Hairy roots biochemical characteristics of vegetable pea’s morphotype with modified leaf

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Abstract

BACKGROUND: In the literature, data on the successful production of high-protein pea root cultures with mutant alleles tl and af tl and their biochemical characteristics are not presented.

AIM: The aim of this study is the biochemical characteristic of the obtained transformed cultures of mutant pea lines with a modified leaf morphotype was. Rol genes clarification of transformed pea mutant lines by wild strains of Agrobacterium rhizogenes and amino acid analysis of the total protein of the obtained roots cultures were objectives of the research.

MATERIALS AND METHODS: Total DNA has been isolated from pea mutant root cultures. The studies were carried out on the equipment of the thermal cycler Tertsik of the company DNA-Technology (Russia). Amplicons were detected by electrophoresis in a 2% agarose gel. The gel was visualized and photographed under ultraviolet light (λ = 312 nm). The quantitative and qualitative amino acid composition of root cultures was determined by ion exchange chromatography on the amino acid analyzer AAA-339 (Microtechna, Czech Republic).

RESULTS: PCR analysis revealed the absence of agrobacterial contamination in the transformed cultures and their stable growth on liquid and agar hormone-free media for 5 years. PCR assay showed the presence of rol C and D genes in culture with the tltl genotype and four rol genes A, B, C, and D in culture with the afaftltl genotype. A differentiated content of a number of amino acids in the biomass of transformed cultures was found depending on the genotype of the culture and the insertion of rol genes. Seven essential amino acids were identified in both cultures.

CONCLUSIONS: Tryptophan turned out to be the limiting essential amino acid for both cultures. In terms of the sum of essential, ketogenic and sulfur-containing amino acids, the culture with the rol genes A, B, C, and D turned out to be the richest and most balanced.

About the authors

Olga O. Timina

Shevchenko State University

Email: otimina@mail.ru
SPIN-code: 7758-9906

Dr. Sci. (Biology), Professor, Faculty of Natural Geography, Department of Botany and Ecology

Moldova, Republic of, 128, 25 October str., Tiraspol, 3300

Oleg Yu. Timin

Research Institute of Ecology and Natural Resources

Author for correspondence.
Email: otimin@mail.ru
SPIN-code: 6471-1117

Cheif of laboratory "Protection and restoration phytocenosises", Cand. Sci. (Agricultural), Assistant Professor

Moldova, Republic of, 3200, Bendery, Kahovskii tupik, 2

Anna Yu. Stepanova

K.A. Timiryazev Institute of Plant Physiology RAS

Email: step_ann@mail.ru
SPIN-code: 6895-2705

Cand. Sci. (Biology), transformation roots chief group, senior staff scientist

Russian Federation, 127276, Moscow, Botanical str., 35

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Supplementary files

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2. Fig. 1. Mutants with different leaf morphotypes: a is a normal wild type, controlled by two dominant genes (Af — development of leaf blades and Tl — presence of tendrils at the end of the leaf); b — multiple-odd-pinnate type with mutant af and tl alleles; c — acacia type with the tl allele

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3. Fig. 2. PCR amplification of the virulence gene virD A. rhizogenes. M is a marker; “–“ — negative control, “+” — positive control (A. rhizogenes, strain A-4), 1 — root culture with tltl genotype, 2 — root culture with afaftltl genotype

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4. Fig. 3. PCR amplification of A. rhizogenes genes from root culture with afaftltl genotype. 1–4 — roots, M — molecular marker (ruler100 bp); C– — negative control; C+ A. rhizogenes strain A-4/B — positive control

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5. Fig. 4. PCR amplification of A. rhizogenes genes from root culture with tltl genotype. Ac — roots, PCR products of the rolC (a) and D (b) genes; 1–6, 9 — mutant plants; M — molecular marker (ruler100 bp); K–, C– — negative control; K+, C+ A. rhizogenes strain A-4/B — positive control. (Data are also provided for another obtained root culture, Spilanthes acmella, the Sp pathway, which was analyzed at the same time.)

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