Characteristics of callusogenesis in oilseed flax under the influence of factors that imitate osmotic stress

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Institute of Oilseed Crops of the National Academy of Agrarian Sciences

Characteristics of callusogenesis in oilseed flax under the influence of factors that imitate osmotic stress

A.I. Soroka

Annotation

The paper was aimed to investigate the impact of substances inducing osmotic stress on flax callus culture characteristics. Two varieties of oilseed flax, Zaporozhsky Bogatyr and Evrika, were used as the material for study.

Callus obtained from flax hypocotyls was cultivated on a modified N6 medium with the addition of substances inducing water deficiency stress, namely, polyethylene glycol (PEG 15000) and mannitol. PEG was added at the concentrations of 10%, 20%, and 30% and mannitol at the concentrations of 0.4M, 0.8M, and 1.2M. Both substances were found to have a significant effect on the development of flax callus. That was manifested in growth inhibition, emergence of necrotic areas, and overall depression. The lower concentrations of osmotic substances did not affect the callus culture as much as the higher concentrations, which caused partial or total growth arrest and cell death. Consequently, intermediate concentrations of osmotic substances, such as 20% PEG or 0.8M mannitol, or values comparable to those, can be considered the most suitable for further research towards developing a system to select drought-tolerant flax genotypes.

Key words: flax, callusogenesis, osmotic stress, polyethylene glycol, mannitol, selection.

Особливості калусогенезу льону олійного під впливом факторів, які імітують осмотичний стрес

A.I. Сорока. Інститут олійних культур Національної академії аграрних наук України

Однією з основних перешкод для стабільного розвитку сільського господарства у сучасних умовах є посуха. Вплив посухи спричиняє низку негативних наслідків для рослин, таких як погіршення росту вегетативної системи, зниження схожості насіння, порушення біохімічних процесів, що в результаті призводить до зниження врожайності (Fahad Shah et al. 2017). Дефіцит зрошувальних земель, відсутність насіння необхідної якості, низький рівень механізації, зловживання хімікатами лише посилюють цю проблему (Dev, 2012). Наприклад, у Сполучених Штатах постійні посухи змушують фермерів переходити на більш стійкі культури, а в деяких країнах Африки нестача води під час посух призводить до загрози продовольчій безпеці цих регіонів (FAO Report, 2015). З огляду на вищевикладене, актуальним завданням є створення нових сортів льону, що мають підвищену стійкість до абіотичних стресів, зокрема до посухи.

Добір in vitro, незважаючи на те, що це відносно недавній підхід до підвищення якості агрономічних ознак у культурних рослин, продемонстрував ефективність на ряді культур (Shehata et al. 2014; Kruglova et al. 2018; Sahu et al. 2023). Ця методика дозволяє створювати цінний агрономічний матеріал, адаптований до умов дефіциту вологи та інших стресових факторів. Однак технології оцінки та відбору посухостійких генотипів на рівні in vitro на льоні ще недостатньо розроблені або навіть невідомі.

Метою даного дослідження було дослідити ефекти формування калусної маси під впливом факторів, що імітують осмотичний стрес в культурі льону in vitro, та виявити особливості дії селективних факторів у калусній культурі.

В якості селективних агентів для індукції осмотичного стресу використовувалися поліетиленгліколь молекулярної маси 15000 (ПЕГ 15000) і маніт (d-манітол). ПЕГ 15000 застосовувався в концентраціях 10%, 20% і 30%, тоді як маніт використовувався в концентраціях 0,4M, 0,8M і 1,2M. Лляний калус культивували на модифікованому штучному поживному середовищі N6 (Chu 1978) з цими осмотиками протягом чотирьох тижнів. У цьому експерименті вивчалася реакція двох генотипів льону олійного різного походження Запорізький богатир і Еврика.

На зазначених середовищах у штучних умовах калус культивували без світла при температурі 25±1°С за стандартними методиками. Контрольну групу культивували на тому ж поживному середовищі без додавання селективного агента. В кінці періоду культивування аналізувався стан розвитку калусу (загибель, виникнення некротичних ділянок, зміна розміру, загальний стан). Ступінь росту калусу оцінювалася в балах за шкалою від 0 до 4, де 0 приймалося за повну відсутність розвитку калусу, 1 не більше 25% експланта, вкритого новоутвореною калусною масою, 2 і 3 поява нових зон проліферації приблизно на 50% і 75% поверхні експланта, 4 новоутворений калус вкриває всю поверхню експланта.

У результаті проведення досліджень встановлено, що на середовищах з додаванням манітолу в концентраціях 0,4 М, 0,8 М і 1,2 М частка калусу з активним ростом знижувалася і спостерігалася поява некротичних зон. Концентрація 0,8 М виявилася найбільш підходящою для забезпечення вибіркового впливу на популяцію клітин для виділення клонів, стійких до осмотичного стресу, оскільки чітко дозволяла диференціювати калуси за життєздатністю.

Introduction

In modern conditions, which are accompanied by global warming, drought is becoming one of the main obstacles to the stable development of agriculture. The effect of drought causes a number of negative effects on plants, such as reduced seed germination, deterioration in the growth of the vegetative part, disruption of biochemical processes, which results in yield reduction (Fahad Shah et al. 2017). The shortage of irrigation systems, lack of quality seeds, low level of mechanization, misuse of chemicals just aggravate this problem (Dev, 2012). For example, in the southwestern United States, persistent droughts have already forced farmers to switch to drought-tolerant crops, and in several parts of Africa, water shortage during droughts have reduced crop yields, jeopardizing food security in those regions (FAO Report, 2015).

In this regard, crops that are more tolerant to low rainfall are becoming increasingly popular. One of those crops is flax a plant that has a significant number of positive characteristics. So, flax can be grown on a wide variety of soils, from heavy clay to light sandy, it is rather hardy, can tolerate short-term periods of drought and has a not long vegetation period. This makes it possible to grow flax both in hot regions and in areas with cool climate and short summer. In addition, flax is known for its versatility its seeds can be used to produce oil, fiber, dietary foods, added directly to bakery products, and flax fiber is a strong and durable material that is used in the textile industry for the manufacture of hypoallergenic clothing and technical fabrics.

Flax is also considered a low-cost crop, since it requires less fertilizers and pesticides to grow, compared to other crops, making its production environmentally friendly. Given the above, the development of new flax varieties characterized by positive traits and possessing increased resistance to abiotic stresses, in particular, to drought, is an urgent task.

In vitro selection, despite being a relatively recent approach for enhancing agronomic traits in plants, has exhibited efficacy in a range of crops (Shehata et al. 2014; Kruglova et al. 2018; Sahu et al. 2023). This technique enables the creation of valuable agronomic material that is adapted to conditions of moisture deficiency and other stressors. However, the technologies for evaluating and selecting drought resistant genotypes at the in vitro level in flax are not yet adequately developed or remain unknown.

The purpose of this study was to investigate the effects behind callus formation under the impact of osmotic stress mimicking factors in flax in vitro culture and to identify the characteristics of the action of selective factors in callus culture.

Materials and Methods. Polyethylene glycol of molecular weight 15000 (PEG 15000) and mannitol (d-mannitol) were employed as selective agents to induce osmotic stress for flax callus. PEG 15000 was used at the concentrations of 10%, 20%, and 30%, while mannitol was used at the concentrations of 0.4M, 0.8M, and 1.2M. Flax calli were cultured on a modified artificial nutrient medium N₆ (Chu 1978) with those osmotics for four weeks. In this experiment, the reaction of two genotypes of oilseed flax of different origin was studied Zaporozhsky Bogatyr and Evrika.

On the indicated media, under artificial conditions, calli were cultured without light at the temperature of 25±1°C according to standard techniques. The control group was planted on the same nutrient medium, however without the addition of a selective agent. At the end of the cultivation period, the state of callus development (death rate, occurrence of necrotic areas, changes in size, visual state) was analyzed. The degree of callus proliferation was scored in points based on the scale from 0 to 4, where 0 was taken as the complete absence of callus development, 1 not more than 25% of the explant covered by newly formed callus, 2 and 3 the appearance of new proliferation zones on about 50% and 75% of the explant surface, 4 newly formed callus covered the entire surface of the explant.

The present study employed the analysis-of-variance method to assess the reliability of divergences observed among the treatments under investigation. Furthermore, we utilized the Plokhinsky method (Lakin 1990) to quantify the degree of influence of the stressful factor on the resistance of callus culture to osmotic stress.

Results and Discussion

Assessment of the influence of mannitol on flax callus culture

selective mannitol polyethylene glycol callus culture flax

Mannitol was utilized as one of the selective agents to simulate osmotic stress at varying concentrations of 0.4M, 0.8M and 1.2M. In the treatment flax calli were cultured on the N6 nutrient medium with that osmotic substance. Control calli were grown on the same medium lacking osmotic stressor. The experiment involved two genotypes of oilseed flax of different origin Zaporozhye Bogatyr and Evrika.

By incorporating mannitol into the nutrient medium at specific amounts, there was a notable impact observed on the growth and development of flax callus. This effect was evidenced by a reduction in the proportion of calli exhibiting active growth and an increase in the occurrence of calli displaying necrotic zones (Table 1).

Table 1

The influence of mannitol as a selective agent imitating osmotic stress on the development of callus in two varieties of oil flax (2022)

*differences from the control are significant atp < 0.05

At the lowest concentration of mannitol, the percentage of calli exhibiting visual enlargement was significantly lower than in the control treatment, yet remained sufficiently high while presenting no noticeable necrotic areas. Increased osmotic pressure within the medium caused cellular death for a subset of calli, leading to the appearance of necrotic lesions. This characteristic was present in treatments with concentrations of mannitol at both 0.8M and 1.2M. Additionally, callus growth was completely impeded at the highest concentration of mannitol. At an osmotic concentration of 0.8M, despite the presence of dead cellular clusters on certain calli, growth continued for the remaining calli.

The extent of callus proliferation exhibited a positive correlation with the occurrence of necrotic regions, and was contingent upon the concentration of osmotic components in the nutrient medium. Notably, under untreated conditions, the proportion of Zaporozhye Bogatyr calli exhibiting continuous growth across the entirety of their surface (as scored by a 4-point scale) was determined to be 93.3%. In contrast, upon exposure to a concentration of 0.4M mannitol, only 10.0% of calli displayed this trait, and complete absence of such calli was observed in subsequent tests where mannitol concentrations were elevated to 0.8-1.2M (Fig. 1).

Such a response of flax callus culture to osmotic challenges at the concentrations studied can be leveraged to select for clones that are resistant to osmotic stress, by exerting a selective effect on the cell population.

Our results are consistent with data from other researchers who have used mannitol as a water stress-inducing agent. So, Hadi et al (2014) showed that an increase in the amount of mannitol in the medium from 0 to 300 g/l causes a progressive decrease in the fresh weight of Ruta graveolens callus when cultivated for 12 weeks. In the experiments of Erst et al (2019), the response of callus culture to stress was somewhat more complex. Thus, calli of Populus pruinosa demonstrated an increase in dry biomass with an increase in the concentration of mannitol in the nutrient medium, and calli of other Popular species and hybrids between them showed.

In relation to the reaction of the flax genotypes studied, it is noteworthy that while all exhibited a comparable response, the Evrika variety displayed a comparatively heightened sensitivity towards the stressor. This is evidenced by the reduction in the proportion of calli with active growth in treatments with low mannitol concentrations, although this difference did not exhibit statistical significance (Table 1).

Fig. 1. The degree of proliferation of oil flax calli of the Zaporozhye Bogatyr variety depending on the concentration of osmotic agent (mannitol) in the medium: 0 no callus development, 1-4 new proliferation zones covering 25%, 50%, 75%, and the entire surface of an explant, respectively

The findings from the test evaluating the extent of the impact of stressful factor have verified that osmotic agent had the primary effect on the capability of callus for subsequent development, contributing to 87.64% of the impact. Conversely, the role of genotype was observed to remain relatively minor, at less than 1%.

The response of calli of two flax genotypes to mannitol addition as a selective agent that simulates osmotic stress has been visually presented in Fig. 2. It is apparent from the figure that the growth of callus cells for both varieties was restricted by mannitol at concentrations between 0.4-1.2M. There was no significant difference in the reaction of the two tested genotypes.

Fig. 2. The reaction of flax varieties Zaporozhye Bogatyr (bottom row) and Evrika (top row) to osmotic stress induced with mannitol (from left to right: control, 0.4M, 0.8M, and 1.2M mannitol in the medium)

Consequently, all three tested concentrations of mannitol adversely affected the growth and development of oilseed flax calli. However, for selective use, the addition of mannitol at the concentration of 0.8 M or close to it can be considered more acceptable, which ensures not only the growth of a certain part of callus cells, but also results in elimination of a significant proportion of them.

Evaluation of the impact of polyethylene glycol on the flax callus culture

The induction of osmotic stress in cells is not limited to the use of mannitol. Other substances that are widely used to create water deficiency are sucrose and polyethylene glycol (PEG). Hydrophilic molecule of PEG can form hydrogen bonds with water molecules, which allows it to reduce the availability of free water molecules and increase the concentration of solutes in the surrounding environment, which imitates water stress conditions. In addition, PEG is relatively inert and very soluble, and that makes it a convenient and effective osmotic agent for in vitro experiments. PEG as an osmotic agent was used with success to differentiate drought tolerant cell lines in several crops, such as rice (Biswas et al 2002), sunflower (Hassan et al 2004), pepper (Nath et al 2005), wheat (Mahmood et al 2012), and others. However, there is no similar research for flax or Linum species.

PEG is commercially available in different molecular weights ranging from 300 to 10 000 000 g/mol (French et al 2009). In our work we used PEG 15000 in concentrations of 10%, 20%, and 30%. The addition of PEG to the nutrient medium at certain concentrations resulted in an effect on the growth and development of flax callus. This effect was demonstrated through a reduction in the proportion of calli with active growth and the appearance of calli with necrotic zones (Table 2).

Table 2

The influence of polyethylene glycol as a selective agent imitating osmotic stress on the callus development in oilseed flax (2022)

*,*** differences from the control are significant atp < 0.05 and 0.001, respectively

At the lowest PEG concentration the proportion of calli that increased noticeably in size, i.e., were characterized by active growth, did not change significantly in comparison with the control treatment for the Zaporozhsky Bogatyr variety and slightly decreased for the Evrika variety. An increase in the amount of osmotic substance in the medium caused cell death in some calli, which was manifested as the necrotic spots. However, those changes were not significant for both studied varieties. This was true for all osmotic concentrations tested.

It should be noted that at high concentration of PEG in the medium the growth of callus was completely arrested. The tested flax varieties exhibited different responses to an intermediate concentration of the osmotic agent, specifically 20%. The variety Zaporozhsky Bogatyr displayed higher tolerance and subsequently a significant proportion of calli with active growth 95.2%, whereas the variety Evrika had only a few calli able to continue active growth, with a frequency of 7.1%. Such reactions of the flax callus culture may allow for future selective pressure on the cell population to identify Linum clones that are resistant to osmotic stress.

With regards to the response of the genotypes utilized in the experiment, it is pertinent to acknowledge that despite being of the same nature, the Evrika variety demonstrated greater susceptibility towards the stressor. Specifically, the proportion of calli with active growth noticeably declined even with exposure to the minimal concentration of PEG, 10%. Moreover, at a PEG concentration of 30%, no calli with active growth were detected for this variety. Conversely, the Zaporozhsky Bogatyr calli exhibited active growth even upon exposure to 20% PEG concentration in the medium and ceased growth solely at the highest PEG concentration (i.e., 30%).

Conclusions

On the other hand, the utilization of polyethylene glycol (PEG) with a molecular weight of 15000 as a selective factor leading to osmotic stress, has demonstrated that for the selection of drought-resistant flax genotypes, a concentration range of 10-20% can be considered optimal.

While the tested genotypes demonstrated a comparable response to the stressors employed in the study, some disparities exist that must be considered while developing selective systems for flax callus culture.

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