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Zontikov D.N., Zontikova S.A., Malahova K.V., Maramohin E.V., Poliykov A.V., Sergeev R.V. Generation of Rubus arcticus L. haploids through in vitro microspore culture technique. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2020, Vol. 55, № 1, p. 128-136.
(how to cite this article)

doi: 10.15389/agrobiology.2020.1.128eng

UDC: 634.71:57.085.23

Acknowledgements:
Supported financially by the Russian Foundation for Basic Research, project No. 18-416-440002 р_а

 

GENERATION OF Rubus arcticus L. HAPLOIDS
THROUGH in vitro MICROSPORE CULTURE TECHNIQUE

D.N. Zontikov1, S.A. Zontikova1, K.V. Malahova1, E.V. Maramohin1, A.V. Poliykov2, R.V. Sergeev3

1Kostroma State University, 17, ul. Dzerzhinskogo, Kostroma, 156005 Russia, e-mail info@kstu.edu.ru, zontikovdn@mail.ru (corresponding author ✉), antennaria@mail.ru, maramokhin91@mail.ru, malakhova.kv1@gmail.com;
2All-Russian Research Institute of Vegetable Growing — Branch of the Federal Scientific Vegetable Center, str. 500, Vereya, Ramenskii District, Moscow Province, 140153 Russia, e-mail vita100plus@yandex.ru;
3Volga State University of Technology, 3, pl. Lenina, Yoshkar-Ola, Republic of Mari El, 424000 Russia, e-mail info@volgatech.net, rsergeyev@yahoo.com

ORCID:
Zontikov D.N. orcid.org/0000-0002-6668-4877
Maramohin E.V. orcid.org/0000-0002-1963-5845
Zontikova S.A. orcid.org/0000-0001-6566-4498
Poliykov A.V. orcid.org/0000-0002-0931-2751
Malahova K.V. orcid.org/0000-0002-7762-8811
Sergeev R.V. orcid.org/0000-0002-5070-9021

Received March 25, 2019

 

Arctic raspberry (Rubus arcticus L.) is a valuable small-fruit crop used as a plantation crop for a relatively short time. R. arcticus is a remontant donor in interspecific hybridization with Rubus idaeus L., though conditioning low yields to hybrids. So R. arcticus is primarily bred for yield enhancement; therefor, the acceleration of the breeding process is of great importance. This can be achieved using plants with a doubled haploid set of chromosomes. This paper is the first to describe the technique of production haploid plants of R. arcticus via in vitro microspore culture. In the experiments we used Finnish cultivars Pima and Mespi and Swedish cultivar Astra. To obtain donor explants, the method of forcing generative shoots was used throughout the year. Microspores were isolated from anthers with the use of manual homogenizer into a 1.5 ml micro test Eppendorf tube. The homogenate was added with 0.5 ml sterile water containing 30 g/l glucose, centrifuged at 4500 rpm, and the microspores were transferred with a microdoser to nutrient medium for morphogenesis initiation. To obtain microspores, the anthers were isolated from buds of 90 to 120 mm long 4-5 days before the flower bloomed. The concentration of the microspores in the suspension was about 40,000 per 0.5 ml sterile aqueous solution with glucose; for this, 60 anthers were crushed. To induce embryoidogenesis, we used the Murashige and Skoog (MS) plant growth medium supplemented with 0.50 to 2.00 mg/l growth regulator 6-benzylaminopurine (6-BAP). After the appearance of embryoids, we used MS, 75 % MS, or 50 % MS growth media, and also the effect of carbohydrate sources, i.e. glucose, sucrose and maltose at a 20, 30 and 40 g/l dosage, was investigated. We have identified the following microspore development stages: tetrads, non-vacuolated microspore, strongly vacuolated microspore, three-cell pollen. It was found that MS nutrient media containing 1.5 mg/l 6-BAP provides for 23±3 embryoids on day 51±2 of culture. We have also found the effect of MS concentration and the source of carbohydrate nutrition on the growth of embryoids. The combination of 0.75 MS and 30 g/l glucose was the most effective leading to embryoid growth on day 12±2 and the appearance of leaflets on day 44±1. On day 40 of culture the embryoids reached 5±0.2 mm in length. The ploidy control of regenerant plants, by counting chromosomes and chloroplasts in the stomata guard cells, confirmed the haploid set of chromosomes (n = 7). These findings allow the use of the proposed technique to generate R. arcticus haploids which, after doubling the chromosome set, may be involved in breeding.

Keywords: Rubus arcticus, haploid, diploid, microspore culture, embryoid, morphogenesis, regenerant plant.

 

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