عنوان مقاله [English]
Plant breeding to created ploidy level is one of the most important parts of plant research in every country, where the breeding of native plants is a priority in every country. Spending a long time on breeding plants with traditional methods has made plant breeding research using in vitro techniques to become more important. In this study, to investigate the callus formation from the endosperm tissue of mature shallot seeds in MS culture medium containing growth regulators with the combination of different concentrations of 2-4, D (1.5 and 2 mg/L) and NAA (0.5 and 1 mg/L) L) of auxins and BAP (0.5 and 1 mg/L) and Kin (0.5 and 1 mg/L) of cytokinins were used. The results showed that using the treatment of 1 mg/liter of NAA and 1 mg/liter of BAP was the best treatment for callus formation. In this research, the concentration of sucrose and different concentrations of BAP hormone were effective for onion production. The results of onion diameter showed that the high concentration of sucrose (50 g/L) and the concentration of 3 mg/L of BAP hormone were suitable for the production of larger onions. By chromosomal analysis of the rooted chives, triploid plantlets were obtained at the end of the experiment. Due to the shortness of triploid plant breeding in this method compared to traditional crossing methods, therefore, this method and the best hormonal compounds are recommended for the production of chives in shallots.
Iranian plateau is known as specific habitat for Persian Shallot (Allium hirtifolium Boiss.) for which some evidences in terms of its domestication are available. It is distributed from North West to central and South West of Iran and grows as a wild plant in the Zagross Mountains. Traditionally, the powder of Persian Shallot is also utilized as highly demanded additive and spice for food relishing by local people. Persian shallot has recently attained arising consideration with respect to its potential in biosynthesizing the valuable secondary metabolites such as allicin, phenolic antioxidants, and ascorbic acid. In diploid plants, the endosperm is a triploid (i.e., having 3 sets of chromosomes) tissue as a result of double fertilization, which is a unique process in higher plants. Undoubtedly, the main source of triploid cells in the plants is endosperm, which has remained as storage tissue in most of monocots up to the final stage of seed formation. From this point of view, endosperm culture technique is known as direct and efficient procedure for micro-propagation of triploid plants which is also categorized as the easy, rapid and low cost way for this purpose. In diploid plants, the endosperm is a triploid (i.e., having 3 sets of chromosomes) tissue as a result of double fertilization, which is a unique process in higher plants (Hoshino et al. 2011; Wang et al. 2016). Triploid plants express the common form of polyploidy at which three complete sets of chromosomes could be distinguished and are traditionally produced through crossing a diploid with an induced tetraploid plant. However, the rate of induction of tetraploids had generally been low and doubling of the diploid chromosome number by the use of spindle inhibitors is lengthy and laborious.
Harvesting of seeds from shallot plants in Khalat Pushan Research Center of Faculty of Agriculture, University of Tabriz was done during the end of June. In this study, for indirect regeneration for callus production, mature endosperm tissue of shallot in MS solid culture medium (Morashik and Skoog, 1962) containing plant growth regulators with the combination of different concentrations of 2-4, D (1.5 and 2 mg/ L) and NAA (0.5 and 1 mg/L) of auxins and BAP (0.5 and 1 mg/L) and Kin (0.5 and 1 mg/L) of cytokinins for a period of 3 months in dark conditions with temperature They were cultivated at 24°C daily and 18°C night temperature. After the production of callus during this period, calluses were divided into smaller pieces in the same previous treatments for one month. After this stage, callus pieces containing primary shoots were transferred to MS culture medium without growth regulators for about 40 days. After producing enough shoots, the shoots were transferred to MS culture medium containing BAP (0.5, 1, 2 and 3 mg/L) and NAA (0.5 mg/L) in two concentrations of 30 and 50 g/L of sucrose.
Ploidy level of the plantlets derived from endosperm cultures was determined by chromosome counting. For this purpose, root tips were excised and pretreated with cooled alpha-bromo-naphthalene (4 °C) for 8 h and fixed in a acetic acid and ethyl alcohol solution (1:3 v/v) overnight. Afterward, the prepared root tips were hydrolyzed in 1 N hydrochloric acid at 60 °C for 8 min and were immediately stained with orcein 2%. The stained samples were squashed in 45% (v/v) acetic acid using traditional method (Panahandeh and Mahna 2011). All samples were inspected and the best ones with clear metaphase plates were selected for photomicrography. Image acquisition was done utilizing a Nikon E400 upright research microscope equipped with a high resolution Nikon DXM 1200 digital camera (Nikon, Tokyo, Japan).
According to the purpose of this experiment, the feasibility of producing callus from endosperm tissue in different hormonal combinations and the modification of Persian shallot plant in the ploidy level was investigated. and in the next step, by examining the effect of sucrose concentration and hormone treatment, it was observed that using a concentration of 50 g/l of sucrose compared to the usual concentration of 30 g/l of MS culture medium for increasing the diameter and length of the bulb is suitable and for similar research. Breeding of plants with using of endosperm culture technique under in vitro condition compared to the traditional method which is done by crossing between diploid and tetraploid plants, and this method has disadvantages such as time-consuming and difficulty in the crossing process. To produce the endosperm of triploid Persian shallot plant under tissue culture conditions and in the next stages after adaptation and cultivation under natural conditions, a comparative process with diploid plants will be done so that by examining important traits including the yield and medicinal properties of this plant we can get positive results to present an important point in this field.