WO2022188557A1 - 一种枇杷果实木质化程度的细胞快速统计评价方法 - Google Patents

一种枇杷果实木质化程度的细胞快速统计评价方法 Download PDF

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WO2022188557A1
WO2022188557A1 PCT/CN2022/072666 CN2022072666W WO2022188557A1 WO 2022188557 A1 WO2022188557 A1 WO 2022188557A1 CN 2022072666 W CN2022072666 W CN 2022072666W WO 2022188557 A1 WO2022188557 A1 WO 2022188557A1
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lignified
lignification
lig
cell
fruit
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吴迪
黄伟男
陈昆松
石艳娜
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浙江大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/025—Fruits or vegetables
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G01N2021/8466—Investigation of vegetal material, e.g. leaves, plants, fruits

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  • the invention belongs to a detection method for agricultural products, relates to a research method for quality evaluation of loquat fruit after harvest, and in particular relates to a rapid statistical evaluation method for cells of loquat fruit lignification degree.
  • Loquat is native to my country and belongs to the Rosaceae, loquat genus. Loquat pulp is soft and juicy, delicious in flavor, rich in nutritional value and rich in flavor, so it is widely loved by consumers. Loquat matures in the high temperature and rainy season, the field heat of the fruit is too large, the postharvest respiration is obvious, the fruit consumption is obvious, it is easy to produce corruption, and the process of fruit senescence and lignification is accelerated. Low temperature treatment is widely used in the storage and transportation of fruits to reduce postharvest consumption and maintain fruit quality.
  • loquat fruit is a cold-sensitive fruit, which is prone to symptoms of cold damage in a low temperature environment, which is mainly manifested as increased fruit hardness, rough taste, difficult peeling of the peel, reduced juice, and increased lignin content in the pulp, which greatly reduces its fruit flavor. cause economic losses. Therefore, it is of practical significance to study the lignification process of loquat fruit for the preservation of loquat fruit during postharvest storage and transportation, the extension of shelf life and the promotion of commodity value.
  • the evaluation of the degree of loquat lignification is mainly by measuring the hardness of the fruit or the lignin content in the pulp.
  • the hardness of the fruit is mainly used to simulate the chewing behavior of people using a texture analyzer, and a probe is used to perform a puncture experiment on the fruit, and a specific peak is extracted from the mechanical curve received by the probe to evaluate the softness and hardness of the fruit.
  • the increase in firmness is one of the symptoms of loquat fruit lignification, and the firmness of the fruit is also affected by many factors.
  • the turgor pressure of the internal cells of the fruit, the different components and structures of the cell walls, and the adhesion between the cell walls are all factors that affect the firmness of the fruit.
  • the hardness index to evaluate the degree of lignification of fruit is not comprehensive and accurate.
  • the lignin content of fruit is generally measured by chemical extraction. First, based on the pulp homogenate, the total amount of lignin in the pulp homogenate sample is obtained through a series of separation, filtration, and purification steps. Compared with the fruit hardness, the lignin content based on chemical extraction method can more intuitively reflect the lignification status of loquat fruit.
  • the chemical extraction method is time-consuming and labor-intensive, and it takes several days to completely obtain the separated and purified lignin components; on the other hand, the sampling of the chemical extraction method is based on the fruit homogenate, which can only reflect the overall average value, while the lignin in the The spatial distribution information in the fruit is hidden.
  • the purpose of the present invention is to solve the problems that the traditional use of texture analyzer or chemical extraction method to evaluate the lignification of loquat has insufficient consideration of the influencing factors and the evaluation standard is not comprehensive and accurate enough, and provides a rapid statistical evaluation method of cells for the lignification degree of loquat fruit.
  • weighted statistics can be carried out according to different lignification conditions and locations, and a more scientific evaluation of the lignification degree of loquat fruit can be carried out.
  • the technical scheme adopted by the present invention to solve the technical problem is: a method for rapid statistical evaluation of cells of loquat fruit lignification degree, which is characterized in that: comprising the following steps:
  • Step a the loquat fruit is cut along the equatorial plane of the fruit, remove half of the fruit, and remove the core, calculate the pulp area S of the entire equatorial plane of the fruit;
  • Step b the equatorial pulp is continuously frozen sectioned, and all sections are dyed with phloroglucinol method
  • Step c Use an optical microscope to obtain a picture of the staining results of the slices, define the degree of lignification according to the different shapes of the lignified cells, and divide the lignified cells into three categories: primary lignified cells P, intermediate lignified cells R and final lignified cells cell U;
  • Step d dividing the equatorial pulp into three annular areas, from outside to inside are respectively the peel area x, the middle area y and the core area z, and the widths of the three annular areas are equal in each radial direction;
  • step e count the total stained areas of the three types of lignified cells in the x, y and z regions of the pulp, respectively, and record them as:
  • Step f Count the staining areas of n lignified cells in the x, y and z regions of the pulp respectively, 2 ⁇ n ⁇ N, and the staining areas of the lignified cells with different n values are counted separately, and recorded as:
  • Step g count the total area of the vascular bundle staining area in the three areas of pulp x, y and z, which are respectively recorded as: And count the number of vascular bundles in each region, denoted as i x , i y and iz ;
  • Step h get a batch of loquat fruits that are just ripe and picked without storage of the same variety, obtain the correction coefficient of the vascular bundle dyeing area in the three regions of pulp x, y and z: the loquat fruit is cut out of the equatorial plane of the pulp according to step a, Perform frozen sectioning and staining according to step b, count the total area of vascular bundle staining in the three areas of pulp x, y and z according to step g, and calculate the staining of each vascular bundle in the three areas of pulp x, y and z respectively.
  • the average value of the area, as the vascular bundle correction constant, is recorded as:
  • Step i Calculate the lignification index Lig x , Lig y and Lig z of each area and the lignification index Lig of the whole fruit:
  • Lig a*(2Lig x +Lig y +0.5Lig z )
  • a is a normalization coefficient that can be set artificially, so that the Lig value is close to 1, and the number of decimal points is reduced to facilitate statistics;
  • the lignification index Lig is used as the lignification evaluation index of the loquat fruit.
  • the loquat pulp is divided into three regions from the inside to the outside, and the more the outer cells undergo lignification, the greater the weight in the lignification evaluation.
  • Loquat lignified cells are classified according to the degree of development of lignification, and are divided into primary lignified cells P, intermediate lignified cells R and final lignified cells U, and their areas are counted respectively. the greater the weight.
  • the aggregation phenomenon of loquat lignified cells was counted, and the more serious lignified cell agglomeration was weighted in the lignification evaluation of the cell group.
  • the area of vascular bundles in loquat pulp was counted and subtracted from the area of vascular bundles in the first-picked fruit to obtain the increase in the area of vascular bundles during storage, which was used in the evaluation of lignification.
  • this method fully considers the spatial distribution factors of lignified cells, the lignification degree factors of lignified cells, the aggregation factors of lignified cells, and the factors of thickening of vascular bundles, and the evaluation system is more reasonable. Weighted calculations were carried out on the causes of fruit lignification and the influence of various factors, which laid a foundation for the subsequent sub-item research on fruit lignification.
  • the cell wall is thickened but the thickened part is not annular, and the cell wall is thickened to an annular shape but the cell wall thickness accounts for less than 1/4 of the cell radius, then it is recorded as the primary lignified cell P; If the cell wall thickens to an annular shape and the cell wall thickness is 1/4 to 3/4 of the cell radius, it is recorded as the intermediate lignified cell R; if the cell wall thickens to the annular shape and the cell wall thickness exceeds 3/4 of the cell radius, it is recorded as the final lignified cell Cell U.
  • the lignification of loquat pulp cells is a gradual process of gradual thickening of the cell wall.
  • Primary lignified cells P, intermediate lignified cells R, and final lignified cells U correspond to the initial, middle, and end stages of cell lignification, respectively.
  • the thickness of each continuous frozen section is 40 ⁇ m.
  • the present invention When evaluating the lignification degree of loquat fruit, the present invention considers both the spatial distribution factors of lignified cells in the pulp, the factors of the development degree of lignified cells, and the factors of lignified cells based on microscopic imaging at the cell level. According to the agglomeration factors, a rapid statistical evaluation method of cells with multiple factors and different weights was established. The evaluation of this method is more comprehensive and accurate, and the lignification evaluation results of loquat have a better correlation with the actual quality.
  • Fig. 1 is a micrograph of primary lignified cells P of the present invention.
  • Fig. 2 is a schematic diagram of primary lignified cells P of the present invention.
  • Figure 3 is an R microscope image of the intermediate lignified cells of the present invention.
  • Figure 4 is a schematic diagram of the intermediate lignified cells R of the present invention.
  • Figure 5 is a U-micrograph of the final lignified cells of the present invention.
  • Figure 6 is a schematic diagram of the final lignified cell U of the present invention.
  • Fig. 7 is a microscopic view of the lignified cells in the aggregate composed of two lignified cells of the present invention.
  • Fig. 8 is a schematic diagram of a group of lignified cells composed of two lignified cells of the present invention.
  • Fig. 9 is a micrograph of the lignified cells in the aggregate composed of three lignified cells of the present invention.
  • FIG. 10 is a microscope view of the vascular bundle W of the present invention.
  • Embodiment a kind of cell rapid statistical evaluation method of loquat fruit lignification degree, comprises the following steps:
  • Step a the loquat fruit is cut along the equatorial plane of the fruit, remove half of the fruit and remove the core, calculate the pulp area S of the whole equatorial plane of the fruit;
  • Step b Continuously freeze the equatorial pulp into sections, and perform the phloroglucinol dyeing treatment on all the sections; the thickness of each continuous frozen section is 40 ⁇ m;
  • Step c Use an optical microscope to obtain a picture of the staining results of the slices, define the degree of lignification according to the different shapes of the lignified cells, and divide the lignified cells into three categories: primary lignified cells P, intermediate lignified cells R and final lignified cells Cell U; for loquat pulp cells: the cell wall is thickened but the thickened part is not ring-shaped, and the cell wall is thickened to a ring shape but the cell wall thickness is less than 1/4 of the cell radius, it is recorded as primary lignified cell P, as shown in Figure 1, As shown in Figure 2, the lignified cells P whose cell wall is thickened to a ring shape but whose cell wall thickness accounts for less than 1/4 of the cell radius are also shown in Figure 2 to distinguish it from the intermediate lignified cells R; the cell wall is thickened to a ring shape and the cell wall is If the thickness is 1/4 to 3/4 of the cell radius, it is recorded as intermediate lignified cell R, as shown in Figures
  • Step d dividing the equatorial pulp into three annular areas, from outside to inside are respectively the peel area x, the middle area y and the core area z, and the widths of the three annular areas are equal in each radial direction;
  • step e count the total stained areas of the three types of lignified cells in the x, y and z regions of the pulp, respectively, and record them as:
  • Step f Count the staining areas of n lignified cells in the x, y and z regions of the pulp respectively, 2 ⁇ n ⁇ N, and the staining areas of the lignified cells with different n values are counted separately, and recorded as:
  • Step g count the total area of the vascular bundle staining area in the three areas of pulp x, y and z, and the microscopic structure of the vascular bundle is shown in Figure 10, which are respectively recorded as: And count the number of vascular bundles in each area, denoted as i x , i y and iz , respectively; among them, each bundle of vascular bundles aggregated together is denoted as one;
  • Step h get a batch of loquat fruits that are just ripe and picked without storage of the same variety, obtain the correction coefficient of the vascular bundle dyeing area in the three regions of pulp x, y and z: the loquat fruit is cut out of the equatorial plane of the pulp according to step a, Perform frozen sectioning and staining according to step b, count the total area of vascular bundle staining in the three areas of pulp x, y and z according to step g, and calculate the staining of each vascular bundle in the three areas of pulp x, y and z respectively.
  • the average value of the area, as the vascular bundle correction constant, is recorded as:
  • Step i Calculate the lignification index Lig x , Lig y and Lig z of each area and the lignification index Lig of the whole fruit:
  • Lig a*(2Lig x +Lig y +0.5Lig z )
  • a is a normalization coefficient that can be set artificially, so that the Lig value is close to 1, and the number of decimal points is reduced to facilitate statistics;
  • Step j take the lignification index Lig as the loquat fruit lignification evaluation index, the larger the Lig value, the more serious the loquat fruit lignification degree.
  • the quality classification of loquat based on the lignified Lig value can guide the storage strategy and sales strategy of loquat.
  • the lignification evaluation system proposed in this scheme can be used as the effect evaluation index of various post-harvest loquat treatments to guide subsequent experimental research.
  • loquat samples Two types were taken, namely "Luoyang Qing" loquat freshly picked and “Luoyang Qing” loquat stored at 0°C for 6 days.
  • Lig x 0.0000595371433787208
  • Lig 0.482368 ⁇ 0.5, no lignification.
  • Lig 1.032566>1, it has been seriously lignified.

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Abstract

一种枇杷果实木质化程度的细胞快速统计评价方法，传统的采用质构仪或化学提取法评价枇杷木质化程度，存在影响因素考虑不全面，评价标准不够全面和不够精确的问题。方法将枇杷果实沿赤道面剖开并去核，并将赤道面果肉冷冻切片并染色，将木质化细胞分为三类：初级木质化细胞P、中级木质化细胞R和终级木质化细胞U，将果肉的赤道面从外到内分为三个环形区域，分别统计三个区域中三种木质化细胞的总面积，再统计不同细胞数的成团木质化细胞面积以及维管束面积，对各项统计结果进行不同权重的加权统计，评价枇杷果实木质化程度。细胞快速统计评价方法考虑了多因素的不同权重，评价全面准确。

Description

一种枇杷果实木质化程度的细胞快速统计评价方法 技术领域
本发明属于农产品检测方法，涉及一种枇杷采后果实品质评价的研究方法，特别涉及一种枇杷果实木质化程度的细胞快速统计评价方法。
背景技术
枇杷原产于我国，是蔷薇科、枇杷属植物。枇杷果肉柔软多汁，风味鲜美，肉营养价值丰富，风味浓郁，因此广受消费者喜爱。枇杷成熟于高温多雨季节，果实的田间热过大，采后呼吸作用明显，对果实消耗明显，极易产生腐败，加速果实衰老木质化进程。低温处理广泛被应用到果实的储运过程中，以减小果实采后消耗，保持果实品质。但是，枇杷果实属于冷敏性水果,在低温环境下容易出现冷害症状,其主要表现为果实硬度上升，口感粗糙，果皮难剥，果汁减少,果肉中木质素含量上升，大大削减其果实风味，造成经济损失。因此，研究枇杷果实木质化过程对枇杷果实的采后果实贮运期间的保鲜,货架期的延长、商品价值的提升具有现实意义。
目前，评价枇杷木质化程度主要是通过测量果实的硬度或者果肉中的木质素含量。果实的硬度，主要是使用质构仪模拟人的咀嚼行为，使用探头对果实进行穿刺实验，在探头所受的力学曲线中提取特定的峰值，用来评价果实的软硬程度。硬度的升高是枇杷果实木质化的症状的表现之一，果实的硬度还受到多种因素的影响。果实内部细胞的膨压，细胞壁的不同的成分和结构，细胞壁之间的粘连性都是果实硬度的影响因素。因此，硬度指标评估果实的木质化程度存在不够全面和精确的问题。果实木质素含量一般通过化学提取法测量，首先基于果肉匀浆，通过一系列分离、过滤、提纯步骤获得果肉匀浆样本中木质素的总量。与果实的硬度相比，基于化学提取法的木质素含量更能直观的反映出枇杷果实的木质化状况。但是化学提取法费时费力，需要几天的时间才能完全得到分离纯化的木质素成分；另一方面，化学提取法的取样基于果实匀浆，仅仅能够反应出整体的平均值，而木质素的在果实中的空间分布信息则被隐藏。
发明内容
本发明的目的在于解决传统的采用质构仪或化学提取法评价枇杷木质化存在影响因素考虑不全面，评价标准不够全面和精确的问题，提供一种枇杷果实木质化程度的细胞快速统计评价方法，基于显微成像对木质化细胞的形态、空间分布进行识别，可以根据不同的木质化情况和位置进行加权统计，对枇杷果实木质化程度进行更科学的评价。
本发明解决其技术问题所采用的技术方案是：一种枇杷果实木质化程度的细胞快速统计 评价方法，其特征在于：包括以下步骤：
步骤a、将枇杷果实沿着果实的赤道面剖开，去除一半果实、并去除果核，计算果实整个赤道面的果肉面积S；
步骤b、将赤道面果肉连续冷冻切片，将全部的切片进行间苯三酚方法染色处理；
步骤c、使用光学显微镜获取切片染色结果图，根据木质化细胞形态的不同，定义木质化程度，将木质化细胞分为三类：初级木质化细胞P、中级木质化细胞R和终级木质化细胞U；
步骤d、将赤道面果肉分为三个环形区域，从外到内分别为靠果皮区域x、中间区域y和靠果核区域z，三个环形区域在每个半径方向的宽度均相等；
步骤e、分别统计果肉的x、y和z区域中，三种类型的木质化细胞的染色总面积，分别记作：
Figure PCTCN2022072666-appb-000001
矩阵中以
Figure PCTCN2022072666-appb-000002
为例，
Figure PCTCN2022072666-appb-000003
表示x区域中初级木质化细胞P的面积；
步骤f、分别统计果肉的x、y和z区域中，n个木质化细胞成团的染色面积，2≤n≤N，n值不同的成团木质化细胞染色面积分开统计，分别记作：
Figure PCTCN2022072666-appb-000004
矩阵中以
Figure PCTCN2022072666-appb-000005
为例，
Figure PCTCN2022072666-appb-000006
表示x区域内2个木质化细胞贴靠成团的成团木质化细胞的染色总面积；
Figure PCTCN2022072666-appb-000007
表示x区域内N个木质化细胞贴靠成团的成团木质化细胞的染色总面积；
步骤g、统计果肉x、y和z三个区域中，维管束染色区域总面积，分别记作：
Figure PCTCN2022072666-appb-000008
并统计每个区域维管束的条数，分别记作i x，i y和i z；
步骤h、取一批同品种刚成熟采摘未经贮藏的枇杷果实，获取果肉x、y和z三个区域中，维管束染色面积的矫正系数：将枇杷果实按照步骤a切出果肉赤道面，并按步骤b进行冷冻切片及染色，按照步骤g统计果肉x、y和z三个区域中，维管束染色区域总面积，分别计算果肉x、y和z三个区域中，每条维管束染色区域面积的平均值，作为维管束矫正常数,分别记作：
Figure PCTCN2022072666-appb-000009
步骤i、计算每个区域的木质化指数Lig x、Lig y和Lig z和整个果实的木质化指数Lig：
Figure PCTCN2022072666-appb-000010
Figure PCTCN2022072666-appb-000011
Figure PCTCN2022072666-appb-000012
Lig＝a*(2Lig x+Lig y+0.5Lig z)
其中，a为可人为设定的归一化系数，让Lig值接近于1，减少小数点位数方便统计；
步骤j、以木质化指数Lig作为枇杷果实木质化评价指数，Lig值越大，枇杷果实木质化程度越严重。
本方法中，将枇杷果肉按照从内到外分成三个区域，越靠外的细胞发生木质化，在木质化评价中的权重越大。枇杷木质化细胞按照木质化发展程度进行区分，分为初级木质化细胞P、中级木质化细胞R和终级木质化细胞U并分别统计其面积，木质化程度越高的果肉细胞木质化评价中的权重越大。并将枇杷木质化细胞的团聚现象进行统计，木质化细胞团聚越严重的细胞群木质化评价中的权重越大。最后对枇杷果肉中维管束的面积进行统计，并与初摘果实的维管束面积进行相减，获取在贮藏过程中维管束的面积增大情况，用于木质化评价中。 本方法在枇杷果实木质化程度评价中，充分考虑了木质化细胞的空间分布因素、木质化细胞的木质化程度因素、木质化细胞的团聚因素，维管束增粗的因素，评价体系更加合理，对于果实木质化的成因和各因素影响均进行了加权计算，为后续的果实木质化各要素分项研究奠定了基础。
作为优选，步骤c中，对于枇杷果肉细胞：细胞壁增厚但增厚部分未成环形、以及细胞壁增厚至环形但细胞壁厚度占小于细胞半径的1/4，则记为初级木质化细胞P；细胞壁增厚至环形且细胞壁厚度为细胞半径的1/4～3/4，则记为中级木质化细胞R；细胞壁增厚至环形且细胞壁厚度超过细胞半径的3/4，则记为终级木质化细胞U。枇杷果肉细胞的木质化是一个逐渐变化的过程，是一个细胞壁逐渐增厚的过程。初级木质化细胞P、中级木质化细胞R、终级木质化细胞U分别对应着细胞木质化的初期、中期、末期，
作为优选，步骤b中，连续冷冻切片的每片厚度为40μm。
作为优选，所述枇杷果实为洛阳青枇杷果实，取a＝1000，Lig＝1000*(2Lig x+Lig y+0.5Lig z)，Lig≤0.5为无木质化，0.5＜Lig≤0.75为轻度木质化，0.75＜Lig≤1为中度木质化，1＜Lig为重度木质化。
本发明在枇杷果实木质化程度评价时，基于细胞级的显微成像，既考虑了木质化细胞在果肉中的空间分布因素，又考虑了木质化细胞的发展程度因素，还考虑了木质化细胞的团聚因素，建立了多因素不同权重的细胞快速统计评价方法，本方法的评价更加全面准确，让枇杷的木质化评价结果与实际品质关联性更好。
附图说明
下面结合附图对本发明做进一步说明。
图1是本发明的初级木质化细胞P显微镜图。
图2是本发明的初级木质化细胞P的示意图。
图3是本发明的中级木质化细胞R显微镜图。
图4是本发明的中级木质化细胞R的示意图。
图5是本发明的终级木质化细胞U显微镜图。
图6是本发明的终级木质化细胞U的示意图。
图7是本发明的2个木质化细胞组成的成团木质化细胞显微镜图。
图8是本发明的2个木质化细胞组成的成团木质化细胞的示意图。
图9是本发明的3个木质化细胞组成的成团木质化细胞显微镜图。
图10是本发明的维管束W的显微镜图。
具体实施方式
下面通过具体实施例并结合附图对本发明进一步说明。
实施例，一种枇杷果实木质化程度的细胞快速统计评价方法，包括以下步骤：
步骤a、将枇杷果实沿着果实的赤道面剖开，去除一半果实、并去除果核，计算果实整个赤 道面的果肉面积S；
步骤b、将赤道面果肉连续冷冻切片，将全部的切片进行间苯三酚方法染色处理；连续冷冻切片的每片厚度为40μm；
步骤c、使用光学显微镜获取切片染色结果图，根据木质化细胞形态的不同，定义木质化程度，将木质化细胞分为三类：初级木质化细胞P、中级木质化细胞R和终级木质化细胞U；如对于枇杷果肉细胞：细胞壁增厚但增厚部分未成环形、以及细胞壁增厚至环形但细胞壁厚度占小于细胞半径的1/4，则记为初级木质化细胞P，如图1、2所示,将细胞壁增厚至环形但细胞壁厚度占小于细胞半径的1/4的木质化细胞P也通过图2统一示意，以与中级木质化细胞R做区分；细胞壁增厚至环形且细胞壁厚度为细胞半径的1/4～3/4，则记为中级木质化细胞R，如图3、4所示；细胞壁增厚至环形且细胞壁厚度超过细胞半径的3/4，则记为终级木质化细胞U，如图5、6所示；
步骤d、将赤道面果肉分为三个环形区域，从外到内分别为靠果皮区域x、中间区域y和靠果核区域z，三个环形区域在每个半径方向的宽度均相等；
步骤e、分别统计果肉的x、y和z区域中，三种类型的木质化细胞的染色总面积，分别记作：
Figure PCTCN2022072666-appb-000013
矩阵中以
Figure PCTCN2022072666-appb-000014
为例，
Figure PCTCN2022072666-appb-000015
表示x区域中初级木质化细胞P的面积；
步骤f、分别统计果肉的x、y和z区域中，n个木质化细胞成团的染色面积，2≤n≤N，n值不同的成团木质化细胞染色面积分开统计，分别记作：
Figure PCTCN2022072666-appb-000016
矩阵中以
Figure PCTCN2022072666-appb-000017
为例，
Figure PCTCN2022072666-appb-000018
表示x区域内2个木质化细胞贴靠成团的成团木质化细胞的染色总面积，如图7、8所示；
Figure PCTCN2022072666-appb-000019
表示x区域内3个木质化细胞贴靠成团的成团木质化细胞的染色总面积，如图9所示；
步骤g、统计果肉x、y和z三个区域中，维管束染色区域总面积，维管束显微镜结构如图10速食，分别记作：
Figure PCTCN2022072666-appb-000020
并统计每个区域维管束的条数，分别记作i x，i y和i z；其中，每一束聚合在一起的维管束记为一条；
步骤h、取一批同品种刚成熟采摘未经贮藏的枇杷果实，获取果肉x、y和z三个区域中，维管束染色面积的矫正系数：将枇杷果实按照步骤a切出果肉赤道面，并按步骤b进行冷冻切片及染色，按照步骤g统计果肉x、y和z三个区域中，维管束染色区域总面积，分别计算果肉x、y和z三个区域中，每条维管束染色区域面积的平均值，作为维管束矫正常数,分别记作：
Figure PCTCN2022072666-appb-000021
步骤i、计算每个区域的木质化指数Lig x、Lig y和Lig z和整个果实的木质化指数Lig：
Figure PCTCN2022072666-appb-000022
Figure PCTCN2022072666-appb-000023
Figure PCTCN2022072666-appb-000024
Lig＝a*(2Lig x+Lig y+0.5Lig z)
其中，a为可人为设定的归一化系数，让Lig值接近于1，减少小数点位数方便统计；
步骤j、以木质化指数Lig作为枇杷果实木质化评价指数，Lig值越大，枇杷果实木质化程 度越严重。以木质化Lig值为依据对枇杷进行品质分级，可以指导枇杷仓储策略和销售策略。同时本方案提出的木质化评价体系，可以作为枇杷采后各种不同处理方式的效果评价指标，指导后续的试验研究。
本实施例中，以洛阳青枇杷果实为例，取a＝1000，Lig＝1000*(2Lig x+Lig y+0.5Lig z)，Lig≤0.5为无木质化，0.5＜Lig≤0.75为轻度木质化，0.75＜Lig≤1为中度木质化，1＜Lig为重度木质化。
取两类枇杷样本，分别为刚新鲜采摘的“洛阳青”枇杷和0℃贮藏6d的“洛阳青”枇杷。
新鲜采摘的“洛阳青”枇杷统计数据如下：
S＝10.20687617cm 2＝1020687617μm 2
Figure PCTCN2022072666-appb-000025
Figure PCTCN2022072666-appb-000026
Figure PCTCN2022072666-appb-000027
Figure PCTCN2022072666-appb-000028
Figure PCTCN2022072666-appb-000029
Figure PCTCN2022072666-appb-000030
Figure PCTCN2022072666-appb-000031
Figure PCTCN2022072666-appb-000032
Figure PCTCN2022072666-appb-000033
Figure PCTCN2022072666-appb-000034
Figure PCTCN2022072666-appb-000035
Figure PCTCN2022072666-appb-000036
i x＝56
i y＝48
i z＝19
最后计算的结果值：
Lig x＝0.0000595371433787208
Lig y＝0.000385471
Lig z＝0.000169979
Lig＝0.482368≤0.5，无木质化。
0℃贮藏6d的“洛阳青”枇杷统计数据如下：
S＝12.50953167902cm 2＝1250953167.902μm 2
Figure PCTCN2022072666-appb-000037
Figure PCTCN2022072666-appb-000038
Figure PCTCN2022072666-appb-000039
Figure PCTCN2022072666-appb-000040
Figure PCTCN2022072666-appb-000041
Figure PCTCN2022072666-appb-000042
Figure PCTCN2022072666-appb-000043
Figure PCTCN2022072666-appb-000044
Figure PCTCN2022072666-appb-000045
Figure PCTCN2022072666-appb-000046
Figure PCTCN2022072666-appb-000047
Figure PCTCN2022072666-appb-000048
i x＝68
i y＝56
i z＝17
最后计算的结果值：
Lig x＝0.0000438954824737649
Lig y＝0.000776167
Lig z＝0.000337217
Lig＝1.032566＞1，已严重木质化。

Claims (4)

  1. 一种枇杷果实木质化程度的细胞快速统计评价方法，其特征在于：包括以下步骤：
    步骤a、将枇杷果实沿着果实的赤道面剖开，去除一半果实、并去除果核，计算果实整个赤道面的果肉面积S；
    步骤b、将赤道面果肉连续冷冻切片，将全部的切片进行间苯三酚方法染色处理；
    步骤c、使用光学显微镜获取切片染色结果图，根据木质化细胞形态的不同，定义木质化程度，将木质化细胞分为三类：初级木质化细胞P、中级木质化细胞R和终级木质化细胞U；
    步骤d、将赤道面果肉分为三个环形区域，从外到内分别为靠果皮区域x、中间区域y和靠果核区域z，三个环形区域在每个半径方向的宽度均相等；
    步骤e、分别统计果肉的x、y和z区域中，三种类型的木质化细胞的染色总面积，分别记作：
    Figure PCTCN2022072666-appb-100001
    Figure PCTCN2022072666-appb-100002
    Figure PCTCN2022072666-appb-100003
    矩阵中以
    Figure PCTCN2022072666-appb-100004
    为例，
    Figure PCTCN2022072666-appb-100005
    表示x区域中初级木质化细胞P的面积；
    步骤f、分别统计果肉的x、y和z区域中，n个木质化细胞成团的染色面积，2≤n≤N，n值不同的成团木质化细胞染色面积分开统计，分别记作：
    Figure PCTCN2022072666-appb-100006
    Figure PCTCN2022072666-appb-100007
    Figure PCTCN2022072666-appb-100008
    矩阵中以
    Figure PCTCN2022072666-appb-100009
    为例，
    Figure PCTCN2022072666-appb-100010
    表示x区域内2个木质化细胞贴靠成团的成团木质化细胞的染色总面积；
    Figure PCTCN2022072666-appb-100011
    表示x区域内N个木质化细胞贴靠成团的成团木质化细胞的染色总面积；
    步骤g、统计果肉x、y和z三个区域中，维管束染色区域总面积，分别记作：
    Figure PCTCN2022072666-appb-100012
    并统计每个区域维管束的条数，分别记作i x，i y和i z；
    步骤h、取一批同品种刚成熟采摘未经贮藏的枇杷果实，获取果肉x、y和z三个区域中，维管束染色面积的矫正系数：将枇杷果实按照步骤a切出果肉赤道面，并按步骤b进行冷冻切片及染色，按照步骤g统计果肉x、y和z三个区域中，维管束染色区域总面积，分别计算果肉x、y和z三个区域中，每条维管束染色区域面积的平均值，作为维管束矫正常数,分别记作：
    Figure PCTCN2022072666-appb-100013
    步骤i、计算每个区域的木质化指数Lig x、Lig y和Lig z和整个果实的木质化指数Lig：
    Figure PCTCN2022072666-appb-100014
    Figure PCTCN2022072666-appb-100015
    Figure PCTCN2022072666-appb-100016
    Lig＝a*(2Lig x+Lig y+0.5Lig z)
    其中，a为可人为设定的归一化系数，让Lig值接近于1，减少小数点位数方便统计；
    步骤j、以木质化指数Lig作为枇杷果实木质化评价指数，Lig值越大，枇杷果实木质化程度越严重。
  2. 根据权利要求1所述的一种枇杷果实木质化程度的细胞快速统计评价方法，其特征在于：步骤c中，对于枇杷果肉细胞：细胞壁增厚但增厚部分未成环形、以及细胞壁增厚至环形但细胞壁厚度占小于细胞半径的1/4，则记为初级木质化细胞P；细胞壁增厚至环形且细胞壁厚度为细胞半径的1/4～3/4，则记为中级木质化细胞R；细胞壁增厚至环形且细胞壁厚度超过细胞半径的3/4，则记为终级木质化细胞U。
  3. 根据权利要求1所述的一种枇杷果实木质化程度的细胞快速统计评价方法，其特征在于：步骤b中，连续冷冻切片的每片厚度为40μm。
  4. 根据权利要求1所述的一种枇杷果实木质化程度的细胞快速统计评价方法，其特征在于：所述枇杷果实为洛阳青枇杷果实，取a＝1000，Lig＝1000*(2Lig x+Lig y+0.5Lig z)，Lig≤0.5为无木质化，0.5＜Lig≤0.75为轻度木质化，0.75＜Lig≤1为中度木质化，1＜Lig为重度木质化。
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CN113049588A (zh) * 2021-03-10 2021-06-29 浙江大学 一种指导枇杷销售策略的枇杷果实品质评价及预测方法

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CN117110124A (zh) * 2023-08-23 2023-11-24 甘肃农业大学 基于质量差法快速测定苹果果肉粉质化的方法

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