Tissue culture of rose and medicinal plants

پر افتخارترين فعالیت مركز بیوتكنولوژي دكتر رستگار، تكثیر به شیوه كشت بافت و طراحي اولین باغ مادري گل محمدي ايران است. خاستگاه و موطن اصلي اين گل كه از نظر اقتصادي و دارويي در جھان به خصوص مغرب زمین جايگاھي ويژه دارد، كوه ھاي جنوب غرب ايران است. اين گل حوالي سال ھاي 1254-1276 میلادي از ايران به دمشق و سپس از آنجا به اروپا برده شده است. گفتني ست گل محمدي كه در دنیا با نام علمي( Rosa Damascen Miller) شناخته مي شود القابي چون سلطان گل ھا( ( King of Flowers) و ملكه گل ھا (Queen of Flowers) دارد. محصولات اولیه گل محمدي عبارتند از: اسانس اول ، اسانس دوم ، اسانس سوم ، گلاب اول ، گلاب دوم ، كانكريت رز (Rose Concrete)اسانس مطلق رز( (Absolute Rose Oil)و اسانس حاصل از استخراج با سیال فوق بحراني (Fluid Extraction Supercritical) از اين میان با ارزش ترين محصول گل محمدي كه به آن لقب طلاي مايع ( (Liquid Gold)داده اند اسانس تام است. از محصولات اولیه در صنایع دارویی، آرایشی و بهداشتی مورد استفاده قرار می‌گیرد.

Production stages of tissue cultured roses in Dr. Rostegar's biotechnology

1- Mohammadi flowers are propagated in the laboratory.
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2- Mohammadi flowers are transferred from the laboratory to acclimatization and are ready to go to the next stage with favorable roots.
3- The bases are transferred from the cultivation tray to the potting bag.
4- The longitudinal growth of the bases and the number of leaves of the center is constantly monitored.
5- The roots grow to several times the length of the branch
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6- The bases are ready to be transferred and the pride and joy of the center's colleagues will take them to Golestan.

 ایجاد گلستان ھای گل محمدی در ایران تا پیش از تأسیس مرکز کشت بافت در کشور از طریق قلمه و پاجوش است که از معایب آنھا می توان به انتقال آلودگی، عدم موزونی و تناسب یکسان، ظرفیت پائین تولید و عدم کنترل تولید اسانس اشاره کرد. این در حالی است که پایه ھای کشت بافتی گل محمدی تولیدی مرکز کشت بافت بیوتکنولوژی دکتر رستگار، دارای ویژگی ھای ذیل ھستند:

1- Production of bases in the tissue culture laboratory
2- Resistant to water shortage and rainfed cultivation
3- Sanitized bases
4- Bases suitable for different climates
5- Subtraction of edible and non-edible roses
6- High production capacity on a large scale
7- The sameness of the base in the created gardens
8- Increasing the power of production management
9- Having a special government subsidy

Introducing the rose flower

There are about 200 species of plants in the Golsarkhian family, of which a limited number have volatile and aromatic substances and are used in essential oils. Among these few essential oils, rose is known as the main product, whose essential oil is used in health, pharmaceutical and food industries (Rusanov et al., 2009). Nowadays, the use of rose essential oil in industry Pharmaceuticals has increased significantly and is increasing day by day. Rosemary cultivation and the production of its essential oil in the three countries of Iran, Turkey and Bulgaria are considered as a strategic product and are of great importance. According to Thomas' studies and co-workers (2000) every year about 7-10% increase in the amount of production of this product. Rose is adapted to a wide range of climatic conditions and is classified as a low-expectation plant. However, very basic information about cultivation, physiology , ecology and biology of this plant are available. In this article, some of the studies done on this plant are mentioned.

Botanical features

Rosa damascena Mill is a branch of flowering plants, sub-branch of plants, dicotyledons, sub-order Rosidea, order Rosales, family Rosaceae, sub-family Rosoideae. Roses are classified as old and non-climbing roses. This plant is in the form of a deciduous shrub, about one to two meters high, with leaves composed of seven to nine serrated and leathery leaves and stems with many branches and thorns, which are brown in color. Or it is red and their tips are straight or bent downwards. The flowers are pink in color with 32 similar pink petals, 5 sepals, at least three of which have large ridges on the edges. There are 100 yellow flags. The surface of the leaves is smooth and green, but the back of the leaves is pale green, the tails of the leaves have a cover of small spines and felt. In some varieties of roses, the stem and leaves are also fragrant. In Iran, only the Kashan rose has leaves with a mild fragrance. The inflorescence of this plant is dihemous with 3-9 flowers and sometimes more.

Goldhi

Rose flowers are formed on annual branches and begin to open in the early morning. This plant has a short flowering period and usually blooms once a year, although some varieties always bloom. At the beginning of the growing season, new leaves appear on the plant for up to six weeks, then vegetative growth continues in the sixth week. Then, in the sixth week from the beginning of growth, flower buds appear on the stems grown in the new season. In other words, it starts 10 to 12 weeks after the beginning of the growing season. The fruit, which is called hip, remains in the form of cups at the end of the branch after the petals fall and changes color when it reaches red color (Pal and Singh, 2013). After the flowering period, the vegetative growth of the plant continues to produce stems for the next year.

Chromosomal characteristics

Roses generally have a ploidy level of 2n to 8n, and most wild roses are diploid (2n=2x=14), while most commercial rose varieties are tetraploid (2n=4x=28). Other ploidy levels such as pentaploid and hexaploid have also been found in rose. However, it has been said about the rose flower that the number of basic chromosomes is 7 (2n=4x=28) (Rout et al., 1999).

The origin, history and distribution of the rose flower

The genus Rosa belongs to the rose family and the Rosoideae subfamily with more than 200 species and 18,000 numbers and is one of the most famous ornamental plants (Gudin, 2000). Most of the rose species are in the form of shrubs and have permanent and deciduous leaves and are usually scattered in the temperate regions of the northern hemisphere. One of the oldest plants of the rose family is the rose flower, which has been used for many years. Initially, the rose was wild and still grows as a car in the Caucasus, Syria, Morocco and Spain (Beales et al., 1998; Rusanov et al., 2005). It seems that the rose is a kind of hybrid obtained from the species R. gallica و R. canina be But in a study conducted in Japan in 2000, three species R. moschata و R. gallica و .R fedschenkoanaThey were known as the parents of Gul Mohammadi (Gault and Synge, 1971). 

Although the origin of this plant has not been clearly clarified yet, most researchers believe that its origin is Bulgaria, Iran, Turkey and India. In general, it seems that the rose, like other rose species, was taken from the Middle East to European countries (Saakov and Rieksta., 1973). Considering the same issue, since the rose was first introduced to European countries from Damascus, it Of course, one should always pay attention to the fact that having the suffix of a region in botanical names does not mean the place of interest of the desired plant, and sometimes it indicates the first identification region where the plant was collected by the botanist. They do the same as the peach tree with a scientific name Rosa persica It is known, but its origin is China. In the researches that investigated the genetic diversity of the flower, it has been proven that the flower had an initial center of origin (most likely Iran) from where it spread to other places (Turkey and Bulgaria). has been transferred.  

The history of Mohammadi flowers in Iran and the world

The origin of the rose has been proven since 30 million years ago according to fossils, also the records show that in China this flower was used for decoration around 600 BC. Rose petals were used as food for centuries. The use of fragrant roses in Iran dates back to more than 2500 years ago. History shows that fragrant roses were used in Sassanid gardens, and for the treatment of diseases, the petals of these flowers were placed in fats and their essence was extracted and used. Rose has a special place in Iranian literature. In the book Bundeshen (Zoroastrian encyclopedia), Rose and Nestern are mentioned with respect. Bo Ali Sina, an Iranian scientist in the 4th century AH, extracted rose water from this plant and used it medicinally. In the Middle Ages, the extract obtained from this plant was used to treat depression. Rose water has been extracted in Iran for centuries, and in the 14th century, it spread from Iran to parts of the Ottoman Empire and Asia Minor. According to historians, rose water is one of Iran's commercial items that have been exported to China and India. Ibn Khaldun mentions in his Kitab al-Abra that the government of Iraq at that time received items as tribute from different cities of Iran, and one of these items given by the city of Shiraz was 30,000 bottles of rose water (Figure 1). According to these historical documents, it can be said that since the beginning of the 10th century, there have been industries related to the production of roses in Iran, especially in Shiraz. Regarding the history of the production of rose oil (essential oil), Iran is known as the first country to produce rose oil (Chevallear, 1996; Beales, 1998; Rusanove et al., 2005). Rosemary oil in Iran dates back to the 7th century AD.

Figure 1- Ibn Khaldun (1332-01406) mentions 30,000 bottles of rose water from Persia as a tribute in her book Al-Abra.

Cultivation of roses

Choosing the right place to grow roses

Above sea level

Most of the flower farms in Iran are located at 1900 meters above sea level or higher. Among the best essential oils produced from the rose, we can mention the essential oil produced in the Lalezar area of Kerman, which is located at an altitude of 3000 meters above sea level. However, it is possible to grow mohammady at lower altitudes, provided that other environmental conditions such as temperature, light intensity, relative humidity, etc. are also available. In general, the suitable climate for the cultivation of mohammadani is a mild climate with moderate weather.

temperature

Almost most of the physiological processes of plants are affected by the ambient temperature and this factor determines the length of the growing season in different geographical areas. All plants thrive in a temperature range between 5 and 40 degrees. In fact, this temperature is known as the critical temperature and the perfect temperature is considered between these two temperatures. 

Air temperature is considered as one of the main environmental factors in the growth and development of plants of the Rosace family, such as roses, and it is very important in the cultivation of these plants. The suitable temperature for the cultivation of this plant is 2 to 30 degrees during the day and 18 to 20 degrees Celsius during the night (Kim and Lieth, 2003). In the rose flower, if the night temperature during the time when the plant enters the flowering phase reaches less than 10 to 12 degrees Celsius, the production of essential oil will decrease significantly; But low temperature during vegetative growth and before flowering will increase the quality and quantity of essential oil produced. However, the suitable temperature range for growing most plants of the rose family is 19 to 27 degrees. According to Weiss (1977), the air temperature between 5 and 15 degrees during the growing season and before flowering of the rose plant will produce larger flowers with more essential oil. In addition, this researcher states that during the flowering period of the flower, a temperature of less than 10 to 12 degrees prevents flowering, while a temperature of 20 degrees increases the production of the essential oil of this flower. Flowering in this plant is directly dependent on the production of lateral buds that later turn into flowers. Temperature is also effective on the production rate of these buds and their transformation into reproductive buds. The amount of temperature during the period of production of lateral buds has a direct effect on the subsequent growth of these buds (Marcelis-van Acker, 1995). When the temperature goes above 24 degrees, the length of the stem and the number of produced flowers will be significantly reduced. (De Vries et al. 1982). At low temperature, the amount of available carbohydrates increases due to the decrease in the growth rate of the plant. Oshio and colleagues (2008) observed that the photosynthetic capacity and the amount of production of different tissues in plants grown at a temperature of 20 degrees during the day and 15 degrees at night increases significantly. In the years with a warmer spring, flowering occurs earlier and in a cool spring, flowering is delayed. In cool weather conditions, as mentioned, the performance and quality of the produced essential oil will be much higher. 

Light intensity and day length

The length of the day is a function of the latitude and position of the sun. In many plants, the length of day and night has a direct effect on vital activities (Waldren, 2003). Although in the case of roses, day length has no effect on flowering time, but on activities that are directly related to plant performance, such as the number of buds. The side effects are the speed and rate of flower abortion, the formation of new branches, the speed of growth and the like. Several reports have investigated the effect of day length on the flowering of roses. In these studies, the mutual effect of temperature and light on the flowering of the rose flower has been investigated. For example, low light intensity at low temperature and relative humidity of 88% has increased the yield of flowers, increasing the quality and quantity of monoterpene compounds in the essential oil of the rose (Misra et al., 2002). High light intensity and low temperature have increased stored carbohydrates. in the leaves (Dieleman and Meinen, 2007) and can reduce the photosynthetic capacity of the plant due to negative feedback. On the other hand, the intensity of low light has also caused a decrease in the intensity of the color of the petals in other species of the rose family. The quality of light has also been studied and it has been determined that it has a direct effect on the increase of productive branches. A high ratio of red to infrared light accelerates the production of new buds, while a low ratio prevents their production (Zieslin and Mor, 1990).

Relative humidity of the air

Relative humidity is one of the important factors that has a direct effect on the quality and quantity of the production of the rose flower. According to the experiments, the most suitable relative humidity during the vegetative growth of the plant is 70% (Arkley, 1963) and 60% when the flowers begin to bloom (Weiss, 1997). 

Soil conditions and its effect on plant growth

Plant growth is a function of the physical and chemical conditions of the soil and choosing the right soil guarantees the best performance. The most suitable soil for the cultivation of roses is deep loamy soil with a pH between 6 and 7.5. Acidic soils cause a decrease in growth and as a result, a decrease in the yield of the rose. On the other hand, in 2003, Brichet reported that rosehips are generally sensitive to calcareous soil conditions and should not be cultivated in such soils. It should be avoided to grow marigolds in calcareous soil, especially when the water conditions are alkaline (Weiss 1997). Rosemary does not like waterlogged conditions and should not be cultivated in clay soil, but in areas with very low rainfall, sandy soil is also unsuitable and the appropriate combination of soil that has the ability to retain water should be used. In heavy soils, root diseases will develop and the problem of food shortage will occur (Karlik et al., 2003). One of the most important issues in the discussion of soil quality can be mentioned salinity. Although there have not been many studies on the resistance of the rose to soil salinity, but it seems that this plant can tolerate salinity up to five decisiemens/meter. Nikbakht and Kafi, quoted by (1382 Dozadeh Emami), in addition to the mentioned items, the amount of heavy elements in the soil, such as lead, cadmium, and the like, is of special importance. For example, by adding phosphorus fertilizers, some cadmium is added to the soil unintentionally and as an impurity in these fertilizers. Here, it is important to mention that heavy metals have not been found in the produced essential oil, but these elements reduce plant growth. On the other hand, if the goal is to use the flower and leaves of the rose flower as food, they should not be cultivated in this kind of soil. 

Fertilizer

The evolution of the flowers of the rose and obtaining a high-quality product depends on the balanced nutrition of the plant. The rose plant requires complete nutrition of all nutrients in order to produce a suitable product. In Bulgaria, 64 kg of nitrogen, 8.7 kg of phosphorus and 36 kg of potassium are consumed annually in each hectare of Golestan, and under these nutritional conditions, the stored amount of these elements in the tested flowers are 0.34, 0.3, and 1, respectively. The percentage was expressed (Koseva et al. 1987). The amount of flower yield and production essential oil in the rose flower is directly related to the levels of these three elements in the leaves and production buds during the development period of the buds (Orlova, 1984). The amount of these elements from the bud development stage to the flowering stage is 67 decreases by 35 and 25% (Weiss, 1997). The appropriate amount of nitrate nitrogen, ammonium nitrogen, phosphorus and potassium in the fields of production of roses is 180-20, 35-180 zero, 50-5 and 300-50 mg/kg expressed by (Karlik et al., 2003) In order to produce a suitable crop, especially in calcareous soils, 200 kg of nitrogen is added in two installments in each hectare, with an interval of 15 to 20 days (Srivastava, 1975). Complete fertilizer containing 32, 18 and 16% nitrogen, phosphorus and potassium is mixed with the soil of the pit, and until the third year, 160-200 kg of nitrogen, 60-90 kg of phosphorus and 40-60 kg of potassium per hectare are added to the soil. But the important point is that all these elements must be mixed with the soil in parts and during several times. In a research conducted in the Faculty of Agriculture of Theran University, the appropriate amount of nitrogen consumption per hectare was stated as 30 to 60 kg per hectare. According to these researchers, these amounts can be different depending on the goal of producing roses, so that if the goal is to produce bigger flowers, 60 kg per hectare of nitrogen can be used, but if the goal is to produce rose water, it can be from lower amounts to 30 kg per hectare. Hectare used. In this research, the recommended amount of nitrogen is 30 kg per hectare in the form of ammonium nitrate and 30 to 60 kg of potassium in the form of potassium sulfate (Daneshkhah et al., 2016). In any case, special attention should be paid to the negative effect of increasing soil potassium on the absorption of other elements. In general, the amount of potassium and phosphorus used in the fields of mohammady is 20 to 50 kg and 50 to 90 kg, respectively. Along with high-use elements such as nitrogen, potassium and phosphorus, low-use elements should also be considered because these elements also play a very vital role in various plant processes. The appropriate amount of these elements in the soil of the rose fields are: boron 0.5 to 5, iron 0.3 to 3, manganese 2 to 3, zinc 0.3 to 3, copper 0.001 to 5, and molybdenum 0.01 to 1 mg/kg. Karlik et al. 2003). The proper use of low consumption elements has in many cases increased the resistance of the rose to adverse environmental conditions. Although the information mentioned above can be fruitful to a great extent, before any proposal to recommend fertilizing programs for mohammadi fields, tests for the decomposition of soil elements and plant organs should be carried out, and based on that, recommendations for fertilizing farms should be made. can be done  

Manure

Perhaps there are few cases in improving the physical and microbial conditions of the soil as vital as the use of animal manures, these manures increase the water retention capacity, balance the pH of the soil, prevent its excessive compaction, and increase the ability to retain nutrients and provide these The elements are available to the roots of the plants. Usually, in the traditional cultivation of mohammadani in Iran, about 10 tons of manure are mixed with the soil of the mohammadani field in the month of March. In the country of India, it is recommended that in the first year, about 18 to 20 tons of animal manure was mixed with the farm soil in the fields where animal manure was used and in the fields where this fertilizer was not used, a significant difference was observed, and in the fields where every year from 20 Tons of manure was used, the rate of flower production was much higher (Mousavi and Faizi, 2010). Here, it should be noted that the manure used must be completely rotten and free of any insect eggs and weed seeds, otherwise the problem of fighting weeds will arise. 

Watering systems

Rosemary is known as one of the plants resistant to water shortage. In traditional crops, the interval between two irrigations varies from 7 to 20 days. Experts recommend watering every 12 to 15 days in the first two years of plant establishment, and after that in the third year, the number of irrigations can be reduced. The first irrigation starts from March and continues until mid-autumn. The most sensitive stage of the mohammedy against water shortage is during their flowering period, during which the irrigation period should be reduced and minimum water stress should occur in this stage. Water stress in this stage will reduce the size of the produced flowers and the quality of the product will decrease. In a research conducted in Kashan, the most appropriate number of irrigations was found to be 5 times. Of course, in this research, the minimum number of irrigations needed to achieve acceptable quality was considered (Mousavi and Faizi, 2018). In any case, it is necessary to try as much as possible to reduce water stress as much as possible on the one hand, and on the other hand, to reduce water wastage as much as possible. In a research that was conducted to compare the efficiency of drip irrigation and flood irrigation in Kashan, the plants that were irrigated with drip irrigation system had higher production efficiency in addition to less water consumption. In this experiment, the amount of freshly harvested flowers in the drip irrigation system is about 1290 kg per hectare and the yield of the plants irrigated with the flood system is about 780 kg, and the amount of essential oil obtained in the flood irrigation method is 29 grams per 100 mg of rose water and 64.6 mg drops per 100 grams of rose water were obtained in plants under irrigation (Faizi et al., 2010). When choosing a place to grow roses, you should be careful to choose a place with a minimum rainfall of 350 mm per year. Of course, if the mohammady flower farm is water-based, he can also cultivate mohamady flowers in other areas that have less rainfall. More care should be taken in selecting suitable areas for fields that are dry and irrigation is not carried out. In such cases, the amount of rainfall should not be less than 350 mm per year.

Soil preparation and plant cultivation

First, in the fall, the land is deeply plowed and about 8 to 10 tons of rotted manure is added to the soil. The plants can be cultivated in two seasons. The first and most important planting time is in winter and after the end of the winter cold in March. It can also be planted in autumn, and in this way, the cultivated plants grow one season ahead. There are two methods of growing mohammedy flowers: atmospheric, piled, and piled, depending on the equipment used, each of these methods can be used. The most suitable distance of the ship that can be seen in most sources is reported as three-row distance (the distance between the plants is three meters and between the rows is four meters). Choosing the right distance is directly related to the type and severity of pruning, so that in India, a distance of 1.5 x 1.5 is used, but the bushes are pruned every year. Dense cultivation causes problems such as the difficulty of passing and passing, reducing the light of the plant and shading them on each other. The size of the cultivation pits has a direct dependence on the type of soil, so that in deep and fertile loamy soils, pits of 5 x 5 can be used, while in areas where the soil is of lower quality, the depth and size of the pit is 60 x 60 and more. It is taken so that in the early years when the shrub produces roots, it allows the plant to increase the volume of the roots and the plant is well established. During cultivation, the amount of 3 to 4 kilos of rotten manure is mixed with 20 to 25 grams of complete manure and the plant is cultivated in this pit. Of course, in areas with high soil alkalinity, a special group of sulfur-decomposing bacteria or phosphorus-releasing bacteria that are available in the market can be used to balance the pH and absorb phosphorus. Before planting the seedlings, in order to reduce the damage of harmful soil organisms, they should be treated with fungicides such as Captan, and then they should be cultivated, and finally, the surface soil of the pit is poured on the roots and irrigation is done. 

Pruning of roses

Rosemary is one of the shrub species of roses, in which the flowers are formed on the branches formed in the current season. Unfortunately, there is a common misconception among the farmers and growers of mohammadii that mohammadii does not need much pruning and only infected or diseased branches of this plant should be removed. If proper pruning in this plant guarantees a good and high quality product. In the plants that are pruned properly and annually, light penetration is more, and because of this, the photosynthesis level of these plants increases and finally a higher quality product is produced. In addition, in such fields, there is also the possibility of easy passage, in the case that in fields where pruning is not done, in addition to reducing the quality of the product, due to the presence of many weeds and the branches collapsing, it becomes more difficult to harvest the product and traffic in these fields. Farms become very difficult. In the case of roses, it is said that any factor that can increase the production of new branches can increase the production of the product. In this plant, if the main branches are cut, due to the predominance of the end of the bud, the end of the stem of the lateral buds will start growing and as a result, more buds will be produced. The time of pruning is different in different climates, but it is generally done after removing the danger of winter cold and before the buds wake up. Based on the research, it is recommended to remove 15 cm from the top branches every year. In this way, a large number of buds are stimulated in all the branches that have been pruned and these buds produce new branches. The results of the conducted tests indicate that the use of this pruning has increased the performance and quality of the essential oil produced. In the flower gardens of Iran, no pruning is done, and only once every 5 to 6 years, the plants are pruned. By doing this, the plant will be removed from the production cycle in the next two to three years. Considering the above-mentioned contents and the negative economic results of not using the proper techniques of pruning of mohammadi flowers, it becomes necessary to familiarize the farmers with this pruning method and train them. For the training of rose seedlings, it is also possible to lift the newly planted plant in the first year. In the second year, two prunings are done, one in the middle of summer and the other before the arrival of winter cold. And in the following years, the plants are removed from the height of one meter every year. In an experiment that was conducted to investigate the effect of the head height and its relationship with the quality of rose essential oil production, it was found that among the three head heights of 90, 60, and 120 cm, the best results were shown by the plants removed at a height of 90 cm. In this plant, all the quality indicators measured were higher than the other two heights and it was reported as the best head height (Pal et al., 2014).

Harvesting, oiling and drying the petals and after harvesting them

From the third year after the planting of the seedling, the harvesting of the rose starts and continues until the ninth year, depending on the density of planting and the way the plant is kept. However, in the traditional farming method that is popular in Iran, the harvest continues until the sixth year, and due to the reduction in the size and quality of the produced product, the plants are removed. The harvesting time of the rose flower varies depending on the cultivated genotype and different climates; But in general, it can be said that it starts in the beginning of May and lasts until the end of June. Harvesting is done by hand, and perhaps the most expensive part of the cultivation of roses is harvesting. The average production in Iran is less than two tons per hectare, while the average production in India is reported to be 3.5 to 4.5 tons.

Effective ingredients in rose essential oil

Rose essential oil contains a set of compounds (95 compounds) that have been found in 94.75% of the extracted essential oils and according to Jalili Haravi et al. Oxides, ethers, aldehydes, geranial, hydrocarbons, eicosan 30%, docosan 14%,  1- Monodecane 7% (flowers obtained in Iran) (Jalali-Heravi et al 2008).

 Essential oil monoterpene alcohols include: citronellol 24 to 43%, geraniol 2 to 18%%، and cis isomer of nerol in the amount of 7 to 18(flowers obtained from Turkey) (Bayrak&Akgu˝l 1994).

 Essential alcohols include: citronellol and Nazul 24%، Geraniol 21% و  لینالول 21برآورد شده است(گل ھای به دست آمده از ھند)(Babu et al 2002 ).

According to the research of Babu and colleagues (2002), the yield of essential oil has a lot to do with its extraction conditions. According to him, the essential oils obtained in natural atmospheric pressure are of higher quality and in addition, the amount of alcohol in rose water is higher than oil essential oil.

The most important compounds that produce rose flower perfume include beta-damasenone, bentaionone and rose oxide. Regardless of the very low amount of two compounds, beta-damasenone and bentaionone, these compounds are one of the key factors in determining the quality of the essential oil produced. 

Based on these results, it is suggested to use concrete semi-solid extract instead of essential oil. In the experiment conducted, the amount of active ingredients in concrete is as follows: Citronellol -4.7%, Phenylethanol 25%%، ان-نونادکان 13%،، ھنیکوسانین 12و ان تریکوسانین 7%به ثبت رسیده است (Reverchon et al .1997).

 Other substances in essential oil include phenolic substances such as gallic acid, flavonoids such as quercetin glycoside (rutin, quercetin, myristin, kaempferol, Kumar et al 2008) and anthocyanins such as cyanidin and 5,3-diglucoside (Velioglu & Mazza 1991). . 

In a research conducted at Suleiman Demirel University in 2002, the effect of harvesting time, the duration of the fermentation stage and the use of towin 20 as a wetting agent on the quality of the flower product were investigated. The flowers were picked at 8 to 10 in the morning, the time of harvesting the flowers was chosen on May 24, June 1, 8 and 15. In order to determine the effect of the harvesting time, the flowers were subjected to chemical analysis immediately after harvesting (experiment one), in the second experiment, in order to investigate the duration of fermentation on the production of essential oil and its constituents, the flowers harvested on May 24 under the treatment of zero, 24, and 12 36 hours of fermentation were placed. For fermentation, the flowers were placed in closed containers at a temperature of 25 degrees. The effect of different concentrations of zero, 2500, 1000 and towin 20 50000 on the quality of essential oil production was investigated. Five important compounds of this essential oil, i.e. citronellol, geraniol, nerol, linalool and phenylethyl alcohol were analyzed by gas chromatography. The results showed that the highest amount of essential oil obtained in the flowers harvested on May 24 was 0.04%. In this experiment, it was found that the amount of essential oil production decreases with increasing temperature in subsequent harvests. With the distance from the harvest time on May 24, the amount of the mentioned 3 substances, except for citronellol, decreased, but the amount of citronellol increased exponentially. The amount of linalool also remained unchanged. The quality index of rose essential oil, i.e., the amount of citronellol to geraniol, increased with increasing time from harvesting on May 24. The best recorded rate is 1/30 to 1/25. In this experiment, the highest rate of this ratio was obtained in the flowers harvested on June 8. In this experiment, the amount of essential oil of flowers that were extracted fresh without fermentation was higher. As the fermentation time increased, the amount of essential oil decreased. 0.027, 0.030, 0.035 and 0.025 at zero, 24, 12 and 36 hours. The fermentation process was also effective in the amount of active substances and the highest change was observed in the case of citronellol and geraniol. As the fermentation time increases, the amount of citronellol increases and the amount of geraniol decreases.

 In the third experiment, the effect of adding Tween 20 was significant and the highest amount of essential oil obtained in the treatment of 2500 mg/liter of this substance was observed. The main reason for this is the effect of this substance in reducing the surface tension of the oils in trichomes. In this test, Tween 20 did not have any side effects on the effective ingredients of the essential oil, and it can be used to increase the essential oil. Finally, it was found that giving short-term fermentation to reach the desired concentration of citronellol 35It is necessary and without doing it, the amount of this article will not be acceptable. 

Another experiment was conducted to investigate the effect of harvesting time during the day and harvesting at different stages of petal development, essential oil quality and production. First, the process of flower development was divided into eight developmental stages (Figures 1 to 8). The essential oil was analyzed by gas chromatography method. The data obtained from gas chromatography showed that the harvesting stage had a noticeable effect on the amount and quality of the essential oil produced, as can be seen in the figure below, the amount of two important substances, citronellol and geraniol, was very low in harvesting the flowers of stage 3, but in Flowers harvested in stage 5, the production rate of these two substances increases significantly (Figure a-1). In the same way, the effect of harvesting time during the day is also significant on the quality of the produced essential oil, and as the harvesting time increases from 10 am to 4 pm, the amount of production of the two mentioned substances decreases rapidly (Figure 1b).

Figure-1: The effect of harvesting time in different stages of development (stage 3 and 5 of development) respectively: citronellol, geraniol, phenylethyl alcohol, henicosanin, nonadecane and heptadecane.
Figure b-1: Effect of harvesting time at different hours of a day. Active ingredients 5, 4, 3, 2, 1 and 6 respectively: citronellol, geraniol, phenylethyl alcohol, henicosanin, nonadecane and heptadecane.
Figure 2: Dividing developmental stages into 8 stages

The obtained results showed that the time of harvesting and the development stage of the flowers had a direct effect on the number of known compounds, so that the highest number of known substances was obtained in the 6th, 5th and 7th stages of development.

Figure: 3 number of compounds known by gas chromatography in a) different stages of flower harvesting and b) different harvesting hours during the day

According to the obtained results, it can be said that for the production of quality essential oil (high level of eugenol), the flowers should be collected in the 5th stage of development and between 8 and 10 in the morning. If the petals are harvested for use as dried flowers, it should be noted that the drying method used will have a direct effect on the quality of the dried petals. According to the available reports, among the three methods of drying in the sun, drying in the shade, drying in the oven, the method of drying the petals in the shade has shown more acceptable quality (Ahmadi et al., 2007).

 In a study, the effect of low-temperature storage of mohammedy flowers was investigated. In this experiment, the harvested flowers were stored at 0 degrees Celsius with four storage methods in plastic bags and elastic bands, Xtend commercial bags, smart bags and polyethylene bags for 60 days. During the storage period, the changes of oxygen and carbon dioxide in the bundles, the weight reduction of the flowers, the color change of the petals and the properties of the essential oil were measured. In this experiment, it was found that the type of storage of flowers has a direct effect on their weight reduction. Among the 4 tested methods, storage in polyethylene bags showed the least weight reduction (1.69%). And the highest observed weight reduction was also observed in Xtend bags by 10.08%. The amount of weight reduction observed in four storage methods was recorded as 0.80, 91.3, 57.7 and 64.3% respectively for storage in plastic bags and elastic bands, Xtend commercial bag, smart bags and polyethylene bags. The effect of this test on the compounds in the essential oil was also significant, as the amount of the most compound in the essential oil, i.e. citronellol, increased compared to the control after 10 days in all storage methods used, while the amount of nerol and Geraniol was reduced compared to the control, and the amount of nonadecane, henicocyanin and eicosan was also higher. In general, in this experiment, it was found that the loss of essential oil and the weight of flowers can be reduced to a large extent by these methods, and in areas where rose-making factories are not available, or to existing factories with a huge volume of flowers harvested in During a short period of time, these storage methods can be used. 

Internal contamination control in tissue culture

After micro-cultivation of plant samples in glass and artificial culture medium due to the availability of nutrients for the growth of microorganisms, these micro-organisms grow rapidly and pollute the culture medium. Internal contaminations, which mainly include bacteria and yeasts, show themselves after a few days of cultivation. The source of internal contamination is from the mother plant material itself and rarely comes from external conditions and cultivation containers. Internal pollution control is usually difficult because the source of pollution is the explant itself, and these pollutions are transferred with the plant and are also with the plant with successive replantings. Different methods are used to control bacteria in tissue culture, which include the use of antibiotics, nano compounds, mercury chloride, and successive recultures. Each of the mentioned methods are briefly described below.

Use of antibiotics

  • Antibiotics are substances that are produced by some microorganisms and can disrupt the growth of other microorganisms and destroy them. Today, artificial substances that are made by humans in this direction are also called antibiotics. Different types of antibiotics are used in plant tissue culture.

1- Penicillins, these types of antibiotics prevent the production of the bacterial cell wall. Penicillins include: amoxicillin sodium, ampicillin sodium, carbenicillium disodium and cephalosporins, etc. 

2- Antibiotics that have an effect on the membrane of bacteria and cause the substances inside them to seep out. Among these types of bacteria, colistin sulfate, polymyxin sulfate, nystatin, etc. can be mentioned.

 3- Antibiotics that prevent protein synthesis in bacteria and ultimately cause their death, such as aminoglycoside antibiotics, among the most widely used antibiotics in tissue culture is carbencillin, which is more effective on the growth of gram-negative bacteria. Is.

 Cefotaxime, which dissolves easily in water and is used at the rate of 300 to 500 mg/liter. 

Nystatin is an antifungal antibiotic that must first be dissolved in dimethyl sulfoxide, which is used at the rate of 500 mg/liter. 

Gentamicin, which is a common antibiotic in tissue culture, which is of great importance for the following reasons, can remain stable in a wide range of pH. It is heat resistant and autoclavable. It can control many bacteria and does not cause toxicity to cell cultures.

 Adding antibiotics to the culture medium also causes problems, such as that if less than the appropriate concentration is used, it does not have much effect, and if more is added, it causes toxicity to the explants. It also prevents the absorption of nutrients by the plant. Antibiotics cannot be used to control surface contamination. Because they cannot control all pathogens by themselves, and usually, in this method, the possibility of controlling contamination is low. But if the explants are washed with antibiotics after disinfection with other sterilizing agents, they can control bacterial contamination to some extent.

 

Use of nano compounds

In general, the results of using nano silver in the culture medium show that this compound plays an important role in reducing bacterial contamination. Immersion of fine pine samples in a concentration of 200 mg/liter of nanosilver has a great role in reducing bacterial contamination, but it has no effect on fungal contamination. Adding nanosilver at the rate of 4 mg/liter to the olive cultivation medium caused severe control of internal pollution and did not have any negative effect on the micro-samples (Rostami and Shahsavar., 2009). The composition of nano silver was used to reduce internal pollution and increase the rate of regeneration in beetroot in vitro cultivation (Ruzbeh et al., 2009). The use of silver silicate nanoparticles plays a very important role in controlling the internal pollution of plants (Park et al., 2006). In micro tissue culture of hybrid rose samples, nano silver was added to the culture medium and the explants were washed with nano silver. The concentration of 100 mg/L nanosilver which was added to the culture medium showed the best result for controlling bacteria and the concentration of 200 mg/L was the best concentration for washing the explants. The use of higher concentrations causes a decrease in the growth of new shoots, and also nano silver does not play a role in controlling fungal contamination (Shokri et al, 2013). On average, the concentration of 50 mg/liter had the greatest effect on the control of bacteria in the cultivation of mohammady. The results showed that the response to different concentrations of nano silver depends on the genotype. 

Pollution control mechanism by nano silver

One of the disinfection mechanisms of nano silver is the catalytic mechanism and production of active oxygen. Nano silver acts as a chemical cell and oxidizing oxygen atom produces -O2 and hydrolyzing water produces -OH, both of which are the most important antimicrobial agents. In the ionic mechanism, the Ag + ion is gradually separated from the nano silver and causes the conversion of the SH bond to the Sag bond in the body wall of microorganisms, which causes the destruction of pathogenic agents. Also, by oxidizing microorganisms, disrupting the respiration of microbes and breaking sulfur molecules in microbes, nano silver particles cause the oxidation and destruction of microbes and viruses.

Mercuric chloride

Mercury chloride has been used by many researchers to disinfect micro samples. Although it is less used in research than sodium hypochlorite, it has more disinfecting effects. Concentrations of 0.1% and 0.2% of mercury chloride were used for 3 and 4 minutes to control bacteria in the culture of the explants. Mercuric chloride has been able to effectively control bacterial contamination, although at a higher concentration and time, it caused the final burn of micro-samples and buds, which caused a delay in bud opening and absorption. Many researchers have used 0.1 mercury chloride for 5 to 7 minutes to disinfect the surface of micro-samples (Rout et al., 1989, Skirvin et al., 1990). In a study conducted in 2013, 0.1% mercury chloride was used for 5 minutes to disinfect small samples of R. damascena (Alsemaan, 2013). In a study that was conducted on the in vitro culture of three genotypes of Golmohamadi, 1% mercuric acid was used for 5 minutes to sterilize the micro-samples (Assare et al., 2016). In order to disinfect small samples of Kazanlik genotype, 1% mercuric acid was used for 5 minutes (Kornova et al., 2000).

در پژوھشی بهمنظور تولید کالوس از برگھای R. damascena از 0.04 درصد کلرید جیوه جھت ضدعفونی سطحی ریز نمونه ھا استفاده شد. بررسی ھا نشان می دھد که در بیشتر پژوھش ھا از غلظت 1/0درصد کلرید جیوه استفاده شده است که می تواند به علت جلوگیری از سوختگی ریز نمونه باشد.  

Consecutive returns

Bacterial contamination can be controlled to some extent by successive and rapid recultivations. After growing the explants in the culture medium, immediately after the appearance of bacterial symptoms, we take out the explants and cut off the end of the explant and transfer it to a new culture medium. We continue this process for several steps until the symptoms subside. Although many methods have been introduced to control bacterial contamination, the best way to prevent contamination is the mother plant. That is, by controlling the conditions, you can have healthy plants that can use healthy explants without the need for treatments to control contamination. The use of 125 to 250 micrograms/liter of oil extracted from the yew tree in tissue culture controlled the contamination of Xanthomonas bacteria to a large extent. Also, the use of single-bud explants in rose tissue culture shows less bacterial contamination compared to those with multiple buds. Although the multi-bud explants had a higher enrichment rate. In an experiment, the effect of 28 known antibiotics (table below) on reducing the growth of gram-negative and gram-positive bacteria in plant tissue culture was investigated. These antibiotics were used separately and in combination, and finally it was found that the combination of imipenem and ampicillin, as well as imipenem and penicillin G at a ratio of 5.5 mg per liter, had the greatest effect in reducing the level of contamination. In this experiment, there was no side effect in reducing plant growth. According to these researchers, these antibiotics can be used commercially to prevent problems related to antibiotics.

References

  • Ahmadi, K; F. White and M.H. extract The effect of different drying methods on the quality and quantity of essential oil of three genotypes of mohammady. Iranian Medicinal and Aromatic Plant Research 176: (2) 1387.24.
  •  Daneshkhah, M.W. Kafi and A. good luck Different levels of nitrogen and potassium on flower performance indicators and essential oil of the rose flower in Kashan. Horticultural Sciences and Techniques of Iran 90-3(2) 1386.8. 
  • Tabai Eghdai, Farhangian, Jafari, Rezaei. Studying the variation in the morphological traits of Golmohamadi genotypes collected from the six central provinces of the country. Iranian Medicinal and Aromatic Plant Research Quarterly. Volume 247.233.2.21 
  • Mousavi, A. and M. Faizi. The effect of organic and chemical nitrogen fertilizers and the number of irrigations on the quantitative and qualitative performance of Mohammedia flowers. Research report of Isfahan Agricultural Research Center 
  • Nikbakht, A. and M. Kafi, Golmohammadi, Iran
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