CN107290395A - A kind of cold-resistant evaluation of lily and screening technique and the application of cold-resistant variety - Google Patents

Abstract

The invention discloses a method and application of lily cold-resistant evaluation and cold-resistant variety screening, which belongs to the high-efficiency agricultural production technology. The method uses the first mature leaf of the second round below the growth point of the lily plant as a material, cuts the mature leaf into leaf blocks of a certain size, and pretreats the leaf blocks. Afterwards, the leaf blocks were processed using a cryogenic cycler, and then the relative electrical conductivity was measured. Finally, the semi-lethal temperature (LT ₅₀ ) of each lily variety was calculated by statistical software, and the cold tolerance of lily varieties was evaluated according to the semi-lethal temperature, and the cold-tolerant lily varieties were screened out. Compared with the traditional evaluation method of cold resistance using lily bulbs or tissue culture seedlings as materials, this method can better reflect the actual cold resistance of lily in the field, and is more representative than the traditional method.

Description

A kind of lily cold-resistant evaluation and screening method and application of cold-resistant varieties

technical field

The present invention mainly relates to the field of lily physiology, and in particular to a method for evaluating lily cold resistance and screening cold-resistant varieties and its application. The method mainly uses low-temperature treatment on the leaves in the vegetative growth period of lily, and obtains the relative electrical conductivity by measuring the electrical conductivity of the leaves. The Logistic regression equation was fitted to obtain the semi-lethal temperature (LT ₅₀ ) of each lily variety, evaluate the cold tolerance of lily varieties, and screen out cold-tolerant lily varieties.

technical background

Lily is a perennial flower bulb belonging to the family Liliaceae. Commonly said lily refers to the general term of various and varieties of Lilium (Lilium spp.) plants. Lily has huge flowers, bright colors, various poses, and pleasant fragrance. As an important ornamental plant, it can be used not only as cut flowers, potted flowers, but also in gardens and green spaces. At the same time, some lily varieties are edible and medicinal, and are widely popular. Consumers love it. At present, lily has become one of the top five fresh cut flowers in the world, and occupies a very important position in the world fresh cut flower market. In recent years, more and more new varieties of lily have been cultivated, and the area of lily planted in China is also expanding rapidly year by year. However, lily has strict temperature requirements during the growth process, and it will stop growing if it is higher than 30 degrees Celsius or lower than 5 degrees Celsius. In the central and northern regions of China, the temperature in winter and spring is low. Although most of the production has adopted solar greenhouses, due to the weak overall foundation, its light, temperature and controllability are poor. Therefore, the low temperature in winter and spring has become the preferred method for the cultivation of lilies in northern regions. Constraints. Therefore, it is particularly necessary to breed cold-resistant lily varieties for cultivation in production, and the prerequisite for breeding cold-resistant lily varieties needs to establish a technical system for evaluating the cold resistance of lily varieties. At present, in the selection of cold resistance of lily varieties, it is mainly through low-temperature treatment of lily bulbs and lily tissue culture seedlings, and by measuring the electrical conductivity, MDA, POD, and protective enzyme activities under low temperature stress to screen lily cold-resistant varieties. There are some disadvantages in these methods, such as bulb low-temperature treatment and screening in the screening process, the processing of each variety will consume a large amount of bulbs, and when there are many varieties, it is time-consuming and labor-intensive; High, and the growth cycle of tissue culture seedlings is long, and it is time-consuming and laborious to start from tissue culture; in addition, the cold resistance evaluation results of lily bulbs and tissue culture seedlings are often quite different from the cold resistance of lily plants in the field. Therefore, it is an urgent problem to establish a screening system for lily varieties that is simple and convenient and meets the actual production needs.

Contents of the invention

The purpose of the present invention is to provide a screening method for lily cold-resistant varieties that is simple and meets the needs of actual production. First, pick the first leaf of the second round below the growth point of the lily plant, clean it, cut it into pieces horizontally and put it in a low-temperature cycler for pretreatment, extract it every other day, and then measure the conductivity before and after boiling (i.e. freezing conductivity and boiling conductivity), calculate the relative conductivity, and calculate the semi-lethal temperature (LT ₅₀ ). According to the semi-lethal temperature, the cold resistance of different varieties of lily was sorted, and the varieties with strong resistance and weak resistance were screened out.

The purpose of the present invention is achieved by the following technical solutions

A screening method for lily cold-resistant varieties, comprising the following steps:

(1) Extract the first mature leaf of the second round below the growth point of the lily plant and clean it;

(2) cutting the washed mature leaves into leaf pieces, and pretreating the leaf pieces;

(3) Utilize low-temperature cycler to process the leaf block after pretreatment;

(4) Take out and thaw the sample after processing in the low-temperature cycler, and extract the thawed sample overnight;

(5) Measure the freezing conductivity of the extract, boil and cool to measure the conductivity again to obtain the boiling conductivity after measuring, according to the formula relative conductivity=frozen conductivity/boiling conductivity×100%, find the relative conductivity;

(6) be: y=K/(1+ae-bx) is analyzed relative conductivity with fitting Logistic regression equation, and wherein Y represents cell injury rate, and x represents processing temperature, and K is the saturation capacity of cell injury rate, a, b are the parameters of the equation; in order to determine the value of a, b, the equation is linearized, ln[(Ky)/y]=lna-bx, so that y=ln[(ky)/y] is transformed into The linear equation of the cell injury rate (Y) and the treatment temperature (x); obtain the a, b values and the correlation coefficient R ² by the linear regression method, and calculate the semi-lethal temperature LT ₅₀ of the lily variety =In[(1/a) ]/b;

(7) Evaluate the cold resistance of lily varieties according to the semi-lethal temperature, and screen out cold-resistant lily varieties.

The process of pretreatment in step (2) refers to wrapping the leaves evenly in one layer with wet gauze, putting them into glass test tubes, and placing them in a refrigerator at 4° C. overnight.

In the step (3), the process of using the low temperature cycler to process the leaves is as follows: when the temperature of the low temperature cycler drops to 4°C, the pretreated sample is put into the low temperature cycler for processing; the processing temperature of the low temperature cycler is 5°C. Each gradient is 0, -3, -6, -9, -12°C; each gradient is treated for 1h, and the cooling time between gradients is 30min.

In step (4), the samples processed in the low-temperature cycler were taken out and put into a refrigerator at 4° C. to thaw, and the thawed samples were taken out and leached overnight.

The time of boiling described in step (5) is 15min.

The lily plant described in the step (1) grows vigorously and has no infection by diseases and insect pests. The size of the first mature leaf in the second round of the same lily variety is the same.

The mature leaves described in step (2) are cut transversely with scissors into leaflets with a width of 1 cm.

The leaching described in step (4) is to add 30ml distilled water in each sample and carry out leaching.

The application of the above-mentioned method in the screening of lily cold-resistant varieties.

The detailed process of the screening method of lily cold-resistant variety of the present invention comprises the following steps:

(1) Pick the first leaf of the second round below the growth point of the lily plant, obtain 10 leaves of the same size for each variety, wash them three times with tap water and distilled water, and then dry the water on the leaves with absorbent paper.

(2) Cut the leaves in step (1) horizontally with scissors into leaflets with a width of 1 cm, and then every 2 leaves are divided into one part, wrapped evenly with wet gauze, and put into a glass test tube of 25 mm × 150 mm, Put it in the refrigerator at 4°C overnight and use it the next day.

(3) Take out the samples in step (2), arrange them in parallel on the test tube rack and tie them tightly with rubber bands, and then put them into the low temperature cycler for processing when the temperature of the low temperature cycler drops to 4°C. The treatment temperature was 5 gradients of 0°C, -3°C, -6°C, -9°C, -12°C. Each gradient was treated for 1 h, and the cooling time between gradients was 30 min. Then take out the sample and thaw it in a refrigerator at 4°C, and take it out for use the next day.

(4) Take out the thawed samples in step 3 and start leaching. The samples in each test tube are divided into three parts and placed in the test tubes respectively. Add 30ml of distilled water to each and leaching overnight.

(5) Measure the electrical conductivity of the sample after step 4). Boil for 15 minutes after the measurement, and measure the conductivity again after cooling down. Then find out its property to electric conductivity according to formula 1:

Relative conductivity (REC, %) = freezing conductivity/boiling conductivity × 100% (I)

(6) In cold resistance research, the Logistic regression equation for relative conductivity fitting is: y=K/(1+ae-bx). Among them, y represents the cell injury rate, x represents the treatment temperature, K is the saturation capacity of the cell injury rate, and a and b are the parameters of the equation. In order to determine the values of a and b, the equation is linearized, ln[(K—y)/y]=lna-bx, and y1=In[(K—y)/y] is converted into cell damage rate (y1) and the linear equation of the treatment temperature (x). The values of a, b and correlation coefficient R ² (fitting degree) were obtained by linear regression method, and the half-lethal temperature was obtained by equation II.

LT ₅₀ =In[(1/a)]/b(II)

(7) According to the semi-lethal temperature, the cold resistance of different varieties of lily was sorted, and the varieties with strong resistance and weak resistance were screened out.

Beneficial effects of the present invention:

The invention provides an effective and convenient method for the selection of cold-resistant varieties of lilies, and lays a certain foundation for the selection and research of cold-resistant varieties in the future. Simultaneously, it also has the following important features: 1) During the vegetative growth process of the lily, the lily leaves are treated at low temperature, which is closer to actual production. 2) The combination of multiple screening methods, progressive screening, is both scientific and convenient.

Description of drawings

Fig. 1 is the relative conductivity fitting curve of 'Trasso orange';

Fig. 2 is the relative conductivity fitting curve of 'wooden door';

Figure 3 is the fitting curve of the relative conductivity of 'Shadow'.

detailed description

The present invention will be further described in detail below in conjunction with specific examples.

The present invention utilizes low-temperature treatment of leaves to carry out experimental screening of 30 cut flower varieties (Table 1).

Table 1 The strains and bulb specifications of the experimental materials

The experimental materials were planted in the glass greenhouse of the chrysanthemum base of Nanjing Agricultural University, and the experiment began at the end of October 2016. The specific implementation examples are as follows:

1.1 Pick the first mature leaf of the second round below the growth point of the lily plant, take 10 leaves of the same size for each variety, wash them three times with tap water and distilled water, and then dry the water on the leaves with absorbent paper, then Use scissors to cut the leaves horizontally into small leaflets with a width of 1cm, and then divide each 2 leaves into 5 parts, wrap a layer evenly with wet gauze, put it into a 25mm×150mm glass test tube, and put it in a 4°C refrigerator Take out the samples overnight and the next day, arrange them in parallel on the test tube rack and tie them tightly with rubber bands, and then put them into the low temperature cycler for processing when the temperature of the low temperature cycler drops to 4°C. The instrument is Polyscience’s PP28 - R30 cryogenic cycler. The treatment temperature is 5 gradients of 0°C, -3°C, -6°C, -9°C, -12°C. Each gradient was treated for 1 h, and the cooling time between gradients was 30 min. After each sample was taken out, it was thawed in a refrigerator at 4°C, and it was taken out for use the next day. The sample after thawing was taken out and started leaching, and the samples in each test tube were divided into 3 parts (3 repetitions) and put into the test tubes respectively, and 30ml of distilled water was added to each of them, and leached overnight. Measure the conductivity of the sample after leaching, boil for 15 minutes after the measurement, and measure the conductivity again after cooling down. Relative conductivity (REC, %)=frozen conductivity/boiling conductivity×100%, so as to obtain the relative conductivity.

1.2 Data processing

The relative conductivity fitting Logistic regression equation is:

y=K/(1+ae-bx)

Semi-lethal temperature (LT ₅₀ )

LT ₅₀ =In[(1/a)]/b

y represents the cell damage rate, x represents the treatment temperature, K is the saturation capacity of the cell damage rate, and a and b are the parameters of the equation.

1.3 Analysis of results

1.3.1 Cell membrane and cold resistance

The results of the electrolyte leakage measurement show that the electrolyte leakage degree of the 30 materials increases with the decrease of the treatment temperature, so the relative conductivity also increases with the decrease of the temperature, and the relative conductivity curve is obviously S-shaped (Fig. 1-3 ), the temperatures corresponding to the five points in the figure are 0, -3, -6, -9, -12°C respectively. It can be clearly seen from the figure that the relative conductivity of 'Trasso Orange' and 'Shady Curtain' showed an obvious upward trend between 0°C and -9°C, and basically stopped rising between -9°C and -12°C and tended to a stable trend; the relative conductivity of 'Trasso Orange' rose sharply between -3°C and -7°C, and the relative conductivity of 'Shady Curtain' rose sharply between -4°C and -8°C, indicating that the 2 This species is the most harmful in this corresponding temperature. The S-type inflection points are their corresponding semi-lethal temperatures. The curve corresponding to the 'wooden door' is basically an upward trend between 0°C and -12°C, a slow rise between 0°C and -5°C, a rapid rise between -6°C and -10°C, and a rising trend between -6°C and -10°C. There is a slowing down trend after 10°C, and its S-shaped inflection point is between -6°C and -10°C.

1.3.2 Comparative analysis of semi-lethal temperature (LT ₅₀ )

The fitting regression equation, fitting degree and half-lethal temperature of 30 lily varieties under different low-temperature treatments according to the relative conductivity are shown in Table 3, arranged in ascending order of half-lethal temperature. The results of the cold tolerance test of the leaves of 30 lily varieties by electrolyte exudation method showed that the electrolyte exudation rate of the experimental materials increased with the decrease of temperature, and the fitting curves of the relative conductivity of all varieties were in an obvious S-type, and The fitting degree is good, which shows that the temperature gradient of the experimental design is reasonable. LT ₅₀ is used as an evaluation index of plant cold resistance, the lower the LT ₅₀ , the stronger the cold resistance, and vice versa, the weaker it is. It can be seen from Table 3 that the semi-lethal temperatures of the 30 experimental materials were between -5°C and -9°C, which indicated that the cold resistance of these lily varieties was relatively weak. Among them, the varieties with relatively weak resistance include 'Trasso Orange', 'Kass Taibai', 'Yellolin', and 'Little Robin', but there are also several varieties with relatively strong cold tolerance such as 'Wumen', 'Red Cardinal' (Table 2, Table 3).

Table 2 Logistic equation and R ² of 30 lily varieties

Table 3 LT ₅₀ of 30 lily varieties

From the above examples, it can be seen that after the leaves of 30 lily varieties are treated at low temperature, the electrical conductivity also increases with the temperature drop, and the relative electrical conductivity curve is obviously S-shaped, and the fitting degree is relatively high. The semi-lethal of each variety The temperature is easy to find out, and then judge its cold resistance. Compared with other screening methods, the method for screening lily cold-resistant varieties is simpler and easier to operate, especially when screening multiple varieties, it can better reflect the superiority of this method. At the same time, this method is more practical than other methods, and it is to screen during the growth period of lily plants, while other methods are either to screen lily bulbs or tissue culture seedlings, which is far from the actual cultivation performance. Therefore, this method can better reflect the actual cold resistance of lily in the field, and is more representative than the traditional method. The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. a screening method for lily cold-resistant varieties, is characterized in that: comprise the following steps: (1) Extract the first mature leaf of the second round below the growth point of the lily plant and clean it; (2) cutting the washed mature leaves into leaf pieces, and pretreating the leaf pieces; (3) Utilize low-temperature cycler to process the leaf block after pretreatment; (4) Take out and thaw the sample after processing in the low-temperature cycler, and extract the thawed sample overnight; (5) Measure the freezing conductivity of the extract, boil and cool to measure the conductivity again to obtain the boiling conductivity after measuring, according to the formula relative conductivity=frozen conductivity/boiling conductivity×100%, find the relative conductivity; (6) be: y=K/(1+ae-bx) is analyzed relative conductivity with fitting Logistic regression equation, and wherein Y represents cell injury rate, and x represents processing temperature, and K is the saturation capacity of cell injury rate, a, b are the parameters of the equation; in order to determine the value of a, b, the equation is linearized, ln[(Ky)/y]=lna-bx, so that y=ln[(ky)/y] is transformed into The linear equation of the cell injury rate (Y) and the treatment temperature (x); obtain the a, b values and the correlation coefficient R ² by the linear regression method, and calculate the semi-lethal temperature LT ₅₀ of the lily variety =In[(1/a) ]/b; (7) Evaluate the cold resistance of lily varieties according to the semi-lethal temperature, and screen out cold-resistant lily varieties. 2. method according to claim 1, it is characterized in that: the process of pretreatment described in the step (2) is described leaf piece is evenly wrapped 1 layer with wet gauze, puts into glass test tube, places 4°C refrigerator overnight. 3. The method according to claim 1, characterized in that: in the step (3), the process of utilizing the low-temperature cycler to process the leaf block is: when the temperature of the low-temperature cycler drops to 4°C, put the pretreated sample into Internal treatment in a low temperature cycler; the temperature of the low temperature cycler is 5 gradients of 0, -3, -6, -9, -12°C; each gradient is treated for 1 hour, and the cooling time between gradients is 30 minutes. 4. The method according to claim 1, characterized in that: in step (4), the sample after processing in the cryogenic cycler is taken out and put into a 4°C refrigerator for thawing, and the thawed sample is taken out and leached overnight. 5. The method according to claim 1, characterized in that: the boiling time in step (5) is 15min. 6. The method according to claim 1, characterized in that: the lily plant described in the step (1) grows vigorously and has no infection by diseases and insect pests. 7. The method according to claim 1, characterized in that: in the step (1), the size of the first mature leaf of the second round of the same lily variety is the same. 8. The method according to claim 1, characterized in that: the mature leaves described in step (2) are transversely cut into leaflets with a width of 1 cm with scissors. 9. The method according to claim 1, characterized in that: the leaching described in step (4) is adding 30ml of distilled water to each sample for leaching. 10. Application of the method according to any one of claims 1 to 9 in screening lily cold-resistant varieties.