Large scale propagation and in vitro weaning for the restoration of Viola palustris to support assisted colonisation of a threatened butterfly
Article Category: Research article
Published Online: Oct 21, 2021
Page range: 170 - 179
DOI: https://doi.org/10.2478/ebtj-2021-0026
Keywords
© 2021 Jonathan P. Kendon, Alzbeta Novotna, Margaret M. Ramsay, Anne Porter, Viswambharan Sarasan, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Serious declines in larval food plant populations, especially that of marsh violet (
Poor seed germination from the available seed stock and time taken to produce quality propagules for large-scale planting led to exploring high through put cloning using in vitro methods. In vitro methods of propagation can provide propagules for conservation when conventional propagation methods are inadequate (6). Large scale micropropagation systems are now being routinely used for diverse restoration activities underpinning the success of different methods available (7, 8).
The costs of tissue culture methods are greater than for conventional propagation, but critical for achieving tangible and rapid conservation outcomes in some species. In such cases it is important to develop methods that are efficient, both in terms of cost and time, in delivering numbers and quality of propagules. Can in vitro systems be improved to cater for the needs of policy makers and conservationists to meet the demands during a period of unprecedented biodiversity loss and decreasing financial resources?
Seeds received from the Durham Wildlife Trust (DWT) were accessioned at the Royal Botanic Gardens, Kew (RBG Kew) as 4009, 4010, 4013, and 4014. Seeds were collected from extant populations of
Murashige and Skoog (MS) medium (13) was used for all in vitro trials with pH set at 5.8 and 0.8% (w/v) agar (Sigma Chemicals, UK) was used as gelling agent for solid culture medium. All the media were autoclaved at 121°C and 15 psi for 15 minutes. Cultures were kept at 22±2°C on a 16/8 photoperiod of 20 μmol m-2 s-1 for seed germination, 35 μmol m-2 s-1 for vegetative multiplication, and 50 μmol m-2 s-1 for one step rooting and weaning using cool white fluorescent lamps.
GA3-treated and untreated seeds of all accessions were germinated on half strength MS medium in Cell Counter vials (25 mL, Alpha Labs, UK). Activated charcoal (AC) at 0.15% (w/v) and sucrose (2%, w/v) were also included in the germination medium. Single seeds were sown in each vial (10 mL medium) and cultured until seedlings with three leaves developed.
Multiplication of
Cloning by temporary immersion system (TIS) called RITA® (Cirad Biotrop, France) was used for the liquid cultures. After preliminary trials for shoot multiplication with thidiazuron (TDZ), 6-benzylaminopurine (BAP) and 6-(γ,γ-dimethylallylamino) purine (2iP), TDZ was selected as the cytokinin for further culture trials. Fresh multiple shoots (4 weeks old) grown on agar medium containing 0.15 mg/L TDZ and either 0, 0.05 or 0.1 mg/ L 1-naphthalene acetic acid (NAA) were harvested for this study. Liquid medium consisted of full MS nutrients with 3% (w/v) sucrose with 0.15 mg/L TDZ at pH5.8. Since liquid culturing carries greater risk of contamination than semi-solid agar culturing the liquid medium also contained 5mL/L PPM™ for the suppression of bacterial growth. For the TIS immersion cycle, explants on the platform were immersed in liquid medium (120 mL) for 5 min. duration every 30 minutes using an aerating pump (Brighton Systems, UK). To achieve a fast multiplication process, explants used consisted of a clump raised on the agar initiation medium with a maximum of three fully developed shoots.
Multiplied shoot clumps, developed from explanted clumps in the bioreactor, were subdivided into sub-clumps and cultured in Magenta jars containing agar-solidified medium with MS salts and vitamins supplemented with 0.05 mg/ L TDZ.
Cultures were grown in this ‘conditioning medium’ for up to 16 weeks or until shoots with leaves were fully developed that could be grown in plug trays for rooting and weaning. Remaining clumps with shoot buds were subjected to further rounds of TIS multiplication. Growth of endophytic slow-growing bacterial contamination in agar culture was controlled using 3 mL/ L PPM™and NaDCC as used for in vitro collecting (14). NaDCC was added at a rate of 0.03% w/v, via stock solution, after autoclaving and once the medium had cooled but before solidification.
Due to the lack of appropriate facilities for handling of in vitro raised propagules at Low Barns (DWT, Durham) where the propagules were planted out, ‘in vitro pre-weaned’ propagules were prepared at RBG Kew. Rooting plugs were made by mixing one-part vermiculite with one-part tissue paper pulp and 0.7 parts coir (weight/weight) in a blender by gently vortexing for 10 seconds with ½ strength Hoagland’s solution (15) (Sigma Chemicals, UK). This substrate was strained using a sieve to remove excess medium so that the substrate retained sufficient moisture for plantlets’ growth. The substrate was autoclaved at 121°C and 15 psi for 50 minutes – the large volume of the substrate and the organic composition (coir) accounting for the increased time for sterilisation. Plastic flip-top food disposable containers measuring 190×140×70 mm (w×d×h) (Polymer UK, London) were used as the rooting and weaning vessel. A longitudinal rectangular hole was made in the lid of the box and Micropore™ (Millipore, UK) tape (soaked in absolute ethanol and air-dried for sterilisation) fixed over it to improve air exchange but maintain sterility of the culture. Seventy percent ethanol was sprayed into the interior of the containers, the lids closed, and vessels left for 10 minutes. Plastic plug trays with plug well dimensions of 18×17×24 mm (w×d×h) and volume of 5 cm3 (H. Smith Plastics Ltd., Essex, UK), cut to size to fit the culture vessel (35 wells per tray) were also sterilised using 70% ethanol, filled with the prepared mix under sterile conditions and plug-tray transferred to the box.
Elongated shoot cultures, cultured over four weeks in shoot elongation medium containing ½ strength MS medium (agar), with at least three leaves were transferred to individual plugs in sucrose-free ½ strength Hoagland’s solution and incubated under cool white fluorescent tubes at 22±2°C on a 16/8 photo-period of 50 μmol m-2 s-1 (higher light intensity than agar cultures to account for photoautotrophic growth, i.e. the lack of sucrose in the liquid medium) for two weeks. Once the shoots had rooted and produced new leaves under photoautotrophic conditions the tape was removed and the plug trays with plant-lets were transferred to shade house conditions.
Different methods of transplantation of in vitro plug propagules into ex vitro conditions were trialled on site. The best outcome was obtained by transplanting all plug propagules from a whole tray into a single two litre pot containing compost, ‘community planting’ as described before. After at least 5 months pots were assessed for survival of plug propagules and establishment ratings attributed (1-5 where 1 is very poor establishment with few good leaves and 5 is fully established with luxuriant, dense leaf growth).
Number of plants established was counted, and a qualitative ‘establishment rating’ of growth quality and density was given of between 1 and 5, where 1 means very small plants with few leaves, low number of plants per pot and little or no obvious fresh growth, and 5 means full establishment of all plants and profuse fresh growth filling the pot.
The propagules were transplanted from the photoautrophic culture boxes in the early spring when water availability was good and average temperature was conducive to survival of propagules. Propagules from a single plug tray (maximum 35) were either transferred to 2 L plastic pots (20 cm diameter) with a generic compost substrate, ‘community planting’, or as single propagules in smaller pots. Once the propagules in the community pots had been grown for at least 5 months they were planted out into the field sites identified as part of the SPBF project. Planting areas chosen were free of standing water and dense ground cover and close to areas planted with nectar-bearing species grown for adult SPBF since the range of the adult butterfly is only around 200 m (2). Each unit of established
Four sites were selected for planting: Middles Farm, Stuartsfield, Byerley House and Black Plantation. These sites were suitable for assisted colonization of SPBF due to presence of nectar plants used by adults and proximity to each other allowing mixing of adults. Black Plantation had a small extant population of both
Number of seeds germinated was collected after periods of 8, 12 and 16 weeks. Percentage of germination was calculated based on the total number of seeds received against total number of seeds germinated. Germination data was analysed using single factor ANOVA to ascertain whether there were differences between pre-treatments and growing media. Multiplication frequency (number of new shoots per explant) of the shoot cultures in both liquid and agar multiplication was analysed using single factor ANOVA followed by Student’s t-test (Microsoft Excel).
A sample of 58 pots (maximum of 35 propagules in each) was selected that had been transplanted for an average of 10 months. Number of plants established was counted, and a qualitative ‘establishment rating’ of growth quality and density was given. Data on number of plants established per pot per number of bioreactor cycles were gathered, grouped according to accession, and analysed by single-factor ANOVA followed by t-test. A 5 m×5 m quadrat was used at the sites to select plantings to record. For each planting, 4 measurements were taken: module spread per pot (i.e. distance between furthest leaf tips of module x the equivalent perpendicular measure); % ground cover (i.e. in plan-view, percentage cover of marsh violet leaves in spread area versus either other plants or bare ground); number of flowers per community pot; number of fruits per community pot. Plantings over two years were assessed to analyse the progress of establishment in the field. Additionally, data were gathered on percentage ground cover for a small, naturally occurring population of marsh violet near one of the plantation sites (Black Plantation). This allowed comparison of introduced and natural plants. Module-spread and percentage ground cover were analysed by single factor ANOVA followed by t-test. soaking in water with different concentrations of GA3. Sample sizes were insufficient to complete statistical analysis for this test, therefore, GA3 pre-soaking concentration was evaluated based on data available from Millennium Seed Bank (MSB) (16). The accessions 4013 and 4014 that were soaked in 300 mg/L GA3 on basal medium germinated and were used for further trials. Combining accessions 4013 and 4014, 10.2% seed germination after 6 weeks was achieved from 1333 seeds.
Initial tests for agar multiplication showed poor growth of seedlings in media containing various types and concentrations of cytokinin except on TDZ containing medium. Total
All stages of in vitro processes, from seed germination to development of transplantable propagules, and transplantation to field are shown in detail in
Figure 1
Flow chart explaining stages from seedling production (highlighted in green)to transplantation to the field (highlighted in red). Substrates/conditions in which the plants are kept at the various stages are marked (agar, liquid medium, plug system subtrate and field conditions) along with the duration of each stage in days, weeks (wks) or months. Average multiplication rates are shown for agar, liquid and repeated bioreactor cycle stages.
Due to the lack of available seeds collected from the wild only 520 seeds of two accessions were available for the study. The two accessions, 4009 and 4010, were sown following overnight average multiplication rate was 2.12 after 8 weeks. For further rounds of multiplication on basal medium solidified with agar was supplemented with 0.15 mg/L TDZ.
For faster multiplication, the temporary immersion system, TIS, RITA® was used. Shoots were cultured for 10 days in the TIS producing clumps of tissue containing small shoots and buds. When transferred these clumps grew, multiplied further and elongated on fresh basal agar medium supplemented with 0.05 mg/L TDZ.
Data were gathered to investigate the differences in multiplication rate between liquid culture and agar culture. The new shoots were counted 4, 8, 12 and 16 weeks after the first bioreactor cycle. The use of the RITA® system markedly improved multiplication rate of the plants (Fig. 2).
Figure 2
Multiplication rate for Viola palustris cultured in semi-solid agar and liquid immersion system. Agar multiplication alone for 16 weeks; multiplication using RITA® for 10 days followed by 16 weeks on agar; 3 – multiplication using a second RITA® cycle for 10 days followed by 16 weeks on agar. Bars represent means of at least 28 explants +/- standard error. Bars with the same letter are not significantly different (P>0.05).
After 16 weeks of growth, one cycle on agar based medium explants produced an average of 14±0.89 shoots. However, after a single bioreactor cycle of 10 days plus 16 weeks in agar an average of 41.2±0.95 shoots were produced. After a second bioreactor cycle, over 10 days, 34.4±1.41 shoots were produced after 16 weeks (Fig. 2). This represents a largeincrease when cultures were grown in the bioreactor before multiplication on agar medium. Additionally, there was no significant decrease in shoot number achieved between single and multiple bioreactor cycles.
Plants were screened for flowering, seed-set and germination and we noticed no changes in morphology and phenology in the clonal propagules. Flow cytometry analysis of clonal in vitro propagules showed no difference in genome size compared to the original seed-derived progenies.
The shoots harvested from the bioreactor were grown for four weeks in the shoot elongation medium (Fig. 1) for the single step rooting and weaning trials. Conventional agar-based media containing low concentrations of minerals and low or no sugar, resulted in poor rooting (Fig. 3a and b) and none of the transplanted plants survived under glasshouse conditions.
Figure 3
Improved rooting of in vitro Viola palustris propagules using photoautotrophic plug substrate and sucrose-free liquid nutrient medium. a – agar culture; b – roots of agar-grown propagule; c – photoautotrophic plug propagule; d – photoautotrophic propagule with substrate removed to show robust root growth. Scale bars 1 cm.
Roots produced using the plug system were numerous and robust following culture of the elongated shoots grown on ½ strength MS agar medium (Fig. 1, Fig. 3c and d).
Data were analysed for number of plants established and establishment rating (Fig.4) for a representative sample of in vitro propagules in boxes transplanted to two litre pots. Average establishment rating for all measured pots was 3.8 and average number of plug propagules established per tray was 23. Average number of propagules produced per tray in vitro had been 32, therefore overall establishment from in vitro to establishment in pots under field conditions was 72%. No significant difference was observed (P > 0.05) between seed accessions for either establishment rating or number of plants established. Establishment was also tested by number of bioreactor cycles to assess whether deterioration in establishment occurred for propagules that had undergone extra bioreactor cycles. No significant difference was observed in either parameter for propagules that had undergone multiple bioreactor rounds.
Figure 4
Establishment rating measure for Viola palustris five months after potting up from photoautotrophic plug trays. A = 2, b = 3, c = 4, d = 5. No photograph is available for rating 1 because no pots achieved an establishment rating of 1, i.e. very poor or no establishment.
The spread and percentage cover of each planted community pot varied across the different sites when analysed after four months and 16 months (Fig. 5). These
Figure 5
Growth and establishment of ex vitro Viola palustris propagules in field sites after 16 months (2014 plantings) and 4 months (2015 plantings). Bars represent the average from a minimum of five colonies +/- standard error. b = significantly (P<0.05) lower ground cover than all other sites.
Within planting years there were no significant differences (P > 0.05) in module spread or percentage cover for each community pot planted. Percentage ground cover was higher in the 2015 plantings (P < 0.05) than the 2014 plantings (Fig.5), possibly due to reduced competition from surrounding vegetation.
Endophytic bacterial contaminants are a serious bottleneck in liquid cultures and eradicating them completely to successfully clone plant material is a challenge. Endophytic contamination of
is available (19, 20, 21). Antibiotic use is common practice for crops and rare plant micropropagation (21, 22) to eliminate bacterial contamination. However, antibiotics were not used in this study because the propagules raised were to be used as food plants by the larvae. Additionally, use of antibiotics in conservation related tissue culture activities cannot be considered as an appropriate practice. Propagules raised in antibiotic/s-containing media should be avoided in native plant conservation,
Figure 6
Establishment of modules planted out in the field. a – 16 months after planting; b – 4 months after planting. Scale bar 5 cm.
especially those that are used in reintroduction and restoration activities. Successful eradication of endophytic bacteria was observed in black kangaroo paw (
Use of propagules from non-meristematic tissue through either callus mediated or direct organogenesis has intrinsic risks in producing plants of altered genotypes. Tissue culture-regenerated propagules are susceptible to somaclonal variations as described in
Another of key finding of our study was the high quality of shoot cultures produced with no apparent presence of hyperhydration. Previously, hyperhydration has been reported during multiplication in liquid media-based large-scale multiplication (29, 30). Juvenile explants of
Photoautotrophic micropropagation using sugar-free medium is widely used based on the success of producing relatively high photosynthetic abilities of plantlets in vitro (31, 32). Our study revealed that uniform high-quality propagules can be raised through this system which needs minimum or no transition period between removal from the photoautotrophic system and survival ex vitro. Shoot proliferation of
When resources for conservation are spread for ever increasing lists of threatened plants, available funds for recovery, propagation and restoration are limited. Lengthy labour-intensive methods are costly and were cited as bottlenecks in delivering success in using in vitro propagules for autumn buttercup conservation (35). There are many reports that highlight the potential application of bioreactor technology to aid conservation (35, 36, 37, 38). However, to our knowledge there is no published report yet on using a bioreactor system to mass propagate native plants to support large scale restoration of butterflies.
Establishment of 72% of propagules is a very satisfactory return given the absence of specialised horticultural facilities on site and inexperience of staff and volunteers handling the plug propagules. Our results show that ‘community planting’ improved survival rate compared to propagules transplanted individually. It was noted that in extant populations,
Both 2014 plantings occurred in tree plantation areas and may have had a lower light level and nutrient availability. Additionally, Stuartsfield site was found to have a drier soil while Black Plantation had a waterlogged soil with little drainage. Both Middles Farm and Byerley House sites appeared more suitable for the plants, with wet but well-drained soil, inclined ground and higher light levels with less tree shading and this observation supports the previous findings. The coppice rate fell by 90% in actively managed woodlands in England between 1905 and 2000 (39). As a result, many woodland gap-dependent butterfly species declined during this period, including the
The ‘community planting’ model used in this study with plants in pots and their subsequent transfer to selected assisted colonisation sites during spring helped establishment of around 14,000 plants. Linking different assisted colonisation sites, mainly farmland, with wild sites in this study has the potential to develop connectivity corridors for marsh violets to recruit naturally and thereby expand the occurrence of SPBF. Recovery programmes that aim to establish new wild populations of species face fresh challenges in the age of climate change (41).
As the geographic ranges of some rare taxa have become increasingly restricted conservation intervention and/or restoration requires highly effective systems (42). Attempts to reintroduce species back to their original wild habitats or introduce them to different areas (translocation/assisted colonisation) requires an assessment of the suitability of potential sites (43, 44) as success is linked to climatic suitability (45). We think the systems we used, propagules developed, and the strategies adopted to establish sustainable colonies of food plants for SPBF were suitable and robust for the large-scale nature of the project.
Complex layers of lengthy, expensive propagation processes, loss of propagules during acclimatisation, and very low establishment under field/wild conditions are driving away policy makers and conservation practitioners from using in vitro methods. Our research showed that high throughput cost effective in vitro methods underpin the success in large scale propagation of genetically diverse germplasm. Simultaneously, propagules raised via in vitro systems should be resilient and easy to handle by untrained people for successful establishment. Ultimately, the results of this study will lead to the successful establishment of populations of both marsh violet and the small pearl-bordered fritillary butterfly as preliminary results are very promising. This shows smart, time-bound and low-cost methods could successfully support large scale projects in a changing climate where biodiversity loss is a serious issue.