Rapid development of homozygous lines through culture of isolated microspores in leafy crops of Brassicaceae Burnett

Relevance
Biotechnological methods are generally used to speed up breeding programs and to enhance genetic diversity, so the culture of isolated microspore in vitro can be regarded as one of very suitable methods. Nontraditional and uncommon vegetable crops belonging to Brassicaceae Burnett. are becoming more popular.
Methods
Accessions of sarepta mustard (Brassica juncea L. Czern.) and rocket salad (Eruca sativa Mill.) were taken for the study with the aim to optimize the basic protocol for these species.
Results
As a result of the study the optimum cultivation conditions have been determined for the species. Sizes of buds 2.5-3.5 mm long for sarepta mustard and 7.0-7.5 long for rocket salad which were used for cultivation had been experimentally defined. It was also shown that the cold pretreatment had improved the embryo yield. The nutritional NLN-13 medium with pH 6.1 and pretreatment at 32°C during a cultivation day had been shown to be more favourable for all accessions. All conditions that had been used were suitable for embryo formation. First divisions had been seen after 4 days of cultivation, while the embryos at primary cotyledonary stage only appeared after 2 weeks of cultivation. The embryo yield per 5 buds reached 25-30 and 5-7 in the sarepta mustard and the rocket salad, respectively. It is worth noticing that the root formation and plant adaptation had passed better and faster in sarepta mustard than in rocket salad. Thus, whole process of homozygous line developing can be completed for 4-5 months, making the breeding program 3 times shorter.

1. Eliseeva O.V., Eliseev A.F. The chemical composition of Eruca sativa (Mill.) and Diplotaxis tenuifolia (L.) DC. // Reports of the TAA. Moscow Timiryazev Agricultural Academy Rep. 2018; v.290, p.4. – P.352-353. (In Russ.)

2. Ludilov V.A., Ivanova M.I. Rare and rare vegetables (biology, cultivation, seed production): Instructional Edition. – M.: FGNU "Rosinformagrotekh", 2009. – 196 p. (In Russ.)

3. Pivovarov V.F. Vegetable crops of Russia. M.: VNIISSOK, 2006. – 384 p. (In Russ.)

4. The state register of breeding achievements approved for use. T.1. "Plant Varieties" (official publication). – M.: RosInformagrotekh, 2019. (In Russ.)

5. Girenko M.M., Zvereva O.A. Green vegetables. M.: Publishing house "NiolaPress"; Publishing house "UNION-Public", 2007. – 176 p. (In Russ.)

6. iFarm project. The Russian market of fresh herbs and vegetables is favorable for investments. URL: http://www.ifarmproject.ru/russiangreenmarket. (In Russ.)

7. ROIF EXPERT, DECEMBER 6, 2018. Market for fresh greens in Russia: imports of fresh greens close to $ 100 million. // РБК Магазин исследований. URL: https://www.marketing.rbc.ru/articles/10602/. (In Russ.)

8. Pechan, P.M., Keller W.A. Identification of potentially embryogenic microspores in Brassica napus. Physiol Plant. – 1988. – Vol.74. – P.377-384.

9. Agnihotri A., Seguin-Swartz G., Dovney R.K. Microspore embryogenesis in B. juncea. In: Pareek L.K., ed. Trends in plant tissue culture and biotechnology. Rajasthan, India: Agrobotanical Publishers; 1996: 218-221.

10. Hiramatsu M.; Odahara K.; Matsue Y. A survey of microspore embryogenesis in leaf mustard (Brassica juncea). Acta Hort. 392:139–145; 1995.

11. Lionneton E.; Beuret W.; Delaitre C.; Ochatt S.; Rancillae M. Improved microspore culture and doubled haploid plant regeneration in the brown condiment mustard (B. juncea). Plant Cell Rep. 20:126–130; 2001.

12. Prem, D., Gupta, K., Agnihotri, A. Effect of various exogenous and endogenous factors on microspore embryogenesis in Indian mustard (Brassica juncea (L.) Czern and Coss). In Vitro Cell.Dev.Biol.-Plant (2005) 41: 266.

13. Leskovsek L., Jakse M., Bohanec B. Doubled haploid production in rocket (Eruca sativa Mill.) through isolated microspore culture. Plant. Cell. Tiss. Organ. Cult. (2008) 93:181–189.

14. Alexander M.P. Differential staining of aborted and non-aborted pollen. Stain technol. – 1969. – Vol.44. – P.117-122.

15. Domblides E.A., Shmykova N.A., Shumilina D.V., Zayachkovskaya T.V., Mineykina A.I., Kozar E.V., Akhramenko V.A., Shevchenko L.L., Kan L.Yu., Bondareva L.L., Domblides A.S. Technology for producing doubled haploids in the culture of microspores of the cabbage family (methodical recommendations) / Publishing house VNIISSOK. – 2016. – 40 p. (In Russ.)

16. Gamborg, O.L. Nutrients requirements of suspension cultures of soybean root cells / Gamborg O.L., Miller R.A., Ojima K. // Exp Cell Res. – 1968. – Vol.50. – P.151–158.

17. Huang B., Bird S., Kemble R., Simmonds D. Effects of culture density, conditioned medium feeder cultures on microspore embryogenesis in Brassica napus L. cv. Topas / B. Huang, // Plant Cell Rep. – 1990. – Vol.8. – P.594-597.

18. Touraev A., Brian P. Forster, S. Mohan Jain. Advances in Haploid Production in Higher Plants. Springer Science, Business Media B.V. – 2009. – P.347.

19. Butenko, R.G. Biology of cells of higher plants in vitro and biotechnologies based on them: a tutorial / R.G. Butenko. – M.: FBK-PRESS, 1999. – 160 p. (In Russ.)

20. Yuan, S.X. Effects of pH, MES, arabinogalactan-proteins on microspore cultures in white cabbage / S.X. Yuan, Y.B. Su, Y.M. Liu // Plant Cell Tiss Organ Cult. – 2012. – Vol.110. – P.69-76.