IN VITRO MICROCLONAL PROPAGATION OF INDIAN LILAC (SYREN) AND MAGNOLIA GRANDIFLORA FROM ORNAMENTAL PLANTS

ABSTRACT

This article presents the results of a study of the cultivation of explants of ornamental plants in DKW medium (Indian Lilac (syren)) and MS medium (Magnolia Grandiflora) with the addition of 30 g/l sucrose and 7 g/l agar, adjusted to pH 5.7+0.1, and sterilized in an autoclave at 121 ºC for 20 minutes. As a result, MS and DKW nutrient media with BAP 2.0 mg/l and IBA 0.02 mg/l, we can see that the results in the explants were as follows, that is, the shoots and buds formed from the callus tissues were fully formed within 18-21 days, and the length of the formed plants was 4.8 cm in Indian Lilac (syren) and 4.5 cm in Magnolia Grandiflora, which is considered to be the best positive result for an ornamental plant.

АННОТАЦИЯ

В данной статье представлены результаты исследований по культивированию эксплантов декоративных растений в среде ДКВ (Indian Lilac (syren)) и МС (Magnolia Grandiflora) с добавлением 30 г/л сахарозы и 7 г/л агара, приведённых к рН 5,7+0,1 и стерилизованных в автоклаве при 121 оС в течение 20 минут. В результате в питательных средах МС и ДКВ с БАП 2,0 мг/л и ИБА 0,02 мг/л результаты в эксплантах были следующими: в течение 18-21 дней полностью сформировались побеги и бутоны, а длина сформированных растений составила 4,8 см у индийской сирени (Syren) и 4,5 см у магнолии Grandiflora, что считается наилучшим положительным результатом для декоративного растения.

Keywords: in vitro, Indian Lilac, Magnolia Grandiflora, explant, microclonal propagation, apical meristem, nutrient media (MS and DKW), citokinin, auxin, phytohormones (BAP and IBA).

Ключевые слова: in vitro, Indian Lilac, Magnolia Grandiflora, эксплант, микроклональное размножение, апиальная меристема, питательные среды (МС и ДКВ), цитокинин, ауксин, фитогормоны (БАП и ИБА).

INTRODUCTION

As a priority area of landscaping worldwide, special attention is paid to research on the identification of ornamental, promising, and pest-resistant plant species and the development of effective methods for their rapid propagation. In this regard, new cultivars and forms of ornamental species have been created. The potential of trees and shrubs in modern landscaping has been assessed, new methods of vegetative propagation have been developed, and optimal methods of seed propagation have been improved.

Ornamental plants are groups of cultivated and wild plants belonging to various botanical families that are used to satisfy the aesthetic needs of people. Ornamental plants are grown for landscaping cities and villages, decorating parks, public buildings and residential areas, and for making bouquets. They are characterized by their beautiful leaves and flowers, the color of their fruits, and the unusual shapes of their stem (with drooping branches, umbrella-shaped, erect). According to a number of characteristics, for example, according to their life form, plants are divided into trees, bushes, semi-bushes and herbs. According to the shape of their stems, they are divided into erect, creeping, low growing, spreading, drooping plants and others.

Trees and bushes are not only a source of raw materials and various products, but also one of the main factors improving the natural environment. The vital activity of the plant world has an impact on the climate, namely, it absorbs and neutralizes CO₂ and other harmful gases and fumes in the air, and reduces the amount of dust in the city air (M. Khakimova, 2021).

In cell biotechnology, in vitro cultivation of cells is based on the ability of plants to reproduce and regenerate, that is, to divide tissues (Davronov 2008).

Plant micro propagation is aimed at protecting endemic species that are at risk of extinction, which have great potential (Bramwell, 1990).

Indian Lilac (syren) is a drought-resistant tree plant belonging to the Lythraceae family. However, it can also be found in the form of a bush. In the wild, this tree is widespread in India. There are about 25 species in the Lythraceae family. Indian Lilac (syren) is a deciduous bush with smooth silver-gray spotted stems, 5-8 m high and up to 8 m wide.

The leaves are oval, 2-7 cm long. The flowers are 2.5 cm in diameter, with wavy edges, white, pink, crimson, red in color, up to 20 cm long, bloom in summer. There are many ornamental varieties of this plant.

In recent years, Indian Lilac (syren) has attracted much attention due to its widespread cultivation, with its products increasingly used in agriculture, animal husbandry, medicine and cosmetics (Bittencourt et al., 2009). It thrives even in nutrient-poor soils, but it cannot tolerate waterlogged or saline areas, and requires a soil pH of 6.2 to 7.0 for good growth. The species can be propagated by seeds, cuttings and tissue culture (Oliveira et al., 2005).

Magnolia Grandiflora, widely used in greening and landscaping, is distinguished by its uniqueness and high appearance, resistance to adverse environmental factors, and it is particularly important because it releases phytoncides that fight disease-causing and infectious bacteria. (L.F. Ding et al. 2018).

Magnolia Grandiflora L. is an ornamental plant belonging to the Magnoliaceae family. It is commonly known as the Southern Magnolia. It is an ornamental tree and is also cultivated as a medicinal plant. Its flowers are used in the treatment of various traditional diseases. An aqueous extract of the seed cones of this plant induces apoptosis against B-cell lymphocytic leukemia (B-CLL). This indicates the therapeutic potential of this species (G.H.Marin et al. 2010).

In recent years, various plant parts of this species have been found to have anticancer, antidepressant, antioxidant, anti-inflammatory, etc. properties. It is known that this species contains about 255 different components, including alkaloids, flavonoids, terpenoids (Y.Lee et al. 2011).

In addition, its leaves have shown antimicrobial properties and phytotoxic effects on beans, corn, and tobacco grown under greenhouse conditions (J.M. Jacynó et al. 1991). The flower extract has antioxidant activity and is also known to reduce melanin content (H. Huang et al. 2012).

Considering the medicinal nature of this species, the present study was conducted to characterize this plant species on morphological basis and screen the seeds and leaves for various phytochemicals.

Different concentrations of sodium hypochlorite were tested for sterilization of explants. Surface sterilization of explants of Indian Lilac (syren) and Magnolia Grandiflora was carried out in a 2.0% NaOCl solution for 20 minutes on a magnetic stirrer. As a result, the number of infected plants was 16%, and the survival rate was 84%. In clonal micropropagation of Indian Lilac (syren) and Magnolia Grandiflora, when 2 mg/l BAP (6-benzyl aminopurine) and 1 mg/l NAA (naphthalene acetic acid) were added to the MS (Murashige and Skoog) nutrient medium, the growth of plants was 86.4% (A.N.Allayarov et al. 2023).

Grafting and microclonal propagation are the only effective methods for propagation of plants that are difficult to root. However, propagation by grafting is more expensive and complicated, requiring specialized nurseries and skilled grafters (Fabbri et al., 2009; Lambardi et al., 2013).

Materials and methods

This research was carried out in 2024 as part of the project “Intensive cultivation of the seedlings of ornamental plants and bushes, and development of technologies for their protection from diseases and pests in order to establish green areas” at the Biotechnology Laboratory of the Scientific Research Institute of Horticulture, Viticulture and Winemaking named after Academician Mahmud Mirzayev.

Explants were taken from actively growing three apical buds and nodal segments of ornamental plants (Indian Lilac (syren) and Magnolia Grandiflora). The collected explants, including the joints with axillary buds, were washed under running water for 1 hour, then washed with liquid soap in plain water for 30 minutes and washed twice in distilled water to remove soap. Then the surface of the explants was sterilized with solutions of Sodium Hypochloride (NaOCl) at different concentrations (1%, 2%, 3%, 4% and 5%) for 10-20 minutes. Then the explants were washed three more times with distilled water.

As nutrient media, Murashige and Skoog (MS) and Driver and Kuniyuki Walnut (DKW) with different content were used.

In the initial stage, explants were cultured in DKW medium (Indian Lilac) and MS medium (Magnolia Grandiflora) supplemented with 30 g/l sucrose and 7 g/l agar, adjusted to pH 5.7+0.1, and autoclaved at 121 ºC for 20 minutes. Cytokinins and auxins were applied to the explants at various concentrations with Murashige and Skoog (MS) and Driver and Kuniyuki Walnut (DKW) media.

For example, to generate callus tissue in Indian Lilac (syren), explants from the tip buds were inoculated into DKW medium containing different concentrations of BAP (6-benzylaminopurine) (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) and IBA (indolylbutyric acid) at 0.01 mg/l:

1) DKW + BAP 0.5 mg/l  + IBA 0.01 mg/l

2) DKW + BAP 1.0 mg/l  + IBA 0.01 mg/l

3) DKW + BAP 1.5 mg/l  + IBA 0.01 mg/l

4) DKW + BAP 2.0 mg/l  + IBA 0.01 mg/l

5) DKW + BAP 2.5 mg/l  + IBA 0.01 mg/l

6) DKW + BAP 3.0 mg/l  + IBA 0.01 mg/l

J. Driver's methodological manual on "Artificial (in vitro) cultivation of tissues and cells in laboratory conditions" (J. Driver 2015) was used to conduct laboratory experiments.

Young seedlings with fully developed roots were removed from the test tube for acclimatization to the soil, i.e., for adaptation of young plants to the soil, and the roots were cleaned of MS and DKW nutrient medium residues. Then the seedlings were planted in a mixture containing coco peat, vermiculite, and perlite in different proportions. The planted plants were placed upside down in closed light-permeable containers and placed in a storage room for 7-10 days. The room temperature and relative humidity were constantly monitored.

Results and discussions

Sterilization of tissues before in vitro culture of explants is the first step crucial for microclonal propagation (Lambardi and Rugini, 2003).

When introducing the apical meristems of plants into the culture, sterilization was carried out and the culture medium was prepared at half the strength of the nutrient medium and the pH was set to 5.45. When we sterilized the explants (50 pieces) in 1% sodium hypochlorite for 10 minutes, the number of surviving shoots was 25 and 50%, while 34 survived for 20 minutes, which was 68%.

When we sterilized 50 explants in 2% sodium hypochlorite for 10 minutes, the number of surviving shoots was 35, an indicator of 70%, while for 20 minutes, 42 shoots survived, an indicator of 84%, showing the highest and most effective result in our experiment.

When we increased the sodium hypochlorite to 3%, 4%, and 5%, the percentage of surviving plants was observed to decrease. When we sterilized 50 explants in 5% sodium hypochlorite for 10 minutes, we found that 20 pieces, that is 40% of our shoots survived, while for 20 minutes sterilization 17 shoots survived, which was the most ineffective result of our experiments, with a rate of 34%.

Table 1.

In vitro external disinfection of ornamental plants (Indian Lilac (syren) and Magnolia Grandiflora)

We can see that our results were ineffective when we sterilized the explants in the control option in distilled water for 20 minutes.

The effects of BAP and IBA phytoregulators on cells and tissues of Indian Lilac (syren) and Magnolia Grandiflora plants in Murashige and Skoog (MS) and Driver and Kuniyuki Walnut (DKW) nutrient media were studied under in vitro condition.

During the propagation process, when the pH of MS and DKW nutrient media was adjusted to 5-6 and phytoregulators were not applied, no bud formation and shooting were observed (Figures 1-2).

Table 2.

The effect of phytohormones on the development of callus tissue under in vitro conditions in ornamental plants (Indian Lilac (syren) and Magnolia Grandiflora)

It was noted that the results of the exposure of MS and DKW media to phytohormones for in vitro microclonal propagation of Indian Lilac (syren) and Magnolia Grandiflora varied.

When MS and DKW nutrient media were treated with BAP 0.5 mg/l and IBA 0.01 mg/l, we can see that the results were as follows: bud swelling was observed in Indian Lilac (syren) at 12 days and in Magnolia Grandiflora at 10 days, bud development stopped at 15 days in Indian Lilac (syren) and 17 days in Magnolia Grandiflora, and the length of the branched plants from the development of the buds was observed to be 2.3 cm in Indian Lilac (syren) and 2.6 cm in Magnolia Grandiflora.

When we exposed MS and DKW nutrient media to BAP 2.0 mg/l and IBA 0.01 mg/l, we can see that the results were as follows: bud swelling was observed in Indian Lilac (syren) at 7 days and in Magnolia Grandiflora at 8 days, bud development stopped at 21 days in Indian Lilac (syren) and in Magnolia Grandiflora plants at 25 days, branched buds’ number was more than 4 pieces in Indian Lilac (syren) and Magnolia Grandiflora, and the length of branched plants was 4.3 cm in Indian Lilac (syren) and 4.1 cm in Magnolia Grandiflora. The plant development, reproduction and callus parts were observed to be in a normal state (Figure 3).

As we increased the concentration of BAP and IBA in MS and DKW nutrient media, negative changes were observed in various parts of the plant. For example, premature senescence was observed in callus parts.

Table 1.

Effect of phytohormones on in vitro development of buds and shoots from callus tissues of ornamental plants (Indian Lilac (syren) and Magnolia Grandiflora) (cm)

Even when we did not expose MS and DKW nutrient media to small amounts of BAP and IBA, no callus formation was observed in the plant, and growth and development did not proceed normally.

When MS and DKW nutrient media were treated with BAP 1.0 mg/l and IBA 0.01 mg/l, we can see that the results in the explants were as follows: buds and shoots formed from callus tissues were fully formed within 22-26 days, and the length of the formed plants was 2.1 cm in Indian Lilac (syren) and 2.5 cm in Magnolia Grandiflora.

When MS and DKW nutrient media were supplemented with BAP 2.0 mg/l and IBA 0.02 mg/l, we can see that the results in the explants were as follows, that is, the buds and shoots formed from the callus tissues were fully formed within 18-21 days, and the length of the formed plants was 4.8 cm in Indian Lilac (syren) and 4.5 cm in Magnolia Grandiflora, manifesting the best positive result for an ornamental plant.

As we increased the concentration of BAP and IBA in MS and DKW media, we observed negative changes in various parts of Indian Lilac (syren) and Magnolia Grandiflora plants. For example, yellowing and shedding of the leaves were observed and branches stopped growing (chart 1).

When MS and DKW nutrient media were exposed to IBA and BAP phytohormones, different root development was observed in plants. IBA hormone was exposed at concentrations ranging from 0.5 mg/l to 3.0 mg/l, and BAP hormone was exposed at concentrations ranging from 0.01 mg/l to 0.05 mg/l. The highest result in the experiment was when MS and DKW nutrient media were treated with 3.0 mg/l IBA and 0.01 mg/l BAP, the number of plant roots was more than 4 and the length of the roots was 4.7 cm in Indian Lilac (syren) and 4.4 cm in Magnolia Grandiflora, and this method was found to be an effective method for rooting ornamental plants (Figure 4).

In order to acclimatize young seedlings with fully developed roots to the soil, i.e., to adapt them to the soil, the seedlings, which were removed from the test tube and whose roots were cleaned of MS and DKW nutrient medium residues, were planted in a mixture containing coconut peat, vermiculite, and perlite in different proportions. The survival rate of plants planted in a mixture of coco peat, vermiculite, and perlite in a ratio of 5:3:2 was 62%. When vermiculite or perlite was not added to coco peat, that is, when the ratio was 9:0:1 or 9:1:0, the survival rate of plants decreased relatively, that is, it was 65-68%.

The highest survival rate of ornamental plants (Indian Lilac (syren) and Magnolia Grandiflora) during the acclimatization process was observed in a mixture of coco peat, vermiculite and perlite in a ratio of 7:2:1, i.e. 78% of the surviving plants. The planted plants were placed upside down in closed containers with light transmission and placed in a storage room for 7-10 days. After ten days, the planted plants were gradually adapted to the external environment. The room temperature and relative humidity were constantly monitored (Figure 5-6).

Conclusion

In conclusion, it should be emphasized that when we carried out external sterilization in Sodium Hypochloride (2%) for 20 minutes during the in vitro microclonal propagation of ornamental plants (Indian Lilac (syren) and Magnolia Grandiflora) into culture, the number of surviving plants showed 84% and was found to be the most effective in our experiments.

In microclonal propagation, when we exposed MS and DKW media to 2.0 mg/l BAP and 0.01 mg/l IBA to form callus tissues in Indian Lilac (syren) and Magnolia Grandiflora plants, callus tissue development was observed in 7 days in Indian Lilac (syren) and 8 days in Magnolia Grandiflora, bud development stopped in 21 days in Indian Lilac (syren) and 25 days in Magnolia Grandiflora, branched buds was found to be more than 4 pieces in Indian Lilac (syren) and Magnolia Grandiflora, and the length of branched plants was 4.3 cm in Indian Lilac (syren) and 4.1 cm in Magnolia Grandiflora. It was also observed that the plant development, reproduction and callus parts were in a normal state.

When we exposed MS and DKW nutrient media to BAP 2.0 mg/l and IBA 0.02 mg/l, we can see that the results in the explants were as follows, that is, the buds and branches formed from the callus tissues were fully formed within 18-21 days, and the length of the formed plants was 4.8 cm in Indian Lilac (syren) and 4.5 cm in Magnolia Grandiflora, and the best positive result was recorded for an ornamental plant.

When MS and DKW nutrient media were treated  with 3.0 mg/l IBA and 0.01 mg/l BAP, the number of plant roots exceeded 4 pieces and the length of the roots was 4.7 cm in Indian Lilac (syren) and 4.4 cm in Magnolia Grandiflora, and it was found to be an effective method for rooting ornamental plants.

For acclimatization of young seedlings with fully developed roots, that is to adapt them to the soil they were removed from the test tube and the roots were cleaned of residues of MS and DKW nutrient media, and when they were transplanted  into a mixture of coco peat, vermiculite, and perlite in a ratio of 7:2:1, the highest survival rate 84% was noted.

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