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Ex-situ conservation of wheat genetic resources from Saudi Arabia

Wheat (Triticum L.) is one of the major food crops of the world, and an important component of food security. The aim of this study was to collect and preserve seeds of wheat growing in eight regions of the Kingdom of Saudi Arabia (Al-Qassim, Asir, Al-Taif, Najran, AL-Baha, Jazan, Al-Madinah and Wadi Al-Dawasir) where wheat has been cultivated since ancient times. Sixty-one accessions/samples of wheat (Triticum aestivum) were collected and placed in dry storage (ex-situ conservation) at −18 °C (i.e. permanent storage). The accessions of local wheat have the ability to grow under harsh environmental conditions such as (high temperature, drought and salinity). Most of these samples were collected directly from farms, but a few were collected from markets. The most important criteria for ex-situ conservation is that seeds need to have a low moisture content (MC) and a high percentage viability. Seed MC was measured for all 61 accessions by the oven-drying method and seed viability was tested in three ways: percentage of germination, tetrazolium chloride testing, and X-ray radiography. The seed MC of the 61 accessions was uniformly very low (0.10–0.12%), and 97 to 100% of the seeds were viable. Thus, all 61 wheat accessions collected in this study have the initial requirements to remain viable for long periods of time in ex-situ conservation in the gene seed bank.


Introduction
Wheat (Triticum L., Poaceae) is one of the major food crops in the world and is important for global food security. As a result of the increasing world population, there is an urgent need to protect this strategic food crop (FAO, 1998;2010). The earliest cultivated forms of wheat were diploid (2n = 2x = 14) and tetraploid (2n = 4x = 28), and genetic evidence indicates that they originated from the south-eastern part of Turkey some 10,00 years ago (Heun et al., 1997;Nesbitt and Samuel, 1996;Dubcovsky and Dvorak, 2007). Subsequently, hybridization gave rise to hexaploid wheats and cultivation of several wheat species spread widely into the near East, Asia and Europe (Feldman and Kislev, 2007). Currently, Triticum aestivum (bread wheat), accounts for about 95% of the world production (Ortiz et al. 2008a).
Much of the land in the Kingdom of Saudi Arabia is not usable for agriculture, due to the topography, insufficient rainfall and high salinity. However, in suitable areas, farmers in the Kingdom have traditionally planted locally selected landraces of Triticum aestivum, notably in Alhaba, Al-Samma, Al-Qaimi, Hanttah Al-Qassim, Hamra, Samira and Madinah (Sayed, 1979). These varieties have been inherited by many generations of farmers in the Kingdom and their seeds represent genetic assets that need to be preserved before a decline in farming, as a result of human and other activities, results in them becoming extinct. The importance of these local wheat varieties lies in their capacity for adaptation to the extreme climatic conditions of heat and drought in the Kingdom. All of these factors contribute to the importance of collecting and conserving agricultural genetic resources in plant gene banks as pure genetic resources for future generations. Therefore, the wheat genetic resources of the Kingdom need to be preserved by ex-situ conservation in gene-banks so that they will be available in the future (Al-Turki, 2002;Al-Turki et al., 2010;Al-Turki et al., 2019). De Carvalho, et al. (2013) reported that 859,472 accessions of wheat germplasm are being maintained in Gene-Banks in 90 countries in Europe, America, Asia, Africa and Oceania. The top six wheat germplasm collections are CIMMYT, NSGC, ICGR-NBPGR, ICARDA and NIAS (de Carvalho, et al. 2013). >850,000 wheat samples are maintained in a living condition in the world, and these samples are stored in 229 collections. Russia, United States, China, India, Italy and Japan have the most significant national wheat collections, as well as CIMMYT in Mexico and ICARDA in Syria (FAO, 1998). In Saudi Arabia, 61 and 126 accessions of wheat germplasm are conserved in the Gene-Banks of King Abdulaziz City for Science and Technology (KACST-BGB) and the Ministry of Environment, Water and Agriculture, respectively. However, despite these germplasm collections, the collection and preservation of the wheat germplasm of Saudi Arabia is far from complete.
The objective of the present work was to collect and conserve the germplasm (as seeds) of wheat (Triticum aestivum L.) from eight geographical regions of Saudi Arabia. Also, we sought to assess the suitability for storage of these seed accessions, before conserving them ex-situ, in long-term storage at about À18°C in the King Abdulaziz City for Science and Technology Gene-Bank (KACST-BGB).

Germplasm collection
We visited numerous wheat farms ( Fig. 1) scattered across the Kingdom of Saudi Arabia and interviewed many wheat farmers about their agricultural experience. Seeds of 61 accessions of Triticum aestivum L. were collected from eight different geographical areas (Al-Qassim, Asir, Al-Taif, Najran, Al-Baha, Jazan, Al-Madinah and Wadi Al-Dawasir) in Saudi Arabia, during 2017-2019 (Table 1). The minimum sample size 1 kg. Some samples were from freshly harvested seeds but others were from seeds that had been stored by farmers. All samples were transferred to the laboratory of the KACST Gene-bank in Riyadh, winnowed by blowing with air to remove debris, and placed in the cold room (5°C short term storage). For each accession seed moisture content and viability were determined.

Determination of seed moisture content
We used four replicates (100 seed each) of each accession. These were weighed, dried at 130°C for 2 h, allowed to cool for 1 h over silica gel and then reweighed (Witte,1995;Touchell et al. 1998;ISTA, 2013). Moisture content percentage = (weight of fresh seeds -weight of dry seeds)/weight of fresh seeds Â 100.

Seed germination tests
Five replicates of 20 seeds each for each accession were placed in 90-mm diameter Petri dishes on two layers of Whatman No. 1 paper moistened with 7 ml distilled water. Dishes were incubated at under two alternating temperature regimes 15/5°C and 20/10°C, with a 12 h light and 12 h dark, cycle. Germination was monitored daily for 30 days, and a seed was scored as germinated when the radicle had emerged to a length of ! 2 mm. The final germination percentage (%) was expressed as G (%) = (A/B) X 100 (Al-Turki, 1992, Baskin andBaskin, 2014;Basahi, 2018), where A is the total number of seeds germinated at the end of experiment and B is the total number of seeds tested.

Tetrazolium chloride tests for viability
According to the International Seed Testing Association protocol (ISTA, 1999), seeds were soaked in a 1% solution of 2,3,5-triphenyl tetrazolium chloride (TTC) for 4 days in a glass vial in the dark at 25°C. A red-stained embryo was counted as viable and a nonstained embryo as nonviable.

X-ray radiography
Four replicates of 100 seeds of each accession were X-rayed with a Faxitron X-ray machine (model MX-20 Dc12) connected to a computer. Seeds were exposed to 18 HV/10 s, and the X-ray plates were evaluated based on presence of the embryo and endosperm. The percentage of seeds with an intact embryo, damaged embryo or no embryo was determined. X-ray images clearly showed the internal structure of the seed, including the embryo. A viable embryo has high homogeneity, but a dead one has dark heterogenous areas, indicating damage to the embryo (Al-Turki and Baskin, 2017; Al-Hammad and Al-Ammari, 2017; Al-Turki et al., 2019).

Statistical analysis
Results from the seed moisture content determinations and viability tests were expressed as the percentages (mean ± standard error). Data were analyzed using one-way ANOVA followed by Duncan post Hoc to compare the means with 5% probability level (Sokal and Rohlf, 1995). SPSS 11.5 for Windows was used for data analysis.

Germplasm collections
Sixty-one accessions of wheat (Triticum aestivum L.) were collected from eight different regions of Saudi Arabia (Al-Qassim, Asir, Najran, Al-Taif, Jazan, Al-Madinah, Al-Baha, Wadi Al-Dawsir) ( Table 1). Twenty-eight accessions were collected from Al-Qassim region, 18 from Asir region, five from Najran, four from Al-Taif, two from Al-Madina, two from Jazan, and one each from Al-Baha and Wadi Al-Dawasir (Table 1). This preliminary study also shows that the geographical distribution of wheat crops is clearly concentrated in the Al-Qassim, Al-Taif and Asir regions, which have relatively low temperatures and high rainfall.

Seed moisture content
The moisture content of the 61 accessions of wheat (Triticum aestivum L.) was uniformly low, ranging from 0.10 ± 0.00 to 0.12 ± 0.00% (Table 2), with no significant differences between accessions (P > 0.05).

Seed viability
Seed viability as assessed by tetrazolium testing for the 61 accessions was uniformly high, ranging from 97 ± 0.10 to100 ± 0.00% (Table 3) with no significant differences between them (P > 0.05). X-ray imaging of seeds of the 61 accession of seeds revealed that almost all had intact, undamaged embryos and endosperms (e.g. Fig. 2) indicating absence of any sign of insect or microbial invasion. Seeds in all the wheat samples studied were long and oval in shape, with a clear embryo at the base of the seed.

Spike morphology
Spike morphology is considered a crucial trait in identifying landraces and for taxonomic studies of them. Morphological Wadi -AL-Dawasir Table 2 Seed moisture content (mean ± se) of 61 samples of Triticum aestivum.
observations were made on flowering spikes of typical examples of the more important landraces.

Discussion
The main aim of the study was to collect some of the genetic resources of local wheat (Triticum aestivum L.) in Saudi Arabia and preserve them in the KACST Gene-bank. Long-term seed storage is a basic and essential step to prevent the ancient wheat Table 3 Seed viability percentage (mean ± se) of 61 samples of Triticum aestivum (as judged by tetrazolium testing (TZ) and X-Ray imaging).

Seed viability % Location
Germination % at TZ % X-Ray % 15/ 5 0 C 20/10 0 C 97 ± 0.10 a 98 ± 0.10 a 97 ± 0.21 a 100 ± 0.00 a Al-Qassim 98 ± 0.12 a 100 ± 0.00 a 97 ± 0.11 a 97 ± 0.21 a Asir 98 ± 0.10 a 100 ± 0.00 a 98 ± 0.10 a 99 ± 0.10 a Najran 99 ± 0.10 a 100 ± 0.00 a 100 ± 0.00 a 100 ± 0.00 a Al-Taif 100 ± 0.00 a 100 ± 0.00 a 100 ± 0.00 a 100 ± 0.00 a Al-Medinah 98 ± 0.10 a 100 ± 0.00 a 100 ± 0.00 a 100 ± 0.00 a Jazan 98 ± 0.11 a 99 ± 0.10 a 100 ± 0.00 a 100 ± 0.00 a Al-Baha 97 ± 0.20 a 100 ± 0.00 a 98 ± 0.10 a 100 ± 0.00 a Wadi -Al-Dawasir  varieties of the Arabian Peninsula from going extinct. Our 61 accessions of the hexaploid Triticum aestivum, belonging to different landraces (varieties) from eight regions of the kingdom, were concentrated in areas with relatively cold climates in winter and high rainfall, particularly Al-Qassim, Asir and Al-Taif regions. Many researchers (e.g. Lazzari, 1988) have pointed out that seed moisture content (MC) is one of the most critical factors that must be controlled to maintain viability of seeds during storage. If seed MC exceeds 12%, seeds are more prone to damage by fungal infection. On the other hand, seed longevity is increased when MC is reduced to 4-6% (Ellis et al. 1991;Witte, 1995;Joshi andSingh, 2004, Ellis andHong, 2006). However, a decrease to as low as < 1% in our work did not have any negative effect on the seed viability. Storing seeds with a high MC (e.g. 15%) at a low temperature (À18°C) would result in seed death. Cell damage would occur in the frozen seeds due to formation of ice crystals that cause damage to the membranes and embryonic axis, resulting in loss of viability. However, values for the moisture content vary among the seeds of different species, and sometimes, even between varieties within the same species and this consequently can affect the longevity of the seeds i.e. their retention of viability (e.g. Joshi and Singh, 2004).
The low seed MC in our 61 accessions of wheat may be due to the preservation of these seeds by farmers who have extensive agricultural experience and are aware of the benefit of dry storage in maintaining seed viability. This low MC is a highly desirable and  important trait that ensures that seeds in the ex-situ seed bank can remain viable for long periods of time. Germination is one of the most important events in the life cycle of plants (Al-Turki 1992;Gutterman, 1993Gutterman, , 2002Basahi, 2018). Stress-inducing factors such as high temperatures and salinity may inhibit germination in the field. In fact, high or low temperatures can play a major role in the inhibition of germination of wheat seeds (Lobell and Ortiz-Monasterio, 2007). Several studies indicate that the optimal temperature for the germination of a wheat crop ranges from 12°C À 25°C (e.g. Acevedo et al., 2002). Buriro et al. (2011) have found that seeds of some wheat varieties from Pakistan have an ability to germinate with high percentages at 10, 20, and 30°C. In our study, final germination percentages were extremely high at both 15/5 and 20/10°C, which is consistent with the results of Buriro, et al. (2011) for wheat varieties from Pakistan.
Our study also shows that seeds of Triticum aestivum from Saudi Arabia do not appear to have genetically fixed (innate) mechanisms of dormancy since untreated seed samples from all 61 accessions of wheat had 97 and 100% germination at 15/5 and 20/10°C, respectively. Freshly harvested seeds of all 61 accessions showed a very high germination percentage (94-100% at 20/10°C). This result shows clearly that neither stored nor fresh seeds of wheat have innate dormancy in Saudi Arabia.
In Saudi Arabia, the King Abdulaziz City for Science and Technology (KACST) has established the botanical Gene-bank (KACST-BGB) along with the KACST-MUZ-Herbarium, and these are important accomplishments in the Kingdom in terms of securing food security and preservation of wheat genetic diversity. We suggest that these achievements must be followed by large national research projects that contribute to the collection and

Conclusion
Seeds of all 61 accessions of wheat (Triticum aestivum) had good and desirable qualities (low MC and high viability) that contribute directly to their preservation in the Gene-Bank of King Abdulaziz City for science and technology (KACST-BGB). We estimate that these seeds will remain viable in the seed bank for ! 25 years. It is clear from visiting different farms that>126 varieties of wheat can be found in Saudi Arabia. For example, Al-Qassim region alone is famous for its cultivation of five landraces of wheat. Thus, more collecting and preservation of wheat genetic diversity is urgently needed before these varieties disappear. This study also concludes that the geographical distribution of wheat crop in the Kingdom is largely concentrated in three different regions (Al-Qassim, Asir and Al-Taif) due to relatively low temperatures in winter in these areas. Also, some varieties of wheat are concentrated in high mountains in Jazan region (south-western of Saudi Arabia), such as in the Jabal-Fayfa and Jabal Al-Hashr mountains, which are also characterized by low temperatures and high rainfall. This study is the first to show the importance of collecting and preserving plant genetic resources (seeds in ex-situ seed banks) of the local wheat varieties in the Kingdom of Saudi Arabia.

Recommendations
Plant genetic resources for food and agriculture are considered one of the most important resources for world food security. Thus, it is urgently important that the genetic diversity of wheat, a staple food source for people, is preserved. To promote this preservation effort, the following recommendations are made: 1-The establishment of several national centres for plant gene banks in various important environmental regions of Saudi Arabia. 2-The establishment of a regional centre for plant genetic banks to preserve plant genetic resources with the aim of collecting plant germplasm in the Arabian Peninsula and the Arabian Gulf region. 3-The establishment of botanical gardens in a strategic environmental region would contribute positively to the work of gene banks by identifying plant biological properties, by cultivating and maintaining many local plants and tracking their life cycle and stages of development. Botanical gardens, herbaria and gene banks have the potential to greatly enhance plant science research in the Kingdom of Saudi Arabia and the Arabian Peninsula. 4-Herbaria, botanical gene banks and botanical gardens will also contribute to the definition and classification of plant species and therefore indirectly the preservation of genetic resources.