Method for cultivating microspores from individual buds to obtain doubled haploids in invitro microspore culture of brassicarapa l. plants

Abstract

FIELD: biotechnology.SUBSTANCE: method for cultivating microspores from individual buds to obtain doubled haploids in an invitro microspore culture of plants of the B. rapa L. species, which consists of isolating microspores from individual buds by cutting them crosswise with a scalpel and immersing the bud halves in Eppendorf-type test tubes with a nutrient medium, 1 bud per test tube, with further shaking of the test tubes and filtration of the resulting suspension through a filter with a pore diameter of 40 μm and culturing the resulting suspension in plate-type Petri dishes with a diameter of 1.2 cm.EFFECT: increasing the efficiency of the method for obtaining doubled haploids of the Brassicarapa L. species in an invitro microspore culture in comparison with standard protocols, where an average sample is cultivated.1 cl, 1 tbl

Description

[0001] The invention relates to the field of obtaining doubled haploids in the culture of isolated microspores in vitro for the species Brassica rapa L.

[0002] Brassica rapa L. belongs to the genus Brassica of the family Brassicaceae. Any breeding is based on the selection and work with aligned material. In traditional breeding, the alignment of the material is achieved through repeated inbreeding. Currently, cell technologies are used to accelerate the breeding process. The production of doubled haploids (DH plants) in vitro microspore culture is an advanced technology for creating aligned material for breeding and genetic research. It allows for several times lower economic and time costs compared to traditional breeding methods (Ferrie and Möllers, 2011; Forster and Thomas, 2005; E. V Kozar et al., 2020).The technology for producing doubled haploids in vitro microspore culture (Dunwell, 2010; Lichter, 1982; Pechan and Keller, 1988) has been introduced into many agricultural plant breeding programs, but for a number of crops it has not yet been developed or has low efficiency, which does not allow the technology to be used on an industrial scale.

[0003] The protocol for producing doubled haploids in vitro microspore culture consists of a large number of stages, each of which has a significant impact on the final result (Ferrie and Caswell, 2011; Maluszynski et al., 2003; Touraev et al., 2009). One of the most important factors is the stage of microspore development. Not all microspores are capable of changing their developmental pathway from gametophyte to sporophyte. Only two stages of microspore development are prone to a change in their developmental pathway: late unicellular vacuolated microspores and early bicellular pollen (hereinafter, for convenience, both these stages are referred to as microspores). Microspores at other stages of development die during microspore culture, respectively, they secrete toxins and poison the nutrient supply, which negatively affects the development of living cellular structures.Therefore, in cell culture, it is important to have a minimum number of microspores at other developmental stages, as well as a maximum number of microspores at the developmental stage responsive to embryogenesis (Kott et al., 1988). However, microspores in buds always mature unevenly, and only the degree of developmental heterogeneity varies from culture to culture. This heterogeneity can play a decisive role in the success of DH technology in cultures where it is high (Kozar et al., 2020).

[0004] In conventional protocols for producing DH plants in microspore culture, microspores are isolated from several buds at once, after which an average sample of the microspore suspension is cultured in Petri dishes (Kozar and Domblides, 2021). To introduce microspores into the culture at an appropriate developmental stage, a reconnaissance cytological analysis is performed before sample preparation. Microspore development stages in buds of various sizes are assessed and the bud size with the highest microspore concentration at the embryogenic stage of development is determined. For some crops, the range of linear sizes of buds with a microspore development stage suitable for embryogenesis varies within 1 mm (Bhatia et al., 2018), but for crops where microspore development is less uniform, the range can narrow to 0.3 mm (Kozar and Domblides, 2021). The narrow selection window for buds significantly complicates the bud selection process and increases the likelihood of selecting buds at the wrong stage. In Brassica rapa L.Uneven development of microspores is also observed, as well as a very narrow linear range of buds in which embryoids are formed (Table 1).

[0005] These factors significantly complicate the application of DH technology, as the optimal bud size varies not only between genotypes but also within a single plant at different stages of its ontogenesis. Reconnaissance analysis to determine the optimal stage of microspore development in buds before each series of experiments is labor-intensive and not always informative enough, as reconnaissance cytological analysis is only an indirect method, and the correlation between the stage of microspore development and the linear size of the bud is subject to error (the microspore population composition may differ among buds of the same size). Therefore, for crops with highly uneven microspore development, selecting the optimal range of linear bud size is a very complex step, which can significantly limit the effectiveness of the technology due to the high probability of error, leading to an unstable and low embryoid yield.On the other hand, the quality and quantity of microspores may vary from bud to bud.

[0006] In order to overcome the problem of the heterogeneity of microspore stages in the preparation, we propose isolating microspores from individual buds by cutting them crosswise with a scalpel and immersing the bud halves in Eppendorf tubes with a nutrient medium (1 bud per tube), followed by shaking the tubes and filtering the resulting suspension through a filter with a pore diameter of 40 μm and cultivating the resulting suspension in plate-type Petri dishes with a diameter of 1.2 cm.

[0007] This reduces the likelihood of variations in the quality of microspore development stages in the specimen. Furthermore, since a range of buds of varying lengths is used in a single experimental series, this reduces the risk of selecting an inappropriate range of bud linear sizes, thereby increasing the likelihood of obtaining embryoids in each experimental series. Taken together, these factors increase the efficiency of the technology for producing doubled haploids of Brassica rapa L. in vitro microspore culture compared to standard protocols that utilize the average sample (Table 1).

[0008] Table No. 1 Effect of the microspore cultivation method on the yield of embryos for in vitro microspore culture in different genotypes of the Brassica rapa L. species.

[0009] Sample number buds, mm Embryoid yield, pcs. / 5 buds using the standard method Embryoid yield, pcs. / 5 buds with the new method Results of two-factor analysis of variance, where: Factors / Value of sums of squares (SS) / Proportion of factor influence, % 1 2,5-2,8 0 0,25 ± 0,45 A nz / 0.83 / 2% B * / 0.33 / 9% A x B nz / 0.08 / 2% random factors / 3.17 / 86% 2,8-3,2 0 0,08 ± 0,29 2 2,5-2,8 2,33 ± 1,15 5,08 ± 1,51 A * / 114.08 / 54% B * / 33.33 / 16% A x B* / 14.08 / 7% random factors / 49.17 / 23% 2,8-3,2 0,33 ± 0,49 0,92 ± 0,79 3 2,5-2,8 3,75 ± 2,80 6,42 ± 1,83 A * / 208.33 / 53% B * / 36.75 / 9% A x B nz / 10.08 / 3% random factors / 138.83 / 35% 2,8-3,2 0,50 ± 0,67 1,33 ± 0,98 4 2,5-2,8 0,83 ± 0,94 1,92 ± 1,38 A * / 16.33 / 28% B * / 6.75 / 12% A x B nz / 1.33 / 2% random factors / 57.92 / 58% 2,8-3,2 0 0,42 ± 0,51

[0010] Standard method – culturing a mixed sample of microspores isolated from several buds in Petri dishes of 6 cm diameter at a rate of 5 buds per Petri dish; new method – culturing a suspension of microspores isolated from individual buds in Petri dishes of 1.2 cm diameter at a rate of 1 bud per Petri dish. Variety samples: No. 1 - rosette cabbage (VIR cat.: k-650 PvBr1051023 Pack choi*harinosa); No. 2 - leafy turnip (VIR cat.: k-215 Uzuki Komatsuna); No. 3 - leafy turnip (VIR cat.: k-330 Torezizoh F1); No. 4 - rosette cabbage (VIR cat.: k-242 Goseki Late). Factor A is the size of the buds, factor B is the cultivation method, A x B is the interaction of the factors of bud size and cultivation method.

[0011] The table presents mean ± SD, *: differences are significant at the 5% probability level, ns: not significant. The influence of factors was calculated as the ratio of the SS of each factor to the total SS.

[0012] Bibliography

[0013] Bhatia, R., Dey, S.S., Parkash, C., Sharma, K., Sood, S., Kumar, R., 2018. Modification of important factors for efficient microspore embryogenesis and doubled haploid production in field grown white cabbage (Brassica oleracea var. capitata L.) genotypes in India. Sci. Hortic. (Amsterdam). 233, 178–187. https: / / doi.org / 10.1016 / j.scienta.2018.01.017

[0014] Dunwell, J.M., 2010. Haploids in flowering plants: Origins and exploitation. Plant Biotechnol. J. 8, 377–424. https: / / doi.org / 10.1111 / j.1467-7652.2009.00498.x

[0015] Ferrie, A.M.R., Caswell, K.L., 2011. Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell. Tissue Organ Cult. 104, 301–309. https: / / doi.org / 10.1007 / s11240-010-9800-y

[0016] Ferrie, A.M.R., Möllers, C., 2011. Haploids and doubled haploids in Brassica spp. for genetic and genomic research. Plant Cell. Tissue Organ Cult. 104, 375–386. https: / / doi.org / 10.1007 / s11240-010-9831-4

[0017] Forster, B.P., Thomas, W.T.B., 2005. Doubled haploids in genetics and plant breeding. Plant Breed Rev 25.

[0018] Kott, L.S., Polsoni, L., Beversdorf, W.D., 1988. Cytological aspects of isolated microspore culture of Brassica napus . Can. J. Bot. 66, 1658–1664. https: / / doi.org / 10.1139 / b88-226

[0019] Kozar, E., Domblides, E., 2021. Protocol of European Radish (Raphanus sativus L.) Microspore Culture for Doubled Haploid Plant Production BT - Doubled Haploid Technology: Volume 2: Hot Topics, Apiaceae, Brassicaceae, Solanaceae, in: Segui-Simarro, J.M. (Ed.), . Springer US, New York, NY, pp. 217–232. https: / / doi.org / 10.1007 / 978-1-0716-1335-1_13

[0020] Kozar, E. V., Domblides, E.A., 2021. Protocol of European Radish (Raphanus sativus L.) Microspore Culture for Doubled Haploid Plant Production, in: Methods in Molecular Biology. pp. 217–232. https: / / doi.org / 10.1007 / 978-1-0716-1335-1_13

[0021] Kozar, E. V., Domblides, E.A., Soldatenko, A. V., 2020. Factors affecting DH plants in vitro production from microspores of European radish. Vavilovskii Zhurnal Genet. Selektsii 24, 31–39. https: / / doi.org / 10.18699 / VJ20.592

[0022] Kozar, E. V, Domblides, E.A., Soldatenko, A. V, 2020. Peculiarities of the sample preparation stage in in vitro microspore culture, in: Actual Biotechnology. pp. 110–111.

[0023] Lichter, R., 1982. Induction of Haploid Plants From Isolated Pollen of Brassica napus. Zeitschrift für Pflanzenphysiologie 105, 427–434. https: / / doi.org / 10.1016 / s0044-328x(82)80040-8

[0024] Maluszynski, M., Kasha, K., Forster, B.P., Szarejko, I., 2003. Doubled haploid production in crop plants: a manual. Springer Science & Business Media.

[0025] Pechan, P.M., Keller, W.A., 1988. Identification of potentially embryogenic microspores in Brassica napus. Physiol. Plant. 74, 377–384. https: / / doi.org / 10.1111 / j.1399-3054.1988.tb00646.x

[0026] Touraev, A., Brian P., F., Jain, S.M., 2009. Advances in Haploid Production in Higher Plants. Springer.