A method for test tube pollination and fertilization of amaryllis ovules

Abstract

The invention provides a method for in vitro pollination and fertilization of amaryllis ovules. The process steps include: sterilizing the transparent flower buds of the male parent of amaryllis, taking the unpolluted anthers and drying them in a sterile beaker to scatter the pollen. Collecting sterile pollen and treating at 4°C for 3 days. After sterilizing, the ovary of the female parent of amaryllis that opened on the same day is separated from the carpel and the ovules are fully exposed, inoculated into a solid and liquid double-layer culture medium, and a large amount of sterile pollen is spread, the medium is sealed with an airtight sealing film, and the medium is cultured in complete darkness for 3 days. The ovules are separated and transferred to a solid MS culture medium, the medium is sealed with a semi-permeable membrane, and the medium is cultured in complete darkness for 45 to 60 days. The fertilized ovules are selected and moved to the medium for culture under light for 30 days. The hybrid zygotic embryos germinate, and finally the hybrid offspring is obtained. The invention obtains the hybrid of amaryllis for the first time through the in vitro fertilization technology, the method is simple and easy to operate, and can effectively overcome the pre-fertilization obstacles of amaryllis distant hybridization and self-pollination with some varieties, and provides technical support for the hybrid breeding of amaryllis.

Description

Technical Field

[0001] The invention belongs to the technical field of plant biotechnology-assisted hybrid breeding, and particularly relates to a method for in vitro test tube pollination and fertilization of amaryllis. Background Art

[0002] Hippeastrum spp. is a perennial bulbous ornamental plant of the Amaryllidaceae family and the genus Hippeastrum. It is native to Central and South America. It has rich flower shapes, bright colors, large flowers and beautiful leaves. It has extremely high ornamental value and is widely used in potted plants, cut flowers and garden viewing, and is deeply loved by people.

[0003] Conventional hybrid breeding is the main method for selecting new commercial varieties of Amaryllis. However, the genetic background of Amaryllis is complex, and hybridization or self-incompatibility often occurs between native species and commercial species. At present, in the work of Amaryllis hybrid breeding, reproductive isolation is mainly broken through technologies such as stigma cutting, assisted pollination, and embryo rescue, but there are still a large number of combinations that cannot successfully obtain hybrid offspring.

[0004] Ovule in vitro pollination and fertilization refers to the method of exposing ovules in vitro, pollinating, fertilizing, and fruiting to obtain hybrids. It is one of the most effective ways to overcome hybridization barriers. Establishing an in vitro pollination and fertilization technology system for Amaryllis can effectively overcome the pre-fertilization barriers of distant hybridization and self-pollination with some varieties of Amaryllis, provide technical support for distant hybrid breeding and germplasm innovation of Amaryllis, have great application value in hybrid breeding and improved variety breeding, and also have important theoretical significance for the study of the physiological and biochemical mechanisms of plant fertilization and early embryonic development.

[0005] Purpose of the Invention

[0006] In view of the shortcomings of the existing amaryllis breeding technology system, the purpose of the present invention is to provide a method for in vitro pollination and fertilization of amaryllis ovules, to solve the problem of hybrid incompatibility between some existing amaryllis varieties, to provide technical support for amaryllis germplasm innovation and development and utilization, and to provide a reference for the establishment of in vitro fertilization systems for other plants. Summary of the invention

[0007] The technical solution adopted by the invention is:

[0008] Collect the fully colored buds of male parent Amaryllis during the flowering period, soak them in 5% sodium hypochlorite for 10 minutes on a clean bench, and rinse them with sterile water three times. Open the buds and take out the anthers that have not yet shed pollen. Use sterile absorbent paper to fully absorb the surface moisture of the anthers, move them into a sterile beaker, seal them with a semi-permeable membrane, and place them in a silica gel desiccator. After 24 to 48 hours, the anthers will split longitudinally and shed pollen. Collect the dried pollen and place them in a sterile centrifuge tube and treat them at 4°C for 3 days.

[0009] After the flower buds of Amaryllis mother plant are fully translucent, remove the anthers and put them in bags for isolation. On the day when the flowers bloom, remove the perianth pieces, stamens, stigmas and styles. On the clean bench, treat the ovary with 5% sodium hypochlorite for 20 minutes and rinse with sterile water three times. Use a sharp sterile scalpel to cut off the outer wall of the ovary to the depth of the dorsal suture, cut the ovary longitudinally, divide it into three carpels, and fully expose the ovules at the ventral suture of the mid-axis.

[0010] Select ovary explants with ovules neatly arranged on the central axis and inoculate them in a solid-liquid double-layer culture medium. Keep the ovary wall downward in contact with the solid culture medium. The central axis and ovules face upward and do not touch the liquid culture medium. Sprinkle sterile pollen that has been dried at low temperature on the surface of the ovules. Seal with airtight sealing film to increase the humidity in the culture dish. The culture room temperature is 25±2℃ and culture is in complete darkness.

[0011] After 3 days of sterile in vitro pollination, the ovary wall and central axis were removed, and the ovules were separated one by one and transferred to the solid medium MS + 30g / L sucrose + 7.5g agar, sealed with a semi-permeable membrane, and cultured in complete darkness. To reduce the browning of the explants, fresh culture medium was replaced every week. As the culture time increased, the ovule volume gradually increased, the middle part gradually bulged, and the color gradually deepened, from colorless and transparent to milky white and brown. After 45 to 60 days of culture, zygotic embryos can be clearly observed in the in vitro ovules.

[0012] The fertilized ovules were selected and transferred to light culture, with 16 h of light and 8 h of darkness per day, and the light intensity was 36 μmol·m -2 ·s -1 After 30 days of continuous culture, zygotic embryos germinated and hybrid offspring were obtained.

[0013] In the above scheme, the solid-liquid double-layer culture medium refers to: solid culture medium composition MS+15g sucrose+7.5g agar. Sterile liquid culture medium with composition of MS+15g sucrose+0.01g / L boric acid+0.15g / LCaCl2 is dripped dropwise on the surface of the solid culture medium to form a thin liquid layer on the surface of the solid culture medium.

[0014] In the above scheme, the large-scale spreading of sterile dry pollen refers to: spreading a large amount of sterile pollen near the ovary axis until the ovary axis and the surface of the ovules are completely covered.

[0015] Advantages and positive advantages of the present invention are:

[0016] 1. The present invention successfully obtained Amaryllis hybrids for the first time through in vitro fertilization, providing technical support for Amaryllis distant hybridization and germplasm innovation;

[0017] 2. During in vitro pollination, the solid-liquid double-layer culture method was used to increase the humidity in the culture dish and the pollen tube germination rate, further improving the fertilization efficiency of the Amaryllis ovules. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 Developmental state of flower bud explants. Among them, A is the partially colored flower buds of the female variety of Amaryllis 'Red Lion', which has not yet matured. Anthers and pollen cannot be collected during this period; B is the fully colored flower buds of the female variety of Amaryllis 'Red Lion', which is the best period to collect male pollen; C is the fully colored flower buds of the male variety of Amaryllis 'Dancing Queen'. At this stage, the ovary is mature and can be used to separate ovules.

[0020] Figure 2 Sterile pollen preparation. Among them, A is the anther inoculated on the surface of MS solid culture medium after sterilization, which cannot shed pollen normally; B is the anther placed in a sterile beaker after sterilization and dried naturally for 48 hours, which can shed a small amount of pollen; C is the anther placed in a sterile beaker after sterilization and dried in a silica gel dryer for 48 hours, which can shed a large amount of pollen.

[0021] Figure 3 In vitro pollination of ovules. A is the ovary explant inoculated on the surface of the culture medium after the ovules are fully exposed; B is the explant in in vitro pollination of ovules.

[0022] Figure 4 Ovule separation and culture. A is the colorless and translucent ovule separated after in vitro pollination; B is the milky white ovule that begins to swell after 30 days of culture; C is the brown and dead ovule.

[0023] Figure 5 Hybrid zygotic embryo formation.

[0024] Figure 6 The hybrid offspring are sterile seedlings. DETAILED DESCRIPTION

[0025] To make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the following is a brief description of the present invention in conjunction with the attached Figures 1 to 6 The present invention is further described in detail with reference to the accompanying drawings and specific embodiments.

[0026] Example 1

[0027] This example uses an in vitro test tube pollination and fertilization method to obtain amaryllis hybrids. The amaryllis variety 'Red Lion' is used as the female parent and 'Dancing Queen' is used as the male parent. After sterile pollen preparation, ovule exposure, sterile pollination, ovule separation and in vitro culture, hybrid test tube seedlings are finally obtained. The specific steps are as follows:

[0028] (1) Sterile pollen preparation

[0029] Select the robust Amaryllis variety 'Red Lion' and collect the fully colored buds during the flowering period (attached Figure 1 A, B). On a clean bench, soak the flower buds in 5% sodium hypochlorite for 10 minutes and rinse with sterile water three times. Open the flower buds and take out the anthers that have not yet shed pollen. Use sterile absorbent paper to dry the surface moisture of the anthers, move them into a sterile beaker, seal with a semipermeable membrane, and dry them in a silica gel dryer for 24 to 48 hours. The anthers will split longitudinally and shed pollen. Different anther treatment methods have a great influence on pollen quality and vitality. The anthers inoculated on the surface of the solid culture medium do not split, and no pollen can be obtained (attached Figure 2 A). The anthers placed in a sterile beaker and dried naturally opened normally, but the amount of pollen was small and a large amount of pollen adhered to the surface of the anthers (attached Figure 2 B), pollen collection is difficult. Place sterile pollen in a silica gel desiccator, split the anthers longitudinally, and obtain a large amount of pollen (attached Figure 2 C). The viability of fresh pollen collected by the two methods was 76.85% and 79.72%, respectively. After the anthers were shed, the dried pollen was collected on a clean bench and placed in a sterile centrifuge tube and treated at 4°C for 3 days.

[0030] Table 1 Effects of pollen treatment methods on pollen yield and vitality

[0031] Pollen treatment methods Anther status Pollen count Pollen vitality (%) Culture medium powder Anthers not dehisced none —— Beaker powder, natural drying Anther dehiscence A small amount of pollen 76.85±9.0 Beaker powder, silica gel drying Anther dehiscence Lots of pollen 79.72±3.95

[0032] (2) Exposing ovules

[0033] The buds of Amaryllis female plant 'Dancing Queen' are fully colored (attached Figure 1 C), remove anthers and bag for isolation. On the day the flowers bloom, remove the perianth, stamens, stigma and style. On a clean bench, soak the ovary in 5% sodium hypochlorite for 20 minutes and rinse with sterile water three times. Use a sharp sterile scalpel to remove the ovary wall to the depth of the dorsal suture, cut the ovary longitudinally to separate it into three carpels, and fully expose the ovules at the ventral suture (attached Figure 3 A).

[0034] (3) Aseptic pollination

[0035] The ovary with ovules neatly arranged on the central axis was selected as the explant and inoculated in a solid-liquid double-layer culture medium. During inoculation, the ovary wall was kept in contact with the solid culture medium, with the central axis and ovules facing upward. A large amount of sterile pollen treated with low temperature drying was spread on the surface of the ovules, completely covering the central axis of the ovary and the surface of the ovules (with attached Figure 3 B). Seal with airtight sealing film, culture in the incubator at 25±2℃ in complete darkness for 3 days.

[0036] The culture medium has a great influence on the in vitro fertilization of Amaryllis ovules (Table 2). The embryo rate of ovules inoculated in solid culture medium is lower than that of solid-liquid double-layer culture. This may be because the liquid culture medium increases the humidity of the culture environment and is more conducive to the elongation of pollen tubes. In solid-liquid double-layer culture, different liquid culture medium components have a certain effect on in vitro pollination and fertilization of ovules. The best liquid culture medium is MS+15g / L sucrose+0.01g / L boric acid+0.15g / LCaCl2, with an embryo rate of 21.67%.

[0037] Table 2 Effect of culture medium on in vitro fertilization of Amaryllis

[0038]

[0039] (4) Ovule isolation and in vitro culture

[0040] After 3 days of sterile in vitro pollination, remove the ovary wall and central axis on a clean bench, separate the ovules one by one and transfer them to a solid medium MS + 30g / L sucrose + 7.5g agar, seal with a semipermeable membrane, culture in complete darkness, and replace with new medium every week. As the culture time increases, the ovule volume gradually increases, the middle part gradually bulges, and the color gradually deepens, changing from colorless and translucent (with Figure 4 A) turns to milky white (with Figure 4 B), brown (with Figure 5 After 45-60 days of culture, zygotic embryos can be clearly observed in the isolated ovules (attached Figure 5 ). During the culture of Amaryllis ovules, the ovules in vitro showed different degrees of browning. Mild browning did not affect IVF and zygotic embryo development. Some severe browning would directly lead to the death of the explants (see Appendix). Figure 4 C). Timely replacement of fresh culture medium is helpful to reduce the degree of ovule browning. Dark conditions can significantly reduce the degree of ovule browning (Table 3). Under completely dark culture conditions, the ovule browning rate was 56.11%.

[0041] Table 3 Effects of light and dark conditions on the browning of Amaryllis ovules

[0042] Culture conditions Browning rate (%) dark 56.11±6.11b Light 94.44±2.77a

[0043] (5) Zygotic embryo germination

[0044] The culture with zygotic embryos was selected and transferred to fresh MS medium and cultured under light conditions, with 16 h of light and 8 h of darkness per day, and the light intensity was 36 μmol·m -2 ·s -1 After 30 days of continuous culture, the embryos germinated and hybrid offspring were finally obtained (see Figure 6 ).

[0045] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for in vitro pollination and fertilization of Hippeastrum ovules, characterized in that: The fully colored buds of Amaryllis were soaked in 5% sodium hypochlorite for 10 min, and the buds were peeled to take the unpolluted anthers; the surface moisture of the anthers was absorbed by sterile absorbent paper and transferred into a sterile beaker, which was sealed with a semipermeable membrane and shed in a silica gel desiccator for 24-48 h; the dried pollen was collected and treated at 4 ℃ for 3 days; Take the ovary of the amaryllis that opened on the same day, sterilize with 5% sodium hypochlorite, use a sharp sterile scalpel to remove part of the ovary outer wall, cut and separate the carpels longitudinally, and fully expose the ovules; inoculate the ovary explants in a solid and liquid double-layer culture medium; spread sterile pollen on the surface of the ovules, seal with an airtight sealing film, and culture in complete darkness at 25±2°C for 3 days; separate the ovules one by one and transfer to a solid culture medium MS+30g sucrose+7.5g agar, seal with a semi-permeable membrane, and culture in darkness; the composition of the solid and liquid double-layer culture medium is: on the solid culture medium MS+15g / L sucrose+7.5g agar, drip a sterile liquid culture medium MS+15g sucrose+0.01 g / L boric acid+0.15g / L CaCl2 drop by drop to form a thin layer of liquid on the surface of the solid culture medium; when inoculating the ovary explants, keep the ovary wall downward and in contact with the solid culture medium, and the central axis and ovules upward and not in contact with the liquid culture medium; After in vitro pollination, the ovules were cultured for 45-60 days until zygotic embryos were visible. The fertilized ovules were transferred to the light environment for culture, with 16 h of light and 8 h of darkness per day, and a light intensity of 36 μmol·m -2 ·s -1 After 30 days of continuous culture, zygotic embryos germinated and hybrid offspring were finally obtained.

2. The method for in vitro pollination and fertilization of Hippeastrum ovules according to claim 1, characterized in that: The specific method for exposing the ovules is as follows: using a sterile scalpel to remove part of the outer wall of the ovary to the depth of the dorsal suture, longitudinally cutting and separating the ovary into three carpels, and selecting explants with ovules neatly arranged on the ventral suture of the mid-axis.

3. The method for in vitro pollination and fertilization of Hippeastrum ovules according to claim 1, characterized in that: The specific method of spreading sterile pollen on the surface of ovules is: spreading a large amount of pollen near the ventral suture of the ovary axis until the ovary axis and the surface of the ovule are completely covered.

Images