AU2020101297A4

AU2020101297A4 - A Method for Storing ‘Zimei’ Pomegranate Pollens

Info

Publication number: AU2020101297A4
Application number: AU2020101297A
Authority: AU
Inventors: Zhaohe YUAN; Xueqing Zhao; Yujie Zhao
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2020-07-09
Filing date: 2020-07-09
Publication date: 2020-08-13
Anticipated expiration: 2028-07-09

Abstract

of Descriptions A method for storing 'Zimei' pomegranate pollens is disclosed in this invention, under the field of plant development biology. The method for storing pomegranate pollens in this invention, includes the following steps: collecting pollens, drying pollens and storing pollens. A pomegranate pollen collection, drying, preservation and vitality detection system is formed in this invention firstly, with simple operation method and stronger repeatability, in order to prolong the storage period of pomegranate pollen effectively, maintain the better vitality of the pollen during storage, and improve the success rate of artificial hybridization pollination of pomegranate. Drawings of Descriptions Figure 1 Figure 2 Figure 3 1/2

Description

Drawings of Descriptions

Figure 1

Figure 2

Figure 3

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Descriptions

A Method for Storing 'Zimei' Pomegranate Pollens

Technical Field The present invention relates to the field of plant development biology, especially a method for storing 'Zimei' pomegranate pollens.

Background Technology Cross-breeding is one of important ways to create new pomegranate varieties. Pollen viability is an important condition to ensure the success of cross breeding. The full florescence of pomegranate in Sichuan and Yunnan is in the middle of April, while the full florescence of pomegranate in Henan and Shandong is in the middle and late of May, which brings some troubles to the hybridization between different varieties in the north and south due to the inconsistent florescence of pomegranate cultivated in the north and south of China. Pollen collection and storage are urgent problems to be solved in the innovation of pomegranate germplasm resources. At present, pomegranate pollen is mostly stored by more complicated drying methods such as silica gel drying and vitrification solution PVS 4 , the processing process is complicated, and the method of directly storing pomegranate pollen without drying treatment can greatly shorten the storage time within the effective viability range of pollen. At room temperature, pomegranate pollen has completely lost its viability after storage for 5 days. Using liquid nitrogen to store pollen, pollen vitality will drop sharply after thawing and low temperature recovery in the later stage. None of these methods of storage are optimal for pomegranate pollen for hybrid test. By taking 'Zimei' pomegranate as a test material, the present invention aims at researching the best processing mode and conditions of storing pomegranate pollens and provides technical support for pomegranate cross-breeding.

'Zimei' pomegranate was introduced into Panzhihua from Israel in 1996, which was a fine variety selected through many years of experiments by Panzhihua Agricultural and Forestry Sciences Institute, Liangshan State Subtropical Crops Research Institute and Sichuan Horticulture Research Institute. 'Zimei' tree is semi-open, with strong tree vigor and branch formation. Leaves are oblong or obovate, young leaves are reddish, old leaves are crimson. Sepals of flower are crimson, receptacle are crimson, and the stype is tubular or bell-shaped. The total amount of flowers is medium, and the proportion of bisexual flowers is higher. Under the subtropical climate in Panxi, 'Zimei' sprout in mid-February, blossom in the first half of March, and flowering from late March to early May. In mid-to-late September, the

Descriptions fruit is ripe. The fruit is nearly spherical, the peel is deep red, and the average weight of a single fruit is 587 g. 'Zimei' pomegranate has strong storability, rough peel texture and 3 mm thickness. The grain is purplish red, the juice is purplish black, and the grain is extremely soft. 'Zimei' has deep red peel, beautiful appearance, soft seeds, high commodity value and great market potential.

Pomegranate pollens must be tested for viability before determining whether it can be used for cross pollination. The hybridization work may fail, or the results cannot be reliably analyzed if pollen with or without viability and viability is used blindly. The pomegranate pollen storage method includes three key technical steps, is simple and easy to operate, effectively maintains the vitality of pomegranate pollen during storage. This invention has important application value in the fields of pomegranate production, breeding, scientific research and the like.

Invention Summary In view of the defects of the prior technology and research, the present invention aims to provide a method for storing 'Zimei' pomegranate pollens, which is simple.

In order to achieve the above purpose, the present invention adopts the following technical solution:

A method for storing 'Zimei' pomegranate pollens, which includes the following steps:

(1) Collecting pollens: mature and budding flowers are collected from 8:00 to :00 in the morning when pomegranate flowers are in full bloom, to ensure the effectiveness of flower organ development;

(2) Drying pollens: the collected flowers that petals are carefully removed, are left indoors for natural drying; when anther dehisces and disperses pollen, the flower is gently vibrated, the pollen is evenly collected on clean white paper, and the pollen is continuously aired until the pollen is dry, and the dry state is that the pollen is completely dispersed and does not form lumps;

(3) Storing pollens: the inside of the glass dryer is cleaned, the inner wall is wiped with 75% alcohol, the discolored silica gel is poured into and cover the bottom of the dryer; dried pomegranate pollen is put into a container, sealed and placed in a sealed dryer, and stored at 4°C.

Descriptions

For a further technical solution, in the step (1), the full flowering period is a period from the end of April to the beginning of May in Sichuan, and 70%-75% of 'Zimei' pomegranate flowers are blooming.

For a further technical solution, in the step (1), the mature flower is a flower with obviously enlarged ovary and vase-shaped flower, and the selected flower is a complete flower.

For a further technical solution, in the step (2), the natural drying is carried out at °C under indoor natural illumination for 24 hours.

For a further technical solution, in the step (3), the container is a 10 mL centrifuge tube, and the preservation is carried out by placing the container in a fresh-keeping layer of a refrigerator for storage at 4°C.

The present invention measures the pollen vitality of 'Zimei' pomegranate using liquid culture medium culture during storage. A method of measuring the viability of pollen is spreading pollen on liquid culture medium and germinating its pollen. The specific operation is as follows: 1-2 drops of liquid culture are based on a glass slide, a small amount of pollen is taken by a pin and evenly sprinkled on a liquid culture medium, wherein the liquid culture medium is sucrose. Slide glass is placed in a culture dish with humidity of 85%-90%, and dark culture is carried out for 12 h at °C. Pollen with normal viability is round when germinating and the pollen tube extends linearly. Pollen tubes with poor viability are bent after germination. Pollen grains without vitality cannot germinate.

The invention has the following beneficial effects: 1. In the method for collecting pomegranate pollens in the present invention, mature complete flowers is selected, in order to ensure the effectiveness of flower organ development, ensures the consistency of pollen fertility in a natural state, and avoids the problem of pollen vitality difference caused by inconsistent development degrees of different types of flower organs.

2. In the method for drying pomegranate pollens in the present invention, it is naturally dried indoors, and it compare with the prior direct sunlight or dryer, the pomegranate pollen drying method ensures pollen activity and objectivity of research under natural conditions.

Descriptions

3. In the method for storing pomegranate pollens in the present invention, the plurality of temperature gradients were respectively placed for storage. Through pollen vitality detection, pollen at room temperature has completely lost vitality after one week. However, if stored at 4°C, the stored pollen can be directly used for experimental research, and the problems of damage to pollen caused by the recovery process after low-temperature treatment such as -20°C, 80°C, liquid nitrogen. The problem in the prior can be avoided, and the method is simple and has strong repeatability.

4. The relative humidity of the pollen culture is 85%-90% when the pomegranate pollen vitality is measured, thus ensuring the water required in the pollen germination culture process.

5. The liquid MS as a basic culture medium is used in the invention, two treatments of sucrose and glucose are arranged on the premise that the basic components are unchanged. Pollen vitality on the glucose culture medium is obviously lower than that on sucrose under the same storage time and storage condition, indicating that the liquid culture medium with sucrose as a sugar source is more suitable for in vitro culture of 'Zimei' pomegranate pollen.

6. For the first time, the invention forms a system for collecting, drying, storing and measuring the vitality of pomegranate pollen. The method has the characteristics of simple operation, rapidness, strong repeatability. At the same time, the method can prolong the effective storage time of pomegranate pollen, improve the success rate of artificial cross pollination of pomegranate, and ensures that cross-breeding is the main way to select excellent varieties of pomegranate.

Brief Description of Drawings Fig. 1 Drying and collecting pollens

Fig. 2 Storing pollens

Fig. 3 Steps of determining pollen viability

Fig. 4 Changes of pollen germination rate of 'Zimei' pomegranate at different storage times.

Descriptions Detailed Description of the Presently Preferred Embodiments In order to make the purpose, technical solution and key points of the embodiment of the present invention clearer, the technical solution in the embodiment of the present invention will be described in detail below with reference to the embodiment of the present invention and the drawings.

Embodiment A method for storing 'Zimei' pomegranate pollens, which includes the following steps:

(1) Collecting pollens: mature and budding flowers are collected from 8:00 to :00 in the morning when pomegranate flowers are in full bloom to ensure the effectiveness of flower organ development; the full flowering period is from the end of April to the beginning of May in Sichuan, and 70%75% of pomegranate flowers in 'Zimei' pomegranate are blooming. The mature flowers have obviously enlarged ovary and vase-shaped flower, and the selected flower is the complete flower.

(2) Drying pollens: petals of collected flowers are carefully removed, the flowers in a room for natural drying are placed, and the natural drying is to dry the flowers at 25°C under indoor natural light for 24 h; when anther dehisces and disperses pollen, the flower is gently vibrated, the pollen is evenly collected on clean white paper, and the pollen is continuously aired until the pollen is dry, and the dry state is that the pollen is completely dispersed and does not form lumps (Figure 1);

(3) Storing pollens: the inside of the glass dryer is cleaned, the inner wall is wiped with 75% alcohol, the discolored silica gel (a granular desiccant, blue, white after absorbing water) is poured into the bottom of the dryer and fill dryer with it. Dried pomegranate pollen is put into a container, sealed, placed in a sealed dryer, and stored at 4°C (Figure 2). The container is a 10 mL centrifuge tube and stored at 4°C in the fresh-keeping layer of the refrigerator.

The dried and subpackaged pollens are stored at multiple temperature gradients. The naturally dried pomegranate pollen is stored at the room temperature of the centrifuge tube, and the pollen completely loses vitality after one week. When pollen is stored at 25°C or higher, the water loss of pollen is serious, which affects the late germination rate. When the storage temperature is below 0°C, the free water in the cell solidifies, the structure of pollen cells is destroyed, and the pollen loses its vitality. Although the antifreeze agents such as glycerin protects cells from low temperature

Descriptions damage to some extent, low temperature will still cause inevitable damage to pollen. The recovery of pollen after low temperature storage makes the test complicated. Through comparison of multiple temperatures, it is found that the pollen activity reaches 42.1% at 4°C after one week of storage. The experimental results are optimal and can effectively avoid the above problems and disadvantages.

The viability of stored pomegranate pollen is regularly tested, and sucrose and glucose are used as the liquid media for culture. Drop 1-2 drops of liquid culture medium on the glass slide and put a small amount of pollen into the solution with a pin. Spread pollen evenly on the liquid culture medium. Slide glass is placed in a petri dish with a humidity of 85%-90% and placed in an incubator at 25°C for cultivation (Figure 3). After 12 h, the number of germinated pollen grains is counted under an optical microscope, and more than 3 representative visual fields are examined under each repeated microscope, and the pollen germination rate is calculated: pollen germination rate (%)= the number of pollen whose pollen tube length is greater than the pollen diameter / the total number of pollen in the visual field x 100%.

The pollen germination rate of glucose (40.7%) culture is significantly lower than that of sucrose (46.2%) by comparing the effects of adding sucrose and glucose to MS culture media respectively (Figure 4) under the same treatment and storage conditions. Through many groups of experiments, it is found that MS + 4.2 g/L sucrose has the best germination rate, and also serves as a carbon source to provide energy source for pollen cells. Sucrose can better regulate osmotic pressure in the culture medium than glucose, which is easy to dehydrate cells and cause poor growth. From the above, it can be seen that the determination of pomegranate pollen viability is suitable for pomegranate pollen germination in sucrose medium.

Based on data analysis, it is found that the method of the invention is particularly suitable for short-term storage of 'Zimei' pomegranate pollen. After storage for 21 days, the germination rate is still kept at 32.48%, within the range of effective pollen germination rate (> 20.0%), and the germination rate can still reach 21.5% after 130 days of storage at 4°C. This indicates that the storage method can effectively maintain the pollen vitality of pomegranate. The pomegranate pollen stored for three months can still be used for hybridization work (Figure 5), in order to satisfy the hybridization breeding and related scientific research work of pomegranates at different regions for one year.

Claims

1. A method for storing 'Zimei' pomegranate pollens, characterized in that, including the following steps:

(1) Collecting pollens: collect mature and budding flowers from 8: 00 to 10: 00 in the morning when pomegranate flowers are in full bloom;

(2) Drying pollens: the collected flowers that petals are carefully removed, left indoors for natural drying; when anther dehisces and disperses pollens, the flower is gently vibrated, the pollens are evenly collected on clean white paper, and the pollens are continuously aired until the pollens are dry, and the dry state is that the pollen is completely dispersed and does not form lumps;

(3) Storing pollens: the inside of the glass dryer is cleaned, the inner wall is wiped with 75% alcohol, the discolored silica gel is poured into and the bottom of the dryer is covered; dried pomegranate pollens are put into a container, sealed and placed in a sealed dryer, and stored at 4°C.

2. A method for storing the pomegranate pollens according to claim 1, characterized in that the vitality of the stored pomegranate pollens are measured, and the specific method includes the following steps of: dropping 1-2 drops of liquid culture on a glass slide, taking a small amount of stored pomegranate pollens by using a pin, uniformly spreading the glass slide in a liquid culture medium, placing the glass slide in a culture dish with a relative humidity of 85-90%, and performing dark culture for 12 hours at 25°C in an incubator to germinate the pollens.

3. A method for storing pomegranate pollens according to claim 1, characterized in that in the step (1), the full flowering period is from the end of April to the beginning of May in Sichuan, and 70%- 7 5 % of 'Zimei' pomegranate flowers are blooming.

4. A method for storing pomegranate pollens according to claim 1, characterized in that in the step (1), the mature flower is a flower with obviously expanded flower ovary and vase-shape flower, and the selected flower is a complete flower.

5. A method for storing pomegranate pollens according to claim 1, characterized in that in the step (2), natural drying is to dry at 25°C under indoor natural light for 24 hours.

Claims

6. A method for storing pomegranate pollens according to claim 1, characterized in that in the step (3), the container is a 10 mL centrifuge tube, and the storage is stored at 4°C in the fresh-keeping layer of the refrigerator.

7. A method for storing pomegranate pollens according to claim 2, characterized in that the liquid culture medium is composed of MS+4.2 g/L sucrose.