CN109247238B - Acer negundo L tissue culture seedling ex-vitro rooting method - Google Patents
Acer negundo L tissue culture seedling ex-vitro rooting method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention belongs to the technical field of plant propagation, and particularly relates to an ex-bottle rooting method for tissue culture seedlings of acer pentalobus, which comprises the following steps: selecting the tissue culture seedling of the five-acer negundo with proliferation of 40 days and good growth condition of 2-4 cm high as an ex-bottle rooting material; inoculating the tissue culture seedling into an induction culture medium of 1/2MS +10mg/L IBA +25g/L sucrose +5g/L agar, and carrying out induction culture for 2-10 days; mixing vermiculite and perlite, sterilizing with carbendazim, and placing into a bottom breathable plug tray; taking the tissue culture seedlings after induction culture, cleaning the base, inserting the tissue culture seedlings into a matrix, compacting, spraying water to leaf surfaces, covering an aperture plate cover, spraying water periodically, uncovering at 25d, and carrying out normal maintenance. The method for rooting the tissue culture seedlings outside the bottles is simple, does not need root washing, avoids tissue culture seedling damage and pollution caused by root washing, and the rooting process is a seedling hardening process, so that the seedling forming time is shortened, and the efficiency is improved.
Description
Technical Field
The invention belongs to the technical field of plant propagation, and particularly relates to an ex-bottle rooting method for tissue culture seedlings of acer negundo.
Background
About 200 aceraceous plants are distributed mainly in the northern temperate zone, the main production places are China and Japan, more than 150 aceraceous plants are distributed in China and various provinces of north and south, but the distribution center is the middle part or the west part, some acer trees are good wood trees, some acer trees are good honey source plants, and some acer trees can be used as shade trees and ornamental trees. Five-petty maple is a rare ornamental tree. So far, only 500 plants are known to be stored in the field of five-leaved maple, the five-leaved maple belongs to a very small population, and the 'very dangerous' grade is listed in 'Chinese biodiversity red famous book, high plant volume'. In 2016, the national government of Sichuan province published 18 major protection wild plant catalogues of Sichuan province, and five acer negundo were listed.
Five-leaf maple (Acer Pentaphyllum), which is a deciduous tree of Aceraceae Acer, can grow as high as 10 meters, but as much as four to five meters, and grows in valley zone with elevation of 2200-. In acer, five-leaflet maple is the only species of five-leaflet maple line. In autumn, the leaves of the five-leaved maple turn yellow from green and finally turn red, the unique chicken claw-shaped leaf shape and the gorgeous color in autumn make the leaves become the objects of attention of global botanic scientists and horticulturists, and the leaves are called as 'two ornamental maples in the world abreast of red maple'.
At present, the propagation technology of the five-leaflet maple is mainly sowing propagation, but the seeds germinate slowly and the germination rate is low. At present, a method for rapidly propagating five-acer negundo branches is also studied, for example, a culture method for rapidly propagating five-acer negundo branches with application number of 201711422694.8 includes the following steps: collecting five-acer negundo branches with shoot lengths of less than 3 years and tender tips of 4-6 cm as explant materials; cutting off leaves of the branch with buds, cutting the branch with buds into stem sections with buds, and disinfecting the stem sections with buds; step three, placing the stem segments with buds into a culture medium for culture, wherein the culture medium is MS + NAA0.05mg/L +6-BA0.05mg/L + GA32.0mg/L + AC 1.0 g/L; step four, strong seedling culture is carried out, and the culture medium is MS + GA31.0 mg/L + NAA0.05mg/L +6-BA0.05mg/L + AC 1.0 g/L; step five, performing rooting culture, wherein a culture medium is White, IBA1.2mg/L, 6-BA1.0mg/L and AC 1.0 g/L; sixthly, moving the tissue culture tank filled with the five-leaf maple seedlings to an outdoor shade shed or a greenhouse for closed-tank hardening seedlings for 15-20 days 7-15 days after rooting culture, wherein the shade degree is 50% -70%; after the tank is closed and the seedlings are hardened, opening a tissue culture tank cover, and opening the tank to harden the seedlings for 3-7 d under natural light; after the hardening off, removing the five-acer negundo seedlings from the tissue culture tank by using tweezers, and cleaning the roots of the five-acer negundo seedlings; seventhly, directly transplanting, or cleaning the five-petiole maple seedlings by using 0.1-0.3% potassium permanganate solution or 500 times of the polyporus frondosus solution; and step eight, periodically fertilizing after transplanting, performing water and fertilizer management, and culturing to obtain five-leaflet maple seedlings. However, the method needs strong seedling culture, rooting culture and other processes, the process is complex, the propagation period is long, the roots of the five-acer palmatum seedlings need to be cleaned after the seedling hardening in the sixth step, and the seedlings are damaged to a certain extent in the cleaning process, and even easily pollute and die if the seedlings are not cleaned in place. Therefore, a propagation method of five-acer negundo with high rooting rate, high survival rate and short seedling time is urgently needed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the five-acer negundo tissue culture seedling ex-vitro rooting method with short seedling time and high rooting rate.
In order to achieve the purpose, the invention adopts the following technical scheme:
an ex-vitro rooting method for tissue culture seedlings of Acer negundo L comprises the following steps:
the method comprises the following steps: preparing tissue culture seedlings: selecting a five-acer negundo tissue culture seedling with proliferation of 40d and good growth condition of 2-4 cm in height as an ex-bottle rooting material, and shearing off the lower and middle leaves of the tissue culture seedling;
step two: rooting and inducing of tissue culture seedlings: inoculating the tissue culture seedling prepared in the step one into an induction culture medium, and carrying out induction culture for 2-10 days, wherein the induction culture medium is 1/2MS + 5-15 mg/L IBA +25g/L sucrose +5g/L agar;
step three: preparing a matrix: sterilizing a mixed matrix of vermiculite and perlite by 800 times of carbendazim, then filling the sterilized matrix into a bottom air-permeable hole tray, filling 3/4 (the quantity of the matrix is the height of the hole tray), then watering thoroughly, and then watering for 1 time by a pot soaking method every 2-3 weeks;
step four: transplanting: and (3) taking the tissue culture seedlings subjected to induced culture in the step two, cleaning the base part, inserting the tissue culture seedlings into the matrix prepared in the step three, compacting, spraying water once per hole with the insertion depth of about 1cm, covering a hole plate cover, spraying water for 2 times in 1-5 d, spraying water once in 5-20 d for a week, uncovering the cover at 25d, and carrying out normal maintenance.
Further, the induction medium was 1/2MS +10mg/L IBA +25g/L sucrose +5g/L agar.
Further, the mass ratio of the vermiculite to the perlite is 1-3: 1.
Further, the mass ratio of the vermiculite to the perlite is 3: 1.
Further, the induction culture of the step two is 2 d.
Further, the tissue culture seedlings transplanted in the fourth step are placed in an environment with the temperature of 22-27 ℃ and the humidity of more than 80%.
The 1/2MS basic culture medium is an MS basic culture medium, the macroelement content is reduced by half, other components are unchanged, the inorganic salt concentration is low, the growth requirement is met, and rooting is facilitated.
The IBA is indolebutyric acid, is a plant growth hormone, is mainly used for rooting of cuttings, can induce the formation of root protomer, promote cell differentiation and division, is beneficial to the generation of new roots and the differentiation of vascular bundle systems, and promotes the formation of adventitious roots of the cuttings.
The rooting condition of the tissue culture seedling of the acer negundo is influenced by the concentration of exogenous hormone, the rooting induction time, the physiological state of the tissue culture seedling and the like. The high-concentration exogenous hormone promotes the rooting and root growth of the tissue culture seedling, but the too high concentration inhibits the rooting and root growth of the tissue culture seedling; the hormone concentration is low, the rooting induction time is too short, the tissue culture seedling sucks a small amount of exogenous hormone, the survival rate is low after transplanting, the rooting induction time is too long, the content of the exogenous hormone in the tissue culture seedling is too high, and the rooting of the tissue culture seedling is inhibited; in addition, there are differences in the response of the same plant to different growth states to hormone concentrations.
Compared with the prior art, the invention has the beneficial effects that:
the tissue culture seedling of the five-leaved maple after propagation is inoculated to an induction culture medium for a period of time, then is transplanted to a matrix for induced rooting and domestication, so that the tissue culture seedling gradually adapts to the external environment, and the rooting rate and the survival rate after transplantation are improved. Meanwhile, the transplanting and rooting process is an acclimatization process, so that the seedling time is shortened. The invention omits the root washing process in the prior transplanting method after the tissue culture seedling takes root, avoids the damage caused by root washing and the pollution caused by the cleaning failure; the method for rooting the tissue culture seedling of the five-acer negundo outside the bottle is simple in process, easy to operate and high in rooting rate, and the survival rate of the five-acer negundo is improved.
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FIG. 1 is a tissue culture seedling of the method for rooting tissue culture seedling of Acer palmatum outside bottle of the present invention;
FIG. 2 is the rooting induction process of tissue culture seedling of the tissue culture seedling bottle external rooting method of Acer nikoense Maxim;
FIG. 3 is the rooted seedling after 40d of transplantation of the method for rooting tissue culture seedling of Acer negundo L in vitro of the present invention.
Detailed Description
The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
An ex-vitro rooting method for tissue culture seedlings of Acer negundo L comprises the following steps:
the method comprises the following steps: preparing tissue culture seedlings: selecting a five-acer negundo tissue culture seedling with proliferation of 40d and good growth condition of 2-4 cm in height as an ex-bottle rooting material, and shearing off the lower and middle leaves of the tissue culture seedling;
step two: rooting and inducing of tissue culture seedlings: inoculating the tissue culture seedling prepared in the step one into an induction culture medium, and carrying out induction culture for 2d, wherein the induction culture medium is 1/2MS +10mg/L IBA +25g/L sucrose +5g/L agar;
step three: preparing a matrix: mixing vermiculite and perlite according to a mass ratio of 3:1, sterilizing by 800 times of carbendazim, filling into a bottom air-permeable hole tray, filling 3/4 of the matrix with the height of the hole tray, then watering thoroughly, watering once by a pot soaking method at 40d, and watering once by a pot soaking method at 60 d;
step four: transplanting: and (3) taking the tissue culture seedlings subjected to induced culture in the step two, cleaning a base part, inserting the tissue culture seedlings into the matrix prepared in the step three, tightly pressing, spraying water once on each hole, covering a hole tray cover, placing the transplanted tissue culture seedlings in an environment with the temperature of 22-27 ℃ and the humidity of more than 80%, spraying water for 2 times in 1-5 days, spraying water once in a week of 5-20 days, uncovering the cover in 25 days, and normally maintaining.
Example 2 Effect of different hormone concentrations of the Induction Medium on rooting of tissue culture seedlings in rooting Induction of tissue culture seedlings
Referring to example 1, the concentrations of the hormone IBA in the induction culture medium in the second step are set to be 0mg/L, 5mg/L, 10mg/L and 15mg/L, the induction culture is carried out for 4d, and the rest steps are the same. And step four, after transplanting, counting the rooting rate, the number of adventitious roots and the root length in the 40 th day, and the results are shown in table 1.
The rooting rate (%) is (number of rooted plants of the tissue culture seedling/total number of the tissue culture seedling) × 100%;
root length: recording the maximum adventitious root length of each tissue culture seedling as the root length of the tissue culture seedling;
TABLE 1 Effect of different hormone concentrations of the induction medium on rooting of tissue culture seedlings of Acer palmatum
As can be seen from Table 1: in the experiment, an induction culture medium of 1/2MS +10mg/L IBA +25g/L sucrose +5g/L agar is used, the rooting effect of the five-leaf maple cultured in an induction way for 4d is the best, the rooting rate reaches 91.67%, the number of adventitious roots reaches 3, and the root length is 4.37 cm.
Example 3 Effect of different days of Induction on rooting of tissue culture seedlings on rooting of Acer palmatum tissue culture seedlings
Referring to the example 1, the number of days for induction culture in the second step is set to be 0d, 2d, 4d, 6d, 8d and 10d, and the rest steps are the same. And fourthly, counting the rooting rate, the number of adventitious roots and the root length after transplanting in the 40 th day, and the results are shown in a table 2.
TABLE 2 Effect of different days of induction on rooting of tissue culture seedlings of Acer palmatum
| Days of Induction | The rooting percentage is% | Indefinite number (strip) | Root length cm |
| 0d(CK) | 53.33bc | 2.26a | 3.36a |
| 2d | 95.83a | 3.33a | 4.82a |
| 4d | 91.67a | 3a | 4.37a |
| 6d | 66.67b | 2.75a | 3.87a |
| 8d | 41.67c | 2.5a | 4.7a |
| 10d | 58.33bc | 3a | 3.5a |
As can be seen from Table 2: the effect of treating the five-leaved maple of 2d by rooting induction is best, the rooting rate reaches 95.83%, the number of the indefinite roots reaches 3.33, and the root length is 4.82 cm.
Example 4 Effect of different substrates on rooting of tissue culture seedlings of Acer nikoense Maxim
Referring to example 1, the medium of step three, namely vermiculite and/or perlite, is arranged into four groups according to the mass ratio of 0:1, 1:0, 1:1 and 3:1, and the other steps are the same. And fourthly, after transplanting, counting the rooting rate, the number of adventitious roots and the root length in the 40 th day, and the results are shown in a table 3.
TABLE 3 influence of different substrates on rooting of tissue culture seedlings of Acer nikoense Maxim
As can be seen from Table 3: the matrix adopts independent vermiculite or perlite, the rooting effect of the five-leaf maple is poor, the test shows that the five-leaf maple with the matrix mass ratio of the vermiculite to the perlite being 3:1 has the best effect, the rooting rate reaches 95.83%, the indefinite number reaches 3, the root length is 4.82cm, but the rooting condition of the five-leaf maple is not obvious different from that of the matrix mass ratio of the vermiculite to the perlite being 1: 1.
Example 5 Effect of five-leaf maple tissue culture seedlings of different heights on rooting of five-leaf maple tissue culture seedlings
The method for the ex-vitro rooting of the tissue culture seedlings of the five-acer negundo refers to example 1, the tissue culture seedlings of the five-acer negundo with good growth conditions of 1.5cm, 2.5cm, 3.5cm and 4.5cm are selected as ex-vitro rooting materials, and the rest steps are the same. And fourthly, after transplanting, counting the rooting rate, the number of adventitious roots and the root length in the 40 th day, and the results are shown in a table 4.
TABLE 4 influence of tissue culture seedlings of different heights on rooting of tissue culture seedlings of Acer nikoense Maxim
As can be seen from Table 4: the rooting rate of the five-petiole maples with the height of 1.5cm reaches 95.83 percent, but the indefinite number of the five-petiole maples is only 1.85. Therefore, the test has the best effect on the five-leaved maple with the height of 3.5cm, the rooting rate reaches 91.67 percent, the indefinite number reaches 3.06, and the root length is 4.07 cm.
The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily implemented by those skilled in the art by means of replacement or modification according to the technical contents disclosed in the specification, and therefore, all changes and modifications that come within the spirit and technical conditions of the present invention should be included in the claims of the present invention.
Claims (4)
1. The method for rooting the tissue culture seedling of the acer palmatum outside the bottle is characterized by comprising the following steps of:
the method comprises the following steps: preparing tissue culture seedlings: selecting a five-acer negundo tissue culture seedling with proliferation of 40d and good growth condition of 2-4 cm in height as an ex-bottle rooting material, and shearing off the lower and middle leaves of the tissue culture seedling;
step two: rooting and inducing of tissue culture seedlings: inoculating the tissue culture seedling prepared in the step one into an induction culture medium, and carrying out induction culture for 2d, wherein the induction culture medium is 1/2MS +10mg/L IBA +25g/L sucrose +5g/L agar;
step three: preparing a matrix: sterilizing a mixed matrix of vermiculite and perlite by 800 times of carbendazim, then filling the sterilized matrix into a bottom air-permeable plug tray, filling 3/4 (the amount of the matrix is the height of the plug tray), then watering thoroughly, and then watering for 1 time by a pot soaking method every 2-3 weeks;
step four: transplanting: and (3) taking the tissue culture seedlings subjected to induced culture in the step two, cleaning the base part, inserting the tissue culture seedlings into the matrix prepared in the step three, compacting, spraying water once per hole with the insertion depth of about 1cm, covering a hole plate cover, spraying water for 2 times in 1-5 d, spraying water once in 5-20 d for a week, uncovering the cover at 25d, and carrying out normal maintenance.
2. The ex-vitro rooting method for the tissue culture seedling of the acer pentaphylum according to claim 1, wherein the mass ratio of the vermiculite to the perlite is 1-3: 1.
3. The ex-vitro rooting method for the tissue culture seedling of the acer pentaphylum according to claim 1, wherein the mass ratio of the vermiculite to the perlite is 3: 1.
4. The ex-vitro rooting method for the tissue culture seedlings of the acer pentaphylum according to claim 1, wherein the tissue culture seedlings transplanted in the fourth step are placed in an environment with the temperature of 22-27 ℃ and the humidity of more than 80%.
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| PCT/CN2019/093768 WO2020107888A1 (en) | 2018-11-30 | 2019-06-28 | Ex vitro rooting method for acer pentaphyllum tissue culture |
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| CN115885844A (en) * | 2021-08-19 | 2023-04-04 | 大庆石油管理局有限公司 | Autumn flame rapid propagation method after northern domestication |
| CN114303780B (en) * | 2022-01-11 | 2023-04-11 | 郑英达 | Red maple planting method adopting container for seedling raising |
| CN114651659B (en) * | 2022-04-15 | 2023-05-23 | 山东省林业科学研究院 | Almond light matrix non-woven fabric container seedling raising method |
| CN116034876A (en) * | 2023-01-10 | 2023-05-02 | 重庆市铜梁区果之王园艺研究院 | GF677 peach stock culture medium and cultivation method thereof |
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| CN104663450A (en) * | 2015-03-05 | 2015-06-03 | 罗焕荣 | Tissue culture and rapid propagation method for Acer rubrum 'Brandywine' |
| CN107455261A (en) * | 2017-09-15 | 2017-12-12 | 安徽农业大学 | A kind of method of Acer saccharum tissue culture quick breeding |
| CN107950398A (en) * | 2017-12-25 | 2018-04-24 | 绵阳师范学院 | The cultural method that a kind of five leaflets maple branch is quickly bred |
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| CN108142284A (en) * | 2016-12-06 | 2018-06-12 | 四川七彩林业开发有限公司 | A kind of tissue culture and rapid propagation method of five leaflets maple |
| CN109247238B (en) * | 2018-11-30 | 2021-09-24 | 四川七彩林科股份有限公司 | Acer negundo L tissue culture seedling ex-vitro rooting method |
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| CN104663450A (en) * | 2015-03-05 | 2015-06-03 | 罗焕荣 | Tissue culture and rapid propagation method for Acer rubrum 'Brandywine' |
| CN107455261A (en) * | 2017-09-15 | 2017-12-12 | 安徽农业大学 | A kind of method of Acer saccharum tissue culture quick breeding |
| CN107950398A (en) * | 2017-12-25 | 2018-04-24 | 绵阳师范学院 | The cultural method that a kind of five leaflets maple branch is quickly bred |
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