Plants, Cells, Algae, and Cyanobacteria In Vitro and Cryobank Collections at the Institute of Plant Physiology, Russian Academy of Sciences-A Platform for Research and Production Center

Abstract

Ex situ collections of algae, cyanobacteria, and plant materials (cell cultures, hairy and adventitious root cultures, shoots, etc.) maintained in vitro or in liquid nitrogen (-196 °C, LN) are valuable sources of strains with unique ecological and biotechnological traits. Such collections play a vital role in bioresource conservation, science, and industry development but are rarely covered in publications. Here, we provide an overview of five genetic collections maintained at the Institute of Plant Physiology of the Russian Academy of Sciences (IPPRAS) since the 1950-1970s using in vitro and cryopreservation approaches. These collections represent different levels of plant organization, from individual cells (cell culture collection) to organs (hairy and adventitious root cultures, shoot apices) to in vitro plants. The total collection holdings comprise more than 430 strains of algae and cyanobacteria, over 200 potato clones, 117 cell cultures, and 50 strains of hairy and adventitious root cultures of medicinal and model plant species. The IPPRAS plant cryobank preserves in LN over 1000 specimens of in vitro cultures and seeds of wild and cultivated plants belonging to 457 species and 74 families. Several algae and plant cell culture strains have been adapted for cultivation in bioreactors from laboratory (5-20-L) to pilot (75-L) to semi-industrial (150-630-L) scale for the production of biomass with high nutritive or pharmacological value. Some of the strains with proven biological activities are currently used to produce cosmetics and food supplements. Here, we provide an overview of the current collections' composition and major activities, their use in research, biotechnology, and commercial application. We also highlight the most interesting studies performed with collection strains and discuss strategies for the collections' future development and exploitation in view of current trends in biotechnology and genetic resources conservation.

Keywords: cryobank; cryopreservation; cyanobacteria; in vitro collection; meristem; microalgae; plant biotechnology; plant cell culture; plant cells; plant germplasm collection; transgenic potato.

Figure 1

Schematic representation of the collections of higher plant material, algae, and cyanobacteria at IPPRAS, storage methods, and interaction with IPPRAS biotechnological facilities, internal collaborators and external users.

Figure 2

Examples of the cell strains maintained in the All-Russian Collection of Plant Cell Cultures: callus cultures of (a) Polyscias fruticosa; (b) Panax japonicus; (c) Ajuga turkestanica; (d) Ajuga reptans. (e) Suspension cell culture of Polyscial fruticosa. (f) 64-year-old callus culture of Panax ginseng. (g–j) Suspension cell cultures under a microscope for the following species: (g) Alcea kusariensis; (h) Ajuga reptans; (i) Polyscias filicifolia; and (j) Nicotiana tabaccum. Photographs © IPPRAS.

Figure 3

Plant families represented in the All-Russian Collection of Plant Cell Cultures (modified from [30]): (a) core collection (43 cell culture strains); (b) core collection plus experimental cell culture strains (117 strains in total). Core collection is composed of comprehensively researched strains with optimized culture conditions and passport data (growth, cytological, biochemical characteristics, etc.) recorded. For (b) percentages below 3% are not labeled in the chart.

Figure 4

Adventitious root propagation: (a) Digitalis lanata Ehrh.; (b,c) Maackia amurensis on solid (b) and liquid (c) medium. (a,b) 25-day-old cultures; (c) 35-day-old culture. Photographs © IPPRAS.

Figure 5

(a) In vitro collection of transgenic potato plants and original varieties; (b) regeneration of transgenic potato plants from leaf explants 86 days after inoculation.

Figure 6

Recovery of various plant species after cryopreservation. Shoot apices were cryopreserved by slow-freezing using the programmed equipment of the IPPRAS cryobank. (a,b): Fragaria x ananassa Duch., cv. Kokinskaya pozdnaya (Russian strawberry cultivar, breeding by Alexander A. Vysotsky); (c,d): Rubus idaeus L., cv. Skromnitza (Russian red raspberry cultivar, breeding by Ivan V. Kasakov); (e,f): Rosa spp. (a) 1999, strawberry plantlets recovered from cryopreserved meristems (90% regeneration) with 40 days of in vitro culture (17 February 1999, protocol according to RF Patent № 2220563 [140,160]); (b) 2001, berries of strawberry plant regenerated from shoot apex cryopreserved in 1999; (c) 1999, raspberry shoots recovered from cryopreserved meristems (80% regeneration) with 40 days of in vitro culture (17 February 1999, protocol according to RF Patent № 2248121 [141]); (d) 2005, the fruiting of raspberry plants obtained from shoot apices after cryopreservation in 1999; (e) 2001, rose shoots recovered from meristems after slow-freezing cryopreservation (10% regeneration) (24 March 2001, protocol according to RF Patent № 2248121) with 30 days of in vitro culture; (f) 2004, the flowering of a rose plant developed from shoot apex after cryopreservation in 2001. Scans from pictures were obtained by film photo camera (Asahi Pentax Spotmatic F, Japan). © O.N. Vysotskaya.

Figure 7

Recovery of rowan (Sorbus spp.) from meristems cryopreserved by fast-freezing technique after air dehydration (protocol according to Eurasian Patent № 036602 [153]): (a) 2006, shoots recovered from apical meristems after fast freezing in LN (21 December 2006); storage at −196 °C for 1 h; thawing and in vitro culture for 39 days (89% regeneration). (b) 2017, flowers on rowan tree developed from shoot apex cryopreserved in 2006. (c) 2021, fruits on rowan tree developed from shoot apex cryopreserved in 2006. (a) Scan from picture obtained by film photo camera (Asahi Pentax Spotmatic F, Japan); (b,c) pictures captured using Sony SLT A37. © O.N. Vysotskaya.

Figure 8

The comparison of strawberry culture regrowth after cryopreservation by the (a) classical slow-freezing method [160] and (b) the new fast-freezing technique developed at IPPRAS cryobank [153]: (a,b) apical meristems isolated from in vitro plantlets of strawberry (Fragaria x ananassa Duch., cv. Kokinskaya pozdnaya, in vitro mericlone from 1987). (a) 60% of meristems recovered growth after slow freezing by programmed equipment (patent RU 2 220 563 C1, [160]); 14 years of cryostorage (17 February 1999–19 September 2013) and cultured in vitro for 52 days. (b) 80% of meristems recovered growth after fast freezing in LN (protocol according to Eurasian Patent № 036602 [153]); 4 years of cryostorage (18 November 2009–19 September 2013) and cultured in vitro for 52 days. Pictures obtained by Sony SLT A37 on 11 November 2013. © O.N. Vysotskaya.

Figure 9

Recovery of lilac (Syringa vulgaris L.) plantlets from apical meristems after fast-freezing technique following air-flow dehydration (protocol according to Eurasian Patent № 036602, [153]) and two months storage at −196 °C (01 July 2022–30 August 2022): (a) cultivar Aucubaefolia; (b) cultivar Polina Osipenko. Shoot regrowth from 100% meristems during 60 days of in vitro culture. Pictures obtained by Sony SLT A37 on 07 October 2022. Photographer: O. V. Koroleva, Main Botanic Garden, Moscow, Russia.

Figure 10

(a) 2016, 20% of bilberry (Vaccinium myrtillus L., Ericaceae) seeds germinated in vitro after storage in LN for over 24 years (1992–2016) [135]; (b) 2016, seedlings of Frailea pulcherrima (Arechav.) Speg. (Cactaceae) developed from 80% seeds germinated after direct freezing in LN and 183 days of LN storage (27 December 2015–19 January 2016). Seeds were germinated in a special soil mixture in the climate-control chamber [142]. Pictures were taken by Sony SLT A37 on 15 July 2016 (a) and 20 July 2016 (b). Photographer: A. Ju. Balekin, IPPRAS.

Figure 11

2022, IPPRAS cryobank specimens: (a) in front-cryotube with lilac shoot apices cryopreserved in 2022; (b) in front-cryotube with strawberry shoot apices cryopreserved in 2019; (c) cryotubes with different plant material in a cryo-rack. Photographs © IPPRAS, 16 February 2023.

Figure 12

Taxonomic diversity of the collection of microalgae and cyanobacteria IPPAS.

Figure 13

Morphological diversity of microalgae and cyanobacteria strains maintained at the collection. Cyanobacteria strains (a–c): (a) Desertifilum tharense Dadheech and Krienitz IPPAS B-1220; (b) Limnospira sp. Nowicka-Krawczyk, Mühlsteinová and Hauer IPPAS B-1526; (c) Dolichospermum sp. (Ralfs ex Bornet and Flahault) P. Wacklin, L. Hoffmann and J. Komárek IPPAS B-1213. Green algae strains (d–f): (d) Chlorella sorokiniana Shihira and R. W. Krauss IPPAS C-1; (e) Ankistrodesmus falcatus (Corda) Ralfs IPPAS A-217; (f) Desmodesmus communis (E. Hegewald) E.Hegewald IPPAS S-313. Red algae strains (g,h): (g) Cyanidium caldarium (Tilden) Geitler IPPAS P-510; (h) Porphyridium cruentum (S. F. Gray) Nägeli IPPAS P-273. Ochrophyta strain (i) Vischeria punctata Vischer IPPAS H-242. Scale bars are 10 µm.

Figure 14

Maintenance and cultivation of microalgae and cyanobacteria strains. (a) slants; (b) flasks; (c) laboratory system for intensive cultivation; (d,e) flat panel photobioreactors, FP-17 and FP-18, respectively. Photographs (a–c)—© IPPRAS; (d,e)— © David Gabrielyan, IPPRAS.

Figure 15

The linkage of plant cells, algae, and cyanobacteria culture collections with biotechnological facilities at IPPRAS. The first three steps of the biotechnological production cycle are accomplished by the collections.