Germination and Seed Viability of <i>Helichrysum maracandicum</i> Popov Ex Kirp. Sterilized under <i>in Vitro</i> Conditions

Most medicinal plants are on the verge of extinction. In this regard, biotechnology is facing the challenge of developing alternative ways to produce biomass with the desired Biological active substance. The maximum yield of high growth performance of the cell culture mainly depends on the selection of optimal ratios and concentrations of growth regulators. This problem, namely the search for the optimal composition of the nutrient medium has become one of the main tasks in the cultivation of H. maracandicum Popov ex Kirp plant cells. Methods and results of seed sterilization of H. maracandicum are discussed in the article. This endemic, rare species of medicinal plant from the flora of Uzbekistan family Asteraceae Dumotr has a unique composition of secondary metabolites. For example, from the biomass of immortelle were isolated flavonoids, coumarins, lipids, phenols, purines, steroids, triterpenoids, glycosides, coumarins, cerines, bitter tannins, essential oils, etc. Used in folk medicine for cholecystitis and diseases of the liver, bladder, and gastrointestinal tract.

• <i>Helichrysum maracandicum
• in Vitro</i>
• Inoculum
• Sterilization
• Seedling Viability

1. Introduction

Plant resources are of importance for agriculture, medicine, and ornamental gardening, but the potential of the beneficial properties of many plants has not yet been sufficiently studied and is of interest in the future. Methodological approaches to the study of collections are based on the principle of maximum coverage of genetic diversity, including wild species, introduced plants, as well as the collection fund of plants cultivated in vitro.

One promising approach is the method of cultured cells in vitro based on somatic cells [1] . Until that time, the authors have studied the bioecological features and cultivation of some rare and introduced medicinal plants in various conditions of Uzbekistan [2] [3] [4] .

At present, much experience has been accumulated in vitro propagation of rare, rare medicinal plants [5] [6] [7] [8] [9] , as well as obtaining useful metabolites from the biomass of their vegetative and generative organs [10] [11] [12] . At the same time, relevant studies on growing H. maracandicum plants from their tissues and organs in vitro have not been conducted. For this purpose, the germination and viability of H. maracandicum seeds under in vitro conditions were studied at this stage of the study. According to the literature, the effect of in vitro sterilization of H. maracandicum seeds on their fertility and seedling viability has not been studied by anyone before in Samarkand or Uzbekistan. In addition, information on seed germination of species of the genus Helichrysum Mill. is poorly reflected in the literature. For example, according to Khujanov [13] , the highest germination of H. maracandicum seeds in laboratory conditions was 82% within 30 days at 25˚C, while the data of this author provide no information on seedling viability.

It is known that seed germination biology includes multifactorial processes of exogenous (temperature, humidity, light, storage conditions) and endogenous (structure of seed coat, physiological state during germination) parameters. According to these factors, exogenous, endogenous, and combined resting are distinguished in plant seeds [14] .

2. Object and Methods of Research

2.1. Seeds Collection and Sorting.

Seeds of plants were collected from the natural population of H. maracandicum in the vicinity of Kyzylbisoy village (Amankutan, Urgut district, Samarkand region, in the northern part of Zeravshan range; 66˚89'03.83"E 39˚29'75.93"N) [13] . Soil is grey soil mixed with fine sand. According to A. N. Khujanov, the mass of 1000 dry seeds of H. maracandicum is 0.08 ± 0.01 g in size, prismatic-brown seeds, called seedpods, have a length of 1.3 - 2 mm and a width of 0.2 - 0.6 mm (Figure 1). One of the unique characteristics of these seeds is their ability to maintain germination for up to 3 years [13] . It is known that the study of seed fertility of plants is one of the main criteria when restoring natural populations of plants and creating their plantations. As the period of seed storage increases, their germinating capacity decreases.

The Germination of H. maracandicum seeds was studied according to Ishmuratov’s method [14] , and their germination capacity was determined according to

Firsova’s method [15] . According to the literature, the germination of freshly harvested seeds is higher when growing medicinal plants from their seeds [10] . Well-matured annual seeds of H. maracandicum were selected for our studies (Figure 1).

2.2. Seed Sterilization.

The method of R. G. Butenko [16] was used for the sterilization of plant tissues and organs. To remove pathogenic microflora, seeds were subjected to surface sterilization with tap water and then distilled water at room temperature. The following seed sterilization scheme was used to remove the internal symbiotic infection, which has a harmful effect when growing seeds in vitro.

To prepare 1 liter of alkaline Domestose solution used for sterilization, 50 ml of Domestose solution was added to 950 ml of water. The antibiotic streptomycin was used at a concentration of 330 µg/ml. After each reagent treatment, the seeds were washed with sterile distilled water.

3. Results Obtained and Their Analysis

Germination of Sterilized Seeds and Seedling Viability

Several different sterilization methods were tried to sterilize in vitro the seeds of H. maracandicum, which is considered the object of the study.

Consistently, the seeds were washed in water for 1 hour, shaken vigorously in a 1% solution of domestose (soapy water), washed 6 times in water, washed in a solution of the antibiotic streptomycin (330 µg/ml), washed in water 3 times, in 70% ethyl alcohol, then washed while shaking for 1 minute, shaken thoroughly, and washed 3 times in clean water. The medium and conditions of sterilization are given in the table below (Table 1).

Based on this scheme, four variants of sterilization were studied. In the first variant, the seeds were shaken intensively in 1% domestose solution for 5 minutes, and the antibiotic streptomycin solution was kept for 6 minutes. In the second variant, the seeds were shaken in 3% domestose solution for 5 minutes and kept for 8 minutes in the antibiotic solution of streptomycin. After treatment with ethyl alcohol, the seeds were washed with intensive shaking for 1.5 min. In the third variant, the seeds were shaken in a 5% solution of domestose

Table 1

Stages and variants of in vitro sterilization of H. maracandicum seeds

№	Stages of sterilization	Sterilization options
		I	II	III	IV
1	Water	60 min.	60 min.	60 min.	60 min.
2	Domestose	1%, 3 min.	3%, 5 min.	5%, 8 min.	8%, 10 min.
3	Water	6 times	6 times	6 times	6 times
4	Streptomycin (330 µg/ml)	6 min.	8 min.	10 min.	12 min.
5	Water	3 times	3 times	3 times	3 times
6	Ethyl alcohol (70%)	1 min.	1.5 min.	2 min.	2.5 min.
7	Water	3 times	3 times	3 times	3 times

for 8 minutes, and in the solution of the antibiotic streptomycin was kept for 10 minutes. The last rinsing with water was carried out under intensive shaking for 2 minutes. In the fourth variant, the seeds were shaken for 10 minutes in 10% domestose solution and incubated in the streptomycin antibiotic solution for 12 minutes. The last rinsing with water was carried out under intensive shaking for 2.5 minutes. As can be seen from the table, the variants used in the experiment differed in the number of stages of cross-sterilization and their duration.

According to A. N. Khujanov, the optimum germination temperature of H. maracandicum seeds in laboratory conditions is 25˚C, and the germination rate is 80% - 82% [10] . The germination of seeds that passed the sterilization stages in the above variants was studied for 30 days in the medium with an air temperature of 25˚C. The results of our studies are shown in Figure 2(a). We can see that seed germination was 23% in the first sterilization option, 31% in the second, 96% in the third, and 16% in the fourth. That is, the highest germination rate observed in the third variant of seed sterilization, seed germination was 14% higher than that noted by A. N. Khujanov [10] .

Observations of the viability of seedlings obtained from seeds after seed sterilization were carried out for 15 days. Analysis of the results of observations of seedling viability at the level of 10%, 15% and 90% was noted in variants 1, 2 and 3. In contrast, in the fourth variant, 16% of the lawns remained on the 5th day of observations, and by the 10th day, all the seedlings died (Figure 2(b)).

4. Conclusions

The results (Figure 3) obtained showed that for in vitro studies with H. maracandicum the most effective sterilization of H. maracandicum seeds, was used in variant 3. Contrary to the other variants, in this case, we used to wash the seeds for 8 minutes with 5% domestose solution, keeping them for 10 minutes in a streptomycin solution, and then for 2.5 minutes in 70% ethanol. Under these conditions, the germination of H. maracandicum seeds reached 96%, and the

viability of the prostrates, 85%.

Thus, the proposed scheme of sterilization of seeds of H. maracandicum Popov ex Kirp allows us to obtain a high yield of viable seedlings of this species to obtain cells and tissues for further work on cell engineering.

Acknowledgements

The authors are grateful to the staff of the SAG AGRO in vitro laboratory for their help with the equipment and the chemical reagent.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

Fund

This article was prepared with the support of an applied grant from the Ministry of Innovative Development of the Republic of Uzbekistan on the topic “Seed propagation and creation of plantations of Helichrysum maracandicum Popov Ex Kirp”.

References

1. Alexandrova, A.A. and Khandy, M.T. (2018) Methods for Obtaining Cell Cultures of Plants of the Genus Artemisia. Prospects of Phytobiotechnology to Improve the Quality of Life in the North Collection of Materials III Scientific-Practical. Conference from International Participation and Scientific School on Cell Biotech, Yakutsk, 4-8 June 2018, 29-31.

2. Tashpulatov, Y.Sh., Khamdamov, I.Kh. and Nurniyozov, A.A. (2019) Water and Coastal Water Vegetation of Various Types of Waters in the Samarkand Region. Eurasian Journal of Biosciences, 13, 1413-1417. https://doi.org/10.5958/2320-3188.2019.00007.X

3. Tashpulatov, Y.Sh. (2020) The Anatomical Structure of the Medicinal Raw Material Acorus calamus L. in the Conditions of Culture of the Samarkand Region (Uzbekistan). Bulletin of Pure and Applied Sciences, 39, 107-115. https://doi.org/10.5958/2320-3188.2020.00013.3

4. Isomov, E.E. and Tashpulatov, Y.Sh. (2022) Influence of Soil Salt on Growth, Development and Seed Productivity of Artichoke Varieties. American Journal of Plant Sciences, 13, 557-563. https://doi.org/10.4236/ajps.2022.135036

5. Novikova, T.I., Nabieva, A.Yu. and Poluboyarova, T.V. (2008) Preservation of Rare and Useful Plants in the in Vitro Collection of the Central Siberian Botanical Garden. Vestnik VOGiS, 12, 594-572.

6. Zaripova A.A. ,et al. (2016)Introduction to in Vitro Culture of Skullcap Baikal Bulletin Botanical Garden Saratov State University 14, 94-98.

7. Muraseva, D.S. (2016) Reproduction and in Vitro Conservation of Rare and Endemic Species of the Genus fritillaria L. Doctoral Thesis, Novosibirsk State University, Novosibirsk, 31 p.

8. Ambros, E.V., Kotsupii, O.V., Novikova, T.I. and Vysochina, G.I. (2018) Clonal Micropropagation of a Rare Species Astragalus sericeocanus Gontsch. and the Content of Phenolic Compounds under in Vitro Conditions. Turczaninowia, 21, 87-99. https://doi.org/10.14258/turczaninowia.21.4.10

9. Erst, A.A., Erst, A.S. and Shmakov, A.I. (2019) In Vitro Propagation of Rare Species Rhodiola Roseafrom Altai Mountains. Turczaninowia, 4, 78-86. https://doi.org/10.14258/turczaninowia.21.4.9

10. Mancini, E., De Martino, L., Marandino, A., et al. (2011) Chemical Composition and Possible in Vitro Phytotoxic Activity of Helichrsyum italicum (Roth) Don ssp. italicum. Molecules, 16, 7725-7735. https://doi.org/10.3390/molecules16097725

11. Mati&#263;, I.Z., Aljan&#269;i&#263;, I., &#381;i&#382;ak, &#381;., et al. (2013) In Vitro Antitumor Actions of Extracts from Endemic Plant Helichrysum zivojinii. BMC Complementary and Alternative Medicine, 13, Article No. 36. https://doi.org/10.1186/1472-6882-13-36

12. Korozhan, N.V. (2018) Influence of Immortelle Flowers Infusion on Mast Cell Degranulation in Vitro. Pharmacy and Pharmacology, 6, 63-72. https://doi.org/10.19163/2307-9266-2018-6-1-63-72

13. Khujanov, A.N. (2020) Biology and Reseurces of Helichrysum maracandicum Popov ex Kirp. Ph.D. Thesis, Botanical Institute Academy of Sciences of the Republic of Uzbekistan, Tashkent, Uzbekistan, 41.

14. Ishmuratova, M.M. (2009) Seeds of Herbaceous Plants: Features of the Latent Period, Use in Introduction and Propagation in Vitro. Gilem, Ufa, 115 p.

15. Firsova, M.K. (1959) Methods for Determining the Quality of Seeds. Selkhoz. Literature, Moskow, 351 p.

16. Butenko, R.G. (1991) Biology of Cells of Higher Plants in Vitro and Biotechnologies Based on Them: Textbook. FBK-PRESS, Moskow, 160 p.