CN101695278A - Method for quickly screening and testing cold resistance of tomatoes - Google Patents
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Abstract
一种快速筛选鉴定番茄耐寒性的方法，首先培养生长3～5周的番茄幼苗，取茎端叶片于3～5℃下处理1～3小时，再于-3～-5℃下处理7～9小时，然后于3～5℃下恢复10～14小时，再于20～22℃下光照14～18小时，最后测定冷处理恢复后的叶片电导率，将叶片投入蒸馏水中抽真空处理后定容、密封条件下于21～23℃下振荡1～3小时，测电导率C1，再密封、沸腾10～20分钟，冷却至室温测电导率C2，计算相对电导率C＝(C1-C0)/(C2-C0)×100％，当相对电导率C的值域≤42％时即为耐寒的番茄品种。本发明整个系统的耐寒性筛选都在可控条件，结果可靠，方法简便，易于掌握，大大缩短了育种周期，同时可用于引种耐寒性的鉴定。A method for quickly screening and identifying tomato cold resistance. First, cultivate tomato seedlings grown for 3 to 5 weeks, take stem leaves and treat them at 3 to 5°C for 1 to 3 hours, and then treat them at -3 to -5°C for 7 to 3 hours. 9 hours, then recover at 3-5°C for 10-14 hours, and then light at 20-22°C for 14-18 hours, and finally measure the electrical conductivity of the leaves after cold treatment, put the leaves into distilled water and vacuumize to constant volume , Shake at 21-23°C for 1-3 hours under sealed conditions, measure the conductivity C 1 , seal again, boil for 10-20 minutes, cool to room temperature and measure the conductivity C 2 , calculate the relative conductivity C=(C 1 - C 0 )/(C 2 -C 0 )×100%, when the value range of the relative conductivity C is ≤42%, it is a cold-resistant tomato variety. The cold resistance screening of the whole system of the invention is under controllable conditions, the result is reliable, the method is simple and easy to master, the breeding period is greatly shortened, and simultaneously it can be used for identification of cold resistance of introduced species.
Description
一、技术领域 1. Technical field
本发明涉及一种果蔬作物耐寒性品种的鉴别方法，确切地说是一个快速筛选鉴定番茄耐寒性的方法。The invention relates to a method for identifying cold-resistant varieties of fruit and vegetable crops, in particular to a method for quickly screening and identifying cold-resistant varieties of tomatoes.
二、背景技术 2. Background technology
低温冻害是农林业生产中一种严重的自然灾害，据统计，世界每年因此造成的损失高达2000亿美元。因此，耐寒作物品种的选育已成为农业生产中一个亟待解决的关键问题。Low temperature freezing damage is a serious natural disaster in agricultural and forestry production. According to statistics, the world's annual losses are as high as 200 billion U.S. dollars. Therefore, the selection and breeding of cold-resistant crop varieties has become a key problem to be solved urgently in agricultural production.
番茄是一种经济效益比较高的蔬菜作物。其果实营养丰富，含多种维生素，是现代人最佳营养食品之一，同时番茄也是保健食品、功能性食品和爱美女士们青睐的美容化妆品。番茄原产自南美洲，我国普遍栽培，一般冬春于保护地育苗，春季栽培为主，冬季温室栽培。虽然我国番茄已获得很多具有优良品质的品种，但耐受寒冷和冻害的品种却很少，加之我国番茄为常年栽培作蔬菜，近年来国内外专家都试图通过基因工程技术和细胞技术工程技术来培育番茄耐寒新品种。因此，寻找一种快速筛选鉴定耐寒番茄品种的方法显得尤为必要。Tomato is a vegetable crop with relatively high economic benefits. Its fruit is rich in nutrition and contains multiple vitamins. It is one of the best nutritional foods for modern people. At the same time, tomato is also a health food, functional food and beauty cosmetics favored by ladies who love beauty. Tomatoes are originally produced in South America and are widely cultivated in my country. They are generally grown in protected areas in winter and spring, mainly in spring and in greenhouses in winter. Although my country's tomato has obtained many varieties with good quality, there are few varieties that are tolerant to cold and freezing damage. In addition, my country's tomato is cultivated as a vegetable all year round. In recent years, experts at home and abroad have tried to use genetic engineering technology and cell technology engineering technology. Cultivate new cold-resistant varieties of tomato. Therefore, it is particularly necessary to find a method for rapid screening and identification of cold-resistant tomato varieties.
传统依靠天然寒冷气候途径筛选鉴定优良耐寒性状的番茄种质资源的方法存在量大，周期长，性状不稳定等缺点。因此，本发明提供一种快速筛选鉴定耐寒番茄品种(系)的方法，可大大缩短耐寒番茄品种的选育周期。The traditional method of screening and identifying tomato germplasm resources with excellent cold-resistant traits by means of natural cold climates has shortcomings such as large quantity, long cycle, and unstable traits. Therefore, the present invention provides a method for quickly screening and identifying cold-resistant tomato varieties (lines), which can greatly shorten the breeding cycle of cold-resistant tomato varieties.
三、发明内容 3. Contents of the invention
本发明针对现有番茄耐寒品种筛选鉴定方法的缺陷，旨在提供一种快速简单有效的筛选鉴定方法以加快番茄耐寒育种的过程，所要解决的技术问题是筛选鉴定方法的可靠性，同时避免优良种质资源的丢失。The present invention aims at the defects of existing screening and identification methods for tomato cold-resistant varieties, and aims to provide a fast, simple and effective screening and identification method to speed up the process of tomato cold-resistant breeding. The technical problem to be solved is the reliability of the screening and identification method, while avoiding the Loss of germplasm resources.
本发明的思路是在特定条件下确立番茄耐寒植株中的组织器官与某种物理量之间的相关性，从而将番茄品种耐寒性的筛选鉴定工作转化为该物理量的检测。通过实验研究，本发明利用在一定的冷胁迫条件下的冷处理，根据番茄离体叶片相对电导率的数值来反映植株耐寒程度，对植株的耐寒能力的强弱能作出快速有效的评价，加快番茄耐寒育种的进程。The idea of the present invention is to establish the correlation between tissues and organs in cold-resistant tomato plants and a certain physical quantity under specific conditions, thereby transforming the screening and identification of cold-resistant tomato varieties into the detection of the physical quantity. Through experimental research, the present invention uses cold treatment under certain cold stress conditions to reflect the degree of cold resistance of plants according to the value of the relative electrical conductivity of tomato detached leaves, and can quickly and effectively evaluate the strength of cold resistance of plants, so as to speed up the production of tomato plants. The process of breeding for cold tolerance.
为确立番茄的品种耐寒性与相对电导率大小之间的相关性，首先在现有番茄品种中分别对耐寒的、中等耐寒的和不耐寒的三种品种离体叶片进行相对电导率分析测定，配合叶片的恢复性试验，观察其外观形态变化特征，对本方法进行优化，通过优化确定叶片的冷处理条件、相对电导率检测条件，同时获得番茄品种耐寒性与相对电导率数值之间的对应关系，然后据此对耐寒性未知的番茄品种进行实验鉴别验证，最后确立本发明的技术方案。In order to establish the correlation between the cold resistance of tomato varieties and the relative electrical conductivity, the relative electrical conductivity of the detached leaves of the three varieties of cold-resistant, moderately cold-resistant and non-cold-resistant tomato varieties were firstly analyzed and measured. Cooperate with the recovery test of the leaves, observe the characteristics of the appearance and shape changes, optimize this method, determine the cold treatment conditions of the leaves and the relative conductivity detection conditions through optimization, and obtain the corresponding relationship between the cold resistance of tomato varieties and the relative conductivity value, Then carry out experimental identification and verification to the unknown tomato varieties of cold resistance according to this, and finally establish the technical scheme of the present invention.
现有番茄品种中耐寒的、中等耐寒的和不耐寒的是指通过传统自然寒冷气候途径，根据其存活率以及产量大小的不同筛选出不同耐寒性状的番茄种质。The cold-resistant, moderately cold-resistant and non-cold-resistant tomato varieties in the existing tomato varieties refer to tomato germplasm with different cold-resistant traits selected according to their survival rate and yield through the traditional natural cold climate approach.
本发明的技术方案包括番茄幼苗的培养、离体叶片的冷处理和恢复、电导率的测定和相对电导率的计算，所述的冷处理和恢复就是取生长3～5周番茄幼苗茎端叶片于3～5℃条件下处理1～3小时，再于-3～-5℃条件下处理7～9小时，然后于3～5℃条件下恢复10～14h，最后在20～22℃下光照14～18小时；所述的电导率的测定就是将冷处理后恢复的叶片在蒸馏水中抽真空15～25分钟，然后用蒸馏水定容、封口，于21～23℃下、振荡1～3小时，测电导率C1，再封口、沸腾10～20min，冷却至室温测电导率C2；相对电导率C＝(C1-C0)/(C2-C0)×100％，式中C0为蒸馏水电导率。当相对电导率C的值域≤42％时则为耐寒品种番茄。The technical scheme of the present invention comprises the cultivation of tomato seedlings, the cold treatment and recovery of detached leaves, the measurement of electrical conductivity and the calculation of relative electrical conductivity. Treat at ~5°C for 1~3 hours, then treat at -3~-5°C for 7~9 hours, then recover at 3~5°C for 10~14 hours, and finally light at 20~22°C for 14~ 18 hours; the measurement of the electrical conductivity is to evacuate the recovered leaves after cold treatment for 15 to 25 minutes in distilled water, then use distilled water to fix the volume, seal, and shake at 21 to 23 ° C for 1 to 3 hours to measure the electrical conductivity Conductivity C 1 , seal again, boil for 10-20 minutes, cool to room temperature and measure conductivity C 2 ; relative conductivity C=(C 1 -C 0 )/(C 2 -C 0 )×100%, where C 0 is Conductivity of distilled water. When the value range of the relative conductivity C is ≤42%, it is a cold-resistant variety tomato.
与传统筛选技术相比，本发明方法简便，易于掌握，大大缩短了育种周期。整个系统的耐寒性筛选鉴定都在可控条件下进行，使得筛选结果更加可靠，利用离体叶片的相对电导率可反映低温对细胞膜的破坏程度，同时结合冷害后的恢复生长实验，既鉴定了细胞对低温伤害的直接耐受能力，又鉴定了材料在解除低温后的生长恢复能力。此外，本发明也可用于引种耐寒性鉴定，可加快新品种鉴定速度，尽早投入生产，可提高番茄耐寒突变体筛选的有效性，避免在生产过程中可能发生的优良品种资源的丢失。Compared with the traditional screening technology, the method of the invention is simple and easy to master, and greatly shortens the breeding cycle. The cold resistance screening and identification of the whole system is carried out under controllable conditions, which makes the screening results more reliable. The relative conductivity of the isolated leaves can reflect the degree of damage to the cell membrane by low temperature. The ability of cells to directly tolerate low temperature damage also identifies the growth and recovery ability of materials after the low temperature is lifted. In addition, the present invention can also be used for the identification of cold resistance of introduced varieties, which can speed up the identification of new varieties, put them into production as soon as possible, improve the effectiveness of screening tomato cold-resistant mutants, and avoid the loss of excellent variety resources that may occur in the production process.
四、附图说明 4. Description of drawings
图1是A、B、C三种发育生长四周后的幼苗表型。Figure 1 shows the phenotypes of three seedlings of A, B, and C after four weeks of development and growth.
图2是A、B、C三种离体叶片冷处理恢复后的叶片表型变化。A品种叶片卷缩程度最小，呈绿色，C品种叶片卷缩程度最大，呈深黑色，B品种介于两者之间。Fig. 2 is the change of leaf phenotype of three kinds of detached leaves of A, B, and C after cold treatment recovery. The leaves of variety A are the least curled and green, the leaves of C variety are the most curled and dark black, and the leaves of B variety are in between.
图3是A、B、C三种相对电导率数据图解。Figure 3 is an illustration of the relative conductivity data of A, B, and C.
图4是验证试验中1#、2#、3#三种幼苗冷处理后恢复生长状况图。Fig. 4 is a diagram of recovery growth status of three kinds of
五、具体实施方式 5. Specific implementation
实验用番茄品种由安徽省农业科学院园艺研究所提供。其中(1)耐寒番茄品种(系)，命名：A1，A2，A3；(2)中等耐寒番茄品种，命名：B1，B2，B3；(3)不耐寒番茄品种，命名：C1，C2，C3。每组三个平行试验。The tomato varieties used in the experiment were provided by the Institute of Horticulture, Anhui Academy of Agricultural Sciences. Among them (1) cold-resistant tomato varieties (lines), named: A 1 , A 2 , A 3 ; (2) medium cold-resistant tomato varieties, named: B 1 , B 2 , B 3 ; (3) non-cold-resistant tomato varieties, named : C 1 , C 2 , C 3 . Each group has three parallel experiments.
1、番茄幼苗的培养：1. Cultivation of tomato seedlings:
将A1、A2、A3、B1、B2、B3和C1、C2、C3番茄种子，播种到土培花盆中，土壤配置为蛭石∶黑土∶珍珠岩＝9∶3∶0.5，使用前黑土需灭菌20min。首次播种浇足植物土壤培养基营养液，以后每周浇水一次。在21℃，16h光照条件下培养4周后，其生长状况见图1。A 1 , A 2 , A 3 , B 1 , B 2 , B 3 and C 1 , C 2 , C 3 tomato seeds were sown into soil culture flowerpots, and the soil configuration was vermiculite: black soil: perlite=9 : 3: 0.5, the black soil needs to be sterilized for 20 minutes before use. Water the nutrient solution of the plant soil medium for the first time sowing, and water once a week thereafter. After culturing for 4 weeks at 21°C under 16h light conditions, the growth status is shown in Figure 1.
2、番茄离体叶片的冷处理：2. Cold treatment of tomato detached leaves:
将培养生长四周的番茄幼苗取出，剪取其茎端叶片，尽量保持所剪取的叶片大小相一致，以A1，B1，C1为一组，放入平皿中，计三组，每组三个平行。将平皿放入4℃冰箱中2h，再放入-4℃冷冻室处理8h，取出后放入4℃冰箱中恢复12h，最后将平皿放置在16h光照培养室(室温21℃左右)中，24h后其叶片的形态变化见图2。Take out the tomato seedlings that have been cultivated and grown for four weeks, cut off the leaves at the stem ends, and try to keep the cut leaves in the same size, put A 1 , B 1 , and C 1 into a group, and put them into a plate, counting three groups, each Group three in parallel. Put the plate in a 4°C refrigerator for 2 hours, then put it in a -4°C freezer for 8 hours, take it out and put it in a 4°C refrigerator for 12 hours, and finally place the plate in a 16-hour light culture room (at room temperature around 21°C) for 24 hours The morphological changes of its leaves are shown in Figure 2.
3、番茄冷处理后离体叶片电导率的测定：3. Determination of electrical conductivity of isolated tomato leaves after cold treatment:
电导率测定方法：将冷处理恢复后的材料从平皿中取出，每种材料3个平行。①用50ml注射器加蒸馏水10ml抽真空20min；②再转入50ml三角瓶中，加蒸馏水40ml(定容50ml)，铝膜封口，放入恒温气/水浴摇床22℃，180r.min-1旋转，定时2h；③取出，测电导率C1；④将所测溶液铝膜封口，煮沸15min，冷却至室温(定容50ml)，测C2；⑤计算相对电导率C＝(C1-C0)/(C2-C0)×100％，其中C0为蒸馏水电导率。三组相对电导率大小范围见表1，数据分析见图3。Conductivity measurement method: take out the recovered material from the cold treatment, and use 3 parallels for each material. ①Use a 50ml syringe and add 10ml of distilled water to vacuumize for 20min; ②Put it into a 50ml Erlenmeyer flask, add 40ml of distilled water (fixed volume 50ml), seal with aluminum film, put it in a constant temperature air/water bath shaker at 22°C, 180r.min -1 rotation , timed 2h; ③Take it out, and measure the conductivity C 1 ; ④Seal the aluminum film of the measured solution, boil for 15min, cool to room temperature (constant volume 50ml), and measure C 2 ; ⑤Calculate the relative conductivity C=(C 1 -C 0 )/(C 2 -C 0 )×100%, where C 0 is the conductivity of distilled water. The relative conductivity ranges of the three groups are shown in Table 1, and the data analysis is shown in Figure 3.
表1Table 1
由此可知，耐寒品种的离体叶片的相对电导率要小于不耐寒品种，中等品种的相对电导率介于耐寒与不耐寒之间。因此，将番茄整株耐寒程度与离体叶片冷处理过后的相对电导率大小建立相关性关系为：相对电导率在42.62％以下的为耐寒品种，42.62％-62.67％之间的为中等耐寒品种，大于62.67％得为不耐寒品种。It can be seen that the relative conductivity of the detached leaves of cold-resistant varieties is lower than that of non-cold-resistant varieties, and the relative conductivity of medium varieties is between cold-resistant and non-cold-resistant varieties. Therefore, the relationship between the cold tolerance of the whole tomato plant and the relative conductivity of the detached leaves after cold treatment is established as follows: those with relative conductivity below 42.62% are cold-resistant varieties, those between 42.62% and 62.67% are medium cold-resistant varieties, More than 62.67% are non-cold resistant varieties.
统计分析得到：-4℃冷处理生长四周的番茄离体叶片后，耐寒品种90％以上的相对电导率在42.62％以下，不耐寒品种90％以上的电导率在62.67％以上：中等耐寒品种90％以上的电导率在52.05％附近，为47.62％-62.67％之间(表1、图3)。Statistical analysis shows that after cold treatment of tomato leaves grown for four weeks at -4°C, the relative conductivity of more than 90% of the cold-resistant varieties is below 42.62%, and the conductivity of more than 90% of the non-cold-resistant varieties is above 62.67%; 90% of the medium cold-resistant varieties The above electrical conductivity is around 52.05%, between 47.62% and 62.67% (Table 1, Figure 3).
4、验证试验4. Verification test
取耐寒性状未知的3个番茄品种，(由安徽省农科院园艺所提供，耐寒性状对申请人来说是未知的)命名为1#，2#，3#；剪取大小相同的茎尖叶片(三个平行)放入平皿中，4℃冰箱中冷处理2h，再放入-4℃冷冻室处理8h，取出后放入4℃冰箱中恢复12h，最后将平皿放置在16h光照培养室(室温21℃左右)中，24h后测其电导率值(表2)。Get 3 tomato varieties with unknown cold-resistant traits, (provided by the Horticultural Institute of Anhui Academy of Agricultural Sciences, the cold-resistant traits are unknown to the applicant) named 1 # , 2 # , 3 # ; cut the same size stem tip Leaves (three in parallel) were placed in a plate, treated in a 4°C refrigerator for 2 hours, then placed in a -4°C freezer for 8 hours, taken out and placed in a 4°C refrigerator for 12 hours, and finally placed in a 16-hour light culture room ( room temperature (about 21°C), the conductivity value was measured after 24 hours (Table 2).
表2Table 2
将验证所得的1#耐寒、2#中等耐寒、3#不耐寒三个品种培养四周的幼苗植株-6℃冷处理8h，4℃恢复12h后，16h光照培养室(室温21℃左右)恢复培养一周，恢复生长状况见图4。图中可见，1#耐寒程度最好，2#次之，3#最差。The seedling plants of the three varieties of 1 # cold resistance, 2 # medium cold resistance and 3 # non-cold resistance obtained from the verification were cultured for four weeks at -6°C for 8 hours, and after 12 hours at 4°C, they were resumed in a 16h light culture room (at room temperature around 21°C) for one week , See Figure 4 for the restored growth status. It can be seen from the figure that 1 # has the best cold resistance, 2 # is the second, and 3 # is the worst.
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| CN102415253A (en) * | 2011-08-25 | 2012-04-18 | 刘慧民 | Method for screening cold-resistant variety of steeplebush |
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| BG51851A1 (en) * | 1993-03-08 | 1993-10-15 | Bg Inst Genetika Doncho Kostov | Method for creating genotype plants tolerant to low temperatures |
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| CN111587745A (en) * | 2020-06-05 | 2020-08-28 | 浙江农林大学 | Cherry tomato variety screening method suitable for overwintering seedling raising |
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