CN113273570A - Pretreatment method for ultralow-temperature preservation of soybean pollen - Google Patents
Pretreatment method for ultralow-temperature preservation of soybean pollen Download PDFInfo
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- CN113273570A CN113273570A CN202010101879.4A CN202010101879A CN113273570A CN 113273570 A CN113273570 A CN 113273570A CN 202010101879 A CN202010101879 A CN 202010101879A CN 113273570 A CN113273570 A CN 113273570A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/40—Fabaceae, e.g. beans or peas
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
Abstract
The invention discloses a pretreatment method for ultralow-temperature preservation of soybean pollen. The invention provides a method for preserving soybean pollen at low temperature for a long time by pretreatment, which can keep the activity of the soybean pollen in vitro for a long time and successfully apply the pollen preserved at the low temperature to hybridization to obtain hybrid seeds. The invention can break through the time and space limitations of soybean hybridization and provide technical support for solving the problems of soybean hybridization breeding flowering asynchronism and cross-region breeding; the method has important significance for expanding the application range of breeding parents of soybean varieties and widening the genetic basis of the soybean varieties; and technical support can be provided for pollen culture and genetic transformation.
Description
Technical Field
The invention relates to a method for keeping activity of plant pollen, in particular to a pretreatment method for ultralow-temperature preservation of soybean pollen.
Background
Sexual hybridization is the main approach of soybean breeding, and hybridization requires the florescence of parents to meet for smooth operation. Because soybeans are sensitive to light and temperature, the difference of flowering phases among varieties is large, and the varieties which can not meet each other in the flowering phases can not be directly hybridized. In addition, due to the short pollen life under natural conditions, soybean crosses are typically only performed during the co-flowering period of the nearby planted parents, resulting in short and inefficient times for suitable crosses. At present, most of soybean crossbreeding is performed by taking varieties with similar geographical sources and flowering phases as parents, and crossbreeding can be performed only in a short time period every year, so that the quantity of crossbreeding combinations is small, the cost is high, and the genetic basis of bred varieties is relatively narrow. If the activity of the soybean pollen can be maintained in vitro for a long time, the limitation of flowering asynchronism of parents can be broken, the hybridization time and space can be widened, the application range of the parents can be expanded, the breeding efficiency can be improved, the genetic basis of soybean varieties can be effectively widened, and meanwhile, technical support can be provided for the realization of work of engineering hybridization, pollen culture and the like.
Cryopreservation is a common technique for plant pollen preservation. The technology can be divided into three categories, namely a program cooling method, a dehydration method and a vitrification method, wherein the dehydration method has the advantages of low cost, simple and convenient operation and the like. The main procedure of the dehydration method comprises two steps of moderate dehydration of plant materials and then rapid liquid nitrogen input for preservation. The dehydration method can be classified into a drying method, a preculture-dehydration method and an embedding-dehydration method according to the manner of dehydration of the material. No matter which method is used, the cells are easily damaged in the cooling process. As the temperature is reduced, the water in the cell freezes, and the formed ice crystals further damage cell membranes and organelles, thus causing the ice crystals to be damaged. In addition, excessive dehydration may increase intracellular electrolyte concentration, form disulfide bonds between protein molecules, destroy the structure of proteins and enzymes, and damage the integrity of cell membranes, thereby causing cell damage. Therefore, for the drying method, it is a key technical point to adjust the water content of the tissue to be preserved according to the species characteristics before cryopreservation and to avoid the tissue cells from being damaged.
Previous researches show that a drying method is relatively suitable for pretreatment of pollen to be stored, but the pretreatment methods of different plant types of pollen before low-temperature storage are greatly different, and the thawing method also has certain influence on the activity of the pollen. For soybean, an important economic plant, the method for carrying out cryopreservation pretreatment on pollen of the soybean is not reported at present.
Disclosure of Invention
The invention aims to provide a method for pretreating ultralow-temperature-stored soybean pollen by integrally dehydrating flowers so as to keep the vigor of the pollen, and provides a successful case for carrying out cross breeding by using the pollen after ultralow-temperature storage.
Firstly, the invention provides a pretreatment method for ultralow temperature preservation of soybean pollen, which comprises the following steps:
(A1) collecting complete flowers of soybean flowers in the initial flowering period;
(A2) and (C) carrying out integral drying treatment on the complete flowers collected in the step (A1) to enable the water content of the flowers to reach 4-45%.
In the step (a2), the water content is more specifically 20% to 42%. In the embodiment of the present invention, the water content is optimally controlled to be 28.78%.
In the step (A1), the flower collection time may be 6-10 am.
The drying method comprises the steps of placing the whole flowers in a drying box for drying treatment, keeping the temperature of the drying box at 35-39 ℃ (more specifically at 37 ℃) and keeping the humidity at 5% -45% (more specifically at 25%).
The drying time is 6-14 h. In the embodiment of the present invention, the optimum time for the drying treatment is 8 hours.
The initial stage of the soybean flower specifically refers to the stage that the elongation of the corolla exceeds the height of the calyx, the top of the flag flap has obvious cracks and the abdominal suture is slightly opened.
In a second aspect, the present invention provides a method for maintaining soybean pollen activity, comprising the steps of:
(B1) obtaining pre-treated soybean flowers according to the method described hereinbefore;
(B2) wrapping the soybean flowers prepared in the step (B1) with tinfoil, and freezing and storing at ultralow temperature.
In the method, the low-temperature cryopreservation is liquid nitrogen preservation, or preservation at-80 ℃ after liquid nitrogen cryopreservation.
The method comprises the step of thawing (B2) the frozen flower to restore soybean pollen activity; the thawing method is as follows (C1), (C2) or (C3):
(C1) washing and unfreezing the soybean flowers wrapped by the tinfoil under tap water at 15-25 ℃;
(C2) thawing the soybean flowers wrapped by the tinfoil in a water bath at 33-37 ℃;
(C3) and naturally thawing the soybean flowers wrapped by the tinfoil indoors at normal temperature.
In a third aspect, the invention also provides the use of any one of the methods described above in soybean cross breeding.
The invention also protects the application of any one of the methods in soybean pollen culture.
The soybean is not limited to a specific variety. In the examples, the soybean is specifically black river No. 43.
The invention provides a technical scheme for pretreatment, pollen low-temperature preservation and unfreezing before soybean pollen ultralow-temperature preservation, and forms a soybean pollen ultralow-temperature preservation technology. The invention can break through the time and space limitations of soybean hybridization, provides technical guarantee for solving the problem of soybean hybridization flowering asynchronism and cross-region breeding, has important significance for expanding the application range of soybean breeding parents and widening the genetic basis of soybean varieties, and also can provide technical support for pollen culture and genetic transformation.
Drawings
FIG. 1 is a graph comparing pollen germination rates of soybean flowers at different development stages.
FIG. 2 is a graph showing the effect of soybean flower drying pretreatment method and time on the germination rate of ultra-low temperature stored pollen.
FIG. 3 is a graph showing the moisture content of the soybean flowers under different drying periods.
FIG. 4 is a graph showing the effect of drying pretreatment time on pollen germination rate before and after cryopreservation of soybean flowers.
FIG. 5 is a graph showing a comparison of germination rates of soybean pollen after drying treatment of flowers at different times before and after cryopreservation.
FIG. 6 is a graph showing the effect of soybean pollen after being stored at ultralow temperature for one year on the survival rate of hybridization.
FIG. 7 is a graph showing the effect of soybean pollen after being stored at ultralow temperature for one year on true breeding rate.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up 3 replicates and the results averaged.
Example 1 establishment of Soybean pollen cryopreservation pretreatment method
Materials and methods
1. Soybean variety
Black river No. 43, approved serial number: bean of black court 2007011; and (4) breeding unit: the black river discharge of the academy of agricultural sciences of Heilongjiang province. Purple flower, long leaf and gray hair in 113d of spring sowing growth period in black river area.
Gold source No. 55, approval serial number: a national audit bean 2013001; and (4) breeding unit: the black river discharge of the academy of agricultural sciences of Heilongjiang province. In the black river, the growth period of the spring sowing is 118d, white flowers, long leaves and grey hair.
2. Planting conditions in field
The field test is carried out at the Heihe academy of agricultural sciences of Heilongjiang province (northern latitude 50 degrees 15 ', east longitude 127 degrees 27', altitude 168.5m), the seeds are respectively sowed in 5 and 9 days in 2018 and 5 and 11 days in 2019, the row length is 1.5m, the plant spacing is 7cm, and the row spacing is 60 cm. 20 rows of seeds are sown in each variety, and the field management is the same as that of a local field.
3. Method for measuring flower water content and pollen germination rate
(1) Method for measuring water content of flower
The Fresh Weight (FW) of the flower to be measured is weighed by a model ZX224ZH analytical balance, and then the flower is dried in an oven at 110 ℃ until the weight is constant (dry weight, DW). The water content (WC,%) was calculated by the following formula.
WC-water content (%), FW-fresh weight, DW-final dry weight.
(2) Method for measuring pollen germination rate
Placing the pollen to be detected in a germination culture medium, culturing for 20min at room temperature (25 +/-2 ℃), observing under a microscope, randomly selecting 6 visual fields, calculating the pollen germination rate of each visual field, and calculating the average value of the 6 visual fields. The standard of pollen germination is that the length of a pollen tube exceeds twice the diameter of pollen, and the calculation formula is as follows:
GR (germination rate) is the pollen germination rate;
a is the total number of pollen visible in each field;
b is the number of germinating pollen.
The formula of the pollen germination culture medium is as follows: 19.2% (mass percent) sucrose, 68.9mg/L GA30.015% (mass percent) H3BO30.05% (mass percent) of CaCl27.5% (mass percentage content) PEG-4000; the solvent is water.
Second, establishment of soybean pollen drying pretreatment method
Determination of optimal flower collection period
After the soybean variety black river 43 is blooming, the following 4 flowers with different degrees of openness are respectively collected, the pollen activity is compared, and the optimal collection period is determined.
Soybean flowers were divided into the following 4 developmental stages according to the degree of openness:
(1) and (3) slightly exposing corolla: the corolla has emerged from the calyx, its color is distinguishable, but is lower in height than the calyx.
(2) And (3) elongation period of corolla: the corolla has elongated and exceeded the height of the calyx, but the flag petals remain tight.
(3) Early blooming period of flowers: the elongation of the corolla exceeds the height of the calyx, the top of the flag valve has obvious cracks, and the ventral suture is slightly opened.
(4) Full bloom stage of the flower: the flag valve, the wing valve and the keel valve are all unfolded.
Pollen activity was tested in the 4 stages without any manual pretreatment, 12 flowers were tested per stage and 3 fields were observed randomly per flower. The result (figure 1) shows that the anther of the corolla in the micro-emergence period has no loose powder, the pollen has not completely developed, and the germination rate is low; the pollen germination rate is still low in the elongation period of the corolla; the pollen maturity of the flower at the initial blooming stage is high, the moisture content is proper, and the germination rate is highest; in the development period of the flower, the pollen is scattered, and the pollen with high germination rate is difficult to collect. The results show that the pollen activity of the flower in the initial flowering phase is the best, and the germination rate is the highest. This stage is the best stage for collecting pollen for long-term storage.
The results of the detection of a large number of collected flowers at different time intervals every day show that the flowers at the early flowering stage at 6-8 am are more in number and high in pollen activity, and are the best period for collecting pollen.
In order to facilitate the operation, a whole flower collection mode is adopted when pollen is collected. The pollen is wrapped by the petals and the calyx, so that the phenomena of too fast dehydration, oxidation inactivation and the like caused by direct exposure in the air can be avoided, and the pollen is convenient to store and hybridize.
In summary, the pollen activity is best when the whole flower at the initial stage is collected at the best time, which is most beneficial for low-temperature preservation. For different regions, the optimal collection time can be flexibly adjusted at different times and months, and is determined according to the flower state (namely, the time with more flowers and higher pollen activity at the initial stage is selected); in the middle 7-8 of the middle of the month in the black river area of the experimental location, 6-8 am is the best time; for Beijing, pollen collected at 6-10 am generally has good activity.
(II) comparison of different drying methods
(A) Drying in an oven: placing the collected complete flowers in an LICHEN101-3BS drying box for 1-14h, monitoring the temperature and humidity in the drying box by using a hygrothermograph, and adjusting the humidity in the drying box by using a silica gel drying agent to control the humidity to be 25% (+ -20%) and the temperature to be 37 ℃ (+ -2 ℃);
(B) drying an incandescent lamp: the whole flower is laid on parchment paper at room temperature of 25 ℃ (± 4 ℃) and humidity of 55% (± 15%), and placed under a 40W incandescent lamp for 1-14h, and the lamp tube is 15cm away from the flower.
(C) Naturally drying: the whole flowers are laid on parchment paper and placed in a shady place for 1-14h at the indoor temperature of about 25 ℃ (± 4 ℃) and the humidity of 55% (± 15%).
Taking the complete soybean flowers collected at 6-8 am and in the initial stage, drying by adopting the three methods, and detecting the pollen germination rate. The results show that the pollen germination rate after oven constant-temperature drying is higher than that of the other two drying modes, and the temperature and humidity controllability of the oven drying method is higher than that of incandescent lamp drying and natural drying, so that the method is the best pollen drying mode (figure 2).
(III) determination of flower drying pretreatment time and optimum moisture content
Taking the complete flowers of the black river 43 collected at 6-8 am and in the initial growth stage, drying the flowers in a drying oven with the humidity of 25% (+ -20%) and the temperature of 37 ℃ (+ -2 ℃), and adjusting the humidity in the drying oven by increasing or decreasing the amount of silica gel drying agent. Samples were taken every 2h and the water content was determined. And (3) respectively taking flower samples while measuring the water content, and measuring the pollen germination rates under the following two states: (1) direct measurement; (2) wrapping with tinfoil, adding liquid nitrogen, quickly freezing with liquid nitrogen for 72h, thawing, and measuring.
The statistical result of the moisture content (figure 3) shows that the moisture content of the soybean flowers rapidly decreases with the extension of the drying time and reaches constant weight after 14 hours.
The result of pollen germination rate measurement (figure 4) shows that the germination rate of the pollen 0-6h before drying treatment is stable above 90%, and is reduced from 8h, but is still maintained above 70% until 14 h. For activity of the pollen thawed after being dried and quickly frozen for 72h by liquid nitrogen, flowers (with water content of 84.49-62.52%) which are dried within 4h completely lose germination capacity after being frozen by liquid nitrogen, and the germination rate is 0. Along with the extension of the drying pretreatment time, the germination rate of the thawed pollen of the flower frozen by liquid nitrogen is rapidly improved, the germination rate of the thawed pollen dried for 6h (with the water content of 41.65%) exceeds 80%, the germination rate of the thawed pollen dried for 8h (with the water content of 21.28%) is highest, and after 8h, the germination rate is in a descending trend along with the extension of the drying time, but is kept at a higher level (> 70.00%) before 14 h.
In conclusion, controlling the moisture content of the pollen before the ultra-low temperature freezing storage is very important for keeping the activity of the pollen after the ultra-low temperature freezing storage; if the water content is too high (for example, the drying time is less than 4h, the water content is 84.49% -62.52%), even if the activity of the pollen before freezing is high, the flowers lose the germination capacity after ultralow temperature storage; if the water content is too low (the drying time is too long), the activity of the pollen before the ultralow-temperature freezing storage is possibly reduced, and the germination capacity of the flowers after the ultralow-temperature freezing storage is also influenced. In the experiment, the pollen is dried for 6-14h before being frozen, the water content is controlled to be 41.65-4.72%, and the germination rate of the thawed pollen is ideal.
(IV) comparison of thawing methods for pollen preserved at ultralow temperature
Pollen is frozen at ultralow temperature and is required to be thawed and restored to activity before being used for hybridization or other tests. In order to screen suitable thawing methods, 3 thawing methods were set up in this experiment: (1) washing and unfreezing the tinfoil wrapped flowers in tap water, namely washing and unfreezing the tinfoil wrapped flowers in the tap water at 20 ℃ (± 5 ℃); (2) the water bath method comprises thawing the flowers wrapped by tinfoil in water bath at 35 deg.C (+ -2 deg.C); (3) naturally thawing at room temperature (25 ℃ +/-4 ℃).
The results show that the difference of the germination rates of the pollens thawed by the three methods is small, wherein the tap water thawing method is more convenient to operate compared with a water bath method and has a higher speed compared with a natural thawing method. The natural thawing method is simple and easy to implement, and is suitable for field hybridization.
In conclusion, the pretreatment, subsequent preservation and utilization method for soybean pollen ultralow temperature preservation, which is established by the invention, comprises the following steps:
(1) collecting the whole flowers: collecting complete soybean flowers at the initial stage; the specific collection time may be 6-10 am.
(2) And (3) drying treatment: drying the collected whole soybean flowers in a drying box to control the water content to be 4-45% (the corresponding drying time can be 6-14h, preferably 8 h); the oven was held at 37 ℃ (± 2 ℃) and 25% (± 20%) humidity.
(3) And (3) ultra-low temperature cryopreservation: wrapping the dried flower with tinfoil, and directly putting into liquid nitrogen (-196 deg.C) for long-term storage or freezing with liquid nitrogen and transferring into-80 deg.C refrigerator for long-term storage.
(4) Taking out the flowers wrapped by the tinfoil, and adopting any one of the following methods for unfreezing: (A) washing and unfreezing the tinfoil wrapped flowers in tap water, namely washing and unfreezing the tinfoil wrapped flowers in the tap water at 20 ℃ (± 5 ℃); (B) the water bath method comprises thawing the flowers wrapped by tinfoil in water bath at 35 deg.C (+ -2 deg.C); (C) naturally thawing at room temperature (25 ℃ +/-4 ℃).
Third, germination rate detection and hybridization verification of long-term cryopreserved pollen
(I) detection of germination Rate
Wrapping 20 parts of flowers which are dried for 6-14h by the method with tinfoil, preserving the flowers in liquid nitrogen for 3 months, transferring the flowers to a refrigerator at-80 ℃ for further preservation for 9 months, then unfreezing the frozen flowers, and detecting the pollen germination rate. And comparing the germination rate data with the germination rate data of flowers which are not frozen and have the same size in the current sampling year. The results (fig. 5) show that the germination rate of the pollen preserved at ultralow temperature for one year is not much different from that before freezing or slightly increases, which indicates that the activity of the soybean pollen is effectively maintained after the pollen is preserved by freezing after being properly dried.
(II) hybridization verification
Wrapping the flower dried for 8h with tinfoil, placing in liquid nitrogen for one year, thawing, performing hybridization experiment between thawed pollen and gold source No. 55 (female parent), and taking pollen pollination result of fresh flower of male parent Heihe No. 43 as control. The calculation formula of the hybridization survival rate (%) is as follows:
F(％)＝(PN/HN)×100％ (3)
f-hybridization survival rate; PN-number of surviving pods; HN-number of hybrid pods.
The results (FIGS. 6 and 7) show that the pollen after one year of cryopreservation was used for hybridization, and the survival rate and true hybrid rate were slightly higher than those of the pollen before freezing, and again, it was confirmed that the activity of soybean pollen was maintained by cryopreservation.
In conclusion, the pollen activity can be effectively maintained by collecting whole flowers of soybeans in the initial flowering stage, drying the whole flowers, and then freezing the whole flowers at ultralow temperature. The method can effectively solve the time and space limitations of soybean hybridization, solve the technical problems of soybean hybridization flowering asynchronism and cross-region breeding supply, and expand the application range of soybean variety breeding parents.
Claims (10)
1. A pretreatment method for soybean pollen cryopreservation comprises the following steps:
(A1) collecting complete flowers of soybean flowers in the initial flowering period;
(A2) and (C) carrying out integral drying treatment on the complete flowers collected in the step (A1) to enable the water content of the flowers to reach 4-45%.
2. The method of claim 1, wherein: in the step (A2), the water content is controlled to be 20-42%.
3. The method of claim 1 or 2, wherein: in the step (A1), the collection time of the flower is 6-10 am.
4. A method according to any of claims 1 to 3, characterized by:
the drying method comprises the step of drying the flowers in a drying box, wherein the temperature of the drying box is kept at 35-39 ℃, and the humidity is kept at 5-45%.
5. The method of claim 4, wherein: the drying time is 6-14 h.
6. A method for maintaining soybean pollen activity, comprising the steps of:
(B1) obtaining pre-treated soybean flowers according to the method of any one of claims 1 to 5;
(B2) wrapping the soybean flowers prepared in the step (B1) with tinfoil, and freezing and storing at ultralow temperature.
7. The method of claim 6, wherein: the low-temperature cryopreservation is liquid nitrogen preservation, or preservation at-80 ℃ after liquid nitrogen cryopreservation.
8. The method of claim 6 or 7, wherein: the method comprises the step of thawing (B2) the frozen flower to restore soybean pollen activity; the thawing method is as follows (C1), (C2) or (C3):
(C1) washing and unfreezing the soybean flowers wrapped by the tinfoil under tap water at 15-25 ℃;
(C2) thawing the soybean flowers wrapped by the tinfoil in a water bath at 33-37 ℃;
(C3) and (4) placing the soybean flowers wrapped by the tinfoil indoors for thawing at normal temperature.
9. Use of the method of any one of claims 1 to 8 in soybean cross breeding.
10. Use of the method of any one of claims 1 to 8 in soybean pollen culture.
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| CN101926323A (en) * | 2010-07-29 | 2010-12-29 | 中国热带农业科学院橡胶研究所 | Ultra-low temperature preservation method for oil palm pollen |
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| Lambrecht et al. | Combined drought and episodic freezing effects on seedlings of low‐and high‐elevation subspecies of sagebrush (Artemisia tridentata) | |
| Gibernau et al. | Pollen viability and longevity in two species of Arum | |
| Szalay et al. | Influence of the changing climate on flower bud development of apricot varieties | |
| Skre | Growth of mountain birch (Betula pubescens Ehrh.) in response to changing temperature | |
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