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Mavlyutova L.I., Elkonin L.A., Kolesova A.Yu. Origin of diploid plants in diploid-tetraploid crosses in maize (Zea mays L.): analysis of endosperm ploidy of plump kernels. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2025, Vol. 60, № 5, p. 827-839.
(how to cite this article)

doi: 10.15389/agrobiology.2025.5.827eng

UDC: 633.15:581.163

Acknowledgements:
The authors express their gratitude to U.Sh. Kuzmina, PhD (Institute of Biochemistry and Genetics, Ufa Federal Research Center RAS) for assistance in flow cytometry experiments, to E.B. Khatefov, DSc (Vavilov All-Russian Institute of Plant Genetic Resources, St Petersburg), and to O.A. Shatskaya, PhD (Lukyanenko National Grain Center, Kraasnodar) for providing seeds of the tetraploid lines Chernaya Tetra and Krasnodar Tetraploid, and to A.N. Zavalishina, PhD (Chernyshevsky Saratov National Research State University) for providing seeds of the AT HPL and KM lines.
Supported financially by the Russian Science Foundation (grant No. 24-13-00063)
The experimental material was created during the implementation of the RF Ministry of Education and Science topic FNWF-2022-0006.

 

ORIGIN OF DIPLOID PLANTS IN DIPLOID-TETRAPLOID CROSSES IN MAIZE (Zea mays L.): ANALYSIS OF ENDOSPERM PLOIDY OF PLUMP KERNELS

L.I. Mavlyutova, L.A. Elkonin✉, A.Yu. Kolesova

Federal Center of Agriculture Research of the South-East Region, 7, ul, Tulaikova, Saratov, 410010 Russia, e-mail lelkonin@gmail.com (✉ corresponding author), lidia.bahteewa@yandex.ru, kolesovaau@yandex.ru

ORCID:
Mavlyutova L.I. orcid.org/0009-0005-8548-5493
Kolesova A.Yu. orcid.org/0000-0002-6330-2052
Elkonin L.A. orcid.org/0000-0003-3806-5697

Final revision received March 31, 2025
Accepted July 07, 2025

An important component of apomictic reproduction in plants is the ability to form unreduced embryo sacs (ES). Heteroploid crosses are an effective tool for identifying such ESs, since when diploid ES is fertilized with tetraploid pollen, two diploid polar nuclei fuse with a diploid sperm, leading to the restoration of the genomic balance in the endosperm (2♀:1♂) and formation of normally developed seeds. Previously, we found that on the ears of the diploid AT maize line, capable of haploid parthenogenesis, as well as on the ears of its hybrids, when pollinated with pollen from tetraploids, plump kernels were formed, from which diploid maternal-type plants and tetraploid hybrids developed. Our aim was to further test the hypothesis on the formation of plump kernels in 2n × 4n heteroploid crosses from unreduced embryo sacs developed in maternal lines by analyzing the endosperm ploidy of such kernels using flow cytometry. In this study, we used the plump kernels developed on the ears of diploid maize lines: AT GPL (2n), which has the ability for haploid parthenogenesis, 18-10-4 (2n), V47 (2n), Korichnevyi Marker (KM) (2n), Krasnodarskaya 2 (2n), Krasnodarskaya 9 (2n), Krasnodarskaya 22 (2n), as well as the F1 and F3 hybrids V47/AT GPL (2n), F1 KM1/Sakharnaya1 (2n), and F1 KM/AT GPL (2n), which were pollinated with pollen of the tetraploid lines Tetra-Pariy (TP) (4n), Krasnodarskiy tetraploid (KrT) (4n), and Chernaya Tetra (ChT) (4n). The crosses were carried out in 2022. The plants were grown in the experimental field of the Federal Agricultural Research Centre of the South-East Region (Saratov, Russia). Pollen from the panicle of each tetraploid plant was shaken off into an individual clean parchment bag and applied to the stigmas of the ears of the diploid maternal plant. On the 18th day after pollination, large, mature kernels were collected from the ears of the diploid lines, sterilized, the endosperm was separated and fixed in a mixture of alcohol (95 %) and propionic acid (3:1) for 3 h, washed in 70 % alcohol and stored in 70 % alcohol at a temperature of -20 °C for subsequent ploidy analysis using flow cytometry. Embryos were placed in test tubes on MS agar nutrient medium, and grown in a growth room (photoperiod 16 h day/8 h night; 26±1 °C). Seedlings with developed roots were transferred to test tubes with water for several days to acclimate to non-sterile conditions, after which they were planted in plastic containers in a greenhouse and then in an experimental field of Federal Research Centre of Agriculture of South-East Region. Root tips from plants grown in plastic containers were used to determine ploidy on the 12th to 14th days after planting in the ground. For each plant, ploidy was determined based on the analysis of 8-10 metaphase plates. When studying ploidy using flow cytometry, nuclei from the leaf of the haploid plant of AT GPL line, and from the endosperms of AT GPL and tetraploid ChT kernels were used as standards. The frequency of ears with normally developed plump kernels varied from 7 % to 75 % among different lines and hybrids; the frequency of plump kernels from the total number of seeds varied from 0.7 to 85.4 % and depended on the pollinator genotype: with TP and ChT, the frequency was significantly higher than for KrT; the frequency of ears with plump kernels was the highest for TP. Cytological analysis of the root meristems of seedlings showed that diploid, triploid, and tetraploid plants developed from the plump kernels. Several fluorescence peaks were detected in the endosperm of the plump kernels, with the first peak observed at values twice higher than those in the endosperm of the diploid line (3C) and corresponding to 6C, similar to the value of the first fluorescence peak of the endosperm of the tetraploid line. This result was observed in the endosperm of kernels from which both tetraploid hybrids and diploid "maternal-type" plants were obtained. Furthermore, peaks corresponding to higher ploidy values (12C, 24C) were also observed in both triploid and hexaploid endosperm, which were attributed to endopolyploidization processes. These data support the hypothesis that normally developed plump kernels develop in 2n × 4n maize crosses on the bases on unreduced ES, an important component of apomixis, and demonstrate the effectiveness of tetraploid pollinators to identify such embryo sacs in maize lines and hybrids, including in studies on the creation of apomictic genotypes using genome editing. Furthermore, the obtained results provide the first direct experimental evidence to support the balance theory of endosperm development, since the ploidy of endosperm of kernels formed in heteroploid crosses has not previously been studied using flow cytometry.

Keywords: apomixis, heteroploid crosses, genomic balance, flow cytometry.

 

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