阅读说明：本技术 一种提高小麦单倍体组培苗加倍移栽结实率的方法 (Method for improving double transplanting maturing rate of wheat haploid tissue culture seedlings ) 是由 刘莹 王立峰 张小娟 谭永强 左珍喜 谭靖 于 2021-08-28 设计创作，主要内容包括：本发明公开了一种提高小麦单倍体组培苗加倍移栽结实率的方法,旨在优化移栽温室内的温度、湿度、光照和气体交换等条件,通过提高组培苗的加倍移栽成活率、加倍成功率和分蘖力及健壮程度,从而达到提高小麦单倍体组培苗的加倍结实率的目的,增加DH系种子的饱满程度和种子数量,获得大量DH新品系。通过拥有特定光谱的LED植物生长灯对小麦植株生长健壮起到了关键作用,大幅提高了小麦单倍体组培苗加倍移栽后的生长健壮程度。按照本发明的方法,移栽的小麦植株分蘖旺盛、茎秆粗壮,成穗率高,加倍成活率为93.2％,结实率为单株平均结实174粒,且籽粒较饱满,可满足大田直播要求。(The invention discloses a method for improving double transplanting maturing rate of wheat haploid tissue culture seedlings, which aims to optimize conditions such as temperature, humidity, illumination, gas exchange and the like in a transplanting greenhouse, and achieves the purpose of improving the double maturing rate of the wheat haploid tissue culture seedlings by improving the double transplanting survival rate, the double success rate, the tillering capacity and the robustness of the tissue culture seedlings, increasing the plump degree and the number of DH line seeds and obtaining a large number of DH new lines. The LED plant growth lamp with the specific spectrum plays a key role in the growth robustness of wheat plants, and the growth robustness degree of wheat haploid tissue culture seedlings after double transplantation is greatly improved. According to the method, the transplanted wheat plant has vigorous tillering, thick and strong stem, high ear forming rate, 93.2 percent of doubling survival rate and full seeds, and the seed setting rate is that the single plant sets up 174 seeds on average, thereby meeting the requirement of direct seeding in fields.)

1. A method for improving the double transplanting maturing rate of wheat haploid tissue culture seedlings is characterized by comprising the following steps:

step 1: (1) 2 temperature control units are arranged in a room, the temperature control range is 10-45 ℃, the temperature control precision is +/-0.5 ℃, and the error of any temperature set point is less than or equal to +/-1.5 ℃; (2) a stainless steel ultrasonic humidifying system is adopted for humidifying inside a room, atomized air of the ultrasonic humidifying system is discharged downwards along with an air outlet of a refrigerating system, moisture is sent into the room through a pipeline at the position of a humidifier, and a water inlet of the pipeline is provided with a filtering device; the dehumidification adopts a freezing type dehumidification unit, and the pipeline type is concealed, so that the internal space is not occupied; the humidity control range in the room is 50-90% RH, the humidity control precision is less than or equal to +/-7% RH, and the humidity control uniformity is +/-8% RH; (3) installing LED lamp beads on the top of a culture frame in a room, and meeting the requirement of indoor plants on illumination in the whole growth period by adopting LED plant growth lamps with specific spectrums; (4) a set of fresh air exchange system is additionally arranged on a room, and a total-heat fresh air exchanger is adopted to discharge turbid indoor air and input outdoor fresh air into the room, so that the influence on plant growth caused by improper gas component ratio in a closed space is avoided; the fresh air volume reaches 400m³/h；

Step 2: taking down the bottle cap of the tissue culture bottle before doubling the wheat tissue culture seedling, and hardening the seedling under the specific greenhouse condition after opening: the temperature is 12 ℃, the humidity is 75% RH, the illumination is 12000Lux, and the fresh air system is started every 6 hours for ventilation for 10 minutes;

and step 3: before transplanting, preparing a special nutrient soil matrix, wherein the nutrient soil matrix is big Chinese peat soil, perlite and vermiculite which are 3: 1;

and 4, step 4: when transplanting, the granular slow release fertilizer is mixed into nutrient soil, and the using amount is 100 g/50L;

and 5: planting the tissue culture seedlings subjected to double treatment into a nutrient soil matrix, wherein the depth is about 1cm, and gradually cultivating soil and promoting the growth of the plants along with the growth of the plants a little;

step 6: and (3) seedling stage cooling and seedling protection measures: keeping low temperature and weak light just after transplanting, gradually heating and lighting after tillering and jointing;

and 7: and (3) supplying water and fertilizer: topdressing is carried out in the jointing stage, and topdressing is carried out simultaneously in the heading and flowering stage, so that the robust growth and good fructification of plants are promoted.

2. The method for improving the double transplantation maturing rate of wheat haploid tissue culture seedlings according to claim 1, wherein the method comprises the following steps: the temperature control unit in the step 1 adopts a scroll compressor; the light source spectrum in the step 1 comprises R, G, B three wave bands, the spectrum structure is 380-780nm monochromatic continuous spectrum, and the three peak wavelength distributions are 660nm, 525nm and 450 nm; the light intensity at the position 15cm below the lamp reaches 1600 mu mol s^-1·m^-2The model of the selected LED lamp is LED-GLM 200B-02.

3. The method for improving the double transplantation maturing rate of wheat haploid tissue culture seedlings according to claim 1, wherein the method comprises the following steps: the model of the big Chinese peat soil in the step 3 is 602, and the specification is 0-10 mm.

Technical Field

The invention relates to the technical field of planting, in particular to a method for improving double transplanting maturing rate of wheat haploid tissue culture seedlings.

Background

Wheat and corn hybridization induced haploid technology belongs to the distant hybridization category, and the obtained wheat haploid young embryo can not grow normally on a plant and can germinate into a seedling only by further tissue culture indoors through young embryo rescue technology. The haploid tissue culture seedling can grow and bear fruit after being subjected to colchicine doubling treatment at a certain stage to obtain homozygous DH line seeds, can be stably inherited, and does not have character separation. The technology is applied to wheat breeding, can shorten the breeding period by at least 7 years, saves the corresponding conventional breeding cost of land, chemical fertilizer, manpower and the like, and is an efficient biotechnical breeding method. The wheat haploid technology also has important application in the aspects of DH colony genetic map construction, gene positioning and the like.

The wheat haploid young embryo is generally subjected to aseptic inoculation in a laboratory about 15 days after wheat blossom, germinates into a seedling about 5-30 days after inoculation, and is subjected to strong seedling culture for 15-30 days after seedling formation, so that manual doubling treatment can be performed. Generally, the wheat haploid tissue culture seedling doubling test is carried out at the beginning of 6 months, the temperature is higher and higher, the survival rate is low after doubling transplantation under natural conditions, and the maturing rate is lower. The method adopted in China mainly comprises two methods, namely that the haploid tissue culture seedlings are placed under the low-temperature condition to go over summer, and then are doubled and transplanted in open field after the temperature drops for 11 months. The method is easy to generate old and stiff seedlings, the root system has poor activity, the doubled survival rate and the success rate are low; and secondly, double transplanting is carried out on the haploid tissue culture seedlings in a simple greenhouse, the DH plants obtained by the method are weak in growth vigor, few in tillering, small in harvested seed amount and not full, and indoor seedling raising is generally needed to improve the field emergence rate. Both methods are not beneficial to improving the doubling setting rate of tissue culture seedlings, directly result in lower production efficiency of DH lines and can not meet the requirements of breeding and tests.

The corn pollen is used for inducing the wheat to generate haploid immature embryos, and a DH new line capable of being stably inherited is obtained through artificial doubling, so that the breeding period is greatly shortened compared with that of a conventional method for breeding the new line. In actual operation, wheat haploid immature embryos generally germinate into seedlings in 5 months, the temperature is higher and higher, and the problem of how to improve the doubling maturing rate of test-tube seedlings becomes the last kilometer of the wheat haploid breeding technology is solved.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a method for improving the double transplanting seed setting rate of wheat haploid tissue culture seedlings. Mainly solves the problem that corn pollen is used for inducing wheat to generate haploid immature embryos, a DH new strain capable of being stably inherited is obtained through artificial doubling, and the breeding period is greatly shortened compared with that of a conventional method for breeding the new strain. In actual operation, wheat haploid immature embryos generally germinate into seedlings in 5 months, the temperature is higher and higher, and the problem of how to improve the doubling maturing rate of test-tube seedlings becomes the last kilometer of the wheat haploid breeding technology is solved.

(II) technical scheme

In order to achieve the purpose, the invention provides the following technical scheme: a method for improving the double transplanting maturing rate of wheat haploid tissue culture seedlings is characterized by comprising the following steps:

step 1: (1) 2 temperature control units are arranged in a room, the temperature control range is 10-45 ℃, the temperature control precision is +/-0.5 ℃, and the error of any temperature set point is less than or equal to +/-1.5 ℃; (2) a stainless steel ultrasonic humidifying system is adopted for humidifying inside a room, atomized air of the ultrasonic humidifying system is discharged downwards along with an air outlet of a refrigerating system, moisture is sent into the room through a pipeline at the position of a humidifier, and a water inlet of the pipeline is provided with a filtering device; the dehumidification adopts a freezing type dehumidification unit, and the pipeline type is concealed, so that the internal space is not occupied; the humidity control range in the room is 50-90% RH, the humidity control precision is less than or equal to +/-7% RH, and the humidity control uniformity is +/-8% RH; (3) install LED lamp pearl at culture frame top in the room, adopt the LED vegetation lamp that possess specific spectrum to satisfy indoor plantingThe requirement of the plant on illumination in the whole growth period; (4) a set of fresh air exchange system is additionally arranged on a room, and a total-heat fresh air exchanger is adopted to discharge turbid indoor air and input outdoor fresh air into the room, so that the influence on plant growth caused by improper gas component ratio in a closed space is avoided; the fresh air volume reaches 400m³/h；

Step 2: taking down the bottle cap of the tissue culture bottle before doubling the wheat tissue culture seedling, and hardening the seedling under the specific greenhouse condition after opening: the temperature is 12 ℃, the humidity is 75% RH, the illumination is 12000Lux, and the fresh air system is started every 6 hours for ventilation for 10 minutes;

and step 3: before transplanting, preparing a special nutrient soil matrix, wherein the nutrient soil matrix is big Chinese peat soil, perlite and vermiculite which are 3: 1;

and 4, step 4: when transplanting, the granular slow release fertilizer is mixed into nutrient soil, and the using amount is 100 g/50L;

and 5: and (4) planting the tissue culture seedlings subjected to double treatment into a nutrient soil matrix, wherein the depth is about 1cm, and the tissue culture seedlings are gradually cultivated and strengthened along with the growth of the plants a little bit.

Step 6: and (3) seedling stage cooling and seedling protection measures: keeping low temperature and weak light just after transplanting, gradually heating and lighting after tillering and jointing;

and 7: and (3) supplying water and fertilizer: topdressing is carried out in the jointing stage, and topdressing is carried out simultaneously in the heading and flowering stage, so that the robust growth and good fructification of plants are promoted.

Preferably, the temperature control unit in the step 1 adopts a scroll compressor; the light source spectrum in the step 1 comprises R, G, B three wave bands, the spectrum structure is 380-780nm monochromatic continuous spectrum, and the three peak wavelength distributions are 660nm, 525nm and 450 nm. The light intensity at the position 15cm below the lamp reaches 1600 mu mol s^-1·m^-2The model of the selected LED lamp is LED-GLM 200B-02.

Preferably, the type of the big Chinese peat soil in the step 3 is 602, and the specification is 0-10mm

(III) advantageous effects

The invention provides a method for improving the double transplanting seed setting rate of wheat haploid tissue culture seedlings. The method has the following beneficial effects:

the invention aims to optimize conditions such as temperature, humidity, illumination, gas exchange and the like in a transplanting greenhouse, and achieves the purpose of improving the doubling maturing rate of wheat haploid tissue culture seedlings by improving the doubling transplanting survival rate, the doubling success rate, the tillering capacity and the robustness of the tissue culture seedlings, increasing the plump degree and the number of DH line seeds and obtaining a large number of DH new lines.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The method for improving the double transplanting maturing rate of the wheat haploid tissue culture seedlings comprises the following steps of 1, 2, 3, 4, 5, 6 and 7; example 3 was carried out;

example 1 transplantation culture for open air method:

the wheat haploid tissue culture seedlings are over summer at low temperature in an illumination incubator, are taken out from the culture medium again for doubling after 11 months are started, are transplanted under the field condition, the soil condition is poor, the survival rate is low, the average rate reaches 53.2%, the maturing rate is 75.1%, and the average maturing rate of a single plant is 38 grains. Furthermore, the harvesting of DH line seeds is delayed by one year.

Example 2 is a method for performing transplantation culture in a common air-conditioned room:

the doubled haploid tissue culture seedlings are transplanted in an air-conditioned room in 6 months, the lowest temperature can only reach 17-18 ℃, the humidity is lower than about 55% RH, the illumination can only reach 6000Lux, the survival rate of the transplanted plants is low, the survival rate is averagely 66.8%, the stems are thin and weak, the leaves are slender and easy to fall down, bamboo sticks are required to be fixed, the number of formed spikes is small, the setting rate is 72.4%, the average setting rate of a single plant is 23, the grains are flat, and the seeds cannot be directly sowed in the field.

Example 3:

accurately controlling the temperature to be 12 ℃, the humidity to be 75% RH and the illumination to be 12000Lux after the haploid tissue culture seedlings are transplanted; gradually increasing the temperature to 15-17 deg.C, humidity to 70% RH, and illumination to 32000Lux in the most selective jointing stage; gradually increasing the temperature to 20-22 ℃ in the heading and flowering stage, controlling the humidity at 75% RH and the illumination at 42000 Lux; the temperature gradually rises to 25 ℃ in the maturation period, the humidity is 70 percent RH, and the illumination is 68000 Lux. Fresh air was turned on every 6 hours, with 10 minutes of ventilation each time.

The required temperature, humidity and illumination are accurately controlled, indoor ventilation is carried out periodically, and doubled haploid tissue culture seedlings are transplanted indoors at the beginning of 6 months.

The LED plant growth lamp with the specific spectrum plays a key role in the growth and the robustness of the wheat plants, the growth and the robustness degree of the wheat haploid tissue culture seedlings after double transplantation are greatly improved, and the method is mainly characterized in that the leaves are dark green, the width of the leaves is close to the level of a field, the stems are thick and strong, the tillering and heading rate is high, the size of the wheat ears is close to the level of the field, the grouting is good, the single-plant single-ear setting rate is high, and meanwhile, as the temperature and the humidity of the wheat plants are controlled in the range of the method, the cultivation effect is good, the seeds harvested after maturation are full, and the double transplantation setting rate is obviously improved compared with that before the method is adopted.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.