Tissue culture and rapid propagation method suitable for ornamental taro

Abstract

The invention provides a tissue culture and rapid propagation method suitable for ornamental taro, and relates to the technical field of plant tissue culture. Comprising the following steps: step 1, preparing materials, namely selecting healthy ornamental taro plants without diseases and insect pests, taking tender lateral buds, stem tips or leaves of the ornamental taro plants as explants, cleaning the explants, disinfecting and cutting the explants; step 2, induction culture: taking MS as a basic culture medium, adding biogenic amine, NAA, TDZ, coconut milk, cane sugar and ascorbic acid to prepare an induction culture medium, and then culturing the explant obtained in the step 1 in a constant-temperature and light-dark culture environment until visible green buds germinate. Through ultrasonic-assisted combined disinfection of the explant, the pollution rate is obviously reduced and the induction culture effect is improved on the premise of reducing the damage of the explant, and through the induction culture of a modulated light source and on the basis of inhibiting spindling, the bud is more robust, the chlorophyll content is increased, and the bud can be continuously subcultured for 8-10 times after multiplication culture without obvious degeneration.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant tissue culture technology, in particular to a tissue culture and rapid propagation method suitable for ornamental taros. BACKGROUND

[0002] Ornamental taros are common Araceae ornamental plants, mainly leaf observation, with rich and varied color, covering a wide color spectrum from colorful, mysterious dark to fresh green, presenting unique artistic quality. With its unique ornamental characteristics and ecological adaptability, this kind of plant shows wide performance in landscape and indoor application.

[0003] The existing ornamental taro planting, such as application number CN202210431563.0, a tissue culture and rapid propagation method for keeping the stable ornamental plant traits of chimeric plants. It belongs to the field of plant nutrition, tissue culture and cultivation technology. The steps are as follows: select the axillary bud carrying the target chimeric ornamental leaf as the explant; after disinfecting the explant, inoculate it into induction medium I, then place it in a culture room to induce the germination of latent axillary buds; control the temperature, light and humidity conditions in the culture room. Acclimatize under natural light, then transplant the bottle. Compared with the current chimeric tissue culture and propagation technology, it can stably carry out tissue culture and rapid propagation, but the explant is prone to browning, and the tissue damage after disinfection is relatively large, and the tissue culture efficiency is low. SUMMARY

[0004] To achieve the above purpose, the present application is realized by the following technical scheme: a tissue culture and rapid propagation method suitable for ornamental taros, comprising the following steps: Step 1, material preparation Select healthy, disease-free ornamental taro plants, and use their tender lateral buds, stem tips or leaves as explants. Wash the explants clean, disinfect and divide them; Step 2, induction culture Use MS as the basic medium, add biological amine, NAA, TDZ, coconut milk, sucrose and ascorbic acid to make the induction medium, then place the explants obtained in step 1 in a constant temperature and light and dark culture environment until green bud points are visible; Step 3, proliferation culture Use liquid MS as the basic medium, add hormones, coconut milk, PVP, ascorbic acid and sucrose to make the proliferation medium, and scale up the bud expansion in the bioreactor; Step 4, rooting culture Use 1 / 2MS as the basic medium, add IBA, NAA, activated carbon, humic acid, sucrose and vermiculite to make the semi-liquid rooting medium, and perform rooting culture on the products obtained in step 3.

[0005] Preferably, in the step one, the explant is washed with flowing clean water, then soaked with 75% ethanol for 30 seconds, immediately washed with sterile water for 2-3 times, disinfected with 2% NaClO combined with 0.1% Tween-20, and washed with sterile water again through ultrasonic assistance.

[0006] Preferably, the explant after being divided in the step one is soaked with 0.5% ascorbic acid, 0.2% citric acid and 0.1% polyvinylpyrrolidone for 2-5 seconds, and then placed in a sterile environment until the surface is dry.

[0007] Preferably, the light and dark culture environment is a dark environment for 6-7 days, and then the explant is cultured in a light environment with LED white light with a PPFD of 30 μmol·m -2 ·s -1 , 16h / d.

[0008] Preferably, the light quality combination of the LED white light is 70% red light (660nm), 20% blue light (450nm) and 10% far red light (730nm).

[0009] Preferably, the hormone is 6-BA 3.0 mg / L, NAA 0.1 mg / L and KT 0.5 mg / L.

[0010] Preferably, the bioreactor is a temporary immersion bioreactor, the immersion frequency is 5 minutes every 4 hours, the aeration is sterile air filtered by 0.1 μm, and the inoculation amount is 200 buds per 5L reactor.

[0011] Preferably, after the rooting culture, the explant is acclimated in an artificial climate environment, and then transplanted into a substrate culture with a weight ratio of peat:coconut husk:perlite:humus soil of 3:3:2:2.

[0012] The application provides a tissue culture and rapid propagation method suitable for ornamental taros. In the application, the ultrasonic assistance combined disinfection of the explant significantly reduces the contamination rate and improves the induction culture effect under the premise of reducing the damage of the explant, the induction culture of the light source inhibits the overgrowth, the bud is more robust, the chlorophyll content is improved, and the explant can be subcultured for 8-10 times continuously without obvious degradation. DETAILED DESCRIPTION

[0013] The technical solutions in the embodiments of the application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the application.

[0014] The embodiment of the present application provides a kind of tissue culture rapid propagation method suitable for ornamental taros, comprising the following steps: Step one, material preparation Select healthy, no disease and pest ornamental taro plants, for example, the plants of Colocasia, Alocasia, use its tender lateral bud, stem tip or leaf as explant, after washing clean, disinfect and divide, specifically, after washing with flowing water, use 75% ethanol to soak for 30 seconds, then immediately wash with sterile water for 2-3 times, realize bacteriostasis, disinfect with 2% NaClO combined with 0.1% Tween-20, and improve permeability by ultrasonic assistance (40 kHz, 5 min), which can improve the disinfection effect, then wash clean with sterile water, after dividing, soak the explant in 0.5% ascorbic acid, 0.2% citric acid and 0.1% polyvinylpyrrolidone for 2-5 seconds, and place in a sterile environment until the surface is dry, 0.5% ascorbic acid, 0.2% citric acid and 0.1% polyvinylpyrrolidone are used to prevent phenolic oxidative browning of the explant, compared with the traditional use of 0.1% HgCl2 disinfection, it is more thorough, avoids the strong toxicity and high risk of HgCl2, is more friendly to plant tissue, and is more environmentally friendly and safe, the ultrasonic assisted disinfection and antioxidant compound system significantly reduces the pollution rate; Step two, induction culture Use MS as the basic culture medium, add 6-BA 2.2 mg / L, NAA 0.08 mg / L, TDZ 0.05 mg / L, coconut milk 50 mL / L, sucrose 30 g / L and ascorbic acid 50 mg / L to prepare an induction culture medium with pH 5.8, then place the explants obtained in step one in a constant temperature and light-dark culture environment until green bud spots are visible, the induction rate is increased to 96.3%, the average number of buds is 7.8 per explant, and the browning rate is reduced to 6.1%, specifically, in a temperature range of 25±1°C, after 6-7 days of dark culture, use LED white light with PPFD 30 μmol·m -2 ·s -1 -2, 16 h / d, the light quality combination of LED white light is red light (660 nm) for promoting cell division and bud proliferation, 70% blue light (450 nm) and 20% for inhibiting overgrowth, promoting chlorophyll synthesis, and 10% far red light (730 nm) for regulating phytochrome and enhancing bud elongation, compared with single white light, the proliferation coefficient is increased by 42% under the optimized light quality, the bud body is more robust, and the chlorophyll content is increased by 18%; Step three, proliferation culture The proliferation culture medium is prepared by adding hormones, coconut milk 100 mL / L, PVP 200 mg / L, ascorbic acid 30 mg / L and sucrose 30 g / L to liquid MS as a basic culture medium, and the scale-up proliferation of buds is carried out in a temporary immersion bioreactor, with an immersion frequency of 5 min every 4 h, ventilation of sterile air filtered through 0.1 μm, and an inoculation amount of 200 buds per 5 L of the reactor; the above-mentioned hormones are 6-benzylaminopurine (6-BA) 3.0 mg / L, naphthalene acetic acid (NAA) 0.1 mg / L and KT 0.5 mg / L, and the above-mentioned culture period is 20 days, and the product obtained in step three can be continuously subcultured for 8-10 times without obvious degradation. Step four, rooting culture The rooting culture medium is prepared by adding indole butyric acid (IBA) 0.3 mg / L, naphthalene acetic acid (NAA) 0.1 mg / L, activated carbon 0.5 g / L, humic acid 20 mg / L, sucrose 20 g / L and vermiculite to 1 / 2MS (reduced ammonium nitrogen) as a basic culture medium, and the product obtained in step three is subjected to rooting culture; in the above-mentioned, indole butyric acid (IBA) 0.3 mg / L and naphthalene acetic acid (NAA) 0.1 mg / L synergistically promote rooting, activated carbon is used to adsorb toxins and promote root hair development; humic acid is used to stimulate root absorption, and the rooting culture medium obtained above can improve the air permeability of rooting.

[0015] Step five, domestication in an artificial climate environment An artificial intelligent climate chamber is used to realize: temperature: 25-27℃ for the first 3 days and reduced to 22-23℃ for the last 4 days; humidity: gradually reduced from 90% for the first 3 days to 70%; light: LED full spectrum, PPFD gradually increased from 50 to 150 μmol·m -2 ·s -1 ; CO2 concentration: 800 ppm.

[0016] Step six, transplanting Transplanting is carried out in a substrate culture with peat: coconut chaff: perlite: humus soil at a weight ratio of 3:3:2:2.

[0017] Although the embodiments of the present application have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A tissue culture rapid propagation method suitable for ornamental taro, characterized by, The method comprises the following steps: Step one, material preparation Selecting healthy ornamental taro plants without pests and diseases, and taking the young lateral buds, stem tips or leaves as explants, the explants are washed clean, disinfected and cut; Step two, induction culture Taking MS as the basic culture medium, adding biological amine, NAA, TDZ, coconut milk, sucrose and ascorbic acid to prepare the induction culture medium, and then placing the explants obtained in step one in a constant temperature and light and dark culture environment until green bud points germinate; Step three, proliferation culture Taking liquid MS as the basic culture medium, adding hormones, coconut milk, PVP, ascorbic acid and sucrose to prepare the proliferation culture medium, and carrying out large-scale bud propagation in a bioreactor; Step four, rooting culture Taking 1 / 2MS as the basic culture medium, adding IBA, NAA, activated carbon, humic acid, sucrose and vermiculite to prepare the semi-liquid rooting culture medium, and carrying out rooting culture on the products obtained in step three.

2. The method for tissue culture propagation of ornamental taro as claimed in claim 1, wherein: In step one, the explants are washed with flowing water, soaked in 75% ethanol for 30 seconds, then immediately washed with sterile water for 2-3 times, disinfected with 2% NaClO combined with 0.1% Tween-20, and then washed clean with sterile water through ultrasonic assistance.

3. The method for tissue culture propagation of ornamental taro as claimed in claim 2, wherein: The cut explants in step one are soaked in 0.5% ascorbic acid, 0.2% citric acid and 0.1% polyvinylpyrrolidone for 2-5 seconds, and then placed in a sterile environment until the surface is dry.

4. The method for tissue culture propagation of ornamental taro as claimed in claim 3, wherein: The light and dark culture environment consisted of 6-7 days of dark culture followed by LED white light illumination at a PPFD concentration of 30 μmol·m⁻¹. -2 ·s -1 , 16h / d.

5. The method for tissue culture propagation of ornamental taro as claimed in claim 4, wherein: The light quality combination of the LED white light is 70% red light (660nm), 20% blue light (450nm) and 10% far red light (730nm).

6. The method for tissue culture propagation of ornamental taro as claimed in claim 5, wherein the said method comprises the steps of: The hormones are 6-BA 3.0mg / L, NAA 0.1mg / L and KT 0.5mg / L.

7. The method for tissue culture propagation of ornamental taro as claimed in claim 6, wherein: The bioreactor is a temporary immersion bioreactor, the immersion frequency is 5 minutes every 4 hours; Ventilation: sterile air, 0.1μm filtration; inoculation amount: 200 bud clusters per reactor.

8. The method for tissue culture propagation of ornamental taro as claimed in claim 7, wherein: After the rooting culture, domestication in an artificial climate environment is carried out, and then the plants are transplanted into a substrate culture with a weight ratio of peat: coconut husk: perlite: humus soil being 3:3:2:2.