Germination of pollen grains of various Linum species on the artificial medium

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Institute of oilseed crops of the NAAS

Germination of pollen grains of various Linum species on the artificial medium

A.I. Soroka

V.A. Lyakh

Abstract

Pollen grains with not-burst (normal) and burst pollen tubes were recorded separately. Results. The media with PEG-2000 in concentrations of 20-30% ensured germination of pollen from both homostylous and heterostylousLinum species up to 40-50%. The addition of sucrose to a medium with PEG disadvantageously influenced pollen grain germination as well as the increase in the PEG molecular weight. Pollen germination on the medium containing PEG-20000 led to the increase in the number of pollen grains with burst tubes. Addition of tris did not influence pollen germination in

L. usitatissimum or negatively affected ability of pollen grains to germinate in L. grandiflorum. The successful in vitro germination of pollen in species of the Linum genus allows to evaluate the quality of flax pollen quickly and efficiently and more successfully implement many genetic and breeding programs. This is especially important for such an important agricultural crop as cultivated flax for which artificial medium for pollen germination was not developed for now.

Key words: Linum, pollen, in vitrogermination, normal and burst pollen tubes, polyethylene glycol, sucrose.

Анотація

Проростання пилкових зерен різних видів роду Linum на штучному поживному середовищі

А.І. Сорока, В.О. Лях

Інститут олійних культур Національної академії аграрних наук України 2 Запорізький національний університет

Мета. Метою даної роботи було вивчити вплив різних компонентів поживного середовища на проростання пилку ряду гомостильних і гетеростильних видів льону.

Методи. Як джерело пилку використовували чотири гомостильні, в тому числі льон культурний, і чотири гетеростильні види льону. Штучне поживне середовище, що використовувалося для проростання пилку in vitro, включало основні компоненти - борну кислоту та хлорид кальцію в концентраціях 200 мг/л, а також досліджувані інгредієнти - поліетиленгліколь (ПЕГ) різної молекулярної маси, сахарозу та тріс (тріс (гідроксиметил) амінометан). Пилок пророщували 3-4 години при температурі 25±1 °С у темряві. У кожному варіанті аналізували від 300 до 400 пилкових зерен у п'яти повтореннях. Пилкові зерна з цілими (нормальними) та пилковими трубками, що лопнули підраховували окремо.

Результати. Середовище з ПЕГ-2000 у концентраціях 20-30% забезпечувало проростання пилку як гомостильних, так і гетеростильних видів Linumз частотою до 40-50%. Додавання сахарози до середовища з ПЕГ, а також збільшення молекулярної маси ПЕГ, несприятливо впливало на проростання пилкових зерен, Проростання пилку на середовищі з ПЕГ-20000 призводило до збільшення кількості пилкових зерен із трубками, що лопнули. Додавання тріс не впливало на проростання пилку L. usitatissimumабо негативно впливало на здатність пилкових зерен проростати у L. grandiflorum.

Висновки. Успішне проростання пилку in vitro у видів роду Linumдозволяє швидко й ефективно оцінити якість пилку льону та більш успішно реалізовувати різні селекційно-генетичні програми. Це особливо актуально для такої важливої сільськогосподарської культури як льон, для якого на даний момент не розроблено штучне середовище для пророщування пилку.

Ключові слова. Linum,пилок, пророщування in vitro,нормальні та лопнуті пилкові трубки, поліетиленгліколь, сахароза.

Main part

Introduction. LinumusitatissimumL. is one of the oldest cultivated plants, whose products have long been used for a variety of human needs. Wild flax species are the donors of many morphological, physiological and biochemical traits for cultivated flax. Some annual and perennial wild relatives are highly ornamental plants and are successfully applied in landscaping, flower bed arrangement, and gardening (Jhala et al, 2008; Lyakh and Soroka, 2008; Lyakh, 2013; Arslanoglu and Aytac, 2020).

A very broad use of various flax species provides for knowledge of the quality of pollen, produced by the plant. Pollen quality influences seed set and ensures high seed yield. Pollen viability is important in breeding programs for inter- and intra species hybridization (Zabicka et al, 2020; Kopecky et al, 2022). Pollen could be also used to estimate some characteristic responses of mature plants to biotic and abiotic stresses and could serve as a sensitive and simple bioassay for environmental. Different pollen breeding technologies require knowledge of pollen quality as well (Hebbar et al., 2018; Lyakh and Soroka, 2022; Patil et al, 2006; Sorkheh et al, 2018).

Nutrient media have already been developed for many agricultural and ornamental plants, which ensure the successful germination of pollen under artificial conditions (Liu, 2013; Jayaprakash, 2018; Wani et al, 2020). However, flax until recently was considered a crop whose pollen did not germinate well on a nutrient medium although some attempts have been made to select a suitable nutrient medium (Pandey and Kumar, 2013).

The purpose of this work was to study the effects of various components of the nutrient medium on the germination of pollen in a number of homostylous and heterostylous flax species to facilitate pollen quality estimation.

Materials and methods.Homostylous wild (L.angustifoliumHuds., L.bienneMill., and L.hispanicumMill.) and cultivated linseed variety Iceberg (L. usitatissimumL.) as well as heterostylous(L.grandiflorumDesf., L.hirsutumL., L.pubescensBanks and Solander, and L.thracicumDegen) Linumspecies, were used in our studies as pollen sources. Pollen was collected at early hours from 7 to 9 a.m., out of 20-40 flowers.

To study the influence of polyethylene glycol and sucrose in a nutrient medium on in vitro pollen germination we took as a basic medium the one containing boric acid and calcium chloride in the concentrations of 200 mg/l. Besides above mentioned components polyethylene glycol (PEG) of various molecular weights, sucrose, or both of them were supplied to the media. A separate medium had also tris (tris (hydroxymethyl) aminomethane) included. Generally the following media were used:

(a) PEG 2000, 20%

(b) PEG 2000, 30%

(c) PEG 2000, 30% + sucrose, 5%

(d) PEG 2000, 30% + sucrose, 15%

(e) PEG 2000, 30% + sucrose, 15% + tris, 1 mg/l

(f) PEG 6000, 30% + sucrose, 5%

(g) PEG 20000, 5% + sucrose, 15%

(h) PEG 20000, 30% + sucrose, 15%

Pollen was germinated for 3-4 hours in a drop of an artificial medium placed on a slide at the temperature of 25±1 °C in the dark. After that, the pollen was viewed under a light Leica microscope (Germany) with a 20^X objective. Pollen grains were counted as germinated if the pollen tube length was more than a pollen grain diameter. Pollen grains with not-burst (normal) and burst tubes were recorded separately. For each 5 replications of each treatment from 300 to 400 pollen grains were analyzed (Lyakh, Soroka, 2008).

The results of the experiments were analyzed statistically using one-way analysis of variance (ANOVA) Table and chart data are presented as mean ± standard error of mean (SEM). A difference between two values was considered significant at p< 0.001, according to Bailey, 2008 and Lyman and Longnecker, 2015.

Results and Discussion. Pollen grains with both normal and burst pollen tubes were detectable among the germinated pollen on all the nutrient media tested in the experiment. The number of normal tubes as a rule considerably exceeded number of burst tubes. At the same time, the ratio of both was significantly influenced by the composition of the nutrient medium (table).

Influence of PEG-2000 concentration on pollen germination in homostylous and heterostylousLinum species (2018-2020) PEG 20% PEG 30%

Table also shows that a medium containing polyethylene glycol-2000 as an osmotic in the concentration of 20% ensured a sufficiently good germination of the pollen for the species under study. The percentage of pollen grains with normal (not burst) pollen tubes ranged from 22.7 in L. bienneto 51.0 in L. grandiflorum. The elevation in concentration of PEG from 20% to 30% reduced this indicator for such species as L. angustifolium, L. bienneand L. hispanicum, and did not affect the pollen germination in all heterostile species, except L. hirsutum, where an increment in the percentage of pollen grains with normal pollen tubes was observed. It should be also noted that regarding the proportion of pollen grains with burst pollen tubes, there was a regularity in decreasing this fraction on a medium containing PEG 30%, compared to PEG 20%, and for three species this difference was even significant. In general, on both media, the number of grains with burst pollen tubes was significantly smaller than the amount of normally germinated pollen grains in all species except for L. usitatissimum, in which they accounted for about 40% of all pollen capable of germination.

Addition to the nutrient medium with PEG-2000 of such an osmotic agent as sucrose adversely affected pollen germination as a whole. And, if the inclusion of sucrose at the concentration of 5% to a medium with PEG 30% reduced the number of pollen grains with normal pollen tubes in only L. usitatissimumand 2 heterostylous species, elevating the sucrose concentration up to 15% reduced the number of germinated grains in all the species (with an exception of L. bienneand L. hispanicum) as demonstrated in fig. 1 for L. grandiflorumand L. hirsutum.

Fig. 1. Influence of sucrose concentration in the medium with PEG-2000 30% on pollen germination in four homostylous (A) and four heterostylous (B) Linumspecies: (b) 0% sucrose, (c) 5% sucrose, (d) 15% sucrose

Fig. 2 and Fig. 3 data grant an idea on the influence of PEG with different molecular weight on pollen germination

Fig. 2. Influence of PEG molecular weight on pollen germination in different Linum species: (c) PEG-2000, (f) PEG-6000

Fig. 3. Pollen germination of L. grandiflorum (1) and L. hirsutum (2) on the media with PEG of different molecular weight: (a) PEG-2000, (b) PEG-6000

When pollen was cultivated on the media, containing PEG at the same concentration (30%) but differing in molecular weight, the number of pollen grains with normal pollen tubes was significantly larger in the case of an osmotic with a lower molecular weight. It is characteristic that the degree of reduction of the investigated indicator on a medium with a larger PEG molecular weight varied for different species. Thus, among the heterostylous flaxes, the number of pollen grains with normal pollen tubes on a medium supplied with PEG-6000, compared to PEG-2000, for L. grandiflorumand L. thracicumdecreased by a factor of 6.6 and 20.5, whereas for L. pubescensand L. hirsutum- the reduction amounted to 34.0 and 24.1% only.

As for the effect of tris it was studied in a limited way for two flax species. This substance at the concentration of 1 mg/l did not affect pollen germination of cultivated flax. At the same time, the pollen of L. grandiflorumgerminated much better on a medium without tris supplement than in the case when it was included as a component of the nutrient medium (fig. 4).

Fig. 5 demonstrates the peculiarities of the germination of pollen for several flax species on a medium containing a high molecular weight polyethylene glycol (PEG-20000) at the concentration of 5%, against the background of 15% sucrose, showing the proportion of pollen grains with normal and burst pollen tubes.

In this case it can be seen that the percentage of pollen grains with burst tubes for all the species was quite large. And for such species as L. pubescensand L. hirsutum, it even exceeded the number of pollen grains with normal tubes. If we talk about the proportion of the grains with normal pollen tubes, it was the closest to that observed when cultivating the pollen on the medium, containing against the background of 15% sucrose polyethylene glycol with a molecular weight of 2000 at the concentration of 30%. When the medium included PEG-20000 at the concentration of 30%, pollen of all the studied species failed to germinate.

Fig. 4. Influence of tris on pollen germination in some Linumspecies: (d) 30% PEG 2000 + 15% sucrose, (e) 30% PEG 2000 +1 mg/l tris

Fig. 5. Proportion of pollen grains with normal and burst pollen tubes after their germination on the medium with PEG-20000 in different Linum species

flax pollen linum

Significant differences in the ability of pollen of the gomostylous and heterostylous species to germinate in vitro on the media studied were not found. At the same time, as can be seen, in our experiment with different flax species, heterostylous flaxes equally well germinated on the media both with PEG 2000 20% and 30%, while homostylous flaxes, except L. usitatissimum,looses ability to germinate with an increase of PEG concentration. Focusing on this issue is based on the revealed differences in the biological activity of lectin-like proteins of generative organs of self- pollinating and cross-pollinated species of plants (Lazareva, 2009). According to data of Southworth (1974), Costa et al. (2013) those proteins can influence the germination of pollen both in vitro and in vivo. Quite possible that wider genetic background of heterostylous species allows them easier adapt on the pollen level to various environmental conditions.

Flax species are economically important crops in many countries around the world cultivating both for oil and fiber production and with ornamental purpose.

Pollen of both homostylous and heterostylous species of the genus Linum germinates well on the media containing, in addition to the basic components boric acid and calcium chloride, an osmotic agent in the form of polyethylene glycol-2000.

Replacing this osmotic with polyethylene glycol of a higher molecular weight or adding sucrose significantly impairs pollen germination rates.

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