ASN, Vol. 8, No 1, Pages 126–137, 2021 126 Corresponding Author: P

ASN, Vol. 8, No 1, Pages 126–137, 2021

Acta Scientifica Naturalis

Former Annual of Konstantin Preslavsky University of Shumen: Chemistry, Physics, Biology, Geography Journal homepage: https://content.sciendo.com/view/journals/asn/asn-overview.xml

Status of a collection from common wheat varieties (Triticum aestivum L.) of Bulgarian origin after a ten-year ex situ storage period

Plamen S. Chamurliyski, Albena M. Ivanova

Department of Crop Science and Plant Protection, Konstantin Preslavsky University of Shumen - College Dobrich, 9302 Dobrich, Bulgaria

Abstract: The purpose of this study is to evaluate the condition of a collection of Bulgarian common wheat varieties under ex situ controlled storage after a ten-year period. The studied materials are characterized by high vitality, without negative changes that will lead to the loss of original germplasm. The established moisture in the seeds is high for the purpose of controlled storage. When it rises above 12,5%, a decrease in the values of germination energy is observed. Higher control of initial moisture and germination is required prior to entering the seeds for storage under ex situ conditions.

Keywords: common wheat, ex situ conservation, germinability, seed moisture

Introduction The dynamic changes in climate over the last sixty years and the intensive human activity have threatened many plant species with extinction [1, 2, 3]. Their preservation is one of the leading priorities of modern society. Effective scientific approaches to the conservation of plant biodiversity have been developed through various conservation methods, two of them are of greater practical importance: in situ - maintaining collections in the natural environment [4, 5] and ex situ - preserving samples at an organismal level. Ex situ storage in seed gene banks is based on the creation of conditions for the reduction of seed metabolic activity, most often as a result of low moisture content and low temperature [6].

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An ex situ system for managing plant genetic resources is operating in Bulgaria. The national Gene bank of the Institute for Plant Genetic Resources (IPGR) - Sadovo was established in 1984 and implements a scientific program for the long-term storage of germplasm under controlled conditions in accordance with the standards of the Food and Agricultural Organization (FAO). The national gene bank maintains three collections: 1) basic collection - long-term storage of the samples in hermetically sealed containers at -18ºC; 2) working collection - newly arrived samples, guarantees seed storage for 3 to 10 years at +6ºC and 50% moisture; 3) exchange collection - samples intended for exchange with partners from the national and international system. According to the EURISCO catalog [7], about 56 082 samples of 2 670 plant species are available in the IPGR - Sadovo under long-term and medium-term storage conditions [8, 9, 10]. The conservation of strategically important varieties and promising lines of common wheat in one of the leading breeding centers in Bulgara is carried out in a similar way. Dobrudzha Agricultural Institute (DAI) - General Toshevo maintains collections with valuable economic qualities from this crop, starting the program in 1996. To date, 4250 samples of cereals are stored there, 3675 of which are common wheat [11]. At the basis of any successful breeding process in cultivated plants is the maintenance of a large set of samples as starting material. On the one hand, rich collections cannot be maintained only in field conditions, and on the other hand, not every created breeding line has the potential to be developed as a variety, but it can be a source of important qualities. Therefore, the existence and management of ex situ collections in breeding programs contributes to the conservation of valuable germplasm over a long period of time with minimal loss of genetic material, labor costs and investment in reproduction [12, 13, 14]. Seed moisture is one of the main indicators of their storage. The ability of the seeds to survive the drying and cooling according to Roberts‘ classification [15] determines their characterization as "storable" (orthodox) and "difficult to store" (recalcitrant). It is subject to continuous monitoring and control throughout the conservation period, using various drying methods [16, 17]. Other important indicators closely related to seed viability during storage are germination energy and germination. Germination energy is a parameter that characterizes the germination capacity of a batch of seeds, while germination is used to determine the number (percentage) of all seeds that can germinate under certain conditions. According to the International Seed Testing Association (ISTA) [18] standards, these two main seed quality indicators are established respectively as 'first date' and 'second date' to account for germination under specific laboratory conditions. Their determination gives a general idea of the condition of the samples after passing through a certain period of conservation. The main objective of this work is to evaluate the condition of a collection of Bulgarian common wheat varieties under ex situ controlled storage after a ten-year period.

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Materials and methods Plant material The study included 25 varieties, 15 of which were developed in DAI – General Toshevo, and 10 in the IPGR - Sadovo. The varieties were stored in a refrigerator at -11°C for more than 10 years (Table 1).

Table 1. Investigated varieties, origins and acquisition date in gene-bank

Varieties of DAI origin Acquisition date Varieties of IPGR origin Acquisition date

Aglika 2004 Momchil 2004

Enola 2004 Boryana 2004

Slaveya 2004 Sadovo 1 2004

Galateya 2004 Pobeda 2004

Ludogorka 2004 Mustang 2004

Ludogorie 2005 Diamant 2004

Pryaspa 2005 Bononia 2004

Iveta 2005 Murgavetz 2004

Medeya 2005 Geya 2005

Progress 2005 Petya 2005

Kristal 2005

Karat 2005

Zlatitza 2005

Merilin 2006

Korona 2006

Investigated traits The assessment of the condition of the stored samples includes an analysis of the indicators: 'first date' germination account (FDGA, %), 'second date' germination account (SDGA, %), 1000 kernel weight (TKW, g) and seed moisture (SM, %). The ratio of 'first date' germination account to seed moisture (FDGA/SM) was also calculated for a more complete assessment of storage.

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Statistical methods Variational, correlation and principal component analyzes were performed. The mathematical processing and statistical analysis was made on XLSTAT-Pro Ver. 7.5.2. Determining FDGA and SDGA According to ISTA regulations [18] a total of 100 seeds are counted out of each sample, which are divided into 4 replications x 25 seeds. They are laid in a paper germination bed BP (germination between papers). Filter paper with a mass of 100 to 120 g/m² and water absorption of 220 to 240% is used. It is pre- moistened to such an extent that no water ring will form around it when pressed with your finger. The seeds are evenly distributed on the germinating bed, oriented tip-to-embryo in one direction and separated sufficiently from each other to avoid contact among the germinated seeds before counting and removal. On the prepared samples, cover strips also made of filter paper are put, and then they are rolled up into rolls, which are put into the germination apparatus - thermostat. Germination conditions are: 20°C, no light. The observation can only be done when the seeds have been developed to such an extent that it can be assessed whether they have all the major organs. The determination of germination energy is carried out on the fourth day (FDGA), establishing the following categories: normally germinated seeds; abnormally germinated seeds; solid seeds; fresh not germinated seeds; dead seeds. After measuring, all normally germinated seeds are separated. The rest are counted on Day 8 (SDGA) on the same grounds for the final determination of germination, which is the percentage of normally germinated seeds of the total number of seeds. When the four replications of 25 seeds in one study were within the limits of the seeds‘ tolerance (1%), the germination rate represents the average of these replications. The average percentage is rounded to the nearest whole number. Measurement of seed moisture It is carried out according to the methodology ISO 712:2009 [19]. According to the methodology, the moisture in the seeds is removed by heating under controlled conditions that allow the moisture loss to be quantified. For common wheat seeds, a 130°C drying method is used. Used equipment:  Mill made of non-moisture absorbent material;  Containers - glass panes of stainless steel, approximately 0,5 mm thick, rounded at the base and flat bottom, with well-fitted lids and recommended sizes to ensure no more than 0,3 g of material per 1 cm³. 3,5 cm³ glass panes were used;  Electric heater oven equipped with a temperature control device in the temperature range 130 ± 3°C;  Desiccator containing appropriate Blaugel (Silica-gel) hygroscopic substance, which guarantees reduced humidity in the desiccator when cooling the glass panes;

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 Precise scales (KERN SOHN 07433) for weight analysis, accurate to the third character. Method of operation: Weigh the glass panes together with the lid before placing the test material. Weigh 1 g of the preliminary fine ground sample. Spread it evenly on the bottom. Insert the glass cover and re-weigh it. Each glass is placed on its lid in the oven preheated to 130°C. Once the temperature in the oven has reached 130°C, a drying time of 60 minutes begins to count. The slides are then slammed and placed in the desiccator for 30 to 45 minutes to cool. Weigh the cooled glasses together with the contents and the lids. All weighings must be accurate to 1 mg. The moisture content is calculated by the formula: SM=(M2-M3)*100/(M2-M1), where SM is the percentage of moisture determined; M1 is the mass of the glass and its lid, g; M2 is the mass of the glass with the lid and the contents before drying, g; M3 is the mass of glass with lid and contents after drying, g. Moisture is determined in two replications at a time. The difference between the results of the two replications shall not exceed 0,2%. If it is larger, the analysis must be repeated in the order indicated.

Results and discussion The mean values of germination energy, germination, moisture and weight per thousand seeds for the samples tested are shown on Table 2. The Pobeda and Bononia varieties from the materials originating from IPGR - Sadovo are characterized by the highest germinating energy and germination at good moisture levels in the seeds. The same parameters are the lowest for the Sadovo 1 variety, where moisture was estimated at 14,4%, and this percentage is unacceptably large for the purposes of controlled storage. The condition of the Karat and Galateya wheat from the selection of DAI - General Toshevo is similar. They also stand out with maximum germination, with Karat germinating energy is also 100%. The moisture measured for this variety is close to the optimum – 10,6%, while in Galateya it is raised to 12,9%, which immediately influenced the value of the germinated energy. The worst results were found in the Ludogorie variety, which is characterized by very low vitality. The variation coefficient is the lowest in germination. This indicates that the collection is still in good condition with regard to ex situ conservation. However, the relatively high variation in humidity is a signal for the occurrence of undesirable conditions. However, at this stage there is no significant effect on the viability of the samples tested. Similar results have been commented on in previous studies [20]. The variation in the weight of one thousand seeds is 8,6%, which is due to the different genetic conditions of the varieties and the conditions of the year in which the materials are harvested. The largest is the weight of one thousand seeds of the Karat variety – 50,9 g and the lowest in the variety Petya – 39,9 g.

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Таble 2. Mean values of germination energy (FDGA), germination (SDGA), 1000 kernel weight (TKW) and moisture (SM) in varieties of DAI - General Toshevo origin

Varieties FDGA, % SDGA, % TKW, g SM, %

Aglika 92,0 95,5 44,2 13,9 Enola 94,5 98,3 44,1 11,2 Slaveya 89,0 92,3 46,0 13,4 Galateya 87,0 100,0 41,9 12,9 Ludogorka 86,0 92,5 41,6 14,2 Ludogorie 85,1 90,8 42,3 14,0 Pryaspa 91,3 97,3 49,7 12,4 Iveta 88,3 94,5 43,5 13,0 Medeya 85,3 91,3 43,8 11,9 Progress 86,3 91,0 48,1 12,1 Kristal 91,3 97,3 50,9 12,4 Karat 100,0 100,0 52,2 10,6 Zlatitza 94,3 99,0 48,2 13,0 Merilin 97,5 99,0 44,3 11,7 Korona 91,5 95,3 48,3 12,2 Momchil 89,0 93,5 47,8 13,0 Boryana 89,8 94,5 49,8 12,9 Sadovo 1 85,0 90,3 45,6 14,4 Pobeda 95,8 100,0 46,2 11,6 Mustang 89,0 93,3 48,2 11,6 Diamant 91,0 97,5 47,9 12,5 Bononia 95,0 100,0 46,1 12,3 Murgavetz 89,3 95,3 42,2 11,5 Geya 93,8 99,0 45,1 11,8 Petya 86,5 91,5 39,7 14,3

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Average 90,6 95,6 45,9 12,6 VC, % 5,1 3,2 8,6 7,7

Figure 1 shows the percentage distribution of materials based on their germination and germination energy in three groups. The impression is that over half of the studied wheat has germination energy below 90%. This is not really a good attestation for proper storage. However, overall germination has not yet shown sharp changes and over 90% of the materials have shown excellent results on this indicator. This means that the collection has successfully survived the 10-year storage period. The high values of moisture in the seeds already mentioned are due either to errors in storage of the collection, or insufficient drying before the seeds enter the chamber (for which no information is available in the database), or due to lack of cyclic monitoring. The requirements of this methodology and storage conditions allow the moisture level to be in the range of 10 -11%, while for example at -18ºC the studies indicate 6-7% as optimal values [21].

52 48 44 40

8 8

85-90 % 91-95 % 96-100 %

FDGA, % SDGA, %

Figure 1. Percentage distribution of materials according to their germination energy and germination

After applying cluster analysis (Figure 2) based on germination (Euclidean distances), three main groups of varieties are clearly formed. Eleven of the wheat are grouped by their high germination and are of excellent vitality. In fact, these are the samples that experienced the best storage, as we found in Figure 1. In the second group (located in the middle of the graph), processes started leading to a decrease in germination. However, these varieties still have good characteristics after ex situ conservation. In the third group (9

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ASN, Vol. 8, No 1, Pages 126–137, 2021 varieties), due to undetermined reasons, there were changes in negative direction, which led to decrease in the values of the studied indicator. After correlation analysis, the correlation coefficient (Table 3) between germinated energy and germination was found to be R=0,78 at the highest level of statistical confidence. This strong positive relationship, as well as the high germination values, is a prerequisite for solidifying the conclusions drawn so far that, after a ten-year storage period, the germplasm under study are in good overall condition. The results reported by other authors are similar to ours [22].

Ludogorka Slaveya Mustang Momchil Progress Ludogorie Petya Medeya Sadovo 1 Murgavetz Merilin

Aglika

Boryana Iveta Zlatitza Korona Гея Енола Karat Galateya Pobeda Bononiya Kristal Pryaspa Diamant

0 0,2 0,4 0,6 0,8 1 1,2 1,4

Figure 2. Cluster analysis and grouping of varieties based on their germination

The negative changes from storage occur earlier in the indicator germinating energy, which characterizes the togetherness of seed germination. This is the reason why a better correlation (0,61) is found between this trait and the moisture in the seeds. However, even with such a relatively small storage period for ordinary wheat, when negative changes are still limited, it is illustrated that better germination energy correlates with lower seed moisture. The weight of a thousand seeds is the other indicator that is relevant to seed survival. Established proven proportional dependence on germinating energy is proven. This trend means that the larger seeds are subject to better storage and must be taken into account before preparing samples for admission to the gene bank.

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Таble 3. Correlation analysis

Variabilities FDGA, % SDGA, % SM, %

FDGA,% 1

SDGA,% 0,78*** 1

SM,% -0,61*** -0,52** 1 TKW, g 0,45** 0,29* -0,42**

*** – p ≤ 0,01; ** – p 0,05; * – p 0,1; n.s. – non significant

The principal component analysis on Figure 3 is made on the basis of the ratio of germinate energy to moisture (FDGA/SM). These are the indicators with the highest correlation coefficient and strong interaction that will give a more complete assessment of storage. First and fourth quadrant varieties with values >0,8 along the axis of the abscissa are defined as having most successfully survived the ten-year period under low temperature conditions without loss or undesirable effects, combining optimal moisture with very good germinating energy. While those in the second and third quadrants with values < -1,6 for F1 have identified processes that would be unfavorable to the duration of their conservation. The prognosis for the Galateya variety in which we found 100% germination is that it will gradually shift even further to the left on the abscissa and fall into the worst performing sample group.

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0,6 Momchil

Korona Kristal Pobeda Ludogorie Iveta Pryaspa Slaveya Medeya Mustang Ludogorka Boryana Мерилин Geya Zlatitza Карат Аглика Bononia -0,2 Sadovo 1 Progress Diamant Enola Petya Galateya Murgavetz

-1 F1 -3,2 -2,4 -1,6 -0,8 0 0,8 1,6 2,4 3,2 4 4,8

Figure 3. Principal component analysis

Conclusion Almost half of the common wheat varieties (44%) in the studied collection retained high germination at optimum moisture levels. They are characterized by excellent vitality, without negative changes that will lead to the loss of original germplasm. Seed moisture is relatively high for the purpose of controlled storage. As it rises above 12,5%, there are negative changes that first affect germination energy. The relationship between germination energy and moisture can be used as an integrated assessment of ex situ storage. Monitoring is recommended to be carried out at smaller intervals, depending on the number of seeds in the sample. Higher moisture and germination control is required before entering storage. It is preferable for the seeds to have a higher weight per thousand seeds, as this indicator is also important for the conservation of common wheat.

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Acknowledgements This publication is realized with the financial support of Project No. RD-08-91/01.02.2021 of the Konstantin Preslavsky University of Shumen.

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