test tube taro as the explant. The test tube taro is easy to induce, convenient to operate, and has uniform and stable size, which can ensure the induction effect and is also convenient for the induction and screening of adventitious buds in the later stage.

[0020] (2) The present invention adds 0.02% of Silwett L-77 to the mutagen, which not only accelerates the absorption, attachment and penetration of the mutagen into the explant, but also avoids the toxic effect of the mutagen on the explant, and can achieve a good mutagenic effect at a lower concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Is the explant germinated after treatment with 0.02% Silwett L-77 + 50 μg / mL PYM.

[0022] Figure 2 Are some mutant tissue culture seedlings selected by the plant morphological identification index method. Note: CK is the control, and the others are mutants.

[0023] Figure 3 Is the UPGMA clustering analysis diagram based on Nei's genetic distance after SSR molecular marker identification. Note: A1 - A19 are the mutants obtained in Example 1, and the other taro are commercially available taro. DETAILED DESCRIPTION OF THE INVENTION

[0024] The following examples are further illustrations of the present invention, rather than limitations on the present invention.

[0025] Example 1:

[0026] (1) Tube taro induction: Cut single plant seedlings of 2 - 4 cm from the proliferated buds and inoculate them into the tube taro induction medium for 60 days. The tube taro induction medium is GS + 60 g / L sucrose + 6 g / L carrageenan, and the culture conditions are: temperature 25 ± 2 °C, 12 h of daily light, and light intensity 2000 lux.

[0027] The GS medium contains the following components: 1200 mg / L NH 4 NO 3 、2100 mg / L KNO 3 、220 mg / L CaCl 2 ·2H 2 O、370 mg / L MgSO 4 ·7H 2 O、255 mg / L KH 2 PO 4 、22.3 mg / L MnSO 4 ·4H 2 O、8.6 mg / L ZnSO 4 ·7H 2 O、6.2 mg / L H 3 BO 3 、0.83 mg / L KI、0.25 mg / L Na 2 MoO 4 ·2H 2 O、0.25 mg / LCuSO 4 ·5H 2 O、0.025 mg / LCoCl、37.3 mg / L FeSO 4 ·7H 2 O、27.8 mg / L Na 2 ·EDTA、2.0 mg / L glycine、0.1 mg / L thiamine hydrochloride、0.5 mg / L nicotinic acid、0.5 mg / L pyridoxine hydrochloride, pH 5.8, and the solvent is water.

[0028] (2) Mutation treatment: Cut off all the roots and leaves on the induced tube taro, cut the leaves to 0.2 cm from the shoot tip, and then put the treated tube taro into a 50 μg / mL PYM solution containing 0.02% (mass fraction) Silwett L - 77 that has been filter - sterilized, and culture it by shaking and soaking on a rotary shaker at 180 rpm and 25 ± 2 °C for 48 h.

[0029] (3) Washing treatment: Wash the tube taro treated by mutation 5 times with sterile water.

[0030] (4) Bud induction culture: The washed taro in test tubes is inoculated into the bud induction medium for culture. The bud induction medium is: YS + 30 g / L sucrose + 6 g / L carrageenan. The culture temperature is 25 ± 2 °C. Dark culture is carried out for the first 7 days, and then it is illuminated for 8 hours every day with a light intensity of 1500 lux;

[0031] The YS medium contains the following components: 1650 mg / L NH 4 NO 3 、1900 mg / L KNO 3 、220 mg / L CaCl 2 ·2H 2 O, 277 mg / L MgSO 4 ·7H 2 O, 255 mg / L KH 2 PO 4 、22.3 mg / L MnSO 4 ·4H 2 O, 8.6 mg / L ZnSO 4 ·7H 2 O, 6.2 mg / L H 3 BO 3 、0.83 mg / L KI, 0.25 mg / L Na 2 MoO 4 ·2H 2 O, 0.25 mg / L CuSO 4 ·5H 2 O, 0.025 mg / L CoCl, 37.13 mg / L FeSO 4 ·7H 2 O, 49.6 mg / L Na 2 ·EDTA, 100 mg / L inositol, 2.0 mg / L glycine, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L nicotinic acid, 0.5 mg / L pyridoxine hydrochloride, pH 5.8, and the solvent is water;

[0032] (5) Proliferation culture: The adventitious buds germinated on the taro in test tubes are cut off and inoculated into the proliferation medium for culture, and each bud is numbered separately. The proliferation medium is: YS + 2 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 6 g / L carrageenan. The culture temperature is 25 ± 2 °C. Dark culture is carried out for the first 7 days, and then it is illuminated for 8 hours every day with a light intensity of 2000 lux;

[0033] (6) Rooting culture: Cut single plantlets of 2 - 4 cm in length from the proliferated buds and inoculate them into the rooting medium for rooting culture. The rooting medium is: 1 / 2 MS + 0.1 mg / L NAA + 30 g / L sucrose + 6 g / L carrageenan + 0.5 g / L activated carbon. The culture temperature is 25 ± 2°C, with 12 hours of daily illumination and a light intensity of 2500 lux;

[0034] (7) Mutant identification: First, conduct a preliminary identification based on morphological indexes such as plant height, leaf morphology, leaf size, petiole color, etc., and screen out mutant single plants that are different from the tissue-cultured seedlings of the original variety; then conduct SSR molecular marker identification on the selected single plants.

[0035] Comparative Example 1

[0036] In this comparative example, during the mutagenesis treatment in step (2), 0.02% Silwett L-77 is not added to the mutagen, and the rest of the operations are the same as those in Example 1, so they will not be elaborated here.

[0037] Comparative Example 2

[0038] The explants used in this comparative example are proliferated buds: The proliferated buds with leaves removed and 0.5 - 1.0 cm of shoot tips retained are used as explants, with 2 - 3 buds in a cluster.

[0039] Except for the different explants, the rest of the operations in this comparative example are the same as those in Example 1, and will not be elaborated here.

[0040] Comparative Example 3

[0041] The explants used in this comparative example are single buds: Select healthy single buds of taro in the current year. First, cut the single buds into shoot tips of 2 cm × 2 cm × 3 cm in size, place them in a laminar flow hood for sterilization treatment, soak them in 75% (v / v) alcohol for 1 minute, wash them 3 - 4 times with sterile water, then take them out and put them into a 0.1% (w / v) mercuric chloride solution for sterilization for 10 minutes, shake continuously during this period, and finally wash them 5 - 6 times with sterile water. Place them on an inoculation tray and cut the explants into shoot tips of 0.5 cm × 0.5 cm × 1 cm in size, and finally blot the surface moisture of the single taro buds with sterile filter paper.

[0042] Except for the different explants, the rest of the operations in this comparative example are the same as those in Example 1, and will not be elaborated here.

[0043] Mortality rate (%) = (Number of dead explants / Number of treated explants) × 100%; Mutation rate (%) = (Number of explants with morphological mutations after germination / Number of treated explants) × 100%

[0044] Table 1

[0045]

[0046]

[0047] The lethality and mutagenesis rates of the statistical examples and comparative examples were counted, and the results are shown in Table 1.

[0048] In the present invention, test-tube taro was used as the explant material, and 0.02% Silwett L-77 was added to 50 μg / mL PYM. The mortality rate of the explant was 52%, and the mutagenesis rate was 46%. Using test-tube taro as the explant material improved the tolerance to chemical mutagens, and adding 0.02% Silwett L-77 to the mutagen significantly increased the mutagenesis rate.

[0049] The explant material used in the present invention was test-tube taro of Zhangxi betel nut taro. Using SSR fluorescence-labeled capillary electrophoresis technology, the mutants and some commercially available betel nut taro varieties were identified and analyzed. The mutant materials did not cluster together, and most of the mutant materials did not cluster in the same group as the parent, indicating a wide range of mutations.

[0050] The above examples are the preferred embodiments of the present invention, but the examples of the present invention are only used to illustrate the technical solutions of the present invention rather than to limit. Within the scope defined by the claims of the present invention, any modifications and changes made by technicians in the art to the present invention should also fall within the protection scope of the claims of the present invention.

Claims

1. A method for chemical mutagenesis of taro, characterized in that, The method comprises the steps of cutting off the roots and leaves of the taro test tube taro, placing the treated test tube taro into a filtered and sterilized bleomycin mutagen containing SilwettL-77, culturing by shaking for mutagenesis, washing the mutagenized test tube seedlings, performing bud induction culture, proliferation culture, rooting culture, and finally obtaining taro mutants through mutant identification.

2. The method according to claim 1, characterized in that The concentration of SilwettL-77 in the Pingyangmycin mutagen containing SilwettL-77 is 0.02% by mass.

3. The method according to claim 1, characterized in that The bleomycin concentration of the SilwettL-77-containing bleomycin mutagen is 50 μg / mL.

4. The method according to claim 1, characterized in that: The mutagenesis was carried out by shaking and immersion culture on a rotary shaker at 180 rpm and 25±2°C for 48 hours.

5. The method according to claim 1, characterized in that The induction method of the test tube seedling is as follows: a single seedling of 2 to 4 cm is cut from a taro proliferation bud, and the seedling is inoculated in a test tube taro induction medium for cultivation. The test tube taro induction medium is GS+60 g / L sucrose+6 g / L carrageenan. The culture conditions are as follows: temperature 25±2°C, daily illumination 12 h, and illumination intensity 2000 lux. The GS medium contains the following ingredients: 1200 mg / L NH4NO3, 2100 mg / L KNO3, 220 mg / L CaCl2·2H2O, 370 mg / LMgSO4·7H2O, 255 mg / L KH2PO4, 22.3 mg / L MnSO4·4H2O, 8.6 mg / L ZnSO4·7H2O, 6.2 mg / L H3BO3, 0.83 mg / L KI, 0.25 mg / L Na2MoO4·2H2O, 0.25mg / LCuSO4·5H2O, 0.025mg / LCoCl, 37.3mg / L FeSO4·7H2O, 27.8mg / L Na2·EDTA, 2.0mg / L glycine, 0.1mg / L thiamine hydrochloride, 0.5mg / L nicotinic acid, 0.5mg / L pyridoxine hydrochloride, pH 5.8, solvent is water.

6. The method according to claim 1, characterized in that The bud induction culture is as follows: the washed test tube taro is inoculated into a bud induction medium for culture, wherein the bud induction medium is: YS+30g / L sucrose+6g / L carrageenan, the culture temperature is 25±2°C, the first 7 days of dark culture, and then the light is exposed for 8 hours per day, and the light intensity is 1500lux.

7. The method according to claim 1, characterized in that The proliferation culture comprises cutting off the adventitious buds germinated on the test tube taro, inoculating them in a proliferation culture medium for culture, wherein the proliferation culture medium comprises: YS+2mg / L 6-BA+0.1mg / L NAA+30g / L sucrose+6g / L carrageenan, the culture temperature is 25±2°C, the first 7 days of dark culture are carried out, and then the light is exposed for 8 hours per day, and the light intensity is 2000lux.

8. The method according to claim 1, characterized in that The rooting culture is as follows: a single seedling of 2 to 4 cm is cut from the proliferation culture bud and inoculated into a rooting medium for rooting culture. The rooting medium is: 1 / 2MS+0.1mg / LNAA+30g / L sucrose+6g / L carrageenan+0.5g / L activated carbon. The culture temperature is 25±2°C, the light exposure is 12 hours per day, and the light intensity is 2500lux.

9. The method according to claim 1, characterized in that: The mutant identification is firstly carried out based on morphological indicators such as plant height, leaf shape, leaf size, petiole color, etc., to screen out mutant plants that are different from the original variety tissue culture seedlings; then the screened plants are identified by SSR molecular markers.

10. The method according to claim 6 or 7, characterized in that: The YS medium contains the following ingredients: 1650 mg / L NH4NO3, 1900 mg / L KNO3, 220 mg / L CaCl2·2H2O, 277 mg / L MgSO4·7H2O, 255 mg / L KH2PO4, 22.3 mg / L MnSO4·4H2O, 8.6 mg / L ZnSO4·7H2O, 6.2 mg / L H3BO3, 0.83 mg / L KI, 0.25 mg / L Na2MoO4·2H2O, 0.25 mg / L CuSO4·5H2O, 0.025 mg / L CoCl, 37.13 mg / L FeSO4·7H2O, 49.6mg / LNa2·EDTA, 100mg / L inositol, 2.0mg / L glycine, 0.1mg / L thiamine hydrochloride, 0.5mg / L nicotinic acid, 0.5mg / L pyridoxine hydrochloride, pH 5.8, solvent is water.

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