CN106172378B - Ultralow-temperature preservation method of grapefruit germplasm pollen - Google Patents
Ultralow-temperature preservation method of grapefruit germplasm pollen Download PDFInfo
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Abstract
The invention relates to a plant ultralow temperature preservation method, in particular to an ultralow temperature preservation method of grapefruit germplasm pollen, which comprises the following steps: (1) drying pollen: drying the pollen to a water content of 10-20%; (2) freezing and storing pollen: sealing and packaging the dried pollen, and storing in liquid nitrogen; (3) and (3) pollen recovery: taking out the pollen from the liquid nitrogen, and thawing for 5-15 min at the temperature of 25-37 ℃; (4) rewetting pollen for 0-3 h; (5) and (5) germinating in vitro. The invention optimizes and defines key elements suitable for ultralow temperature preservation of the pomelo germplasm pollen, such as water content range, thawing conditions, rewetting conditions, in-vitro germination conditions and the like. By optimizing and limiting the conditions, the germination rate of the grapefruit germplasm pollen after ultralow-temperature preservation is obviously improved.
Description
Technical Field
The invention relates to a plant ultralow temperature preservation method, in particular to an ultralow temperature preservation method of grapefruit germplasm pollen.
Background
pomelo germplasm (Citrus grandis (L.) Osbeck) is an important germplasm resource, is a plant of the genus Citrus in the family rutaceae, and is native to asia. The pomelo fruit has beautiful shape, delicious taste, rich nutrition and high economic value, and the development and utilization of the pomelo fruit are promising at home and abroad in recent years. The natural environment of the shaddock producing area in China is superior, and the ecological conditions are complex. In addition, the single embryo property of the pomelos is easy to generate variation in seedling propagation, and extremely rich germplasm resources are formed for thousands of years. According to incomplete statistics, the variety, the strain, the type and the strain of the national pomelo germplasm resources are more than 200. The pomelo species in different regions have wide difference in form and maturation period, and show abundant genetic diversity. In order to protect the genetic diversity of pomelos, collection and preservation of germplasm resources of different pomelo varieties are urgently needed.
at present, pomelo germplasm is preserved mainly in a nursery position, namely in a field planting mode, but the mode not only occupies cultivated land, but also consumes a large amount of manpower and material resources for cultivation and management, and natural disasters such as drought, flood, low temperature, heat damage, plant diseases and insect pests can cause germplasm resources to be lost, so that the pomelo germplasm is not suitable for long-term preservation in the field mode. The commonly used conventional low-temperature seed preservation mode is not suitable for the pomelo germplasm, and researches show that the pomelo seeds are intermediate seeds and have extremely sensitive dehydration tolerance. For the test-tube plantlet preservation mode of asexual propagation crops, there has been a research report, when adult-state pomelo stems are used as materials to induce axillary buds to germinate so as to establish a test-tube plantlet tissue culture technical system, since pomelos are woody plants and are exposed outdoors for a long time, a large amount of bacteria are stored, and thorough disinfection is difficult to realize. Moreover, multiple tissue subcultures may also result in genetic variation, which is labor and material intensive. Therefore, cryopreservation becomes an important way for possible long-term backup preservation of pomelo germplasm.
Cryopreservation, i.e., the storage of plant cells, tissues or organs and other materials in vapor or liquid nitrogen at a temperature of below-150 ℃, is considered to be a safe, economical and effective long-term storage method suitable for asexual propagation crops. The ultra-low temperature preservation technology has good application prospect in the preservation of plant germplasm resources, so that the ultra-low temperature preservation technology is more and more emphasized by people. To date, over 200 types of plant material have been subjected to cryopreservation, using types of material including shoot tips, dormant buds, pollen, cells, protozoa, and the like. In recent years, there have been many reports of cryopreservation of plant materials, and germplasm preservation of horticultural plants mainly based on vegetative propagation has been studied most.
Research reports that the survival rate of the pomelo stem tip can reach about 60 percent through the steps of pre-culture, vitrification, restoration culture and the like by taking the pomelo stem tip as a storage carrier and initially establishing a vitrification method ultralow-temperature storage technical system.
in the research of pollen preservation, it has been reported that the germination rate of Shatian pomelo pollen after thawing can reach 53.92% by drying the Shatian pomelo pollen until the water content reaches 13.5% and preserving the Shatian pomelo pollen at-80 ℃. But the developed plant pollen cryopreservation research shows that the pollen is preserved under the liquid nitrogen condition, so that the preservation time can be obviously prolonged, the invasion of plant diseases and insect pests can be effectively avoided, the breeding efficiency can be effectively improved, and convenience is provided for germplasm exchange between regions and countries.
Chinese patent publication No. CN101982046B discloses a long-term reliable storage method for pear pollen, which comprises drying pear pollen, loading into a color-changing silica gel box, wrapping with tinfoil, sealing, freezing with liquid nitrogen, storing at ultralow temperature, standing at-73 deg.C, -20 deg.C and 4 deg.C for 24 hr for gradually reviving when needed in the next year, and recovering to room temperature in dark environment. The method is safe and reliable, can effectively maintain the vigor of the pear pollen, is simple and easy to operate, saves space, can be used for reference to the preservation of other germplasm resources, and has wide application value. However, the inventor finds out through experiments that when the technical scheme is applied to ultralow temperature preservation of the pomelo germplasm pollen, the moisture returning is insufficient during the ultralow temperature preservation recovery of the pollen, and the pollen after thawing can not germinate in vitro on a culture medium; secondly, the pollen is not tightly packaged in a sealing way, so that the pollen is possibly soaked in liquid nitrogen to cause freezing damage and lose pollen germination vitality; and thirdly, when the revived pollen is preserved at ultralow temperature, the temperature gradient gradual reviving mode is adopted, so that the defects that ice crystal damage in pollen cells is caused by temperature rise to cause pollen germination activity loss and the like are possibly increased, and therefore, the method is not suitable for the ultralow-temperature preservation of the grapefruit germplasm pollen.
Therefore, it is highly desirable to provide an ultra-low temperature preservation method for grapefruit germplasm pollen, and to improve the germination rate after preservation as much as possible.
disclosure of Invention
Aiming at solving the problems in the prior art, the invention aims to provide a proper ultralow temperature preservation method for grapefruit germplasm pollen.
in order to realize the purpose of the invention, the technical scheme of the invention is as follows:
The invention firstly provides an ultralow temperature preservation method of grapefruit germplasm pollen, which comprises the following steps:
(1) Drying pollen: drying the pollen to a water content of 10-20%;
(2) Freezing and storing pollen: sealing and packaging the dried pollen, and storing in liquid nitrogen;
(3) And (3) pollen recovery: taking out the pollen from the liquid nitrogen, and thawing for 5-15 min at the temperature of 25-37 ℃;
(4) rewetting pollen for 0-3 h;
(5) And (5) germinating in vitro.
The pollen in the steps (1) to (3) may be pollen or pollen-containing anther.
In order to better improve the germination efficiency of the thawed pollen, the invention develops a special pollen in-vitro germination culture medium, namely the step (5) is as follows: placing the pollen after rewetting on a pollen in-vitro germination culture medium containing 100-150g/L of sucrose, 0.1g/L of boric acid, 0.1g/L of potassium nitrate, 0.3-1.0g/L of calcium nitrate, 0-0.3g/L of magnesium sulfate and 0-10g/L of agar for dark culture at 24-26 ℃ for 16-20 h.
preferably, the water content required to be controlled in the drying step is optimized for the pomelo germplasm pollen, namely the step (1) is specifically to dry the pollen at the temperature of 25-35 ℃ until the water content is 10-20%. Under the water content, the grapefruit germplasm pollen can keep the pollen activity under the ultralow temperature preservation condition, so that better ultralow temperature in-vitro germination rate is shown after the defrosting and rewetting treatment.
Further, the step (2) can be a conventional cryopreservation means, for example, pollen is wrapped with parchment paper, sealed and packaged in a cryopreservation tube, and put into a liquid nitrogen tank for preservation.
On the basis, the invention particularly introduces a moisture regaining step, namely the step (4) is that the pollen after being revived is taken from the freezing storage tube by a dissecting needle and put into an ampere bottle, the ampere bottle is put into a closed container containing supersaturated copper sulfate, and the pollen is rewetted for 0-3 h, preferably 3h at room temperature. Experiments prove that the germination rate of the thawed grapefruit germplasm pollen can be obviously improved by introducing the moisture regaining step.
furthermore, the research shows that different ultralow-temperature preservation methods are more specifically adopted for different pomelo germplasm pollens on the basis of providing the ultralow-temperature preservation method for the pomelo germplasm pollens, so that the germination rate of the thawed pomelo germplasm pollens can be more specifically improved.
The method comprises the following specific steps:
when the grapefruit germplasm pollen is grapefruit germplasm pollen, the method comprises the following steps:
(1) Drying pollen: drying the pollen to a water content of 10-20%;
(2) freezing and storing pollen: sealing and packaging the dried pollen, and storing in liquid nitrogen;
(3) and (3) pollen recovery: taking out pollen from liquid nitrogen, and thawing at 37 deg.C for 5 min;
(4) And (3) pollen rewetting: putting the revived pollen into an ampere bottle, putting the ampere bottle into a closed container containing supersaturated copper sulfate, and rewetting for 2-3 h at room temperature;
(5) In vitro germination: placing the pollen after rewetting on a pollen in-vitro germination culture medium containing 100-150g/L of sucrose, 0.1g/L of boric acid, 0.1g/L of potassium nitrate, 0.3-1.0g/L of calcium nitrate, 0.0-0.3g/L of magnesium sulfate and 10g/L of agar for dark culture at 25 ℃ for 16-20 h.
When the grapefruit germplasm pollen is Kui grapefruit germplasm pollen, the method comprises the following steps:
(1) drying pollen: drying the pollen to a water content of 10-20%;
(2) Freezing and storing pollen: sealing and packaging the dried pollen, and storing in liquid nitrogen;
(3) And (3) pollen recovery: taking out pollen from liquid nitrogen, and thawing at 37 deg.C for 5 min;
(4) And (3) pollen rewetting: putting the revived pollen into an ampere bottle, putting the ampere bottle into a closed container containing supersaturated copper sulfate, and rewetting for 2-3 h at room temperature;
(5) In vitro germination: and placing the pollen after rewetting on a pollen in-vitro germination culture medium containing 150g/L of sucrose, 0.1g/L of boric acid, 0.1g/L of potassium nitrate, 1.0g/L of calcium nitrate, 0.3g/L of magnesium sulfate and 0-10g/L of agar for dark culture at 25 ℃ for 16-20 h.
When the grapefruit germplasm pollen is northern brick pomelo germplasm pollen, the method comprises the following steps:
(1) Drying pollen: drying the pollen to a water content of 10-20%;
(2) Freezing and storing pollen: sealing and packaging the dried pollen, and storing in liquid nitrogen;
(3) And (3) pollen recovery: taking out pollen from liquid nitrogen, and thawing at 37 deg.C for 5 min;
(4) in vitro germination: putting the recovered pollen in a pollen in-vitro germination culture medium containing 150g/L of sucrose, 0.1g/L of boric acid, 0.1g/L of potassium nitrate, 0.3-1.0g/L of calcium nitrate, 0.2-0.3g/L of magnesium sulfate and 0-10g/L of agar, and carrying out dark culture at 25 ℃ for 16-20 h.
On the basis, the invention further provides application of the method in improving the ultralow temperature preservation quality of the pomelo germplasm pollen.
the invention has the beneficial effects that:
The invention optimizes and defines key elements suitable for ultralow temperature preservation of the pomelo germplasm pollen, such as water content range, thawing conditions, rewetting conditions, in-vitro germination conditions and the like. By optimizing and limiting the conditions, the germination rate of the grapefruit germplasm pollen after ultralow-temperature preservation is obviously improved.
drawings
FIG. 1 shows the effect of different thawing modes and germination medium components on germination rate of grapefruit pollen after cryopreservation in example 1 of the present invention.
FIG. 2 shows the effect of different rewetting conditions on the germination rate of grapefruit pollen after ultralow temperature preservation in example 2 of the present invention. Wherein: a represents the water content of the grapefruit-10%; b represents a grapefruit-water content of 20%; c stands for Kui pomelo-water content 10%.
Detailed Description
preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 Effect of thawing mode and Germination Medium on pollen germination Rate after ultra-Low temperature preservation
First, experimental material
The material used in the experiment is 3 parts of pomelo germplasm pollen of 'left pomelo', 'Kui pomelo' and 'North brick pomelo' provided by the national fruit tree germplasm citrus orchard (Chongqing).
Second, experimental method and concrete steps
Drying pollen: and (3) drying the collected anthers under an incandescent lamp (at the temperature of about 30 ℃) until the water content is 10-20%.
sealed package liquid nitrogen preservation: after the pollen is properly dried, the pollen is put into a 1.8ml cryopreservation tube (Nunc in the United states) and is put into liquid nitrogen (CBS 3000-AB SERIES) for preservation.
-thawing: the freezing tube that will be equipped with the pollen takes out from the liquid nitrogen container, carries out the thawing, two kinds of modes: (1) washing with tap water at 25 deg.C for about 15min to thaw; (2) rapidly thawing in 37 deg.C water bath for about 5 min.
counting the pollen germination in vitro after ultralow temperature preservation: carrying out in-vitro germination culture on a pollen in-vitro culture medium by adopting a hanging drop germination method or a solid culture medium germination method, wherein the formula of the pollen in-vitro culture medium specifically comprises the following steps: 100-150g/L of sucrose, 0.1g/L of boric acid, 0.1g/L of potassium nitrate, 0.3-1.0g/L of calcium nitrate, 0.0-0.3g/L of magnesium sulfate and 0-10g/L of agar (in vitro germination culture media are numbered as M2, M3, M4 and M5, see Table 1). Isolated pollen cultures were grown overnight at 25 ℃ in the dark. Pollen in vitro germination rate statistics and photographs were performed using a microscope (OLYMPUS SZ X16). When the pollen tube is counted, the pollen tube with the length exceeding half of the diameter of the pollen grains is regarded as viable pollen, 4 fields are randomly selected each time, about 100 pollens are averagely selected in each field, each group is repeated for 4 times, and the pollen germination rate is calculated.
TABLE 1 pollen Germination culture medium formula table
Third, test results
Experiments show that the components of the thawing mode and the recovery culture medium have obvious influence on the in vitro germination rate of the pomelo germplasm pollen in the ultralow-temperature preservation, and the results are shown in figure 1, wherein after a sample with 20% of water content of the pomelo germplasm pollen is preserved by liquid nitrogen and thawed at 37 ℃, the average in vitro germination rate of the pollen is 32-40% on in vitro germination culture mediums (M3 and M5) of 100-doped 150g/L sucrose, 0.1g/L boric acid, 0.1g/L potassium nitrate, 0.3-1.0g/L calcium nitrate, 0.0-0.3g/L magnesium sulfate and 10g/L agar; the sample with the moisture content of 10% Kui pomelo germplasm pollen is preserved by liquid nitrogen, thawed at 25 ℃, and then on an in-vitro germination culture medium (M2 and M3 culture media) containing 150g/L of sucrose, 0.1g/L of boric acid, 0.1g/L of potassium nitrate, 1.0g/L of calcium nitrate, 0.3g/L of magnesium sulfate and 0-10g/L of agar, the in-vitro average germination rate of the pollen is 39-43%; after a sample with the water content of 10% of northern brick pomelo germplasm pollen is preserved by liquid nitrogen, the in-vitro average germination rate of the pollen is 34-42% after the pollen is thawed at 37 ℃ on in-vitro germination culture media (M3 and M4 culture media) containing 150g/L of sucrose, 0.1g/L of boric acid, 0.1g/L of potassium nitrate, 0.3-1.0g/L of calcium nitrate, 0.2-0.3g/L of magnesium sulfate and 0-10g/L of agar.
example 2 Effect of rewetting on the germination Rate of grapefruit pollen
First, experimental material
the variety used in the experiment is 2 parts of pomelo germplasm pollen of 'left pomelo' and 'Kui pomelo' provided by the national fruit tree germplasm orange garden (Chongqing).
Second, experimental method and concrete steps
drying pollen: and (3) drying the collected anthers under an incandescent lamp (at the temperature of about 30 ℃) until the water content is 10-20%. 3 samples of "left grapefruit-water content 10%", "left grapefruit-water content 20%", "Kui grapefruit-water content 10%", were obtained.
sealed package liquid nitrogen preservation: after the pollen is properly dried, the pollen is put into a 1.8ml cryopreservation tube (Nunc in the United states) and is put into liquid nitrogen (CBS 3000-AB SERIES) for preservation.
-thawing: the freezing tube that will be equipped with the pollen takes out from the liquid nitrogen container, carries out the thawing, two kinds of modes: (1) washing with tap water at 25 deg.C for about 15min to thaw; (2) rapidly thawing in 37 deg.C water bath for about 5 min.
-rewetting: taking 10mg pollen from the frozen tube into an ampere bottle by using a dissecting needle, placing the ampere bottle into a closed container containing supersaturated copper sulfate, and rewetting for 3h at room temperature.
Counting the pollen germination in vitro after ultralow temperature preservation: carrying out in-vitro germination culture on pollen in-vitro culture media (M3 and M5) by adopting a hanging drop germination method or a solid culture medium germination method, wherein the formula of the pollen in-vitro culture media is as follows: 100-150g/L of sucrose, 0.1g/L of boric acid, 0.1g/L of potassium nitrate, 0.3-1.0g/L of calcium nitrate, 0.0-0.3g/L of magnesium sulfate and 10g/L of agar. Isolated pollen cultures were grown overnight at 25 ℃ in the dark. Pollen in vitro germination rate statistics and photographs were performed using a microscope (OLYMPUSSZ. times.16). When the pollen tube is counted, the pollen tube with the length exceeding half of the diameter of the pollen grains is regarded as viable pollen, 4 fields are randomly selected each time, about 100 pollens are averagely selected in each field, each group is repeated for 4 times, and the pollen germination rate is calculated.
Third, test results
experiments show that the ultralow temperature preservation of the pomelo germplasm pollen is obviously influenced by the thawing at 37 ℃ and the rewetting treatment. The maximum in vitro average germination rate of the variety pollen used in the experiment can reach 70 percent after ultralow temperature preservation. The result is shown in figure 2, after the sample with the water content of 10 percent of the grapefruit germplasm pollen is preserved by liquid nitrogen, the sample is thawed at 37 ℃ and is subjected to rewetting treatment, and the in-vitro average germination rate of the pollen is 40-60 percent; preserving a sample with 20% of water content of the grapefruit germplasm pollen by using liquid nitrogen, thawing at 37 ℃ and carrying out rewetting treatment, wherein the in-vitro average germination rate of the pollen is 45-52%; the sample with the moisture content of Kui pomelo germplasm pollen of 10% is preserved by liquid nitrogen, thawed at 37 ℃ and subjected to rewetting treatment, and the in vitro average germination rate of the pollen is 70%.
In addition, experiments show that the northern pomelo is thawed for 5min at 37 ℃, the resurrection pollen is placed on a pollen in-vitro germination culture medium containing 150g/L of sucrose, 0.1g/L of boric acid, 0.1g/L of potassium nitrate, 0.3-1.0g/L of calcium nitrate, 0.2-0.3g/L of magnesium sulfate and 0-10g/L of agar for dark culture at 25 ℃ for 16-20 h without a rewetting step, and the highest germination rate can be achieved.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (2)
1. an ultralow temperature preservation method of pomelo germplasm pollen is characterized by comprising the following steps:
When the grapefruit germplasm pollen is the grapefruit germplasm pollen, the method comprises the following steps:
(1) Drying pollen: drying the pollen to a water content of 10-20%;
(2) Freezing and storing pollen: sealing and packaging the dried pollen, and storing in liquid nitrogen;
(3) And (3) pollen recovery: taking out pollen from liquid nitrogen, and thawing at 37 deg.C for 5 min;
(4) And (3) pollen rewetting: putting the revived pollen into an ampere bottle, putting the ampere bottle into a closed container containing supersaturated copper sulfate, and rewetting for 2-3 h at room temperature;
(5) In vitro germination: placing the pollen after rewetting in a pollen in-vitro germination culture medium containing 100-150g/L sucrose, 0.1g/L boric acid, 0.1g/L potassium nitrate, 0.3-1.0g/L calcium nitrate, 0.0-0.3g/L magnesium sulfate and 10g/L agar for dark culture at 25 ℃ for 16-20 h;
(II) when the pomelo germplasm pollen is Kui pomelo germplasm pollen, the method comprises the following steps:
(1) Drying pollen: drying pollen to water content of 10%;
(2) freezing and storing pollen: sealing and packaging the dried pollen, and storing in liquid nitrogen;
(3) and (3) pollen recovery: taking out pollen from liquid nitrogen, and thawing at 37 deg.C for 5 min;
(4) And (3) pollen rewetting: putting the revived pollen into an ampere bottle, putting the ampere bottle into a closed container containing supersaturated copper sulfate, and rewetting for 2-3 h at room temperature;
(5) in vitro germination: placing the pollen after rewetting on a pollen in-vitro germination culture medium containing 150g/L of sucrose, 0.1g/L of boric acid, 0.1g/L of potassium nitrate, 1.0g/L of calcium nitrate, 0.3g/L of magnesium sulfate and 10g/L of agar for dark culture at 25 ℃ for 16-20 h.
2. the method of claim 1, wherein the method is used for improving the ultralow temperature preservation quality of grapefruit germplasm pollen.
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| CN107361057A (en) * | 2017-07-12 | 2017-11-21 | 梁晓 | A kind of Chinese date pollen cryopreservation method |
| CN107593689A (en) * | 2017-10-26 | 2018-01-19 | 中国林业科学研究院亚热带林业研究所 | A kind of thin shell mountain pecan peach flower powder processing and freezing and storing method |
| CN109769801A (en) * | 2019-01-25 | 2019-05-21 | 南京林业大学 | A kind of method that ilex verticillata pollen saves |
| CN110432139B (en) * | 2019-09-12 | 2021-01-15 | 河北省农林科学院昌黎果树研究所 | Method for rapidly collecting sweet cherry pollen and storing sweet cherry pollen at ultralow temperature for long time |
| CN111493065B (en) * | 2020-05-12 | 2021-09-28 | 云南省热带作物科学研究所 | Ultralow-temperature preservation method of moringa oleifera pollen |
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