阅读说明：本技术 一种诱导辽东楤木体细胞胚发生及植株再生的方法 (Method for inducing somatic embryogenesis and plant regeneration of Aralia elata ) 是由 程瑶 于锡宏 蒋欣梅 佟雪姣 刘在民 史绪梅 韩兆平 白晓丽 高照亮 于 2019-05-05 设计创作，主要内容包括：本发明公开了一种诱导辽东楤木体细胞胚发生及植株再生的方法,包括以下步骤：S1、外植体的选择与处理；S2、愈伤组织的诱导；S3、愈伤组织的增殖培养；S4、胚性愈伤组织的诱导；S5、胚性愈伤组织分瓶培养；S6、再生根培养；S7、驯化移栽,本发明培养速度快,经济适用性高,可用于优异野生种质资源的保存与繁殖,满足园艺栽培和中草药的规模化种植。(The invention discloses a method for inducing somatic embryogenesis and plant regeneration of Aralia elata, which comprises the following steps: s1, selection and treatment of explants; s2, inducing callus; s3, propagation culture of callus; s4, induction of embryogenic callus; s5, culturing the embryonic callus in bottles; s6, culturing regenerated roots; s7, domestication and transplantation, the method has the advantages of high culture speed and high economic applicability, can be used for preserving and propagating excellent wild germplasm resources, and meets the requirements of horticultural cultivation and large-scale planting of Chinese herbal medicines.)

1. A method for inducing somatic embryogenesis and plant regeneration of Aralia elata seem is characterized by comprising the following steps of:

s1, selection and treatment of explants: taking young leaves, stems and petioles of plants in the vigorous growth period of 5-7 months as explants for callus induction, washing the picked explants with running water, soaking and disinfecting the explants on a super-clean workbench by 70% alcohol, soaking the leaves for 30-60 seconds, soaking the petioles and stems for 2-3 minutes, washing the explants with sterile water for 2-4 times, disinfecting the explants with mercuric chloride for 7-10 minutes, washing the explants with sterile water for 2-4 times, sucking water by sterile filter paper, and using a scalpel to perform callus induction on the explantsCutting the leaf to about 1cm²Cutting petiole and stem into 0.2-0.5cm small segments, and inoculating to callus induction culture medium;

s2, induction of callus: taking an MS culture medium added with 2,4-D, sucrose (30g/L) and agar (6.5g/L) as an induction culture medium, wherein the 2,4-D concentration of leaf-induced callus is 0.5mg/L-1.0mg/L, the 2,4-D concentration of stem-and petiole-induced callus is 1.0mg/L-3.0mg/L, and the induction conditions of callus are as follows: the temperature is 25 +/-1 ℃, the humidity is 60-70%, dark culture is carried out for 10-15 days, then 12h/12h (2000lx) light culture is carried out, light yellow and soft callus is generated after 5 days, and the callus is transferred to a proliferation culture medium after 30-45 days;

s3, multiplication culture of callus: subculturing the callus on a culture medium added with 0.5-1.0mg/L2,4-D and IBA (indolebutyric acid), and subculturing once every 15 days; the culture conditions are temperature 25 +/-1 ℃, humidity: 60% -70%, light period of 12 h/dark 16h, and illumination intensity of 20001 x.

S4, induction of embryogenic callus: transferring 1g of the callus after propagation culture to an induction culture medium, wherein the induction culture medium takes MS as a basic culture medium, and IBA1.0-4.0mg/L, 6-BA0.5-1.0mg/L, sucrose 30g/L and agar 6.5g/L are added, and the induction conditions are as follows: the temperature is 25 +/-1 ℃, the humidity is 60-70%, the light period is 8 h/16 h in darkness, and the illumination intensity is 2000 lx.

S5, culturing the embryogenic callus in bottles: after culturing on a somatic embryo induction culture medium for 10 days, quickly proliferating the callus by 3-5 times to form a large embryogenic callus group, averagely distributing the proliferated callus on a new culture medium for continuous culture, inoculating 1-2g of the callus in each bottle, spreading the callus on the culture medium, and starting to generate a somatic embryo after 13 days;

s6, regeneration root culture: cutting 2 cm-long adventitious buds with a scalpel and cutting the adventitious buds to a regeneration root culture medium, wherein the rooting culture medium takes WPM as a basic culture medium, 0.5-1.0mg/L of IBA, 0.2-1.0mg/L of NAA, 30g/L of sucrose and 6.5g/L of agar are added, and the induction conditions are as follows: the temperature is 25 +/-1 ℃, the humidity is 60-70%, the photoperiod is 12 h/dark 12h, the illumination intensity is 2000lx, and after 45 days, somatic embryos are developed into complete plants with strong growth vigor;

s7, domestication and transplantation: after rooting culture for 45 days, selecting plants with strong root systems and strong growth potential for acclimatization culture, firstly taking out tissue culture seedlings by using tweezers, washing the root substrates completely by using tap water to avoid damaging the roots, then transplanting the seedlings into a 10 multiplied by 10cm nutrition pot, and mixing the volume of nutrient soil with the volume of sterilized field soil: turfy soil: vermiculite is (1:1:1), air humidity is kept at 80-90% after transplanting to prevent tissue culture seedling leaves from being dried, the tissue culture seedlings are placed in an original incubator or a tissue culture room for culture under the condition of 14h/10h photoperiod, the temperature is 25 +/-1 ℃, the illumination intensity is 2000lx, after one week, the air humidity is reduced to 60-70%, after 2 weeks, the domesticated tissue culture seedlings are transferred to a greenhouse for culture, a sunshade net is used for appropriately shading, a plastic film is covered to keep the air humidity at 60-70%, after 30 days, the sunshade net and the plastic film are gradually removed, the survival rate is counted, and the tissue culture seedlings are transplanted to the open field.

2. The method of inducing somatic embryogenesis and plant regeneration of Aralia elata seem as claimed in claim 1, wherein the optimal choice of explant in S1 is stem.

3. The method of claim 1, wherein the optimal 2,4-D concentration for inducing leaf-derived callus is 0.5mg/L, the optimal 2,4-D concentration for inducing stem and petiole-derived callus is 1.0mg/L, and the culture conditions are dark culture for 15 days and then light culture.

4. The method for inducing callus proliferation according to claim 1, wherein IBA in S3 is 0.5mg/L and 2,4-D concentration is 0.5mg/L, and culture conditions are temperature 25 ± 1 ℃, humidity: 60% -70%, light 12 h/dark 12h photoperiod.

5. The method for inducing somatic embryogenesis and plant regeneration of Aralia elata seem as claimed in claim 1, wherein IBA concentration in S4 is 3.0mg/L, 6-BA concentration is 0.5mg/L, and culture conditions are 25 ± 1 ℃, humidity is 60% -70%, and light is 8 h/dark for 16h photoperiod.

6. The method for inducing somatic embryogenesis and plant regeneration of Aralia elata seem according to claim 1, wherein the embryogenic callus proliferated in S5 is cultured in bottles, wherein 1-2g of embryogenic callus is inoculated to each bottle, and the callus is uniformly spread on a culture medium.

7. The method of claim 1, wherein the root-regenerating culture medium of S6 is WPM medium supplemented with IBA1.0mg/L, NAA0.2mg/L, sucrose 30g/L, and agar 6.5 g/L.

8. The method for inducing somatic embryogenesis and plant regeneration of Aralia elata seem according to claim 1, wherein in S7, the volume ratio of the preferred nutrient soil to the sterilized soil is as follows: turfy soil: vermiculite is (1:1:1), the preferable culture condition is 14h/10h photoperiod, the temperature is 25 +/-1 ℃, the humidity is 80-90%, and the illumination intensity is 2000 lx.

Technical Field

The invention relates to the technical field of planting, in particular to a method for inducing somatic embryogenesis and plant regeneration of Aralia elata.

Background

Aralia elata seem is a small perennial deciduous tree or shrub of Aralia in Araliaceae, the tender bud of which is a rare edible wild vegetable, the wild vegetable is crisp and tender in texture, unique in smell and rich in nutrition, is known as the king of the wild vegetable, and the root, stem bark, leaf and other tissues of which contain rich araloside, so that the Aralia elata seem has high edible value and medicinal value. In recent years, with the export of large amounts of buds of Aralia elata seem from Japan, which causes serious damage to wild Aralia elata seem resources in northeast forest regions, people begin to research an artificial cultivation technology of Aralia elata seem. The propagation of Aralia elata can adopt 4 methods: firstly, utilizing seeds to perform sexual propagation and culture seedlings; secondly, carrying out vegetative propagation by using the root section to produce cutting seedlings and root-burying seedlings; thirdly, the branch propagation is carried out by utilizing the biological characteristic of strong sprouting capacity of the Aralia elata seem; fourthly, tissue culture is carried out by utilizing totipotency of plant cells to produce tissue culture seedlings. As the dormancy stage of Aralia elata seem seeds is longer, the problems of irregular seedling emergence and long seedling time exist in seedling propagation, and the propagation coefficient and survival rate of cuttings and root-buried seedlings produced by asexual propagation are lower. The rapid breeding of the seedlings can be realized by utilizing the tissue culture technology, and the storage problem of rare wild resources is solved.

There are two ways for aralia elates plant regeneration, one is callus formation and the other is embryoid formation. Aralia elata seem is regenerated through a callus, the induction rate is low, multiple subcultures are needed, generally 6-10 months are needed, and the propagation period is long. There are two ways to form embryoid bodies, one is to form somatic embryos directly from explants (Dai J L, TanX, Zhan Y G, et al, Rapid and regenerative Plant regeneration of Araliaelata Seem. vitamin organic embryo production [ J ]. Plant Cell Tissue and organic Culture,2011,104(1): 125-. Secondly, callus is formed firstly, and then somatic embryos (CN 108064697A) are formed by induction, the method has high propagation coefficient, but still has the following defects that the hormone concentration of the callus induced by different explants is not clear, and the propagation coefficient of the callus is low; IBA is added independently, more callus is differentiated into adventitious roots, a mixture of the callus, the adventitious roots, adventitious buds and complete somatic embryos is formed after differentiation, the somatic embryos are difficult to separate, and the utilization rate is low; after the somatic embryos are inoculated on a growth culture medium, callus clusters and secondary somatic embryos are easily formed on the base part, and domestication and transplantation are difficult. Aiming at the defects of the prior art, the invention aims to provide a more effective method for somatic embryogenesis and plant regeneration of Aralia elata, which effectively improves the induction and proliferation efficiency of somatic embryos, thereby establishing a more efficient Aralia elata plant regeneration system.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a method for inducing somatic embryogenesis and plant regeneration of Aralia elata.

A method for inducing somatic embryogenesis and plant regeneration of Aralia elata seem comprises the following steps:

s1, selection and treatment of explants: taking young leaves, stems and petioles of plants in the vigorous growth period of 5-7 months as explants for callus induction, washing the picked explants with running water, soaking the explants on a superclean workbench for disinfection by 70% alcohol, soaking the leaves for 30-60 seconds, soaking the petioles and stems for 2-4 minutes, washing with sterile water for 2-4 times, then disinfecting with mercuric chloride for 7-10 minutes, washing with sterile water for 2-4 times, sucking water with sterile filter paper, cutting the leaves into about 1cm by a scalpel²Cutting petiole and stem into 0.2-0.5cm small segments, and inoculating to callus induction culture medium;

s2, induction of callus: taking an MS culture medium added with 2,4-D and sucrose (30g/L) agar (6.5g/L) as an induction culture medium, wherein the 2,4-D concentration of leaf-induced callus is 0.5mg/L-1.0mg/L, the 2,4-D concentration of stem-and petiole-induced callus is 1.0mg/L-3.0mg/L, and the induction conditions of callus are as follows: the temperature is 25 +/-1 ℃, the humidity is 60-70%, dark culture is carried out for 10-15 days, then 12h/12h (2000lx) light culture is carried out, light yellow and soft callus is generated after 5 days, and the callus is transferred to a proliferation culture medium after 30-45 days;

s3, multiplication culture of callus: subculturing the callus on a culture medium added with 1.0mg/L2,4-D and 1.0mg/LIBA (indolebutyric acid), and subculturing once every 15 days;

s4, induction of embryogenic callus: transferring the callus after propagation culture to an induction culture medium, wherein the induction culture medium takes MS as a basic culture medium, and IBA1.0-4.0mg/L, 6-BA0.5-1.0mg/L, sucrose 30g/L and agar 6.5g/L are added, and the induction conditions are as follows: temperature 25 ± 1 ℃, humidity: 60% -70%, a photoperiod of 8 h/dark 16h and illumination intensity of 2000lx, and mature somatic embryos are induced after 13 days;

s5, culturing the embryogenic callus in bottles: after culturing on a somatic embryo induction culture medium for 10 days, quickly proliferating the callus by 3-5 times to form a large embryogenic callus group, averagely distributing the proliferated callus on a new culture medium for continuous culture, inoculating 1-2g of the callus in each bottle, spreading the callus on the culture medium, and starting to generate a somatic embryo after 13 days;

s6, regeneration root culture: cutting 2 cm-long adventitious buds with a scalpel, cutting the adventitious buds to a rooting culture medium, adding 0.5-1.0mg/L IBA, 0.2-1.0mg/L NAA, 30g/L sucrose and 6.5g/L agar to the rooting culture medium by taking WPM as a basic culture medium, wherein the induction conditions are as follows: temperature 25 ± 1 ℃, humidity: 60% -70%, the photoperiod is 12 h/dark 12h, the illumination intensity is 2000lx, and after 45 days, somatic embryos are developed into complete plants with strong growth vigor;

s7, domestication and transplantation: after rooting culture for 45 days, selecting plants with strong root systems and strong growth potential for acclimatization culture, firstly taking out tissue culture seedlings by using tweezers, washing the root substrates completely by using tap water to avoid damaging the roots, then transplanting the seedlings into a 10 multiplied by 10cm nutrition pot, and mixing the volume of nutrient soil with the volume of sterilized field soil: turfy soil: vermiculite is (1:1:1), air humidity is kept at 80-90% after transplanting to prevent tissue culture seedling leaves from being dried, the tissue culture seedlings are placed in an original incubator or a tissue culture room for culture under the condition of 14h/10h photoperiod, the temperature is 25 +/-1 ℃, the illumination intensity is 2000lx, after one week, the air humidity is reduced to 60-70, after 2 weeks, the domesticated tissue culture seedlings are transferred to a greenhouse for culture, a sunshade net is used for appropriately shading, a plastic film is covered to keep the air humidity at 60-70%, after 30 days, the sunshade net and the plastic film are gradually removed, the survival rate is counted, and the tissue culture seedlings are transplanted to the open field.

Specifically, the optimal choice of explant in S1 is stem.

Specifically, the optimal 2,4-D concentration of the leaf-induced callus is 0.5mg/L, the optimal 2,4-D concentration of the stem-and-petiole-induced callus is 1.0mg/L, and the culture condition is that the dark culture is changed into the light culture after 15 days.

Specifically, the concentration of IBA and 2,4-D in S3 is 1.0mg/L, the culture condition is that the temperature is 25 +/-1 ℃, and the humidity is as follows: 60% -70%, light 8 h/dark 16h photoperiod.

Specifically, the concentration of IBA in S4 is 3.0mg/L, the concentration of 6-BA in S4 is 0.5mg/L, the culture condition is that the temperature is 25 +/-1 ℃, and the humidity is as follows: 60% -70%, light 8 h/dark 16h photoperiod.

Specifically, the embryogenic callus proliferated in S5 needs to be cultured in bottles, each bottle is inoculated with 1-2g, and the callus is evenly spread on a culture medium.

Specifically, the rooting culture medium in S6 is WPM culture medium added with IBA1.0mg/L, NAA0.2mg/L, sucrose 30g/L and agar 6.5 g/L.

Specifically, in S7, the preferable volume ratio of the nutrient soil to the sterilized field soil is: turfy soil: vermiculite is (1:1:1), the preferable culture condition is 14h/10h photoperiod, the temperature is 25 +/-1 ℃, the air humidity is 80-90%, and the illumination intensity is 2000 lx.

The invention has the beneficial effects that: the regeneration method of the aralia elates somatic embryogenesis way has the following advantages:

firstly, the culture speed is high, the proliferated callus is inoculated on an induction culture medium, a cotyledon type embryo is formed after 23 days, the mature somatic embryo is transferred to a rooting culture medium, and the seedling can be formed after 1-2 months.

Secondly, the economic applicability is high, and each gram of callus can be differentiated into more than 200 embryoids or continuously expanded on a new callus culture medium.

And thirdly, the method can be used for the preservation and reproduction traits of excellent wild resources, and meets the requirements of horticultural cultivation and large-scale planting of Chinese herbal medicines.

Fourthly, the invention can also be used as a bridge of genetic engineering, shortens the breeding period, greatly shortens the breeding and reproduction period of forest trees and lays a foundation for the genetic engineering improvement work of the tree species.

And fifthly, the method is easy to implement and convenient to popularize.

Drawings

FIG. 1 is a dendrogram of the effect of hormone ratio and hours of light on somatic embryo induction;

FIG. 2 is a dendrogram showing the influence of hormone ratio on the rooting rate and the rooting amount of Aralia elata.

Detailed Description

The invention is further illustrated with reference to specific embodiments in the following with reference to figures 1-2.

A method for inducing somatic embryogenesis and plant regeneration of Aralia elata seem comprises the following steps: