CN102277435A

CN102277435A - Plant ploidy identifying method and use thereof

Info

Publication number: CN102277435A
Application number: CN2011102335773A
Authority: CN
Inventors: 崔群香; 郝振萍; 王倩
Original assignee: Jinling Institute of Technology
Current assignee: Jinling Institute of Technology
Priority date: 2011-08-16
Filing date: 2011-08-16
Publication date: 2011-12-14

Abstract

The invention belongs to the field of biotechnology and particularly relates to a new method for counting chloroplasts in a leaf lower epidermis pore protective cell and use of the method in plant ploidy identification of cruciferae crops such as green Chinese cabbage. In the method, the chloroplasts are counted by using the characteristic that chloroplast can emit red fluorescence in ultraviolet exciting light without treatment such as dyeing, the chloroplast ploidy demarcation of green Chinese cabbage is determined by the number of the chloroplasts of a plant of which the ploidy is known and consequently the ploidies of plants from different sources can be determined. Tests prove that the method has the advantages that: the material preparation is free from limitation of growth stage of plants on materials and is quick and accurate; and the cost is low. And the method overcomes the drawbacks of material preparation limitation, complex technique and high cost of the prior art.

Description

A kind of plant methods for ploidy determination and application

Technical field

The invention belongs to biological technical field, be specifically related to a kind of ploidy evaluation of counting the novel method of blade lower epidermis Stomacal guard cell number of chloroplast and being applied to Plantula Brassicae chinensis, the present invention utilizes chloroplast(id) need not count number of chloroplast through the characteristic that red fluorescence is promptly sent in processing such as dyeing under uv excitation light, and judges the ploidy of different sources plant with this.

Background technology

Plantula Brassicae chinensis is a kind of typical different flower (freedom) pollination crop, hybrid vigour is obvious, utilize microspores culture or anther culture technique to obtain double haploid colony (DH) and can obtain pure lines fast, quicken breeding process, but the regeneration plant colony that anther culture or microspores culture obtain is the population mixture of Different Ploidy level often, further utilize and must carry out the ploidy evaluation; The part tetraploid plant that Plantula Brassicae chinensis nature or artificial induction produce has higher economic value, also can directly apply to production, is the population mixture of Different Ploidy level equally but induce the plant of generation, also must carry out the ploidy evaluation and just can effectively utilize.

Belonging to the common method of carrying out the ploidy evaluation on the crop rapes such as Plantula Brassicae chinensiss has: root tip chromosomes counting process, morphology identification method and stream Schwann Cells instrument assay method.Because karyomit(e) is less, chromosome counting method is more loaded down with trivial details, and time-consuming; The morphology identification method just can be reached a conclusion after will waiting until plant blossom, and the cycle is long; Though stream Schwann Cells instrument assay method efficiently and accurately, required instrument costliness, cost height.

Wang Xianxue (1980), Yang Jinhou (1990), Zhang Lingyuan etc. (2005), a chrysanthemum equality (2007), Yuan Suxia the studies confirm that Stomacal guard cell number of chloroplast of scholar in watermelon, mulberry leaf, loquat, capsicum and cabbage vegetables such as (2009) are controlled by karyomit(e), chloroplast(id) is counted significant difference between ploidy, and the ploidy that can be applied to plant is identified.These investigators observe after handling with certain density iodine monoiod(in)ate potassium solution dyeing again, and the principle of this method utilization is that the starch small grain that contains in the chloroplast(id) can be dyed atropurpureus by iodine monoiod(in)ate potassium.But chloroplast(id) is less among the one side guard cell in Plantula Brassicae chinensis, may be that therefore low being difficult to of starch content be difficult to count by the dyeing of iodine monoiod(in)ate potassium solution on the other hand.And need not any processing under uv excitation light, the chlorophyll that contains in the chloroplast(id) promptly sends red fluorescence, can see the chloroplast(id) of different sites among the guard cell clearly by focusing, can very clearly under 400 times of mirrors, count, overcome iodine monoiod(in)ate potassium solution staining pretreatment time prolong cause chloroplast(id) to disintegrate and chloroplast(id) between overlapping etc. and influence the problem of counting accuracy.

Summary of the invention

The technical issues that need to address of the present invention are: a large amount of plant ploidy appraisal cost height, slow-footed problem, and invent chloroplast(id) counting in a kind of blade Stomacal guard cell and be used to identify the method for the plant ploidy of cresss such as Plantula Brassicae chinensis.

Technical scheme: according to relevant novel method---the chloroplast(id) several times boundary method of setting up a kind of Plantula Brassicae chinensis ploidy evaluation of ploidy with number of chloroplast; Chloroplast(id) in plant leaf (comprising cotyledon, lotus throne leaf and a kind of sedge leave) Stomacal guard cell of different sources and different developmental phases is observed counting under fluorescent microscope, determine the ploidy of plant according to chloroplast(id) mean number, the chloroplast(id) number is less than 10 the monoploid that is, be less than 16 the diploid that is more than or equal to 10, more than or equal to 16 be polyploid such as tetraploid.

A kind of plant methods for ploidy determination and application comprise:

(1) number of chloroplast is observed and counting among the blade guard cell:

(2) determine the ploidy of plant to be measured according to chloroplast(id) mean number and chloroplast(id) several times cut off value.

Beneficial effect: the present invention has set up a kind of number of chloroplast rapid counting method and chloroplast(id) mean number in the Stomacal guard cell has been applied to the ploidy evaluation of Plantula Brassicae chinensis, set up a kind of technical system of chloroplast(id) mean number boundary method Rapid identification Plantula Brassicae chinensis ploidy, compare with present technology, its advantage is:

(1) count number of chloroplast quick and precisely, cost reduces; (2) no matter draw materials conveniently, be not subjected to plant source and the restriction of etap of living in, be the test-tube plantlet or the plant of field growing, no matter is that cotyledon, lotus throne leaf or a kind of sedge leave all can be used for the ploidy evaluation.

Four, description of drawings

Fig. 1 is a Plantula Brassicae chinensis diploid adjoining tree blade Stomacal guard cell chloroplast(id)

Fig. 2 is a Plantula Brassicae chinensis tetraploid adjoining tree blade Stomacal guard cell chloroplast(id)

Fig. 3 is a Plantula Brassicae chinensis haplobiont blade Stomacal guard cell chloroplast(id)

Fig. 4 is a Plantula Brassicae chinensis liploid plant blade Stomacal guard cell chloroplast(id)

Fig. 5 is a Plantula Brassicae chinensis tetraploid plant blade Stomacal guard cell chloroplast(id)

Five, embodiment

A kind of plant methods for ploidy determination provided by the present invention and application comprise:

1, materials and methods: to the seedling from seed of Plantula Brassicae chinensis liploid variety Vantage, No. one, capital hat, SUZHOUQING, Su Xiu, tetraploid SUZHOUQING, and Hua Guan, Hua Jing, green grass or young crops that ploidy knows obstruct and green No. 2 microspores culture regeneration plant of summer, take 5-7 joint blade, tear and take off epidermis, (* 400) observe chloroplast(id) and the counting in the Stomacal guard cell under fluorescent microscope, every blade is observed 10 pores, asks its mean value.

Utilizing pollen mother cells process chromosome number and behavior variation to carry out ploidy after all plant boltings are bloomed determines, monoploid pollen mother cells I in mid-term forms 10 univalents, first anaphase 5/5 is separated into the master, and the pollen of generation is flat, the degree of depth three is split or out-of-shape; The diploid pollen mother cells diakinesis stage and mid-term I form 10 bivalents, 10 karyomit(e)s of 2 every utmost points in mid-term, the pollen of generation are 3 to split shape; The tetraploid pollen mother cell is big than diploid, and reduction division is normal, and mid-term, I formed 20 bivalents, and the pollen three of generation splits or four splits.

2, chloroplast(id) several times boundary: distribution and mean value thereof according to number of chloroplast in 30 strain monoploid, 80 strain diploids and the 30 strain tetraploid blade lower epidermis Stomacal guard cells have determined that the cut off value of guard cell's number in the Different Ploidy plant leaf lower epidermis pore is respectively: the chloroplast(id) number is less than 10 the monoploid that is, be less than 16 the diploid that is more than or equal to 10, more than or equal to 16 be polyploid such as tetraploid.

3, the plant ploidy is identified: plant the 5th joint and the above blade of taking ploidy the unknown, tear to take off in epidermis and the distilled water on slide glass and launch, covered, number of chloroplast in the counting Stomacal guard cell under fluorescent microscope, 10 pores of every blade counting are asked its mean value and are determined the ploidy of plant according to known ploidy cut off value.

4, result and analysis: chloroplast(id) is counted in the 124 strain plant that the boundary method identifies has 36 strain monoploid (Fig. 3), 76 strain diploids (Fig. 4) and 12 strain tetraploids (Fig. 5).Each ploidy plant and florescence, the result according to reduction division chromosome number and behavior variation evaluation coincide.

The present invention is directed to and to make the Plantula Brassicae chinensis plant that ploidy is judged fast, especially microspores culture regenerated plant, set up blade Stomacal guard cell chloroplast(id) counting number and Analysis and Identification system that suitable Plantula Brassicae chinensis ploidy is identified, improved Plantula Brassicae chinensis ploidy breeding process greatly.

Claims

1. plant methods for ploidy determination is characterized in that being made of following two steps:

(1) number of chloroplast is observed and counting among the blade guard cell:

Take plant the 5th joint and the above blade of ploidy the unknown, tear to take off in epidermis and the distilled water on slide glass and launch, covered, number of chloroplast in the counting Stomacal guard cell under fluorescent microscope, 10-20 pore of every blade counting asks its chloroplast(id) to count mean value;

(2) determine the ploidy of plant to be measured according to chloroplast(id) mean number and chloroplast(id) several times cut off value:

The cut off value of number of chloroplast is respectively in the Plantula Brassicae chinensis blade lower epidermis Stomacal guard cell: the chloroplast(id) number is less than 10 the monoploid that is, be less than 16 the diploid that is more than or equal to 10, more than or equal to 16 be polyploid such as tetraploid, chloroplast(id) is counted the ploidy that plant to be measured is determined in mean value and the comparison of above-mentioned cut off value.

2. a kind of plant methods for ploidy determination according to claim 1 is characterized in that utilizing fluorescent microscope to carry out number of chloroplast and observes fast and counting.

3. the described a kind of plant methods for ploidy determination of claim 1 is characterized in that in breeding time in each stage Plantula Brassicae chinensis or brassica plant being carried out the evaluation of plant ploidy.

The described plant methods for ploidy determination of claim 1 identify Plantula Brassicae chinensis or plant ploidy that cress is spontaneous or the artificial induction produces plant or flower pesticide and microspores culture produce in application.