doi: 10.15389/agrobiology.2018.3.531eng

UDC 633.18:631.527:581.143.6:575.2

The authors would like to deeply thank colleagues from the Laboratory of Rice Breeding (Primorskii Research Institute of Agriculture) for the hybrid rice material provided.

Supported in part by Far East Program for Basic Research of the Far Eastern Branch RAS (grant  15-I-6-005)




M.V. Ilyushko1, M.V. Skaptsov2, M.V. Romashova1

1Primorskii Research Institute of Agriculture, Federal Agency for Scientific Organizations, 30, ul. Volozhenina, pos.  Timityazevskii, Primorskii Krai, 692539 Russia, e-mail (✉ corresponding author);
2South Siberian Botanical Garden of Altai State University, 61, prosp. Lenina, Barnaul, 656049 Russia, e-mail

Ilyushko M.V.
Skaptsov M.V.
Romashova M.V.

Received January 30, 2017


Rice is an important food crop grown in the south of the Russian Far East. Therefore, breeding new varieties with high harvest and crop quality is relevant. Anther in vitro culture is successfully applied in breeding programs in rice-growing countries, including Russia. In anther in vitro culture, flow cytometry is applicable to select haploid, dihaploid and polyploid regenerants. Cytological studies show genome variations from haploids to hexaploids in plant tissue in vitro culture, and also chromosome changes which result in aneuploidy or endopolyploidy leding to an inconstant nuclear DNA content. In the work, we followed the aims i) to evaluated nuclear DNA content by flow cytometry in an androgenic rice regenerant population, and ii) to estimate the applicability of the combination of two approaches, the anther in vitro culture technique and flow cytometry, in rice breeding. A total of 1099 regenerants from in vitro anther culture of a single F2 (UkrNIIS 3435 × Ukr 96) rice (Oryza sativa L. ssp. japonica Kato) hybrid plant were separated into four groups with regard to morphological features. Haploids were sterile plants with very small flowers, dihaploids were fertile plants, tetraploids were the plants with very few large seeds, an expressed keel and the ribbed floral scales. Also, there were the plants without seeds which flowers were normal in size but formed two or more sterile panicles. In the last group of the regenerants the plants died during early development. A total of 176 regeneranats were estimated by flow cytometry. It was revealed that nuclear DNA content varied greatly (Cv = 32 %) in the plants without seeds. This group seems to include plants with double set of chromosomes, triploids, tetraploids, and pentaploids. Additionally, in this group we found the regenerants with endopoyploidy since five of the plants had two nuclear DNA content peaks like those for haploids and diploids. In 23 plants nuclear DNA content approximated to dihaploid chromosome set and averaged 2.00 pg. Obviously, an aneuploidy characteristic of rice anther in vitro cultures could lead to aliquant changes in chromosome set in the regenerants, causing a loss of fertility. The dihaploid and tetraploid plants were low variable (Cv of 10.5 and 5.3 %) and had nuclear DNA content of 1.88 and 3.75 pg, respectively, whereas the haploids were high variable (Cv = 29 %) with an average nuclear DNA amount of 0.89 pg. Our findings indicate that flow cytometry, together with production index, may be applied to reveal tetraploid regenerants and to remove haploids in rice breeding. That allows avoiding ex vitro trials of unpromising regenerants.

Keywords: Oryza sativa L., anther culture in vitro, flow cytometry, regenerant, haploid, dihaploid, tetraploid.


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