Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2018, Vol. 53, ¹ 3, p. 521-530.
(how to cite this article)

doi: 10.15389/agrobiology.2018.3.521eng

UDC 635.655:581.143.6:58.085

Acknowledgments:
Supported financially by a grant from the President of the Russian Federation for the state support of young Russian scientists in the framework of the project MK-9241.2016.11

 

NDIRECT SHOOT ORGANOGENESIS OF SOYBEAN Glycine max (L.) Merr.
FROM STEM SEGMENTS AND USE OF THE EXPLANTS FOR Agrobacterium-MEDIATED TRANSFORMATION

N.V. Varlamova1, M.A. Rodionova1, L.N. Efremova1, P.N. Kharchenko1, D.A. Vysotskii1, M.R. Khaliluev1,2

1All-Russian Research Institute of Agricultural Biotechnology, Federal Agency of Scientific Organizations, 42, ul. Timiryazevskaya, Moscow, 127550 Russia, e-mail marat131084@rambler.ru (✉ corresponding author), nv_varlamo-va@rambler.ru, marrod54@gmail.com, laraefremova@mail.ru, kharchenko@iab.ac.ru, den_vis@mail.ru;
2Timiryazev Russian State Agrarian University—Moscow Agrarian Academy, 49, ul. Timiryazevskaya, Moscow, 127550 Russia

ORCID:
Varlamova N.V. orcid.org/0000-0003-2339-0120
Kharchenko P.N. orcid.org/0000-0001-5074-0531
Rodionova M.A. orcid.org/0000-0003-1540-5644
Vysotskii D.A. orcid.org/0000-0003-2650-1236
Efremova L.N. orcid.org/0000-0002-1579-438X
Khaliluev M.R. orcid.org/0000-0001-7371-8900

Received December 11, 2017

 

Soybean Glycine max (L.) Merr. is an important oil, food and fodder crop for human and animals fodder. Currently, soybean lines genetically modified for improved resistance to herbicides and pests and for reduced linolenic acid content are widely grown. More than 85 % of transgenic soybean plants are obtained using Agrobacterium-mediated transformation method. Developed Agrobacterium-mediated protocols are based on somatic embryogenesis and direct or indirect shoot organogenesis. Cotyledons, cotyledonary nodes, hypocotyl and epicotyl segments, immature or mature embryos serve as explants. Despite the large number of Agrobacterium-mediated protocols, stable transformation of soybeans is still not a routine procedure because it depends on the genotype. Surprisingly, the data on the use of soybean stem segments in genetic transformation is practically absent, although stem segments widely and efficiently serve as explants in the production of most transgenic monocotyledonous plants. Thus, the purpose of the study was to develop a protocol for shoot organogenesis from stem segments of soybean and their application as explants for the production of transgenic plants by Agrobacterium-mediated transformation. Stem segments of aseptic soybean seedlings of breeding lines 1476 and 1477 were used for callus induction and shoot organogenesis. The explants were cultured on four various MS-based growing media which differed in 6-benzylaminopurine (BA) concentrations (0.5 and 1.0 mg/l) in combination with i) 0.1 mg/l indole-3-acetic acid (IAA), or ii) 0.1 mg/l indole-3-acetic acid (IAA) and 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2.4-D). It was shown that the studied soybeans genotypes differ significantly in morphogenetic ability. Out experiments confirmed that the addition of 2.4-D resulted in inhibition of shoot organogenesis in the both genotypes. It was found that 1 mg/l BA in combination with 0.1 mg/l IAA are the best growth regulators providing the highest frequency of indirect shoot organogenesis. As a result, an effective protocol of indirect shoot organogenesis from soybean stem segments of line 1476 seedlings was developed which ensures more than 50 % frequency of organogenesis. This protocol was applied in genetic transformation of soybean line 1476 by Agrobacterium tumefaciens strain AGL0 carrying the plasmid pCambia1381Z-pro-SmAMP1-771. By gradual selection on the induction medium supplemented with hygromycin B (1-10 mg/l), 8 independent lines of putative primary transformants were selected. PCR analysis confirmed the presence of the selective (hpt) and marker (uidA) genes in 4 independent transgenic lines. The transformation efficiencies calculated based on the results of PCR analysis was 2.0 %. These results indicate the successful involvement of stem segments as explants for genetic transformation of soybean.

Keywords: soybean, Glycine max (L.) Merr., in vitroculture, shoot organogenesis, Agrobacterium-mediated transformation.

 

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