CN102870612B - Evaluation method for cotton bollworm resistance of Bt-transgenic cotton - Google Patents

Abstract

The invention relates to the field of biological control, in particular to an evaluation method for cotton bollworm resistance of Bt (bacillus thuringiensis)-transgenic cotton. The method comprises the following steps: leaves are collected in emergence periods of a second generation, a third generation and a fourth generation of cotton bollworms; and non-transgenic cotton is adopted as a control material; the leaves are placed in a box; hatched larval cotton bollworms are inoculated; the dead quantity and instar of the larval cotton bollworms are investigated after the hatched larval cotton bollworms are inoculated; statistical analysis is conducted; and the instar inhibition rate of the cotton bollworms is calculated. According to the method, the sample quantity is relatively less; only one cotton bollworm can be inoculated, so that labor is saved and cotton bollworm tests are reduced, that is to say, the labor and the time are saved. According to the method, evaluations are conducted separately on the emergence periods of the second generation, the third generation and the fourth generation of the cotton bollworms, so that impacts and effects of foreign protein on the cotton bollworm resistance in the cotton growth and development processes can be reflected accurately, and the life of the cotton capable of resisting the cotton bollworms is prolonged. With the adoption of the method, the use quantity of the cotton bollworms is reduced greatly; and the method is easy to operate, simple, accurate, objective and reliable.

Description

Transgenic Bt cotton bollworm resisting evaluation method

Technical field

The present invention relates to biological control field, relate to particularly transgenic Bt cotton bollworm resisting evaluation method.

Background technology

The nineties cotton bollworm broke out and caused harm year after year last century, to Cotton in China, produced and brought grave danger, and output of cotton loss reaches 40%～60%, and local cotton region reaches more than 80%, even total crop failure, more than 400 hundred million yuan of national cotton losses.Transgenic cotton against pests in 1997, in China's large-area applications, has effectively been controlled breaking out of cotton bollworm and has been caused harm, and has promoted the sustainable and stable development of Cotton in China.To 2006, national Insect Resistant Cotton area reached 3,500,000 hectares, accounts for 66% of Cotton in China area, and wherein Insect Resistant Cotton area in the Huanghe valley accounts for more than 98%, and province's Insect Resistant Cotton such as Henan, Hebei, Shandong, Anhui realize 100% kind and plant.

Due to the toxin of Bt transgenic cotton can be in cotton plant continuous expression, but the g and D along with cotton plants, the expression of foreign protein in plant body generally presents progressively downward trend, is also consistent trend with cotton field bollworm generating capacity and the extent of injury simultaneously.Cotton in seedling stage, flower bud phase and flowering and boll-setting period exogenous protein expression amount show inconsistent on different cotton materials, and fall is not identical yet, and cotton bollworm is at two generations, three generations and the generating capacity in four generations and aggregation extent the degree progressively alleviating.Therefore cotton should change and carry out overall merit according to the occurrence and harm degree of the expression characteristic of transgene cotton foreign protein and cotton bollworm the evaluation of resistance of cotton bollworm.

Due to existing Resistance Strain of Cotton cotton bollworm property bioassay method, sampling amount and to connect worm amount all very large, time-consuming taking a lot of work.Existing Resistance Strain of Cotton cotton bollworm property bioassay method sampling amount is minimum is to establish 20 blades of repetition, repeats for three times, and every blade connects 5 examination worms.Because existing raw survey method is the lethality of trying worm is judging quota, if 5 of access examination worms, every dead 1 examination worm, lethality is 20%, also has all the other 4, illustrates that this blade is 20% to the resistance lethality of examination worm, if every blade only connects 1, the lethality of this blade is 100%, and this blade is 100% to the resistance lethality of examination worm, can not correctly, objectively react the resistance level of blade to examination worm.Method of the present invention is that every blade connects 1 examination worm, only registers the length of time of the growth of examination worm, if examination worm is dead during investigation result, be designated as 0 the length of time, more can be embodied under adverse environmental factor, the growth abnormal condition of examination worm, thus reflection is except the resistance level of cotton to cotton bollworm.In addition, two generations, three generations and four generations of cotton bollworm emergence period, cotton plants exogenous Bt insecticidal proteins expression all presents the trend of reduction, and existing Resistance Identification method is same criterion, for whole decline of later stage cotton foreign protein, causes the error in evaluation.

Summary of the invention

The present inventor proposes and has completed the present invention in order to address the above problem.

The object of this invention is to provide that a kind of sampling quantity is few, method is easy, easy to operate and objective Resistance Strain of Cotton cotton bollworm property evaluation method.

Resistance Strain of Cotton cotton bollworm property evaluation method of the present invention comprises the following steps:

1, Resistance Strain of Cotton cotton bollworm property evaluation method, is characterized in that, said method comprising the steps of:

(1) in the second generation, the third generation and four generations of cotton bollworm emergence period, gather respectively second of top of cotton and launch blade completely, take non-transgenic cotton variety as control material;

(2) the fresh blade gathering is placed in experiment box;

(3) in putting vaned experiment box, connect cotton bollworm newly hatched larvae, tighten lid, be placed under room temperature;

(4) connect worm " Invest, Then Investigate " cotton bollworm larvae death toll and the length of time; If examination worm is dead, be designated as 0 the length of time;

(5) carry out statistical analysis, calculate cotton bollworm inhibiting rate in the length of time, take two, three generations and the length of time four generations of cotton bollworm emergence period inhibiting rate be to judge resistance level index, wherein the length of time, inhibiting rate computing formula was as follows:

According to the specific embodiment of the present invention, aphid resistance authentication method of the present invention comprises the following steps:

(1) in the second generation, the third generation and four generations of cotton bollworm emergence period, gather respectively second of top of cotton and launch 10-20, leaf completely, take non-transgenic cotton variety as control material;

(2) the fresh blade gathering is placed in the experiment box with preservation to every box a slice;

(3) in putting vaned experiment box, connect 1 1d cotton bollworm in age newly hatched larvae, tighten lid, be placed under room temperature;

(4) connect 5d investigation cotton bollworm larvae death toll and the length of time after worm; If examination worm is dead, be designated as 0 the length of time;

(5) carry out statistical analysis, calculate cotton bollworm inhibiting rate in the length of time, take two, three generations and the length of time four generations of cotton bollworm emergence period inhibiting rate be to judge resistance level index.

For example, according to the specific embodiment of the present invention, other criterion of resistance level is as follows.

The method according to this invention, sampling quantity is relatively less, can only connect 1 cotton bollworm, both can saving of labor, also economize examination worm, be equivalent to saving of work and time.Method of the present invention was evaluated separately from two generations, three generations and four generations of cotton bollworm emergence period, and the effect of foreign protein bollworm resisting in energy accurate response Developmental of Cotton process, simultaneously for extending the Insect Resistant Cotton life-span.The method significantly reduces by worm amount, and method is easy, easy to operate, accurate, objective, reliable.

Embodiment

Embodiment 1

(1), in the second generation, the third generation and four generations of cotton bollworm emergence period, gather respectively second of top of cotton and launch 10-20, leaf completely; Take non-transgenic cotton variety as control material;

(2) the fresh blade gathering is placed in the experiment box with preservation to every box a slice;

(3) in putting vaned experiment box, connect 1 1d cotton bollworm in age newly hatched larvae, tighten lid, be placed under room temperature;

(4) connect 5d investigation cotton bollworm larvae death toll and the length of time after worm; If examination worm is dead, be designated as 0 the length of time;

(5) carry out statistical analysis, calculate cotton bollworm inhibiting rate in the length of time, take two, three generations and the length of time four generations of cotton bollworm emergence period inhibiting rate be to judge resistance level index (referring to table 1);

(6) experimental result is as shown in the following Table 2:

Other criterion of table 1 resistance level

The different cotton material indoor bioassay of table 2 result

In the present embodiment, the transgene cotton materials A 2, B4, C4 and the D2(that use are referring to table 2) be from 2011, the national region test material of the Huanghe valley, to select at random and evaluate its resistance, these four parts of these evaluation results of transgenic cotton floral material all reach anti-level, consistent with Net evaluation result in 2011.Show that the method significantly reduces by worm amount, method is easy, easy to operate, and evaluation result is accurate, objective, reliable.

Embodiment 2

Within 2012, by existing Resistance Strain of Cotton cotton bollworm authentication method < < genetically modified plants and products thereof Environmental security, detect

Pest-resistant cotton part 1 (No. 953 bulletin-12-2007 of the Ministry of Agriculture) > >, in his-and-hers watches 2, A2, B4, C4 and tetra-cotton materials of D2 have carried out biologicall test, and experimental procedure and qualification result are as follows;

1, sampling method

At target pest two, three, four generations occurance peak, respectively from the 3rd cotton leaf that transgenic pest-resistant product till the land, sense worm reference substance tills the land the expansion of collection top, 20 of the every community of every processing random acquisitions.

2, operation

The agar medium that adds 20 mm in Boiling tube (35 mm * 120 mm), by fresh-keeping in blade (reservation petiole) insertion medium, every test tube is put a blade, and every blade connects 5 of cotton bollworm 1 d instar larvaes.After connecing worm, use the absorbent cotton jam-pack mouth of pipe, in case cotton bollworm escapes; Be put in the insectary of 25～28 ℃ or incubator and raise.

3, outcome record

After connecing worm, the 5th d checks larval feeding situation and larva death state.Record the dead borer population of larva and the borer population of living, range estimation larval feeding situation, larval feeding condition level is pressed appendix Table A-1, A-2 carries out.

Larval mortality is pressed formula (1) and is calculated:

$X=\frac{n}{A}\&times;100---\left(1\right)$

In formula:

X: larval mortality, unit is %;

N: dead borer population, unit is head;

A: connect borer population, unit is head.

Corrected mortality is pressed formula (2) and is calculated:

$\mathrm{Xt}=\frac{X1-X0}{1-X0}\&times;100---\left(2\right)$

In formula:

Xt: larva corrected mortality, unit is %;

X1: process lethality, unit is %;

X0: contrast lethality, unit is %.

4, interpretation of result

Judge the resistance to insects of transgenic cotton against pests.The criterion of transgenic cotton against pests resistance to insects rank is pressed appendix A and is carried out.

Appendix A

Table A-1 transgenic cotton against pests resistance to insects evaluation criteria

Resistance rank	Larva corrected mortality Y (%)	Blade mined grade
			High resistance (1 grade)	Y>80	1
Anti-(2 grades)	50<Y≤80	2
			In anti-(3 grades)	30<Y≤50	3
Low anti-(4 grades)	Y≤30	4
			Sense (5 grades)	Same check variety	5

Table A-2 cotton bollworm larvae takes food transgenic cotton against pests blade situation range estimation grade scale

Blade mined grade	The situation that takes food is described
		1	Be injured and be syringe needle shape not in flakes
2	Be injured and be small pieces, but be no more than 25% of leaf area
		3	Killed in blocks, surpass 25% of leaf area, but be no more than 50% of leaf area
4	Killed in blocks, surpass 50% of leaf area
		5	Blade is taken food in a large number, killed with conventional check variety

5, qualification result

Table 3 transgene cotton bollworm resisting qualification result

Embodiment 3

Within 2012, with the existing Bt of turning cotton exogenous protein expression amount examination criteria < < genetically modified plants and products thereof the pest-resistant transgenic Bt cotton exogenous protein expression of composition detection amount inspection specifications (No. 1485 bulletin-14-2010 of the Ministry of Agriculture) > >, A2, B4, C4 and tetra-cotton material exogenous protein expression amounts of D2 are detected, testing result is as follows:

1, detection method

This test operation all strictly carries out by detection kit specification, and detection kit is the Cry1Ac/Cry1Ab detection kit that U.S. Envirologix company produces.

2, testing result

Transgene cotton exogenous protein expression amount testing result

Claims (1)

1. transgenic Bt cotton bollworm resisting evaluation method, is characterized in that, said method comprising the steps of:

(1) in the second generation, the third generation and four generations of cotton bollworm emergence period, gather respectively second of top of cotton and launch blade completely, take non-transgenic cotton variety as control material;

(2) the fresh blade gathering is placed in experiment box;

(3) in putting vaned experiment box, connect 1 cotton bollworm newly hatched larvae, tighten lid, be placed under room temperature;

(4) connect worm " Invest, Then Investigate " cotton bollworm larvae death toll and the length of time, if examination worm is dead, be designated as 0 the length of time;

(5) carry out statistical analysis, calculate cotton bollworm inhibiting rate in the length of time, take two, three generations and the length of time four generations of cotton bollworm emergence period inhibiting rate be to judge resistance level index, wherein the length of time, inhibiting rate computing formula was as follows: