CN110771508B

CN110771508B - Preparation method of artificial blueberry seeds

Info

Publication number: CN110771508B
Application number: CN201911172973.2A
Authority: CN
Inventors: 侯义龙; 蔡军
Original assignee: Dalian University
Current assignee: Dalian University
Priority date: 2019-11-26
Filing date: 2019-11-26
Publication date: 2022-07-05
Anticipated expiration: 2039-11-26
Also published as: CN110771508A

Abstract

The invention belongs to the technical field of plant biology and discloses a preparation method of artificial blueberry seeds. On a clean bench, putting the stem segment with a bud in embedding medium, mixing, sucking one propagule with a suction pipe each time, and dropping into coagulating agent for ion exchange reaction for 5-15 min; then rinsing with sterile water for 10-20min, and stopping reaction; fishing out and drying; the proportion of the three substrates of the wood chips, the soil and the perlite is 1-5: 1-5: 1-5, keeping the substrate wet, spraying water for 4-5 times per day with the air humidity of 70-90%, and transplanting indoor hardening seedlings. Based on the modern plant tissue culture technology, a top-grade artificial seed technical system of the excellent blueberry variety, which has the advantages of stable system, high emergence rate, simplicity, convenience, practicability and the like, is established, and a foundation is laid for the rapid propagation of blueberry seedlings.

Description

Preparation method of artificial blueberry seeds

Technical Field

The invention belongs to the technical field of plant biology, and relates to a preparation method of artificial blueberry seeds.

Background

Plant Artificial seeds (Artificial seeds) or Synthetic seeds (Synthetic seeds) are seeds produced artificially. The artificial seed technology is a new technology developed on the basis of the in vitro plant culture technology. The artificial seed is an artificial capsule containing plant embryoid or bud, nutrient, hormone and other components. The concept of artificial seeds was first proposed by Murashige in 1978 at the 4 th International society for plant tissue cell culture. Murashige considers that with the continuous development of plant tissue culture technology, a plurality of embryoids can be synchronously cultured by a small amount of explants, and the embryoids are embedded in certain capsules to have the function of seeds and can be directly used for field sowing. In 1985, Kamada, a Japanese scholar, extended the concept of artificial seeds, and he thought that granules produced by tissue culture with meristematic tissue (shoots, callus, embryoid and growing point, etc.) developing into whole plants, which can be used as substitutes for natural seeds for sowing, were all called artificial seeds, using an appropriate method for embedding. In 1995, Chinese scientists Chendefu et al further extended the concept of artificial seeds, and they thought that the somatic embryos produced in the in vitro culture of plants or the meristematic tissues (such as embryoid, bud and stem segment, etc.) capable of developing into complete plants were embedded in the shells containing nutrients and having protective functions, and the formed granules capable of germinating and emerging under proper conditions were called artificial seeds. In recent years, this concept has been developed from the coating of somatic embryos in the narrow sense to the coating of any suitable plant propagules, such as adventitious buds, tubers, axillary buds, shoot tips, protocorms, callus and hairy roots.

Blueberries, also known as bilberries, are plants of the genus Vaccinium of the family Ericaceae and are emerging fruit tree species with higher economic value and broad development prospects. The blueberry propagation mode usually adopts cutting propagation and tissue culture propagation, the propagation speed is slow, and the requirements of the rapidly-developed blueberry industry cannot be met. The artificial seeds can provide a way for rapidly propagating the nursery stocks.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides the blueberry artificial seed preparation method, which is based on the modern plant tissue culture technology, establishes a technical system of the blueberry fine variety top-grade artificial seed preparation method with the advantages of stable system, high emergence rate, simplicity, convenience, practicability and the like, and lays a foundation for the rapid propagation of blueberry seedlings.

The above purpose of the invention is realized by the following technical scheme:

a preparation method of artificial blueberry seeds comprises the following specific steps:

on a clean bench, putting the stem segment with a bud in embedding medium, mixing, sucking one propagule with a suction pipe each time, and dropping into coagulating agent for ion exchange reaction for 5-15 min; then rinsing with sterile water for 10-20min, and stopping reaction; fishing out and drying; the proportion of the three substrates of the wood chips, the soil and the perlite is 1-5: 1-5: 1-5, keeping the substrate wet, spraying water for 4-5 times every day with the air humidity of 70-90%, and performing indoor hardening seedling transplantation.

The embedding medium is artificial endosperm, and the formula is as follows: WPM + IAA1.0-3.0mg/L +2ip 0.05-0.2mg/L +1-7 wt% maltose, pH 5.8.

The coagulant is artificial seed skin and has the following formula: 1-5 wt% of sodium alginate and 1-3 wt% of CaCl₂+ 1-5% by weight of cellulose + 1-5% by weight of hemicellulose.

The embedding medium is artificial endosperm, and the formula is preferably as follows: WPM + IAA2.0mg/L +2ip 0.1mg/L +7 wt% maltose, pH 5.8.

The coagulating agent is artificial seed coat, and the preferable formula is as follows: 3 wt% sodium alginate and 2 wt% CaCl₂+5 wt% cellulose + wt 1% hemicellulose.

The proportion of the three substrates of the wood chips, the soil and the perlite during transplanting and hardening is preferably 1: 1: 1.

the WPM is fully called woody plant culture medium; the IAA is fully called indoleacetic acid; the 2ip are all referred to as 2-isopentenyl adenine.

The blueberry seeds are artificially synthesized according to the main structure and the functions of the seeds and based on the basic principle of plant tissue culture.

Compared with the prior art, the invention has the beneficial effects that:

1. the top grade of the blueberry variety is a new high-quality blueberry variety in China, the resource is novel, and the top grade stem with the leaf buds of the blueberry variety is taken as a propagule, and an artificial seed technical system is established for the first time; the research on artificial blueberry seeds can provide effective measures for quickly propagating blueberry seedlings in production.

2. The preparation method of the top-grade artificial seeds of the high-quality blueberry varieties has the advantages of good technical system stability and simple and convenient preparation; the emergence rate is high. The perlite has good air permeability and water absorbability, the humus soil is also called nutrient soil, contains relatively rich organic matter core mineral elements, the wood chips are loose and air permeable, the water permeability is strong, the effects of the three are complementary, and the cooperation of the three can provide a good growing and developing environment for plants. Wood chip: humus soil: perlite is 1: 1: 1 has the highest survival rate and the best effect when being used as a transplanting matrix.

Drawings

FIG. 1 is a top-level artificial seed map of a blueberry variety prepared by the method.

FIG. 2 is a germination diagram of "top-grade" artificial seeds of the blueberry variety prepared by the invention.

Detailed Description

The invention is described in more detail below with reference to specific examples, without limiting the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be obtained from commercial sources.

Example 1

Placing sterile stem segment with a bud in embedding medium (artificial endosperm, formula: WPM + IAA1.0 mg/L +2ip 0.05mg/L +4 wt% maltose, pH 5.8) on superclean bench, mixing, sucking one propagule each time with suction tube, and dripping into coagulating agent (artificial seed coat, formula: 5 wt% sodium alginate +3 wt% CaCl₂+3 wt% cellulose +5 wt% hemicellulose) for 5 min. The reaction was then stopped by rinsing with sterile water for 10 min. Fishing out and drying. The proportion of the three substrates of the wood chips, the soil and the perlite is 5: 1: 1, keeping the substrate wet, spraying water for 4-5 times every day with the air humidity of 80%, and performing indoor hardening seedling transplantation.

Example 2

Placing sterile stem segment with a bud in embedding medium (artificial endosperm, formula: WPM + IAA 3.0mg/L +2ip 0.2mg/L +1 wt% maltose, pH 5.8) on superclean bench, mixing, sucking one propagulum each time with suction tube, and dripping into coagulating agent (artificial seed coat, formula: 1 wt% sodium alginate +1 wt% CaCl)₂+1 wt% cellulose +3 wt% hemicellulose) for 10 min. The reaction was then stopped by rinsing with sterile water for 15 min. Fishing out and drying. The proportion of the three substrates of the wood chips, the soil and the perlite is 1: 1: and 5, keeping the substrate wet, spraying water for 4-5 times every day when the air humidity is 80%, and performing indoor hardening seedling transplantation.

Example 3

Putting a stem segment with a bud in an embedding medium (artificial endosperm, formula: WPM + IAA2.0mg/L +2ip 0.1mg/L +7 wt% maltose, pH 5.8) on a clean bench, mixing, sucking a propagule with a suction tube, and dripping into a coagulant (artificial seed coat, formula: 3 wt% sodium alginate +2 wt% CaCl)₂+5 wt% cellulose +1 wt% hemicellulose) for 15 min. The reaction was then stopped by rinsing with sterile water for 20 min. Fishing out and drying. Three substrates of wood dust and soil are crushed during transplanting and hardening seedlingsThe proportion of soil to perlite is 1: 1: 1, keeping the substrate wet, spraying water for 4-5 times every day with the air humidity of 80%, and performing indoor hardening seedling transplantation.

Experimental study

1. Screening of Artificial endosperm

Firstly, screening the most suitable rooting culture medium, and removing agar from the most suitable rooting culture medium to obtain the artificial endosperm. The artificial endosperm takes WPM as a basic culture medium, and the IAA concentration is set as follows: 1.0-3.0 mg/L; the 2ip concentration is: 0.05-0.2 mg/L; the maltose concentrations were: 1 to 7 wt%. The artificial endosperm formula was screened by orthogonal assays according to the 3-factor 3 level above, without considering interaction. The rooting rate is used as a screening index (see table 1-1).

Experiment design: in the artificial endosperm, the IAA concentration is designed to be 1.0mg/L (A1), 2.0mg/L (A2) and 3.0mg/L (A3); the 2ip concentration is designed as: 0.05mg/L (B1), 0.1mg/L (B2), 0.2mg/L (B3); the maltose concentration is designed as follows: 1% (C1), 4% (C2), 7% (C3). The orthogonal design method is adopted, the experimental treatment combinations are 9, and each treatment is provided with 5 times of repetition. Namely:

A1B1C1 treatment 1

A1B2C2 treatment 2

A1B3C3 treatment 3

A2B1C2 treatment 4

A2B2C3 treatment 5

A2B3C1 treatment 6

A3B1C3 treatment 7

A3B2C1 treatment 8

A3B3C2 treatment 9

TABLE 1-1 rooting percentage (%)

TABLE 1-2 analysis of variance of the results of TABLE 1-1

Tables 1-3 test for significance of differences between the data of Table 1-1

Treatment of	Mean number of	Difference of 5%	1% difference
				Treatment 5	92.00	a	A
Treatment 6	88.00	ab	AB
				Treatment 7	76.00	bc	ABC
Treatment 4	72.00	c	ABC
				Treatment 8	72.00	c	ABC
Treatment 3	68.00	c	BC
				Process 1	64.00	c	C
Treatment 2	64.00	c	C
				Process 9	64.00	c	C

The experimental results are as follows: the treatment combination A2B2C3 (i.e., WPM + IAA2.0mg/L +2ip 0.1mg/L +7 wt% maltose) was the optimal rooting medium on which the rooting rate was the greatest.

2. Screening of artificial seed coats

The method is characterized in that a stem section with a leaf bud at the top level of a blueberry variety is taken as a propagule, and WPM + IAA2.0mg/L +2ip 0.1mg/L +7 wt% maltose is taken as artificial endosperm, and proper artificial seed coat screening is carried out. The artificial seed coat is prepared from 1-5 wt% sodium alginate and 1-3 wt% CaCl₂The cellulose with the concentration of 1 wt% -5 wt% and the hemicellulose solution with the concentration of 1 wt% -5 wt% are matched with each other. 4, a factor 3 horizontal orthogonal design is made, and the artificial seed coat is screened by an orthogonal test without considering the interaction. Using artificial seeds in WPM + 7% maltoseThe germination rate on the medium was used as an index for screening (see Table 2-1).

Experiment design: in the artificial seed coat, the concentration of sodium alginate is 1% (A1), 3% (A2) and 5% (A3); CaCl₂Concentrations were 1% (B1), 2% (B2), 3% (B3); the cellulose concentration is 1% (C1), 3% (C2), 5% (C3); the hemicellulose concentration was 1% (D1), 3% (D2), 5% (D3). The orthogonal design method is adopted, the experimental treatment combinations are 9, and each treatment is provided with 5 times of repetition. Namely:

A1B1C1D1 treatment 1

A1B2C2D2 treatment 2

A1B3C3D3 Process 3

A2B1C2D3 treatment 4

A2B2C3D1 treatment 5

A2B3C1D2 treatment 6

A3B1C3D2 treatment 7

A3B2C1D3 treatment 8

A3B3C2D1 Process 9

TABLE 2-1 Germination ratio (% of different treatments)

TABLE 2-2 analysis of variance of the results of TABLE 2-1

Source of variation	Degree of freedom	Sum of squares	Mean square	F value	F0.05	F0.01
							Treatment room	8	4320.00	540.00	3.47**	2.21	3.05
Error of the measurement	36	5600.00	155.56
							Total variation	44	9920.00

Tables 2-3 test for significance of differences in the data of Table 2-1

The experimental results are as follows: treatment combination A2B2C3D1 (i.e., 3 wt% sodium alginate +2 wt% CaCl)₂+5 wt% cellulose +1 wt% hemicellulose) is the most optimal artificial seed coat formulation.

3. Screening of the transplanting Medium

The wood chips, the humus soil and the perlite are mixed according to different proportions (1-5: 1-5). Transplanting and hardening seedlings, keeping the matrix wet, spraying water for 4-5 times every day when the air humidity is 80%, and transplanting indoor hardening seedlings.

Experiment design: in the transplanting matrix, three kinds of matrixes, namely wood chips, humus and perlite, adopt three kinds of proportions: humus soil: perlite 5: 1: 1. wood chip crushing: humus soil: perlite is 1: 1: 1 and wood chips: humus soil: perlite is 1: 1: 5. the survival rate of the transplanted artificial seeds in the three proportions is examined (see table 3-1). 10 seeds were transplanted per pot per treatment, setting 5 replicates.

TABLE 3-1 survival rate of transplantation on substrate of different ratios (%)

TABLE 3-2 analysis of variance of TABLE 3-1

Sources of variation	Degree of freedom	Sum of squares	Mean square	F value	F0.05	F0.01
							Treatment room	2	2813.33	1406.67	24.82**	3.89	6.93
Error of	12	680.00	56.67
							Total variation	14	3493.33

TABLE 3-3 significance test of differences in the data of TABLE 3-1

Treatment of	Mean number of	Difference of 5%	1% difference
				Treatment 2	80.00	a	A
Treatment 3	52.00	b	B
				Process 1	50.00	b	B

The experimental results are as follows: wood chip crushing: humus soil: perlite is 1: 1: the proportion of 1 is the best matrix, and the survival rate of the transplanting on the matrix is the maximum.

The embodiments described above are only preferred embodiments of the invention, and are not all possible embodiments for the practical implementation of the invention. Any obvious modifications to the above would be obvious to those of ordinary skill in the art, but would not bring the invention so modified beyond the spirit and scope of the present invention.

Claims

1. A preparation method of artificial blueberry seeds is characterized in that blueberry varieties are top grade; on a clean bench, putting the stem segment with a bud in embedding medium, mixing, sucking one propagule with a suction pipe each time, and dropping into coagulating agent for ion exchange reaction for 5-15 min; then rinsing with sterile water for 10-20min, and stopping reaction; fishing out and drying; the proportion of the three substrates of the wood chips, the soil and the perlite is 1: 1: 1, keeping the substrate moist, spraying water for 4-5 times per day with the air humidity of 70-90%, and performing indoor hardening seedling transplantation;

the embedding medium is artificial endosperm, and the formula is as follows: WPM + IAA2.0mg/L +2ip 0.1mg/L +7 wt% maltose;

the coagulant is artificial seed skin and has the following formula: 3 wt% sodium alginate and 2 wt% CaCl₂+5 wt% cellulose +1 wt% hemicellulose.