Pretreatment method for observing or removing embryos from tomato ovules

Abstract

To provide a pretreatment method for observing or extracting embryos in tomato ovules, which enable easy extraction and observation of embryos in tomato ovules.SOLUTION: A pretreatment method for observing or extracting embryos in tomato ovules involves immersing tomato ovules in an aqueous solution of sodium hypochlorite in an amount of 0.2 mass% or more and 0.6 mass% or less.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present invention relates to a pretreatment method for observing or extracting embryos in tomato ovules.

Background Art

[0002] The technology of artificial ovule and embryo culture has been an important technology conventionally practiced in the field of crop breeding. In particular, in distant hybridization for introducing useful traits of different species or genera into the target line, since it frequently occurs that hybrid embryos do not grow normally due to the reproductive isolation mechanism, the ovule and embryo culture technology is particularly required (see, for example, Non-Patent Document 1). In order to efficiently obtain the target line by ovule and embryo culture, it is necessary to select embryos at an appropriate growth stage and use a large amount for culture. Therefore, it is important to observe and discriminate the embryo morphology before directly culturing the ovules or extracting and culturing the embryos from the ovules. Generally, as a method for observing embryo morphology, a method of dissecting ovules under a stereomicroscope can be mentioned (see, for example, Non-Patent Document 2). In tomatoes, immature embryos and abnormal embryos have strong adhesion to the endosperm part, and skilled techniques are required to isolate the embryos without damage. In addition, there are methods for observing embryo morphology by tissue fixation and sectioning methods (see, for example, Non-Patent Document 3) and tissue clearing methods (see, for example, Non-Patent Documents 4, 5, and 6), but these methods require time for sample preparation, take several days to several weeks until the embryos can be observed, and have the disadvantage that the embryos die during the sample preparation stage.

[0003] Sodium hypochlorite is widely used for the purpose of disinfecting seeds and the like. As an example of using a sodium hypochlorite solution to obtain immature embryos, after removing the husk of immature rice seeds and treating them with 70% ethanol for 1 minute, treating them with a 1.5% sodium hypochlorite solution containing 1 drop of Tween-20, and then washing them with sterilized water, a method of aseptically excising immature embryos from the seeds using sterilized forceps under a microscope and using the immature embryos for transformation is known (see, for example, Patent Document 1).

Prior Art Documents

[0004] [License 1] Special Announcement No. 2022-531146 [Non-licensed literature]

[0005] [Non-licensed Document 1] BR Thomas, No. 1, "Efficient Hybridization Between Lycopersicon esculentum and L. peruvianum via Embryo Callus", Theoretical and Applied Genetics, (ドイツ), Springer, July 1981, Volume 59, No. 4, p. 215-219 [Non-licensed Document 2] Lanzhuang Chen, 1 other author, "Cross-compatibility between the Cultivated Tomato Lycopersicon esculentum and the Wild Species L. peruvianum, L. chilense Assessed by Ovule Culture in vitro", Japanese Journal of Breeding, Japan Breeding Society, June 1, 1991, Vol. 41, No. 2, pp. 223-230 [Non-licensed Document 3] Yoji Nitta, "Method for studying crop morphology: Advantages of the method of making slicing of maize のロからミクロまでパラフィンとその", Chronicle of the Japan Crop Society, Japan Crop Society, July 7, 2009, Volume 74, No. 1, p. 95-97 [Non-licensed Document 4] ALEKSANDRA PONITKA, et al., "Cleared-ovule technique used for rapid access to early embryo development in Secale cereale × Zea mays croses," ACTA BIOLOGICA CRACOVIENSIA Series Botanica, (Poland), Polish Academy of Sciences, March 11, 2004, Vol. 46, pp. 133-137. [Non-Patent Document 5] Monika Kwiatkowska and 4 others, “Refinement of a clearing protocol to study crassinucelate ovules of the sugar beet (Beta vulgaris L., Amaranthaceae)”, Plant Methods, (UK), BioMed Central, July 8, 2019, Vol. 15, No. 71 [Non-Patent Document 6] Yuki Sakamoto, et al., "TOMEI (A Novel Clearing Method “TOMEI”)," Microscope, The Japanese Society of Microscopy, September 29, 2016, Vol. 51, No. 3, pp. 150-153. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] As mentioned above, observation and extraction of embryos within ovules are being performed. The object of the present invention is to provide a pretreatment method for observing or removing embryos from tomato ovules, which makes it easier to remove the embryos from the ovules and to observe them. [Means for solving the problem]

[0007] As a result of diligent research to achieve the above objectives, the inventors have discovered that immersing tomato ovules in a specific concentration of sodium hypochlorite alleviates adhesion between the endosperm and the embryo, making it easier to remove the embryo from the ovule. This allows for easy removal of embryos at various developmental stages, from globular embryos to mature embryos, without damaging them. Furthermore, the ovules become transparent, allowing for visual observation of the embryo within. Based on these findings, the inventors have completed the present invention. Accordingly, the present invention is a pretreatment method for observing or removing embryos from tomato ovules, characterized by immersing the ovules in an aqueous sodium hypochlorite solution containing 0.2% to 0.6% by mass of the ovules. [Effects of the Invention]

[0008] The present invention provides a pretreatment method for observing or removing embryos from tomato ovules, which allows for observation of the embryos within the ovules or easy removal of the embryos from the ovules. The extracted embryos can be cultured, germinated, and callus-formed. [Brief explanation of the drawing]

[0009] [Figure 1] This photograph shows a fruit, ovule, and embryo 10 days after pollination. [Figure 2] This photograph shows a fruit, ovule, and embryo 12 days after pollination. [Figure 3] This photograph shows the fruit, ovule, and embryo 14 days after pollination. [Figure 4] This photograph shows the fruit, ovule, and embryo 17 days after pollination. [Figure 5] This photograph shows the fruit, ovule, and embryo 21 days after pollination. [Modes for carrying out the invention]

[0010] The present invention will be described in detail below. In this invention, tomato ovules refer to immature seeds from the time a fertilized egg begins to develop after pollination until it becomes a seed. In the present invention, after pollination, the pericarp of the enlarged fruit is incised and excised with a scalpel or the like, and the ovules adhering to the placenta are detached with forceps or the like, whereby the ovules can be taken out from the fruit. The taken-out ovules are immersed in an aqueous sodium hypochlorite solution containing 0.2% by mass or more and 0.6% by mass or less. When the concentration of the aqueous sodium hypochlorite solution is less than 0.2% by mass, the effect is not sufficient, and when it exceeds 0.6% by mass, the taken-out embryo becomes difficult to grow, which is not preferable. The immersion time is appropriately adjusted according to the size of the ovules. Since the ovules of tomatoes are substantially elliptical as shown in FIGS. 1 to 5, when the length in the longitudinal direction (major axis of the ellipse) of the ovules is L mm and the concentration of the aqueous sodium hypochlorite solution is M% by mass (M is in the range of 0.2 or more and 0.6 or less), (3KL 2 ) / (5M) seconds (K is a constant of 150 or more and 200 or less) is preferable. When the immersion time is short, the effect is not sufficient, and when it is long, the taken-out embryo becomes difficult to grow, which is not preferable. The immersion method is not particularly limited as long as the ovules can be immersed in the aqueous sodium hypochlorite solution. For example, the immersion treatment is performed using a beaker, a tube or the like. At this stage, the ovules become transparent, so the shape of the embryo to be taken out can be observed. However, it is possible to observe the embryo more reliably by taking out the embryo from the ovule and observing it. The method of taking out the embryo from the ovule may be a method of splitting the integument of the ovule and extruding the contents. For example, a method of making a hole in the ovule with forceps and extruding the embryo can be mentioned. When culturing the taken-out embryo, the embryo can be washed with sterilized water or the like and grown using a medium. As the culturing method, a conventionally known method for culturing plant embryos can be used, and there is no particular limitation. For example, a method of aseptically placing and culturing the embryo on a solid medium such as MS medium, LS medium, White medium, B5 medium or the like can be mentioned. The germinated embryo can be grown into a plant body by culturing. Furthermore, embryos that do not germinate during culture and instead form callus can be differentiated again to develop into plants. [Examples]

[0011] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Example Test] (1) On the 10th, 12th, 14th, 17th, and 21st day after pollination, the pericarp of tomato fruits (variety name: Micro-Tom (non-parthenocarpic)) was incised and removed with a scalpel, and the ovules attached to the placenta were detached with tweezers and removed. The tomatoes used are shown in Figures 1 to 5(A). The fruit size was approximately 10mm-12mm on day 10, 12mm-14mm on day 12, 14mm-16mm on day 14, 16mm-17mm on day 17, and 17mm-18mm on day 21. (2) Dispense a 0.6% by mass sodium hypochlorite aqueous solution into a 1.5 ml tube, immerse the removed ovules in it, tap them several times, and let them stand. The immersion times were 600 seconds on day 10, 850 seconds on day 12, 1100 seconds on day 14, 1900 seconds on day 17, and 2400 seconds on day 21 after pollination, and the ovules were removed from the tubes. The size of the ovules (longitudinal direction) was approximately 1.7mm to 2.0mm on day 10, approximately 2.0mm to 2.4mm on day 12, approximately 2.4mm to 2.6mm on day 14, approximately 3.0mm to 3.6mm on day 17, and approximately 3.6mm to 3.8mm on day 21. Figures 1 to 5(B) show the ovules before immersion, and Figures 1 to 5(C) show the ovules after immersion. After immersion, the ovules became transparent, allowing their contents to be observed. (3) Using tweezers, a hole was made in the ovule, and the embryo and endosperm (contents) were squeezed out. (4) The embryo contained within the endosperm of the extruded contents was separated with tweezers and washed with sterile water. (5) This embryo was observed using a stereomicroscope (Olympus Corporation: product name SZ61). The results are shown in Figures 1 to 5(D). (6) These embryos were placed on Murasigeskoog medium and cultured in a cultivation room at a temperature of 25°C with a light period of 16 hours and a dark period of 8 hours. The embryo germination rate is affected by the number of days elapsed since pollination. However, in the method of the present invention, the embryo germination rate was 16 out of 30 samples at day 14, 6 out of 6 samples at day 17, and 14 out of 14 samples at day 21, which were good results. Furthermore, the callus formation rate of embryos is also affected by the number of days elapsed since pollination. In the method of the present invention, callus formation occurred in 8 out of 35 samples on day 10, 1 out of 24 samples on day 12, and 2 out of 30 samples on day 14.

Claims

[Claim 1] A pretreatment method for observing or removing embryos from tomato ovules, characterized by immersing the ovules in an aqueous sodium hypochlorite solution containing 0.2% to 0.6% by mass of the ovules.

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