CN107581040A - The multifarious high flux culture systems of one kind identification Root inheritance and application - Google Patents

Abstract

The invention relates to a high-throughput culture system for identifying root system genetic diversity and its application, and belongs to the technical field of plant culture and identification of root system traits. The main technical scheme is as follows: including a container, the container is provided with a culture unit, a fixed bracket, and a spray system; the bottom of the container is provided with movable wheels, and the bottom of the container is a nutrient solution; the culture unit is placed On the fixed support, the culture unit includes a transparent plate, a stalk clamp and a light-shielding cover, and the light-shielding cover is covered on the outside of the transparent plate through the stalk clamp; the spray system includes a submersible pump, a connecting Pipe, nozzle, spray controller, the submersible pump is placed in the nutrient solution, the submersible pump is connected to the nozzle through a connecting pipe, and the submersible pump is connected to the spray controller. The high-throughput culture system of the invention is used for non-destructive and dynamic observation, identification and screening of root growth characteristics, digital photo collection of root system morphology and analysis of root system properties.

Description

A high-throughput culture system and application for identifying root genetic diversity

technical field

The invention belongs to the technical field of plant cultivation and identification, and specifically relates to a high-throughput cultivation system for identifying root system genetic diversity and its application.

Background technique

The root system is the main organ for plants to absorb water and nutrients. Root traits directly affect the absorption of water and nutrients by the root system, and play a key role in the growth and yield of crops. The root system is called the "hidden half" of the plant. This nature of the root system determines the difficulty of observation and research; on the other hand, it is difficult to accurately observe the dynamic changes of root system traits and its response to soil environmental conditions with traditional root system research methods, which restricts people's understanding of root system structure to a certain extent. Awareness of form and function.

Root system research has become a research hotspot in agriculture and even the entire plant science community, and is recognized by the academic circles as the second "green revolution" in agricultural production. Numerous studies have shown that crop varieties with different root traits have different adaptability to nutrients, drought and other environmental stresses. Therefore, optimizing root traits and functions and breeding crop varieties with excellent root traits is one of the important measures to solve agricultural production in arid and barren soil areas.

In recent years, the root system research has made great progress, but due to the constraints of the "hidden" characteristics of the root system and the lack of effective research methods, the root system research lags behind the research on other plant organs. Traditional research methods include whole root excavation method, partial sampling method, root window method, hydroponics method, root box method, soil column method, aeroponics method, mesh bag method, etc. Field research mostly uses excavation method and local sampling method to carry out , Research in artificially controlled environments mostly uses hydroponics, soil column methods, aeroponic methods, and mesh bag methods, among which hydroponics are mostly carried out in greenhouses or climate chambers.

Among the above-mentioned traditional methods, the excavation method and local sampling method commonly used in the field have a large workload and take a long time, and will cause great damage to the root system structure and shape during the processing process, and it is impossible to obtain samples of the complete root system; artificially controlled environments are commonly used. The hydroponic method, root box method, etc. Under hydroponic conditions, the structural and morphological characteristics of plant roots are very different from their growth in the soil. The root box meets the needs of dynamic observation to a certain extent, but it is not intuitive enough, and some root systems It will still be covered by soil and other media, and it is impossible to observe and record the root structure and morphological characteristics in an all-round way. Using traditional root research methods, it is difficult to conduct dynamic observation and identification of root traits on a large scale for multi-genotype varieties (eg, 200 or more varieties) at the same time.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention proposes a high-throughput culture system for identifying the genetic diversity of the root system, which can measure the dynamic development process of the root system and its response to the growth environment, and can obtain photos of the dynamic growth of the root system and carry out Quantitative analysis is suitable for batch screening of multiple varieties of crops (such as 1000 varieties), and can be used to study the response of roots to environmental stresses such as water, nutrition, and temperature.

The technical scheme of the present invention is as follows: a high-throughput culture system for identifying the genetic diversity of roots, including a container, in which a culture unit, a fixed support, and a spray system are arranged; the bottom of the container is provided with movable wheels, The inner bottom of the container is a nutrient solution;

The culture unit is placed on a fixed support, and the culture unit includes a transparent hard board, a stalk clip and a light-shielding cover, and the light-shielding cover is sealed on the outside of the transparent hard board by the stalk clip;

The spray system includes a submersible pump, a connecting pipe, a nozzle, and a spray controller. The submersible pump is placed in the nutrient solution. The submersible pump is connected to the nozzle through a connecting pipe, and the submersible pump is connected to the spray controller. The spray head is installed on the fixed bracket. The water control of the plants to be observed is realized through an automatic water supply system. A water pump and a water supply pipeline are installed in the cultivation unit. The power supply of the water pump is controlled by a timing device to start or shut down. At the same time, the time period of the water pump switch can also be adjusted according to actual needs;

The vertical support of the fixed support is provided with support points for the culture unit.

Further, the shading cover is black cloth.

Further, the connecting pipes are PVC pipe I and PVC pipe II.

Further, the number of culture units is 20.

Further, the container is made of light-shielding plastic.

Further, the formula of the nutrient solution is as follows:

_K2SO4 : _400-1200 μM;

MgSO ₄ 7H ₂ O: 50-600 μM;

CaCl ₂ ·2H ₂ O: 200-1400 μM;

KH ₂ PO ₄ : 15-70 μM;

H ₃ BO ₃ : 2-20 μM;

ZnSO4 _{· 7H2O} : 0.5-2.5μM;

MnSO ₄ ·H ₂ O: 0.1-1.6 μM;

CoSO ₄ ·7H ₂ O: 0.1-1.8 μM;

CuSO ₄ 5H ₂ O: 0.1-1.5 μM;

Na ₂ MoO ₄ ·H ₂ O: 0.01-0.2 μM;

FeNaEDTA: 20-120 μM;

NH ₄ NO ₃ : 800-3000 μM.

The present invention also claims the application of the high-throughput culture system for identifying multi-genotype root genetic diversity in root shape analysis, dynamic observation, identification, screening and its application to water, nutrient, salinity, and heavy metal stress.

The beneficial effects of the present invention are as follows: (1) for non-destructive and dynamic observation, identification and screening of root growth characteristics, digital photo collection of root system morphology and analysis of root system traits; (2) a large number of genotypes can be carried out in a small space It is especially suitable for root system analysis and screening research on multiple different genotypes or varieties/strains of the same crop; (3) There are movable wheels at the bottom of the cultivation system for easy movement, and an adjustable automatic water supply system for easy watering management, suitable for studying the effects of water stress on root growth; (4) suitable for long-term observational research and relatively deep root (1.2m deep root) research; (5) obtained high-quality root traits parameters can be used for root model Parameterized so that the model simulates and reconstructs the root model. This system builds an efficient and accurate research platform for revealing the genetic diversity of root traits and breeding varieties with excellent root traits.

Description of drawings

Fig. 1 is a schematic structural view of the high-throughput culture system of the present invention;

Fig. 2 is a schematic diagram of the internal structure of the present invention.

Among them: 1. Container, 2. Culture unit, 3. Fixed bracket, 4. Spray system, 1a, nutrient solution, 1b, movable wheel, 2a, transparent hard board, 2b, black cloth, 2c, stalk clamp, 3a, Vertical support, 3b, supporting point of culture unit, 4a, submersible pump, 4b, PVC pipe I, 4c, nozzle, 4d, spray controller, 4e, PVC pipe II.

detailed description

The present invention will be further described below by specific embodiment:

Example 1

Carry out research on genetic diversity of chickpea (Cicerarietinum L.) multi-genotype (variety) roots. Chickpea is an edible legume crop rich in high economic value and rich in protein. The global planting area is about 1.2 million hectares, of which 72% are planted in the southern subtropical and subtropical regions. In this example, 270 different genotypes (variety) of chickpeas from 29 countries were studied on root genetic diversity. The dynamic observation device adopted, as shown in Fig. 1-Fig. 2, comprises container 1, and described container 1 is provided with culture unit 2, fixed support 3, spraying system 4; Described container 1 bottom is provided with movable wheel 1b, the inner bottom of the container 1 is a nutrient solution 1a; the culture unit 2 is placed on a fixed support 3, and the culture unit 2 includes a transparent hard board 2a, a stalk clamp 2c and a light-shielding cover. The light-shielding cover is clamped and covered on the outside of the transparent hard board 2a through the stalk clamp 2c; the vertical support 3a of the fixed support 3 is provided with a culture unit support point 3b; the spray system 4 includes a submersible pump 4a, a connecting pipe , nozzle 4c, spray controller 4d, described submersible pump 4a is placed in nutrient solution 1a, described submersible pump 4a is connected nozzle 4c by connecting pipe, described submersible pump 4a is connected spray controller 4d, described The shower head 4c is installed on the fixed bracket 3 . The shading cover is black cloth. The connecting pipes are PVC pipe I4b and PVC pipe II4e. The number of culture units 2 is 20. The container 1 is made of light-shielding plastic.

The formula composition and concentration of above-mentioned nutrient solution are as follows:

_K2SO4 : 600 μM _;

MgSO ₄ ·7H ₂ O: 200 μM;

CaCl ₂ ·2H ₂ O: 600 μM;

KH ₂ PO ₄ : 20 μM;

H ₃ BO ₃ : 5 μM;

ZnSO ₄ ·7H ₂ O: 0.75 μM;

MnSO ₄ ·H ₂ O: 0.2 μM;

CoSO ₄ ·7H ₂ O: 0.2 μM;

CuSO ₄ 5H ₂ O: 0.2 μM;

Na ₂ MoO ₄ ·H ₂ O: 0.03 μM;

FeNaEDTA: 20 μM;

NH ₄ NO ₃ : 1000 μM.

Each incubator is placed with 20 culture units 2 with transparent hard plates, and each culture unit 2 is composed of a 4mm transparent hard plate 2a and a black cloth 2b, and the black cloth 2b is covered on the transparent hard plate 2a, sealed left and right (partial culture The left and right sides of the unit are sealed with stalk clamps 2c to facilitate the opening of the cultivation unit 2 for dynamic observation), and the upper and lower sides are opened for the growth of 20-40 seedlings. Provide water and nutrient supply for plant growth through a controllable automatic spraying system. The pH of the nutrient solution was tested every other day, and the nutrient solution was replaced once a week.

After sterilizing (soaking in 10vt% H ₂ O ₂ for 10 minutes) and imbibition (saturated CaSO ₄ solution, 12 hours), seeds of the same size were selected for each tested variety, and placed in plug trays filled with washed river sand for 36 hours. Select seeds that germinate consistently, and carefully transplant them into the culture unit 2 of the root culture system, with 2 plants per room (1-2 plants can be transplanted depending on the plant type).

Regularly change the position and orientation (movable wheels 1b are arranged at the bottom to facilitate movement), so as to reduce the impact of the greenhouse environment on plant growth. The growth of the root system is regularly observed with a camera, drawn with a marker pen, etc., and the image data is saved. It can be harvested at any stage of the root system growth, and the harvested samples are further processed for research.

Example 2

The response of barley (Hordeum vulgare L.) roots to different phosphorus treatments was developed. Barley is an important crop with a global production of 11.4 million tons in 2014, making it the fourth largest crop after maize, rice and wheat. In this example, the responses of roots of 9 different genotypes (variety) of barley to different phosphorus treatments were studied. Nine cultivars were selected from a high-throughput root trait screening and identification study of 190 barley genotypes as representatives of different root architectures.

The formula composition and concentration of nutrient solution, except nitrogen and phosphorus, other are all identical with embodiment 1,

_NH4NO3 : 2000 μM _;

KH ₂ PO ₄ : 4 μM (low phosphorus treatment), 20 μM (optimal phosphorus treatment); 100 μM (high phosphorus treatment).

The steps of seed disinfection and transplantation are the same as in Example 1. This embodiment includes 9 barley varieties, 3 phosphorus treatments, 3 repetitions for each treatment, and 1 independent culture system for each repetition, 9 culture systems in total. This embodiment is carried out in a greenhouse (day and night temperatures are 22° C. and 16° C. respectively), and the experiment management is the same as the observation method in Example 1.

Example 3

Carry out research on the genetic diversity of multi-variety roots of maize (Zea mays L.). Corn is one of the main food crops in the world. The total global corn output exceeded 1 billion tons in 2013. my country is the second largest corn producer after the United States. my country's total corn production, yield per unit area and planting area all exceed wheat, ranking second only to rice. The northern region is the main producing area of corn in my country, which is generally affected by different degrees of drought, which seriously restricts the growth and yield of corn. In recent years, important progress has been made in the research of breeding of fine varieties of maize, drought-tolerant physiology, efficient water absorption and mechanism. In terms of research on corn root system, Chinese scholars have focused on the response and adaptation of root system to nitrogen/phosphorus supply, temperature and light stress, drought and water use, soil microorganisms (mycorrhiza or pathogenic bacteria), and cultivation measures (planting density, farming methods), etc. properties, and the relationship between root cap structure and lodging resistance and yield. In the breeding of drought-resistant and water-saving maize varieties, it is urgent to carry out research on the diversity of root traits of multiple varieties, and reveal the relationship between root traits and water absorption and their response to drought stress.

In this study, through root trait diversity identification test (variety screening experiment) and root box experiment, maize genotypes with different root traits in my country were selected to study the diversity of maize root traits. Representative varieties of root architecture, used to study the response of maize varieties with different root architecture to drought stress.

This embodiment is except that the concentration of nutrient solution is different from embodiment 1, and other conditions are all identical with embodiment 1.

The concentration of the nutrient solution is as follows:

_K2SO4 : 1000 μM _;

MgSO ₄ ·7H ₂ O: 700 μM;

CaCl ₂ ·2H ₂ O: 200 μM;

KH ₂ PO ₄ : 15 μM;

H ₃ BO ₃ : 2 μM;

ZnSO ₄ ·7H ₂ O: 0.5 μM;

MnSO ₄ ·H ₂ O: 0.1 μM;

CoSO ₄ ·7H ₂ O: 0.1 μM;

CuSO ₄ 5H ₂ O: 0.1 μM;

Na ₂ MoO ₄ ·H ₂ O: 0.01 μM;

FeNaEDTA: 100 μM;

NH ₄ NO ₃ : 2000 μM.

Example 4

The root responses of six representative crops with different root types, wheat, corn, soybean, rapeseed, white lupine and narrow-leaved lupine, to phosphorus deficiency were studied. Use this equipment to observe the change of root system architecture under low phosphorus stress conditions, and analyze the change characteristics of root system architecture, rhizosphere environment, plant physiology and root anatomical structure of various crops under different phosphorus supply levels. In this example, except that the concentration of N and P is different from that of Example 1, other conditions are the same as in Example 1.

_NH4NO3 : 2000 μM _;

KH ₂ PO ₄ : 4 μM (low phosphorus treatment), 20 μM (optimal phosphorus treatment); 100 μM (high phosphorus treatment).

Claims (7)

1. one kind identification multifarious high flux culture systems of Root inheritance, it is characterised in that including container (1), described appearance Culture unit (2), fixed support (3), spraying system (4) are provided with device (1)；Described container (1) bottom is provided with castor (1b), described container (1) inner bottom part are nutrient solution (1a)；

Described culture unit (2) is placed on fixed support (3), and described culture unit (2) includes transparent hardboard (2a), takes out stalk (2c) and shading covering are pressed from both sides, described shading covering is covered on the outside of transparent hardboard (2a) by taking out stalk folder (2c) clamp；

Described spraying system (4) includes immersible pump (4a), connecting tube, shower nozzle (4c), sprayer controller (4d), described diving Pump (4a) is placed in nutrient solution (1a), and described immersible pump (4a) connects shower nozzle (4c), described immersible pump by connecting tube (4a) connection sprayer controller (4d), described shower nozzle (4c) are arranged on fixed support (3)；

Culture unit supports point (3b) is set on the vertical support frame (3a) of described fixed support (3).

2. the identification multifarious high flux culture systems of Root inheritance as claimed in claim 1, it is characterised in that described screening Light covering is black cloth (2b).

3. the identification multifarious high flux culture systems of Root inheritance as claimed in claim 1, it is characterised in that described company Take over as pvc pipe I (4b) and pvc pipe II (4e).

4. the identification multifarious high flux culture systems of Root inheritance as claimed in claim 1, it is characterised in that described training It is 20 to support unit (2).

5. the identification multifarious high flux culture systems of Root inheritance as claimed in claim 1, it is characterised in that described appearance Device (1) is made up of shading plastics.

6. the identification multifarious high flux culture systems of Root inheritance as claimed in claim 1, it is characterised in that described battalion The formula of nutrient solution (1a) is as follows：

K₂SO₄：400-1200μM；

MgSO₄·7H₂O：50-600μM；

CaCl₂·2H₂O：200-1400μM；

KH₂PO₄：15-70μM；

H₃BO₃：2-20μM；

ZnSO₄·7H₂O：0.5-2.5μM；

MnSO₄·H₂O：0.1-1.6μM；

CoSO₄·7H₂O：0.1-1.8μM；

CuSO₄·5H₂O：0.1-1.5μM；

Na₂MoO₄·H₂O：0.01-0.2μM；

FeNaEDTA：20-120μM；

NH₄NO₃：800-3000μM.

7. one kind identification the multifarious high flux culture systems of polygene type Root inheritance root system shape analysis, dynamic observation, Identification, screen and its to the application in terms of moisture, nutrient, saline and alkaline, heavy metal stress.