Apomixis-prone winter rape (Brassica napus L.) forms: biological and economic features

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Apomixis-prone winter rape (Brassica napus L.) forms: biological and economic features

N.А. Hlukhova, D. К. Ehorov

Plant Production Institute named after V. Y. Yuriev National Academy of Agrarian Sciences of Ukraine

We studied the features of development, morphology and economic characteristics of winter rapeseed forms capable of partial apomixis (apomictic form), an original approach to the study of apomixis has been applied. Based on these results, we explained the existing differences in the morphology and development of lines of winter rapeseed with partial apomixis (80-100 % of plants set seeds without pollen) and varieties propagated exclusively by amphimixis. The studies were carried out in the field and laboratory conditions of the Institute of Plant Production Institute named after VYa Yuriev NAASU (Kharkiv, Ukraine). Apomictic forms were characterized by high germination energy (p=0.000001), early maturity (p=0.000524), long winter dormancy (p=0.000001) and long flowering (p=0.000044). Apomictic forms had sprawling rosettes during the autumn vegetation period, branched roots and deep winter dormancy. On average, over five years of research, apomictic plants produced 23-30 % more pollen than the control variety Black Veleten. In terms of productivity, apomictic forms ambivalently reacted to the conditions of the year.

Key words: apomixis, amphimixis, pollen-producing ability, rapeseed, winter hardiness, yield, oil content. pollen producing ability rapeseed

ФОРМИ РІПАКУ ОЗИМОГО (BRASSICA NAPUSL.), СХИЛЬНІ ДО АПОМІКСИСУ: БІОЛОГІЧНІ ТА ГОСПОДАРСЬКІ ОСОБЛИВОСТІ

Н. А. Глухова, Д. К. Єгоров

Інститут рослинництва імені В.Я.Юр'єва Національної академії аграрних наук України

Метою дослідження було вивчення особливостей розвитку, морфології та формування господарських якостей форм ріпаку озимого, здатних до часткового апоміксису (apomixes-form). Дослідження проводили в польових та лабораторних умовах Інституту рослинництва імені В.Я. Юр'єва НААН, м. Харків. Вивчали самозапилені лінії, що були виділені з сортів Liradjet, Світоч, Северянин та селекційну лінію Сівер, у яких 80100 % рослин зав'язували насіння за безпилкового режиму (далі apomixes-form). Стандартом слугували сорти Чорний велетень та Антарія, які розмножуються виключно за рахунок амфіміксису і не здатні зав'язувати насіння за безпилкового режиму. Спостереження за зимостійкістю, ростом та розвитком рослин проводили згідно «Методики проведення кваліфікаційної експертизи сортів рослин на придатність до поширення в Україні» (2016). Пилкоутворювальну здатність визначали за методикою Савченка М.І. (1980), олійність - на приладі ЯМР-аналізатор MQC 5-12 фірми Oxford instruments, проводили перерахунок даних на абсолютно суху речовину. Адаптивність розраховували за методикою Eberhart S.A., Rassel W.A. (1966) на підставі вивчення коефіцієнту регресії (b). Рівень самонесумісності визначали шляхом примусового запилення квіток, що розцвіли, та бутонів з наступним аналізом зав'язуваності насіння. Використовували пакет програм STATISTIKA 10.

Apomixes-form, порівняно зі стандартами Чорний велетень і Антарія, характеризувались високою енергією проростання (р=0,000001) повні сходи можна отримати протягом 3-4 днів із дати початку проростання насіння, у порівнянні - 6-8 днів у стандартів. Apomixes-form є ранньостиглими (р=0,000524), дозрівання основної кількості насіння цих форм в роки спостережень проходило на 5-7 днів швидше стандартів. Для Apomixes-form притаманний тривалий період зимового спокою (р=0,000001), відновлення весняної вегетації в умовах затяжної весни проходило на 7-8 днів пізніше, в умовах ранньої весни - на 4-5 днів пізніше сортів-стандартів, що розмножуються виключно за допомогою амфіміксису. Для apomixes-form характерна розлога форма розетки восени та розгалужений корінь: перший тип - із добрим розвитком 3-4 бічних корінців, розташованих у нижній частині головного кореня; другий тип - наявність більш тонких бічних корінців, розташованих по всій довжині головного кореня, а також більшої кількості додаткових тонких корінців.

Apomixes-form мають тривалий період цвітіння (р=0,000044). Рослини apomixes-form на 29,0 % продукують більше пилку, життєздатність якого

на 14-21 % вище стандарту Чорний велетень. Apomixes-form є самонесумісними. Apomixes-form мали найменшу варіабельність олійності насіння. За урожайністю apomixes-form неоднозначно реагували на умови року. Можливими причинами низької врожайності apomixes-form були: 1) глибокий період зимового спокою, що у випадку стрімкого наростання температури навесні та весняних суховіїв спричиняє зрідженість посівів; 2) крупний пилок, який у певних умовах вологості повітря ускладнює вітрозапилення; пилкові зерна на 23-30 % (174,7-210,3 мкм²) крупніші пилкових зерен стандартного сорту Чорний Велетень; 3) розташування генеративних органів квітки, маточка приймочки знаходиться вище пиляків, що ускладнює процес самозапилення, особливо у вологу погоду або за відсутності льоту бджоли.

Ключові слова: ріпак, апоміксис, амфіміксис, зимостійкість, врожайність, олійність, пилкоутворювальна здатність.

Intoduction

A plant, as a rule, follows one habit of seed reproduction, but can use both apomixes and amphimixis at the same time (Maletskii 2013). According to Savidan's (2000) and Spillane et al. (2004) data, in agricultural plants, most apomicts are facultative or partial, that is, they are capable of switching from sexual reproduction to asexual reproduction and vice versa. Naumova (2008) and Barcaccia et al. (2020) assert, despite comprehensive theoretical and practical studies, apomixis remains less thoroughly studied compared to amphimixis.

Reviewing literature, we came across an opinion that partial or facultative apomixis is considered as an additional reproduction system. However, information on the effect of partial apomixis on the physiological processes and morphology of an apomixis-prone plant (carrier) is very limited. Most often, physiological and morphological deviations are interpreted as an integral part of the hybrid itself.

Thompson (1979) called apomixis-prone rapeseed forms “forms with fertile cytoplasm” and indicated that these forms were highly winter hardy. However, it is not possible to elucidate from this publication which type of apomixis the author had in mind. In garlic (Alium sativum) breeding, a lot of researchers compared bolting and nonbolting forms in different years and showed that clones differed in the color and inclination of leaves, in the shape and color of the outer scales of the bulb, in the clove color, number, size and location, in the ripening time, in the dependence of the bulb formation on temperature, and photoperiod, as well as in cold tolerance and winter hardiness (Senula and Keller 2000; Pavlova et al. 2018; Helmey 2020). In carrot breeding, genotypes derived via gynogenesis and androgenesis (Semenova et al. 2003) and differing in the contents of sugars, carotenoids, and dry matter and in the fruit weight were compared. However, it should be noted that, in these studies, the researchers compared forms obtained by irregular sexual reproduction, but not plants (carriers) capable of developing these forms under certain conditions. There is not available information on the effect of partial apomixis on the morphology and development of angiosperms.

In view of the aforesaid, the following question arises: Are there morphological and physiological peculiarities in plants that are capable of partial (facultative) apomixis compared to plants that reproduce exclusively by amphimixis? Such studies are supposed to expand the scope of using these forms. This became the main objective of our study.

Material and methods

The study was carried out in the field and laboratory of the Plant Production Institute named after V.Ya. Yuriev of NAASU (Kharkiv, Ukraine). The predecessor was black fallow; the soil was typical chernozem. Winter 00-rapeseed accessions were studied. Self-pollinated lines derived from varieties Liradjet, Svitoch, Severianin and the breeding line Siver were taken as test material. These accessions, depending on the year, have 80 % to 100 % of plants capable of apomixis. The check varieties were Chorniy Veleten and Antariia, which are not prone to apomixis. To identify forms with partial apomixis, a pollen-free regimen was applied - emasculation of flowers without further pollination followed by isolation of emasculated flowers. We analyzed 20 plants, castrating 8 flowers per raceme.

Winter hardiness as well as growth and development of plants were evaluated in accordance with "Methods of Qualifying Evaluation of Plant Varieties for Suitability for Dissemination in Ukraine", 2016. Pollen-producing ability was determined by Savchenko's method (1980). The oil content was determined using an MQC 5-12 NMR analyzer (Oxford instruments) and adjusted for absolutely dry basis. The adaptability was calculated from the regression coefficient (b) by Eberhart and Rassel's method (1966). The self-incompatibility was assessed by controlled pollination of opened flowers and buds followed by analysis of the set seed number. Data were statistically processed in STATISTICA 10.

Results and discussion

We compared the physiological and morphological characteristics, pollen-producing ability, yield capacity and oil content of forms with partial apomixes with the check accessions that did not possess this feature.

In the development of winter rapeseed plants, autumn is of great importance, as a favorable autumn positively affects overwintering and, as a result, the yield. In our experiment, seedlings of apomictic forms emerged 2-3 days later than in the check varieties (the difference was significant, p=0.00099) and this further negatively influenced the development of rapeseed plants.

The apomictic forms have a high germination energy in comparison with the check varieties; seeds fully germinated in a shorter period. While in the apomictic forms seeds fully germinated within three days in 2017 (favorable year) and within four days in 2015 (unfavorable year), in the check varieties - within five days in 2017 and within seven days in 2015. This feature of the apomictic forms was confirmed at the 5 % significance level (p=0.000001).

The duration of the interphase periods of the initial stages of ontogenesis (autumn) in the apomictic forms did not differ significantly from Chorniy Veleten (form that reproduce by amphimixis). Analysis of the growing period length showed that the apomictic forms were early ripening. Compared to Chorniy Veleten, most of the seeds in these forms ripened by 5-7 days faster, which meant a significant difference (p=0.000524). Thus, this feature of the apomictic forms can be used to create early ripening rapeseed varieties and hybrids.

It is known that some forms of rapeseed are able to bury deeper their roots, others to shallow up, and some are deprived of these features. Some scientists (Becka et al. 2004; Tyutyunnik and Zhatova 2015) associate this feature with a genetic predisposition and plant response to changes in environmental factors, including physical changes in soil structure during drastic temperature shifts.

In our experiments, the main feature of the autumn vegetation of all the apomictic forms under investigation consisted in the fact that plants buried in the ground on average 5-6 days before the cold weather onset. Thus, the root collar was covered with soil, which can be considered an element of plant protection against low temperatures. The root collar was buried on average by 1-2 cm and the depth of the root collar depended on the microlandscape.

We made an attempt to explain the mechanism of winter rapeseed plant burying. For this, we investigated the morphological characteristics of roots. It was found that the apomictic forms had two types of roots (fig. 1): a) with 3-4 well- developed lateral roots located in the lower part of the main root; b) with thinner lateral roots located along the entire length of the main root as well as with a large number of additional roots.

ab

Fig. 1. Types of roots in the apomixis-prone rapeseed forms (a - a root with 3-4 well-developed lateral roots and b - a root with a large number of lateral roots)

Thus, a large number of roots along with their good development allow the plant to increase its hold in the soil and resist physical changes in the soil occurring during sharp temperature fluctuations throughout the day (day-night) in late autumn. However, it should be noted that similar types of roots are also seen in forms that reproduce exclusively by amphimixis. It can be assumed that these types of roots are additional elements of plant protection against winter stress.

In our study, it was also characteristic that the apomictic forms had a spreading rosette of leaves in the autumn. This feature can be considered as additional protection against negative factors of overwintering due to the fact that the leaves of one plant cover the root collar of a neighboring plant. In addition, the spreading rosette of leaves allows reducing the impact of sharp drops in air temperature in the autumn-winter period on the root collar.

The renewal date of the spring vegetation characterizes the awakening of a winter plant under certain light and thermal conditions. Likhochvor, Prots (2005), by analogy with winter wheat, consider the temperature of resumption of spring vegetation for rapeseed to be 5°C, while Solesca (2008) and Williams (2010) mentioned 2°С. The reason for this is the difficulty in determining the renewal date of the spring vegetation in rapeseed. Therefore, we documented the moment of regrowth of the rosette leaves as the renewal date of the spring vegetation. During the period when the air temperature steadily rose above 2°C, we measured the rosette height in the same plants using an arithmetic ruler with 2-day intervals. We observed that the apomictic forms had a deeper dormancy than the check accessions. During an extended spring, the spring vegetation renewed by 7-8 days later than in the check accessions; when the spring was early - by 4-5 days later. This difference was significant at the 5 % significance level (p=0.000001). It was beneficial that the apomictic forms did not resume their growth during deep thaws, but, at the same time, this feature had a negative effect, as during early spring, with a rapid rise in above-zero temperatures, plants grew too slowly or died. With a rapid increase in the average daily temperature in the springs of 2015 and 2016, we observed that some plants were dehydrated and died without winter stress-induced maceration of root tissues.

We noticed that the apomictic forms had longer anthesis than that in Chorniy Veleten (p=0.000044). This is attributed to a larger number of formed flowers in the apomictic forms. Lines Liradjet and Svitoch consistently formed significantly larger numbers of flowers during the experiment. On average, the apomictic forms produced the minimum number of flowers in 2016 (273±34 flowers); the maximum number - in 2018 (356±28 flowers).

The peculiarity of all the apomictic forms under investigation lied in the fact that the flower was macrostylous, indicating the priority of cross-pollination.

Over the years of our research, the data on the pollen grain size were ambiguous and varied not only within the experimental group, but also from year to year (table 1). On average across six years, the apomictic forms produced larger pollen grains (by 2330 %) compared to the check variety, Chorniy Veleten, but the difference was not statistically significant (p=0.089).

The pollen grain size of the accessions under investigation depended on year conditions (table 1). The check varieties differed in adaptability. Thus, Chorniy Veleten can be recognized as a most suitable form growing under unfavorable climatic conditions (h, < 1), while Antaria, on the contrary, as a form that is most demanding to growing conditions (h, > 1). Among the apomictic forms, there were both environmentally tolerant accessions (lines Liradjet and Severianin) and demanding ones (lines Svitoch and Siver). From the data in Table 1, it can be concluded that 2017 with a gradual increase in the average daily temperature and moderate precipitation in the spring and summer was the most favorable year for the formation of large pollen grains in the Liradjet and Severianin lines. Lines Svitoch and Siver were the most environmentally demanding. 2017 and 2019 with a gradual increase in the average daily temperatures and the spring-summer precipitation amount close to the average long-term value were the most favorable years for line Svitoch. 2018 with an excessive precipitation in the spring and a high average daily temperature during anthesis (>20°C) was favorable for line Siver. Our results confirm the data obtained by Senechal et al. (2015) and Reinmuth-Selzle (2017), who studied the atmosphere effect on pollen.

Pollen grain size [pm²]

Table 1

The values designated by the same letter do not differ significantly at p <0.05 (Duncan's test). b_j - regression coefficient (b) by Eberhart and Rassel's method.

To compare the apomictic forms with the check varieties, we identified homogeneous groups (table 1). According to the pollen grain size, four homogeneous groups were distinguished. Line Liradjet turned out to be the most remote from Chorniy Veleten and Antariia; in 4 of 5 years of the experiments, it was in other groups.

According to Geodakyan's data (1977), the pollen amount is determined by environmental conditions, namely, favorable conditions increase the pollen amount, while an insufficient amount of pollen, on the contrary, gives information about unfavorable environmental conditions. In this regard, we studied the pollen-producing ability of a flower and a whole plant. According to Ejsmond et al. data (2015), there was a relationship between the air temperature and pollen amount: the higher the temperature was, the less the pollen amount was. In our study, the pollen production per flower in the rapeseed accessions depended on year conditions and no common pattern was seen (Table 2). It can be explained by different eco-geographical origin of the studied forms. From the calculated coefficient of adaptability of the pollen© Н. А. Глухова, Д. К. Єгоров

producing ability of a flower, lines Severianin and Siver can be referred to the most demanding to growing conditions forms (b_i > 1), while line Svitoch - to the least demanding (b_i < 1). Based on the data summarized in table 2, it can be seen that a gradual increase in the average daily temperature in the early spring and the precipitation amount exceeding the optimum (2018) were favorable for the pollen production per flower in line Severianin. A rapid increase in the average daily temperature combined with the precipitation deficit (2015) was favorable for line Svitoch. As to line Siver, the conditions in 2015, 2018, and 2019 were favorable. In general, these facts are predictable. The initial varieties, from which the lines under investigation were derived, had been created under various eco-geographical conditions.

Pollen amount per flower (pollen grain number) [ths. ps.]

Table 2

The values designated by the same letter do not differ significantly at p <0.05 (Duncan's test) bj - regression coefficient (b) by Eberhart and Rassel's method.

Thus, variety Severianin had been bred in the non-chernozem zone of Russia, which is characterized by later spring, excessive precipitation and higher temperatures in summer than in the East of Ukraine. Varieties Svitoch and Siver had been bred in the Southwest of Ukraine, where spring is early and precipitation deficit is hasher than in the East of Ukraine. Taking into account the above, one can conclude that the pollen-producing ability per flower of winter rapeseed plants will be significantly influenced by eco-geographical origin of a breeding accession, which can be considered as a factor contributing to selection and fixation of a certain trait in a plant population.

Large pollen grains may not always be an indicator of its quality. Our study showed that a greater amount of fertile and viable pollen was produced in the dry year (2015). Nevertheless, pollen of the apomictic forms was the most viable, regardless of the year. When germinating pollen on a sucrose solution, the minimum amount of viable pollen (78 %) was observed in inbred line derived from variety Siver in 2016 and the maximum amounts (91 % and 93 %) were was recorded in inbred lines originated from varieties Liradjet and Severianin, respectively, in 2015. Compared to the check variety (Chorniy Veleten), pollen of the apomictic forms was by 14 % to 21 % more viable.

For successful fertilization and enhanced seed setting, sufficient amounts of pollen are required. Our study demonstrated that the apomictic forms produced more pollen (by 29.0-71.0 %) than the check variety (Chorniy Veleten). Therefore, apomictic forms can be used in the breeding of winter rapeseed for heterosis.

Testing for sporophytic and gametophytic self-incompatibility showed that the apomixis-prone forms prone were self-compatible. It is a beneficial feature when it is desirable to obtain sufficient numbers of seeds under bags (restricted conditions) in order to maintain the varietal purity.

We showed that the oil content in the studied accessions changed depending on the year conditions, with the 5 % significance level of differences (p=0.0000003). 2017 was the most favorable year for this trait (Table 3). The check varieties (Chorniy Veleten and Antariia) were highly differentiable in terms of the oil content in seeds (Fig. 2): in 2015, compared to 2017, which was favorable for growth and development, seeds contained by 9.1 % and 8.5 % less oil, respectively (table 3). The oil content in the apomictic forms, in comparison with the check varieties, is less affected by the conditions of the year; the difference between unfavorable and favorable years was from 4.6 % in inbred line Svitoch to 7.1 % in inbred line Liradjet.

The oil content in seeds of the apomixis-prone forms [%]

Table 3

Thus, if conditions change sharply, apomictic forms can produce seeds with a relatively high content of oil. This is an important feature, since a 1 % decrease in the oil content in seeds corresponds to a loss of about 10 kg of oil per ton of seeds. But, Duncan's test for the oil content placed the studied accessions in one group, and the adaptability index indicated tolerance of the “oil content” trait to environmental conditions (Ь=1). It is seen from figure 2, that the interquartile range (LQR) oil content of the test accessions is within 43-44 %, indicating a high genetic stability of this trait. At the same time, the oil content in seeds of the apomictic forms is less variable in comparison with the check varieties (Chorniy Veleten and Antariia). There was an ambiguous response of the apomictic forms to the year conditions. Under the dry conditions of 2015 and under close to the optimum conditions of 2016, the studied apomictic forms contained more oil. In 2017, with an optimal amount of precipitation and an average daily air temperature of +20 °C during seed-filling, plants of all experimental samples accumulated the greatest amount of oil.

It is known that the seed weight is highly dependent on external conditions. Taking cereal embryogenesis as an example, Bannikova et al. 1991, proved that at 30°C the growth of cells was inhibited and they did not reach the optimal size by the end of development, but the final number of cells did not depend on temperature.

Abranyi and Pletser 1987 believe that extending the grain filling time, especially under extreme conditions, has a positive effect on the grain weight. We found no significant differences on the 1000-seed weight between the apomictic forms and the check varieties (Chorniy Veleten and Antariia) (table 4).

Table 4

Yield of the 14pomixes-prone forms (2015-2019)

Fig. 2. Box-and-whiskers plot of the oil content in seeds, 2015-2019

All test samples produced the heaviest seeds in the wet year of 2019.

The 1000-seed weight is in the focus of winter rapeseed breeding. Therefore, it could be foreseen that the studied samples would have an adaptability coefficient equal to one and a single homogeneous group, which indicates the plasticity and stability of this trait in the studied samples.

Table 4 shows that the yields of the apomictic forms and the check varieties (Chorniy Veleten and Antariia) differently depended on the year conditions. Based on the adaptability coefficient for the "yield" trait, lines Severianin and Siver can be considered as the most demanding to growing conditions (b, > 1), while line Svitoch - to the least demanding (b, < 1).

Conclusions