Morphological Evaluation of Asparagus Azerbaijanensis Wild Accessions and Drought Tolerance Assessment of Iranian Asparagus Under Greenhouse Condition

Some of the most cultivated commercial cultivars of asparagus are susceptible to abiotic stresses. Different species of asparagus plant are distributed in different regions of Iran which could be useful genetic resources for applying in asparagus breeding programs. The purposes of the current study were to evaluate morphological variation of A. azerbaijanensis accessions and drought tolerance assessment of Iranian asparagus in order to introduce superior accessions. Two different accessions of A. azerbaijanensis species were collected. A number of 30 plants were selected and 22 morphological traits were then evaluated. A factorial experiment based on completely randomized design was performed in order to drought tolerance assessment of 9 Iranian asparagus accessions. The average of spear length and diameter in Ahar and Makou plants were 48.94 cm and 4.70 mm, respectively. Based on results, plants of Ahar accession produced short and thick spears but on the other hand, Makou accession plants produced longer and thinner spears. Score 8 (80% similarity rate) was averagely chosen by panel members in terms of similarity rate of A. azerbaijanensis accessions spears taste to that of Mary Washington commercial cultivar. The highest percentage of seed germination was recorded in Baladeh and Gazanak accessions (71.66 and 70.83%, respectively). Baladeh accession showed the highest carotenoid content under drought stress conditions (0.43 mg g Fw − 1 ). Suitable size and delicious taste of spear in A. azerbaijanensis species has made it a suitable candidate for commercial cultivation and also improving spear characteristics by using selection method. Baladeh and Gazanak accessions could be introduced as superior accessions with high drought tolerance rate. High resistance rate of these accessions can be related to their ploidy level and distribution areas characteristics. accessions rst The values of dry of


Introduction
Asparagus is a perennial plant which belongs to Asparagaceae family. Fleshy stem is edible part of asparagus which is called spear (Kanno and Yokoyama, 2011). Some of the most cultivated commercial cultivars of asparagus are susceptible to abiotic stresses such as drought and salt stress (Drost and Wilcox-Lee 1997). Asparagus o cinalis is the most economically important species and Mary Washington is the most cultivated commercial cultivar of asparagus worldwide (Amian et al. 2018). Drought stress is one of the most important environmental stresses which can seriously affect quality and productivity rate of agriculture crops (Ghahremani et al. 2020). Iran is the second largest country in the Middle East which is climatically part of the Afro-Asian belt of deserts and has a unique plant and animal diversity (Ebrahimi et al., 2021). According to some comprehensive researches which were conducted by Sarabi  The main purposes of the current study were to evaluate morphological variation of A. azerbaijanensis accessions collected from western regions of Iran, drought tolerance assessment of Mary Washington commercial cultivar under greenhouse condition and also evaluation of drought resistance rate of Iranian asparagus different accessions in order to introduce superior accessions for applying in future asparagus breeding programs.

Materials And Methods
Morphological evaluation of A. azerbaijanensis wild accessions In rst experiment, two different accessions of A. azerbaijanensis species were identi ed and collected from Ahar (a city located in East Azerbaijan province, Iran) and Makou (a city located in West Azerbaijan province, Iran) cities (Fig. 1). The locations of distribution regions were detected according to Hamdi and Asadi, (2017) and also based on local people guidance. A number of 30 plants containing male and female plants (in equal numbers) were selected and 22 morphological traits were evaluated according to Mousavizadeh et al. (2015) (Fig. 2).
Soil samples were collected from four different locations in distribution areas at a depth of 0 to 60 cm and some of the most important soil characteristics were then measured. The climate characteristics were collected from weather stations located in Ahar and Makou cities and exact geographical location of distribution regions was recorded by using GPS software (Table 1).

Drought tolerance assessment of Iranian asparagus
In second experiment, Iranian asparagus accessions belong to three different species including A. azerbaijanensis, A. o cinalis and A. verticillatus were collected from different regions of Iran (distribution areas were identi ed according to Mousavizadeh et al. (2015) and Hamdi and Asadi (2017) and also based on local people guidance) (  Fig. 3).
Asparagus fruits were harvested from late spring to early summer 2020. Seeds were then separated and stored under cold condition until beginning of greenhouse experiment. Seeds sterilization was carried out by sodium hypochlorite (1.5 % (v/v)) for 10 min and rinsing in distilled water for three times (5 min for each time). Mary Washington commercial cultivar was applied as control. Based on eld water capacity (FC), four levels of drought stress including 100% FC (control), 75% FC (mild drought stress), 50% FC (moderate drought stress) and 25% FC (severe drought stress) were simulated. Gravimetric method was used to determine FC. First, the soil (5 kg) was oven dried for 48 h at 103 0 C and then weighted. Then, a plastic pot (18×22 cm) was lled with dried soil and then saturated with distilled water. Soil weight was recalculated after 48 h of drainage. Finally, FC was evaluated by calculating of difference between drained soil weight and dried soil weight.
Sterilized seeds were sown at depth of 1 cm. First time irrigation was carried out immediately after sowing and irrigation was then continued at 6-days interval. The greenhouse temperature and relative humidity were adjusted on 27 ± 2 0 C and 65%, respectively. Tables 3 and 4 show some important characteristics of applied soil and irrigation water and Fig. 4 shows experimental units and Iranian asparagus seedling grown under greenhouse condition.
Three different groups of asparagus traits including a) seed germination indices b) morphological traits and c) physiological traits were measured to determine drought resistance rate of each accession.

a) Seed germination indices
Seed germination percentage and speed were calculated by using of following formula: (1) (Czabator 1962) (2) Germination speed = n 1 /d 1 + n 2 /d 2 + n 3 /d 3 +… (Czabator 1962)  Total chlorophyll and carotenoid content, electrolyte leakage and proline content were the evaluated physiological traits. Total chlorophyll and carotenoid content was evaluated according to spectrophotometric method (spectrophotometer-SAFAS UVmc2) as descripted by Witham et al. (1971). Electrolyte leakage was evaluated based on electrical conductivity changes measurement as descripted by Lutts et al. (1996). Proline content was measured by using method descripted by Bates (1973). Sulfosaicylic acid was used to extract free proline. proline content was determined by a spectrophotometric absorption measurement (spectrophotometer-SAFAS UVmc2) at 520 nm and expressed as  Table 5).
Results of correlation analysis between evaluated morphological traits and spear properties showed that there is signi cant correlation between spear length and diameter and number of primary and secondary branches, internode length of secondary branch, leaf length and diameter and scale number (Fig. 5). According to results, spear length increased and spear diameter decreased with increasing the number of primary and secondary branches. In contrast, increasing of leaf length and diameter led to producing shorter and thicker spears in A. azerbaijanensis accessions. Among the evaluated properties, number of primary and secondary branches showed the highest correlation with diameter of spear (r = -0.75) and leaf length was the highest correlated trait to length of spear (r = -0.85) ( Table 6).
Taste panel analysis showed that no one of panel members detect bitter taste in spear harvested from A. azerbaijanensis accessions plants.
In the other hand, all of panel members detected astringent taste in spears harvested from both Ahar and Makou accessions plants. Score 8 (80% similarity rate) was averagely chosen by panel members in terms of similarity rate of A. azerbaijanensis accessions spears taste to that of Mary Washington commercial cultivar.  Table 7).
The lowest values of dry weight of aerial and underground parts were recorded in plants grown under highest level of drought stress (0.06 and 0.09 g respectively). Increasing of drought stress severity from 100% FC to 25% FC led to a signi cant decrease in seed germination percentage and speed. No signi cant difference was observed between asparagus plants grown under 100, 75 and 50% FC in terms of plant height (Table 8).
Increasing of drought severity from 100% FC to 75% FC did not cause a signi cant decrease in total chlorophyll content of asparagus accessions. The lowest asparagus plants chlorophyll content was recorded under stress level of 25% FC (0.96 mg g Fw − 1 ) (Fig. 7b).
Baladeh accession showed the highest carotenoid content under drought stress conditions (0.43 mg g Fw − 1 ) and in the other hand, Ahar and makou accessions were the least valuable plant materials in terms of this measured trait. There was no signi cant difference between Chalaki, Mahmoudabad and Taleghan accessions in total carotenoid content under different levels of drought stress (Fig. 8a). There was no signi cant difference between asparagus plants grown under stress levels of 100% and 75% FC in terms of total carotenoid content but increasing of drought stress severity to 50% FC led to a signi cant decrease in this trait. The lowest carotenoid content was recorded in plants grown under highest applied drought stress level (0.352 mg g Fw − 1 ) (Fig. 8b).
The lowest electrolyte leakage was recorded in Gazanak and Baladeh accessions under drought stress conditions. Electrolyte leakage in Mary Washington asparagus plants (64.86%) was higher than that of Gazanak, Baladeh, Mahmoudabad, Chalaki, Talrghan and Alborz accessions and lower than that of Ahar. Makou and Shiraz accessions (Fig. 9a). According to obtained results, electrolyte leakage of asparagus plants cells increased with increasing of drought stress severity. Electrolyte leakage increased by 27.32% with increasing of drought severity from 75% FC to 50% FC (Fig. 9b).
Gazanak was the superior accession in terms of free proline content under highest applied level of drought stress (4. assessment showed the highest level of tolerance rate to applied drought stress conditions (Fig. 10).

Discussion
Iran is one of biggest countries located in Middle East. Based on reports, more than 8  accessions of Cenchrus species were higher than those of tetraploid (4x) accessions under drought stress conditions. But in the other hand, free proline content of tetraploid (4x) plants cells was higher than that of diploid (2x) plants.

Conclusion
In conclusion, results of our study showed that wild accessions of A. azerbaijanensis species, due to their high morphological variation and desired spear taste and size, are suitable candidates to apply in asparagus breeding programs with the aim of producing hybrids plants with superior vegetative growth. Also, the superiority of Iranian asparagus different accessions (specially Baladeh and Gazanak accessions) over the commercial cultivar in tolerating of water de cit stress, promises the possibility of further using of Iranian asparagus accessions in breeding projects with aim of producing dry resistant cultivars.

Declarations
Funding: This work was supported by university of Zanjan (grant number 00.01.360).

Con icts of interest:
We have no con icts of interest to disclose.
19. Zinkernagel J, Kahlen K (2017) Water relations of asparagus crop under different water supply and implications for plant-based irrigation scheduling. Acta Hortic 1150: 385-390. Tables   Table 1 Climate     In each row having at least one common letter are not statistically different at 1% level according to t test   In each row having at least one common letter are not statistically different at 1% level according to Tukey's test Table 9 Effect of interaction between accession and drought stress on measured characteristics In each row and column having at least one common letter are not statistically different at 1% level according to Tukey's test Accessions code is based on Table 2  Table 10 Effect of interaction between accession and drought stress on free proline content In each row and column having at least one common letter are not statistically different at 1% level according to Tukey's test Accessions code is based on Table 2 Figures Figure 1 Distribution regions of A. azerbaijanensis wild accessions  Correlation between evaluated morphological traits and length and diameter of spear Effect of accession (a) and drought stress (b) on total chlorophyll content of asparagus plants Effect of accession (a) and drought stress (b) on electrolyte leakage percentage (Accessions code is based on Table 2)

Figure 10
Cluster analysis based on measured characteristics under drought stress conditions (Accessions code is based on Table 2)