Method of induced organogenesis of shoot callus tissue of white cabbage explants

Abstract

FIELD: biotechnology.SUBSTANCE: method for induced organogenesis of shoots from callus tissue of white cabbage explants. In particular, a highly effective and universal method was developed for inducing shoot formation in vitro using hypocotyl and cotyledon explants obtained from 6-day-old aseptic donor sprouts of white cabbage (B. oleracea L. convar. capitata (L.) Alef.) of the Dh line, as well as the Podarok and Parus varieties. Using this method, multiple organogenesis with a frequency of 73.0% was achieved in three subcultures of juvenile callus obtained from hypocotyls and cotyledons on the basal MS medium supplemented with 2 mg / l 6-BAP, 0.02 mg / l NUK and 5 mg / l AgNO3.EFFECT: this method can be used to induce shoot formation in various genotypes of white cabbage, for the purposes of genetic transformation or genomic CRISPR / Cas editing of cabbage plants to obtain plants with improved economically valuable traits.3 cl, 1 dwg, 3 tbl, 2 ex

Description

[0001] Field of technology to which the invention relates

[0002] The invention relates to the field of biotechnology, in particular to the field of introduction into in vitro culture, obtaining callus cultures and regenerated plants on solid agar media, using hypocotyl and cotyledons of the genotypes of white cabbage Brassica oleracea L. convar. capitata (L.) Alef. as primary explants.

[0003] Prior Art

[0004] White cabbage (Brassica oleracea L. convar. capitata (L.) Alef.) is one of the most important crops of industrial vegetable growing in the world. According to the Food and Agriculture Organization of the United Nations (FAO), in 2023, the area under cabbage was about 2.36 million hectares, and the gross production was 73.8 million tons with an average yield of 31.3 t / ha. China and India are the largest cabbage producers in the world (more than 62% of the world gross harvest). Russia ranks 3rd in the world gross harvest (~ 2.6 million tons or 3.5% of the total gross harvest) with cultivation on an area of ​​70.3 thousand hectares [1].

[0005] For successful cultivation of white cabbage in a specific ecological and geographical zone, a range of F1 varieties and hybrids with high adaptive potential to the abiotic and biotic conditions of the region is required. The most important factor for realizing the potential yield of cabbage is increasing its resistance to a number of fungal (Alternaria brassicicola, Botrytis cinerea, Pythium spp. Plasmodiophora brassicae) and bacterial (Xanthomonas campestris pv. campestris, Erwinia carotovora subsp. carotovora) phytopathogens [2-4], as well as to abiotic stress [4, 5]. The creation of modern highly productive genotypes with increased resistance to stress factors of various etiologies can be achieved using traditional [5-7], modern biotechnological [6, 8, 9] and genetic engineering approaches [10-13], including the CRISPR / Cas9 genome editing method

[14] .

[0006] For the successful application of the above-mentioned biotechnological approaches, it is necessary to obtain plants in vitro. In vitro shoot regeneration of white cabbage is achieved through somatic organogenesis or embryogenesis. Somatic embryogenesis is induced to obtain haploid plants from the culture of isolated anthers [16, 17] and microspores [18, 19]. Cotyledons and hypocotyl segments obtained from juvenile aseptic seedlings are most often used as explants for the induction of somatic organogenesis of cabbage in vitro [20-24].

[0007] Induction of callus formation and subsequent indirect shoot organogenesis is carried out mainly on a basal nutrient medium formulated according to the Murashige and Skoog (MS) formula

[29] with the addition of various types and amounts of plant growth regulators (PGRs). The most commonly used cytokinin and auxin components are 6-benzylaminopurine (6-BAP) and 1-naphthylineacetic acid (NAA) in a wide range of concentrations from 0.1 to 4.0 mg / L [15, 20, 21, 22, 30]. Much less frequently, thidiazuron (TDZ) [24, 25], zeatin

[30] or kinetin

[28] are used instead of 6-BAP. Alternative sources of auxins can be 2,4-D

[28] , 3-indolylbutyric [26, 27] or 3-indoleacetic acid (IAA)

[30] . In general, the choice of the type and concentration of PGRs in the nutrient medium depends significantly on the genotype and explant of B. oleracea L. convar. capitata (L.) Alef.

[0008] However, the widespread use of modern biotechnology and genetic engineering methods is limited by the low regenerative and transformation potential of white cabbage plant cells in vitro. This problem is so pressing that increasing the morphogenetic potential of cabbage is achieved through genetic engineering

[14] .

[0009] Thus, the objective of the present invention is to develop a highly effective method for induced organogenesis of shoots from explants of hypocotyls and cotyledons of white cabbage to obtain transgenic or genome-edited plants with improved economically valuable traits.

[0010] Essence of the invention

[0011] To solve this problem, a method is proposed that involves obtaining aseptic donor explants from 6-day-old aseptic seedlings, culturing the obtained explants on a basic nutrient medium MS with the addition of 5 mg / l AgNO3 as an inhibitor of ethylene biosynthesis and 6-BAP and NUK as a source of cytokinin and auxin to induce callus formation and somatic organogenesis, and rooting the regenerated shoots in vitro and their adaptation to soil conditions.

[0012] Brief description of drawings

[0013] Figure 1. Highly efficient protocol for multiple in vitro organogenesis of somatic shoots from juvenile cotyledon and hypocotyl explants of three cabbage genotypes.

[0014] The description of the method of the present invention will now be continued by giving specific examples of its implementation.

[0015] Examples

[0016] Example 1. Obtaining aseptic donor explants

[0017] The plant material used in the method of the present invention consisted of three modern industrial genotypes of B. oleracea var. capitata bred in Russia: the Dh selection line, as well as the Parus and Podarok varieties. Aseptic seedlings were obtained in vitro from seeds after their surface treatment with sterilizing solutions. The seeds were pre-rinsed with running tap water for 10 minutes, after which they were surface sterilized with a 12.5% ​​sodium hypochlorite solution for 20 minutes at room temperature. Then, the sterilized seeds were washed with autoclaved distilled water 3 times for 10 minutes and transferred to culture vessels with a basic nutrient medium based on mineral components and vitamins, formulated according to the MS recipe (product ID: M519, PhytoTech Labs, Lenexa, USA), supplemented with 3% sucrose and 0.7% agar (product ID: P1001, Duchefa Biochemie, Haarlem, Netherlands), pH 5.7-5.8.Autoclaving of the nutrient medium was carried out for 20 min at a temperature of 121°C and a pressure of 1.1 atm. Donor seedlings were cultured under factorostatic conditions of a WLR-351H climate chamber (Sanyo, Japan) at a temperature of 24 / 22°C (day / night) in diffused light (30-40 μmol m -2 With -1 ) with a photoperiod of 16 hours light / 8 hours dark.

[0018] Example 2. Induction of callus formation and somatic organogenesis

[0019] Morphogenesis was induced by culturing cotyledon explants, including 2-3 mm petioles, and approximately 10 mm long segments of the mid-part of hypocotyls, obtained from 6-day-old aseptic seedlings. Intact explants were cultured on the basal MS nutrient medium supplemented with 5 mg / L AgNO3 as an ethylene biosynthesis inhibitor, 2.0 mg / L 6-BAP, and 0.02 mg / L NUK as a source of cytokinin and auxin. Stock solutions of 6-BAP, NUK, and AgNO3 were sterilized by passing through sterile nitrocellulose filters with a pore diameter of 0.22 μm (Jet Biofil, Guangzhou, China) and added after autoclaving to the nutrient medium cooled to 45°C. Hypocotyl and cotyledon explants were placed on the surface of the nutrient medium horizontally and abaxially, respectively, and cultured in a WLR-351H climate chamber (Sanyo, Japan) at a temperature of 24 / 22°C (day / night), 70-80 μmol m -2 With -1illumination intensity and a photoperiod of 16 h light / 8 h dark. Subcultivation on fresh nutrient medium was carried out every 15 days. The efficiency of morphogenesis processes was assessed based on the frequency of callus formation (after 30 days of cultivation) and the frequency of shoot / root organogenesis. The frequency of callus formation (%) was defined as the ratio of the number of explants that formed callus tissue to their total number (n=30). The frequency of shoot and root organogenesis (%) was defined as the ratio of the number of explants in which shoot or root organogenesis occurred to the total number of explants (n=30).

[0020] The data on the evaluation of the efficiency of morphogenesis processes are given below in Tables 1-3.

[0021]

[0022] The average values ​​for three biological replicates (n=30) are shown.

[0023] From the data presented in Table 1, it can be concluded that the explants of cotyledons and hypocotyls of the studied genotypes were characterized by a high ability to form calluses when cultivated on the nutrient medium of the present invention with a frequency of 80.6 to 100%.

[0024]

[0025]

[0026]

[0027] The average values ​​for three biological replicates (n=30) are shown.

[0028] The frequency of indirect somatic organogenesis of shoots during cultivation of cotyledon and hypocotyl explants of the studied genotypes was found to vary widely (from 55.2 to 89.1%) (Table 2). Cotyledon explants of the Dh line were characterized by a significantly higher frequency of somatic organogenesis of shoots (80.6%) compared to hypocotyl segments (55.2%). In contrast, for the Parus and Podarok varieties, hypocotyl segments were distinguished by a greater capacity for somatic organogenesis of shoots. Nevertheless, on average, across all the studied genotypes and nutrient media, no reliable differences in the frequency of somatic organogenesis of shoots between cotyledon and hypocotyl explants were found.

[0029]

[0030] The average values ​​for three biological replicates (n=30) are shown.

[0031] When cultivating cotyledon and hypocotyl explants of the studied cabbage genotypes, organogenesis of not only shoots but also roots was observed in almost all cases, with rates ranging from 3.7 to 34% (Table 3). The only exception were hypocotyl segments of the Parus variety, which were completely incapable of root production. The frequency of root regeneration from cotyledon explants was significantly higher. However, no significant differences were found between the different cabbage genotypes for this parameter.

[0032] Example 3. Rooting of regenerated shoots in vitro and their adaptation to soil conditions

[0033] Cabbage shoots regenerated from callus tissue of each genotype in Example 2 were separated and transferred to MS nutrient medium supplemented with 0.5 mg / L NUK for rooting. The in vitro-rooted regenerates were then adapted to soil conditions. For this purpose, the roots were thoroughly washed from the nutrient medium with running tap water, after which the regenerates were transferred to plastic pots filled with sterilized garden soil and cultivated under the following conditions: temperature 22°C, humidity 60-70%, and illumination of 150 μmol m -2 With -1 .

[0034] Thus, using the model of Russian white cabbage genotypes, a universal, highly effective protocol for multiple somatic organogenesis of shoots in vitro from juvenile explants of cotyledons and hypocotyls has been developed, which, in 95 days, allows for the complete cycle to be completed from the stage of introducing seeds into in vitro culture to growing adapted seedlings under ex vitro conditions (Figure 1).

[0035] The work on creating this invention was carried out with the financial support of the Ministry of Science and Higher Education of Russia, project No. 075-15-2023-582.

[0036] Bibliography

[0037] 1. Food and Agriculture Organization (FAO). World's Cabbage Production. Available online: http: / / www.fao.org / faostat / en / #data / QCL (accessed on 15 June 2025).

[0038] 2. Lu, L.; Monakhos, S. G.; Lim, Y. P.; Yi, S.Y. Early Defense Mechanisms of Brassica oleracea in Response to Attack by Xanthomonas campestris pv. campestris. Plants 2021.10, 2705.

[0039] 3. Das Laha, S.; Kundu, A.; Podder, S. Impact of Biotic Stresses on the Brassicaceae Family and Opportunities for Crop Improvement by Exploiting Genotyping Traits. Planta 2024, 259(5), 97.

[0040] 4. Prajapati, N. K.; Meena, R. C.; Kumar, S.; Kumar, P.; Chand, K. Cabbage Breeding Tools for Biotic and Abiotic Resistance. Roman. J. Hort. 2024, V, 23-32.

[0041] 5. Parkash, C; Kumar, S.; Thakur, N.; Singh, S.; Sharma, B.B. Cabbage: Breeding and Genomics. Vegetable Sci. 2023, 50, 231-243.

[0042] 6. Jabeen, A.; Mir, J.I.; Malik, G.; Yasmeen, S.; Ganie, S.A.; Rasool, R.; Hakeem, K.R. Biotechnological Interventions of Improvement in Cabbage {Brassica oleracea var. capitata L.). Sci. Hort. 2024, 329, 112966.

[0043] 7. Bursakov, S.A.; Karlov, G.I.; Kharchenko, P.N. Marker Breeding of White Cabbage. RUDN J. Agronomy and Animal Industries 2024, 19(4), 578-591.

[0044] 8. Pivovarov, V.F.; Bondareva, L.L.; Shmykova, N.A.; Shumilina, D.V.; Mineikina, A.I. New Generation Hybrids of White Cabbage (Brassica oleracea L. convar. capitata var. alba DC) Based on Doubled Haploids. Sel'skokhozyaistvennaya biologiya 2017, 52(1), 143-151.

[0045] 9. Thakur, P.; Kumari, N.; Kumar, A.; Sharma, P.; Chadha, S. Recent Advances in Development and Utilization of Double Haploids (DHs) in Economically Important Vegetable Crops. Plant Cell Tissue Organ Cult. 2023,156, 15.

[0046] 10. Lee, Y.H.; Yoon, I.S.; Suh, S.C.; Kim, H.I., Enhanced Disease Resistance in Transgenic Cabbage and Tobacco Expressing a Glucose Oxidase Gene from Aspergillus niger. Plant Cell Rep.2002, 20, 857-863.

[0047] 11. Bhattacharya, R.C.; Maheswari, M.; Dineshkumar, V.; Kirti, P.B.; Bhat, S.R.; Chopra, V.L. Transformation of Brassica oleracea var. capitata with Bacterial betA Gene Enhances Tolerance to Salt Stress. Sci. Hort. 2004, 100, 215-227.

[0048] 12. Rafat, A.; Abd Aziz, M.; Abd Rashid, A.; Abdullah, S.N.A.; Kamaladini, H.; Sirchi, M.T.; Javadi, M. Optimization of Agrobacterium tumefaciens-mediated transformation and shoot regeneration after co-cultivation of cabbage (Brassica oleracea subsp.capitata) cv. KY Cross with AtHSP101 gene. Sci. Hort. 2010, 124, 1-8.

[0049] 13. Xing, Y.; Raza, M.A.; He, Y.; Song, J.; Song, J. BoPRR9, a Pseudo-Response Regulator Protein from Cabbage, Plays a Negative Regulatory Role in the Response to Cold Stress. Environ. Exp.Bot. 2024, 224, 105801.

[0050] 14. Zhang, Y.; Liu, J.; Li, Y.; Ma, H.; Ji, J.; Wang, Y.; Zhuang, M.; Yang, L.; Fang, Z.; Li, J.; Zhang, C; Liu, L.; Lebedeva, M.; Taranov, V.; Zhang, Y.; Lv, H. Generation of Novel bpm6 and dmr6 Mutants with Broad-Spectrum Resistance Using a Modified CRISPR / Cas9 System in Brassica oleracea. J. Integrative Plant Biol. 2025, 67(5), 1214-1216.

[0051] 15. Gerszberg, A. Tissue Culture and Genetic Transformation of Cabbage (Brassica oleracea var. capitata): an Overview. Planta 2018, 248, 1037-1048.

[0052] 16. Kirakosyan, R.; Kalashnikova, E. Morphogenesis of Anthers Isolated from Cabbage (Brassica oleracea L.) in vitro. KnE Life Sci. 2022, 385-393.

[0053] 17. Mineykina, A.; Bondareva, L.; Soldatenko, A.; Domblides, E. Androgenesis of Red Cabbage in Isolated Microspore Culture In Vitro. Plants 2021, 10, 1950.

[0054] 18. Kozar, E.V.; Kozar, E.G.; Domblides, E.A. Effect of the Method of Microspore Isolation on the Efficiency of Isolated Microspore Culture In Vitro for Brassicaceae Family. Horticulturae 2022, 8, 864.

[0055] 19. Kozar, E.V.; Domblides, E.A. Protocol for Obtaining Doubled Haploids in Isolated Microspore Culture in vitro for Poorly Responsive Genotypes of Brassicaceae Family. Biol. Methods and Protocols 2024, 9(1), bpae091.

[0056] 20. Dogru, S.M.; Balkaya, A.; Kurtar, E.S. In vitro Micropropagation of Maintainer White Head Cabbage Lines Using Cotyledon and Hypocotyl Explants. Black Sea J. Agric. 2022, 5, 180-188.

[0057] 21. Gerszberg, A.; Hnatuszko-Konka, K.; Kowalczyk, T. In vitro Regeneration of Eight Cultivars of Brassica oleracea var. capitata. In Vitro Cell. Dev. Biol. Plant 2015, 25, 80-87.

[0058] 22. Munshi, M.K.; Roy, P.K.; Kabir, M.H.; Ahmed, G. In vitro Regeneration of Cabbage (Brassica oleracea L. var. capitata) through Hypocotyl and Cotyledon Culture. Plant Tissue Cult. Biotech. 2007,17(2), 131-136.

[0059] 23. Hasan, A.M.; ElKaaby, E.A.; AL-Jumaily, R.M.K. In Vitro Effects of Different Combinations of Phytohormones on Callus Induction from Different Explants of Cabbage Brassica oleracea var. capitata L. Seedlings. Iraqi J. Sci. 2021, 3476-3486.

[0060] 24. Gambhir, G.; Kumar, P.; Srivastava, D. High Frequency Regeneration of Plants from Cotyledon and Hypocotyl Cultures in Brassica oleracea cv. Pride of India. Biotechnol. Rep.2017,15, 107-113.

[0061] 25. Gambhir, G.; Srivastava, D.K. Thidiazuron Induces High Frequency Shoot Regeneration in Leaf and Petiole Explants of Cabbage (Brassica oleracea L. var. capitata). J. Biotechnol. Biomater. 2015, 5(172), 172.

[0062] 26. Danaila-Guidea, S.; Rosu, A.; Ionica, M.; Visan, L.; Dobrinoiu, R. Caulogenesis Approaches Applied in vitro Micropropagation Varieties-Cabeza Negra 2, Arena and Red Amager-of Brassica oleracea var. capitata rubra Form. Curr. Trend Nat. Sci. 2012,1(1), 75-79.

[0063] 27. Pavlovic, S.; Adzic, S.; Cvikic, D.; Zdravkovic, J.; Zdravkovic, M. In vitro Culture as a Part of Brassica oleracea var. capitata L. Breeding. Genetika 2012, 44(3), 611-618.

[0064] 28. Pavlovic, S.; Vinterhalter, В.; Zdravkovic-Korac, S.; Zdravkovic, J.; Cvikic, D.; Mitic, N. Recurrent Somatic Embryogenesis and Plant Regeneration from Immature Zygotic Embryos of Cabbage (Brassica oleracea var. capitata) and Cauliflower (Brassica oleracea var. botrytis). Plant Cell Tissue Organ. Cult. 2013, 113, 397-106.

[0065] 29. Murasnige, Т.; Skoog, F.A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiol. Plantarum 1962,15, 473-197.

[0066] 30. Qamar, Z.; Jahangir, G.Z.; Nasir, I.A.; Husnain, T. In-vitro Production of Cabbage and Cauliflower. Advancements Life Sci. 2014,1(2), 112-118.

Claims

1. A method for induced organogenesis of shoots from callus tissue of white cabbage explants, including: surface sterilization of cabbage seeds with a 12.5% ​​sodium hypochlorite solution for 20 minutes at room temperature; transfer of sterilized seeds into culture vessels with 0.7% agarized basic MS medium with pH 5.7-5.8 and subsequent cultivation for 6 days at a temperature of 24 / 22°C day / night, illumination of 30-40 μmol m -2 With -1 and a photoperiod of 16 h light / 8 h dark to obtain 6-day donor seedlings; obtaining explants from 6-day-old donor seedlings; cultivation of the obtained explants on 0.7% agarized basic MS medium with the addition of 5 mg / l AgNO3, 2.0 mg / l 6-BAP and 0.02 mg / l NUK for 45 days at a temperature of 24 / 22°C day / night, illumination of 70-80 μmol m -2 With -1and a photoperiod of 16 hours light / 8 hours dark for multiple organogenesis of shoots from callus formed on explants; rooting of regenerated shoots in vitro on 0.7% agarized MS base medium with the addition of 0.5 mg / l NUK under the same cultivation conditions; and adaptation of rooted regenerated shoots to soil conditions in plastic pots filled with sterilized garden soil, at a temperature of 22°C, humidity of 60-70% and illumination of 150 μmol m -2 c -1 .

2. The method according to paragraph 1, characterized in that the explants are cotyledon explants with petioles 2-3 mm long, obtained from 6-day-old aseptic sprouts.

3. The method according to claim 1, characterized in that the explants are explants of segments of the middle part of hypocotyls approximately 10 mm long, obtained from 6-day-old aseptic sprouts.

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