Document Type : Original Article
Authors
1
PhD candidate. Department of Horticultural Sciences and Landscape Engineering, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
2
Associated Prof. Department of Horticultural Sciences and Landscape Engineering, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
Abstract
1. Introduction: All the species belonging to the Asparagus subgenus are dioecious with the basic number of chromosomes (=10x), and the number of chromosomes varies depending on the type of species and due to changes in the ploidy level. Genotypes of Asparagus subgenus with chromosome number diploid (x2=20), triploid (x3=30), tetraploid (x4=40), pentaploid (x5=50), hexaploid (x6=60), octaploid (x8=80), decaploid (x10=100), and dodecaploid (x12=120) can be found. Asparagus is widely distributed in different climates of Iran. Asparagus officinalis L. is an edible herbaceous perennial dioecious crop species with different ploidy levels. One of the problems of asparagus production is the decrease in fertility due to the increase in ploidy levels, resulting in a reduction in seed production. The limitation of genetic resources in asparagus makes it necessary to use methods such as vegetative embryogenesis for the clonal propagation of this plant. One of the main uses of vegetative embryo is its use in artificial seed production. Hydrated artificial seeding was first achieved by coating alfalfa embryos. These seeds are prepared by encapsulating vegetative embryos or other vegetative organs of the plant in a hydrogel. So far, several methods have been used to produce artificial seeds, such as potassium alginate, sodium alginate, agar, gelrite, etc., among which the use of sodium alginate and calcium alginate has been introduced as the most successful methods.
2. Materials and Methods: The current research was carried out to encapsulate tissue culture-originated vegetative embryos. For this purpose, the seeds of A. officinalis L. with two different ploidy levels (diploid and octaploid) were prepared. After washing the seeds with water and detergent, the seeds were disinfected in 70% ethanol under a laminar hood for one minute and then placed in 30% sodium hypochlorite for 15 minutes. In the next step, the seeds were washed three times with sterile distilled water. Then they were established in the MS basic culture medium. The resulting seedlings were used for collecting five to seven-cm single-node explants. These single-node explants were cultured on B5 liquid media supplemented with 2 mg/L 2,4-D. Then they were kept in the axophyton device for 14 days in the induction phase. Afterwards, the explants were grown in B5 media without 2-4-D. After the emergence of vegetative embryos, the embryos were encapsulated using 2% sodium alginate in half-strength B5 medium containing 1 ppm of Kinetin. To harden the coverings of somatic embryos, a 100 mM calcium chloride solution was applied at three different time points of 30, 40, and 60 minutes.
3. Results and Discussion: The lowest and highest percentage of germination rate among artificial seeds with Kinetin was recorded as T40 (25.2) and T30 (39.9), respectively. Also, the lowest and highest percentage of germination rate among artificial seeds without Kinetin was observed in C40 (21.7) and C60 (47.3), respectively. The comparison of the average data on the germination speed between the mutual effects of mass and encapsulation treatment showed that the highest rate was related to the diploid seedlings obtained from artificial seeds treated with Kinetin, with a time of 60 minutes (AO6T60), as 14.4, and the lowest rate was related to the diploid mother plant (AO6P), as 9.7. Comparison of the average data of the effects of encapsulation treatment related to the average time required for germination shows that the highest was related to the 30-minute treatment (c30), as 8.1, and the lowest was related to the c40 treatment, as 2.4, and no significant difference was observed between the other treatments (p>0.05). Comparison of the average effects of encapsulation treatment in terms of length and number of stems shows that the longest stem length after the mother seedlings was related to Kinetin treatment 30 minutes (T30), as 6.8 cm, and this result was also observed for the trait number of stems. The lowest value of these traits was observed in the C60 treatment, as 4.61 cm for stem length and the C30 treatment for stem number (4 numbers). The results of the average comparison between two plant populations show that the highest amount of carotenoid, total chlorophyll, chlorophyll a, and chlorophyll b is related to octaploid plants. Based on the results of comparing the averages related to the encapsulation treatment, the highest amount of carotenoids was observed for 40 minutes with kinetin (T40) as 1.69 (mg/g fresh weight), and the lowest was observed for 40 minutes without Kinetin (C40) as 1.09 (mg/g fresh weight). In terms of chlorophyll b and total chlorophyll, the highest amount was observed in the mother seedlings, and the lowest was observed for chlorophyll b in C40 treatment as 1.02 (mg/g fresh weight) and for total chlorophyll in T30 treatment as 1.08 (mg/g fresh weight).
4. Conclusion: The results of the comparison of the averages related to the mutual effects between plant mass and encapsulation treatments showed that in terms of carotenoid, chlorophyll b and total chlorophyll, the highest amount was related to the octaploid seedlings obtained from artificial seeds without 60 minutes Kinetin, and the lowest amount of carotenoid was related to the diploid seedlings obtained from artificial seeds with 60 minutes Kinetin, and in terms of chlorophyll b, it corresponded to diploid seedlings obtained from artificial seeds without 60 minutes Kinetin, and in terms of total chlorophyll, corresponding to diploid seedlings obtained from synthetic seeds without 40 minutes Kinetin.
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