T. C.
SELÇUK ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ
PROPAGATION OF ARONIA (Aronia
melanocarpa) WITH TISSUE CULTURE
Huda Mohammed Mansoor ALMOKAR
MASTER OF SCIENCE Horticulture Department
April-2019 KONYA All Rights Reserved
iv ABSTRACT
MS/Ph.D THESIS
PROPAGATION OF ARONIA (Aronia melanocarpa) WITH TISSUE
CULTURE
Huda Mohammed Mansoor ALMOKAR
THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCE OF SELÇUK UNIVERSITY
THE DEGREE OF MASTER OF SCIENCE OF SCIENCE Advisor: Prof. Dr. Lütfi PIRLAK
2019, 71 page Jury
Prof. Dr. Lütfi PIRLAK Prof. Dr. Ahmet EŞİTKEN Prof. Dr. Halil Ibrahim OĞUZ
Aronia (Aronia melanocarpa), a small fruit in the same time considered ornamentally valuable, could be propagate by seeds but this process is not recommended. The most easily method to propagate in the in vitro by using micro propagation technology, where considered one of the most common methods to propagate by using vegetative propagation method especially Viking cultivar. In this study used buds semi-hardwood from aronia plant the explant length 1cm and, each of explant contains one lateral bud figure 2 (explant –a). Based in this study to reproduce aronia in in vitro by tissue culture technology and by using micro propagation was obtained on suitable culture medium, where the highest shoot length (14.60 mm) achieved at MS basal medium which combination of different concentrations of growth regulators (1.0 mg l-1 BA +0.02 mg l-1 IAA 0.1 mg l-1 GA3) at treatment 8.
The highest shoot number (64 unit) were obtained at MS basal medium containing a combination of growth regulators at different concentrations (2.0 mg l-1 BA +0.01 mg l-1 IAA+0.1 mg l-1 GA3) at
treatment 6. The highest callus rate achieved at treatments (3, 4, 10) respectively, all the procedures in in vitro was obtained within 4 weeks, and so this include rooting process where the highest root number (9.5 unit) at MS control at 0.0 mg l-1 concentration of IBA. The most suitable MS basal medium for root
length containing 1.0 mg l-1 concentration of IBA in length (18 mm) and the highest plant length (33
mm) at 2.0 mg l-1 concentration of IBA. Concerninig results of infection, two subcultures considered best
subculture compared to other subcultures from where number of death plants and infected plants particularly free of bacterial infections, where the percentage of healthy plants (99%).
v ÖZET YÜKSEK LİSANS
ARONİA’NIN (Aronia melanocarpa) DOKU KÜLTÜRÜ YÖNTEMİYLE ÜRETİMİ
Huda Mohammed Mansoor ALMOKAR Selçuk Üniversitesi Fen Bilimleri Enstitüsü Ziraat Fakültesi Bahçe Bitkileri Anabilim Dalı
Danışman: Prof. Dr. Lütfi PIRLAK
2019, 71 Sayfa Jüri
Prof. Dr. Lütfi PIRLAK Prof. Dr. Ahmet EŞİTKEN Prof. Dr. Halil Ibrahim OĞUZ
Aronia (Aronia melanocarpa), dekoratif olarak değerli olduğu düşünülen küçük bir meyvedir. Aronia bitkisi tohumla çoğaltılabilir ancak fidan üretiminde bu yöntemtavsiye edilmez. Kültür çeşitlerinde genellikle vegatatif üretim metotları kullanılır. Bu çalışmada aronia bitkisinin doku kültür yöntemi ile üretimi araştırılmıştır.Farklı büyüme düzenleyici konsantrasyonlarının kombinasyonu olan MS ortamında en yüksek sürgün uzunluğu (14.60 mm) uygulama 8’de sağlanmıştır. En yüksek sürgün sayısı (64 birim), muamele 6'da farklı konsantrasyonlarda (2,0 mg 1 -1 BA + 0.01 mg 1 -1 IAA + 0.1 mg 1 GA3) bir büyüme düzenleyici kombinasyonu içeren MS bazal ortamında elde edilmiştir. Kontrol MS ortamında IBA’nın 0.0 mg l-1 konsantrasyonunda en yüksek kök sayısının (9,5 ünite) elde edilmesi dahil in vitro’daki bütün prosedürler 4 haftada sağlanmış ve en yüksek kallus oranı sırasıyla 3. 4. ve 5. uygulamalardan elde edilmiştir. Kök uzunluğu için en uygun MS bazal ortamı, 1,0 mg l-1 IBA’nın konsantrasyonunda (18 mm) elde edilirken en yüksek bitki uzunluğu (33 mm) ise IBA’nın 2,0 mg l-1 konsantrasyonundan sağlanmıştır. Enfeksiyon sonuçlarıyla ilgili olarak, bakteriyel enfeksiyonlardan ari olarak sağlıklı bitki oranının %99 olduğu iki alt kültür diğer alt kültürlerle karşılaştırıldığında en iyi alt kültürler olarak tespit edilmiştir.
Anahtar Kelimeler: Aronia melanocarpa, mikro üretim, Viking, Bitki büyüme düzenleyicileri.
vi
PREFACE
I would like to acknowledge Turkey first and YTB second that they give me scholarship for three years as well as they accepted my re-search which was sup-ported by them. I would like acknowledge webmaster Prof. Dr. Lütfi PIRLAK particularly which supported me morally during my research and I acknowledge Dr. Muzaffer İPEK and Dr. Osman DOĞAN generally where they supported me in write research. Thanks to the Yalova Atatürk Central Horticultural Research Institute for the supply of plant materials.
Huda Mohammed Mansoor ALMOKAR KONYA-2019
vii CONTENTS
ABSTRACT ... iv
CONTENTS ... vii
SYMBOLS AND ABBREVIATIONS ... ix
1. INTRODUCTION ... 1
1.1. Aronia description ... 3
1.2. Studies on micropropagation of Aronia ... 5
2. LITERATURE SEARCH ... 7
2.1. What is Aronia medical benefits ... 8
2.1.1. Anthocyanin ... 9
2.2. Chemical compound in different cultivars of Aronia ... 11
2.3. Tissue culture ... 13
2.4. Micropropagation ... 15
2.5. The most important plant hormones and her importance for growth and develops plants ... 17
2.5.1. Plant growth regulators (PGRs) ... 17
2.6. Synthesize and Transport of plant hormones ... 25
2.6.1. ABA ... 25
2.7. Translocation ... 26
2.7.1. Gibberellins ... 26
2.7.2. Auxin ... 27
2.7.3. Cytokinin ... 28
2.8. How to work hormones in the plant ... 29
2.8.1. Auxins ... 30
2.8.2. Cytokinins ... 31
2.8.3. Gibberellins ... 31
2.8.4. Ethylene ... 31
2.8.5. Abscisic Acid ... 32
2.8.6. Other Plant Hormones ... 32
2.9. Effect of plant hormones on bacteria human gut and human health generally .... 33
2.10. The importance of rhizosphere microbiome in plant health ... 33
2.11. The occurrence and possible functions for Melatonin in higher plants ... 34
2.12. Possible melatonin functions in higher plants, as well as regulation of circadian rhythms and photoperiodic reactions ... 34
2.13. The functions specific for the melatonin to higher plants ... 36
3. MATERIAL and METHODS ... 37
3.1. Plant material ... 37
3.2. Tissue culture conditions and media ... 38
viii
4. RESULTS AND DISCUSSION ... 41
4.1. Results ... 41
4.1.1. Shoot proliferation ... 41
4.1.2. Rooting ... 45
Figure 4. Effect of IBA on the rooting of Aronia melanocarpa ... 48
4.1.3. Sterilization ... 48 4.2. Discussion ... 52 4.2.1. Sterilization ... 52 4.2.2. Proliferation ... 53 4.2.3. Rooting ... 54 5.1. Conclusion ... 56 5.2. Suggestions ... 57 REFERENCES ... 58 RESUME ... 71
ix SYMBOLS AND ABBREVIATIONS
Symbols % : percent
μm : Micrometre
mg l−1 : Milligram Per Litre Ng/g : Nanogram/Gram Abbreviations
MS : Murashige and Skoog PGRs : Plant Growth Regulators IAA : Indole-3-Acetic Acid IBA : Indole-3-Butyric Acid BA : 6-Benzyladenine ABA : Abscisic Acid TDZ : Thidiazuron
NAA : Naphthaleneacetic Acid NOA : 2-Nophthoxyacrtic Acid
ACC : 1-Aminocyclopropane-1-Carboxylic Acid CKs : Cytokinins
NPA : Naphthylphthalamic Acid
2,4,5D : 2, 4, 5-Trichlorophenoxyacetic Acid P-CPA : 4-Chlorophenoxyacetic Acid
2, 4-D : 4-Chlorophenoxyacetic Acid Ca : Calcium
2 ip : 2-Isopentenyl
BAP : 6-Benzylaminopurine DMSO : Dimethylsulfoxide
BZR1 : Brassinosteroid Signaling Positive Regulator TIR2 : Transport Inhibitor Response2
UV : Ultraviolet
UTIs : Urinary Tract Infections HDL : High-Density Lipoprotein CSCs : Cancer Stem Cells
HPLC : High-Performance Liquid Chromatography CA : Coal
IPT : Iso-Pentenyl-Transferasem SA : Salicylic Acid
FW : Fresh weight HCL : Hydrochloric U.S : United States
AC : Activated Charcoalhas
1. INTRODUCTION
Among the rest of berry fruits, aronia (Aronia melanocarpa) is one of the most important berry fruits in terms of containing the highest percentage of antioxidants.
Aronia melanocarpa is a Rosacea family species and has two types commonly of native
North American shrubs: Aronia arbutifolia (L.) which is (red chokeberry) and Aronia
melanocarpa (Michx.) Ell. which is (black chokeberry) (Kokotkiewicz et al., 2010).
There are another species of aronia called Aronia prunifolia (Purple chokeberry) contained the highest amount of total anthocyanins, phenolics, and proanthocyanidins (Bräunlich, 2014). It is original home North America. Aronia melanocarpa classified as ornamental's shrub as well as decorative autumn coloration that making very popular (Hirvi and Honkanen, 1985). The use of tissue culture for production of commercial plants especially for plants that has economically importance is due to modern and sophisticated developments of this field in biotechnology. Consider new methods to produce uniform planting material of high quality and diseases free plants. Biotechnology tools such as micropropagation and in vitro culture provide a valuable alternative in the fight against viruses and management of genetic resources. The micropropagation method is the most recent and widely used in the spread of commercial plants for horticulture and forests (Isac et al., 2010). Aronia berry classified between most fruit in terms of antioxidants, where the antioxidants percentage in Aronia is higher than that is in apple, banana, and others. The high levels of flavonoids and anthocyanin in Aronia is higher than those found in cranberries and five times more. In addition to their containment on an antioxidants and polyphenols and they also contain on mineral and vitamins. As well as the chemicals content in Aronia it has been alleged that it reduce some disease such as the potential cancer and heart diseases. To supply a natural red color in products with poor color stability, Aronia juice has been increasingly used in the food industry. Commercially, Aronia is fundamentally used for juice either alone or blended with other fruit juices such as apple or grape. Mainly Aronia juice in commercially, used either alone or blended with other juices such as apple and grape. Food coloring, tea, syrup and fruit spread coloring all these uses includes other uses for Aronia. The uses of Aronia juice have different from zone in Europe to other. In Russia apple and Aronia juices are combined and fermented to producing or giving red wine. Either in Europe the juice often blended with apple juice to give juice a color. In Lithuania, they have been using Aronia juice alone or blended
with other fruits so as to produce dessert wines. Reports from the Ukraine describe aronia as improving the color, tannin level and sugar of grape wines. There is a small commercial grower in Iowa and another in Oregon, but the main commercial source for the juice comes from fruit grown in Europe, but there is a small commercial grower in Iowa and another in Oregon. Test plantings have been established by the USDA Plant Materials Program at 11 sites in North Dakota, South Dakota and Minnesota (Smith and Ringenberg, 2003). For aronia propagation methods if there are one or more type from aronia plants, can propagate those plants by vegetative methods. The most common species that reproduce by vegetative reproduction are Viking and Nero. This includes propagation by micropropagation, and semi-hardwood and hardwood stem cuttings, layering. As long as aronia produce a high percent of apomictic seed, its cultivars can also be grown from seed, they will come true from seed. Most cultivars are also nearly seedless, so it is difficult to get many seed (Anonymous, 2018a). For aronia benefits prevent urinary tract infection and weight control, there are others benefits relate in aronia fruit such as treat inflammations, hypertension as well as can be very beneficial in cases of arthritis, cardiovascular conditions and other diseases. Aronia also contributes to strengthening immunity, blood vessels, lower blood pressure levels, and aronia also delays the natural aging process (Anonymous, 2018b). Therapeutically they show positive effect in the anti-inflammatory and anti-oxidative activity and, also in the treatment several of neoplasms (Kowalczyk et al., 2003).
The purpose of the research was to obtain homogeneous plants from aronia as well as high rate of shoot production and obtain on plant free of fungal and bacterial infections. The search aim is to find an efficient way to multiply the aronia in large scale numbers for expanding the areas cultivation in order to meet our local needs. In addition, the search also aims to determine the best media culture for large scale numbers at low cost. During the 90th, the main target the expanded researches were to obtain plants free of bacterial infections and prevent the infection of viruses. That research included many types of fruit, especially small fruits such as an aronia and strawberries, which can be produced in a laboratory in micropropagation method. Achievements and trends in the use of tissue culture for the mass micropropagation represent most often a rapid economical and safe technique for multiplication of valuable varieties of trees and shrubs (Isac et al., 2010). In order to obtain search results in vitro micropropagation process was analyzed the influence of the culture medium
composition on the number of shoots regenerated and their lengths in successive (Şuţan et al., 2017).
1.1. Aronia description
Aronia is a new variety and usually produce as an outdoor landscape plant. Aronia plant has ability to adapting a variety of landscape uses which including mass planting or hedging. The purpose of the breeding program was to produce new Aronia cultivar like ‘Viking ' but in shorter and narrower shape (Brand and Connolly, 2018). The Aronia shrubs grow to the height of 2 meters almost. Usually shrubs produce umbels containing many small white flowers which are 30 flowers during May and June months and after that this flowers ripen to black berries covered by a waxy coating (Oszmiański and Wojdylo, 2005). Descriptions of Aronia different according cultivars and Aronia have many cultivars that are distinguished by their colors and sizes, the cultivars are Viking, Nero, Hugin, Arbutifolia, Purnifolia, Galicjanka, and Fertödi. For arbutifolia the leaves of this cultivar are characterized in their large orbicular shape, where greater than 7 cm long and 5 cm wide, as well as the diameter of fruits larger than 10 mm, the fruits maturing in late July through September, the fruit colors usually purple to purple black (Connolly, 2014). In addition to the fruits of Aronia arbutifolia taller compared to black chokeberry where reaching almost 5 m as well as narrow elliptical/lance late to narrowly ovate or obovate leaves. Aronia arbutifolia fruits be smaller compared to Aronia melanocarpa fruit, the fruits are ripen from October until November, whereas into the winter the bright red pomes persisting (Gleason and Cronquist, 1991; Dirr, 1998; Rossell and Kesgen, 2003). According to historical sources
Aronia melanocarpa cv.‘Nero’ or cv ‘Viking’ appear to be identical, where these two
cultivar derived from an interspecific hybrid of aronia (Anderson, 1980). Work has been done on improved black chokeberry Aronia mitschurinii or ‘Viking’ type cultivars. After that limited work evaluation has been conducted and the antioxidant capacity of wild Aronia. In the state of Illinois using local plants of two populations the medicinal properties of non-cultivated Aronia has been studied (Sueiro et al., 2006). Chokecherries are similar to plums and cherries therefore they are stone fruit. Whereas aronia are related to Saskatoon’s, apple, and pears they are therefore considered pome fruit and this similarity produced from old German word for aronia translates to “apple berries”. The leaves are glossy with dark green color in the fall turns to the bright red
where, the leaf shape is elliptical and simple. After the leaves emerge the flowers are small forming pretty clusters. Usually aronia plants be tall shrubs selected for purpose fruit production. From base of the plant the branches are sprout. As well as some spreading branches producing from the plants, these branches have ability to live over 20 years approximately. Most time aronia plant selected to two purposes that are ornamental plant and fruit quality particularly. All ornamental types contain on edible fruits some fruit be small also yield are lower compared to fruiting cultivars. In the nursery trade "black chockberry" sometimes called Aronia berry, and also known as
Photinia melanocarpa (Rosendahl, 1955). The most commercial two cultivars from
aronia cultivars growing by growers are Viking’ and ‘Nero’ but Viking produce higher yields and taller compared to Nero cultivars, but Nero probably better than Viking for growers that they must harvest by hand. The growers noted a few or no different between two cultivars that relating in fruit qualities. In U.S both of black and red aronia were planted especially to be as ornamental shrubs. Whereas, Europeans developed each of Viking and Nero as large fruited for fruit production. There are some evidence reported that the varieties Viking is an hybrid uncompounded with mountain ash (Sorbus aucuparia), which indicate explain the Viking cultivars has high sorbitol contents and an large fruit (Anonymous, 2018c). Where the resulting new plant is similar to the mother plant (Brand, 2010). Aronia can grow in soil which pH should be between 5.5 and 7.0 where Aronia thrive in acidic soil as well as wet area. Also can grow in both soil clay and sandy as well as in most cases can grow in very poorly soil free from fertilizer or need little fertilizer. There are some cases Aronia grew in dry soil but the result was no recommended off because of low nitrogen in that sandy soil of Minnesota. The plant must grow in soil which contain high nitrogen fertilizer in order to plants grow more than a foot each year. Can addition one cup of granular fertilizer like calcium nitrate, onto the ground by conventional producers. In order to keep Aronia healthy and productive there must be proper pruning as is essential in addition to facilitate picking .The purpose of pruning Aronia is to remove older branches and keep new fruiting branches sprouting to growing for next year’s (Jensen, 2014).
1.2. Studies on micropropagation of Aronia
The micropropagation method is the most recent and widely used in the spread of commercial plants for horticulture and forests (Isac et al., 2010). However, there are several studies about aronia growth with tissue culture by using micropropagation technology. One of these studies about studies of the in vitro micropropagation ability of Aronia melanocarpa where was used during this study two cultivars from aronia ‘Melrom’ and ‘Nero’, the result was chokeberry cultivar ‘Nero’ have higher ability of regeneration as well as the greatest number of shoots had obtained with the basal medium containing MS, LF microelements and LF vitamins, supplemented with 4,5 mg×dm-3 BA and 0,6 mg×dm-3 IBA. Compared with ‘Melrom’ (Şuţan et al., 2017). There are another study about, micropropagation of mature Crataegus aronia L., a medicinal and ornamental plant with rootstock potential for pome fruit ,showed that the cytokinin was only among plant growth regulators that promoted both of shoot elongation and shoot proliferation, where at 5.0 and 7.5 µM BA, were obtained higher shoot numbers compared with lower concentrations of BA (Nas et al., 2012). There are several searches about ornamental plants that near from Aronia melanocarpa such as ace (Mrytus communis L.). Where there is a study on effect of plant growth regulators on in vitro micropropagation of ace (Mrytus communis L.). Where, this study proved that the suitable medium for micro propagation was MS medium with a concentration of BA 4.44 μm and IBA at a concentration of 1.47 μm and GA3 with a concentration of 0.58 μm. After that were obtained on the vegetative result, the average number of vegetative growth was 12.8 (MS) plus and the rooting rate was 95% where IBA at concentration of 4.9 μm. Then the seeded growths were transferred to a pot containing perlitus and bitmus by 2: 1 in order to quantify and quantify it during 5 weeks followed their growth naturally. The plants then finally transferred to the field. This technique allows the production of homogenous and bacteria free plants on a large scale. Ace (Mrytus communis L.) can be multiplied in laboratory by using tissue culture technology. To preserve this important local origin in terms of environmental and medical reasons, we recommend the laboratory multiplication method while using the MS medium supplemented with the concentration of BA μM 4.44 and GA concentration of 0.58 μM. For rooting, we recommend to use the IBA supplementation medium with a concentration of 4.90 μM. It should also be noted that this study should be followed for vascular and fertile vascular plants that are difficult to propagate in
traditional ways (Anonymous, 2018d-a). As well as there are several search about aronia one from them about bioactive compounds in hyperhydric and normal micropropagated shoots of Aronia melanocarpa, in this search were used different concentrations of plant hormones, where used thidiazuron (TDZ) and (NAA) in certain proportions for the purpose of proliferation and rooting, for shoot multiplication Aronia
melanocarpa were cultured on Murashige and Skoog (MS) medium supplemented with
0.25, 0.5, 1.0 or 2.0 mg l−1 thidiazuron (TDZ) alone or in combination with 0.1 or 0.5 mg l−1 _ naphthaleneacetic acid (NAA) multiple shoot formation was observed and among the different concentrations of TDZ studied, the maximum frequency of shoot multiplication (85.2%), mean number of (10.4) shoots per explant and mean shoot length (3.2 cm) were obtained on MS medium fortified with 0.5 mg l−1 TDZ. The culture media was containing low concentration of NAA with TDZ, where shoot produce was more than using high concentration of NAA but when using low concentration of TDZ (1.0–2.0 mg l−1) decreased the number of shoots per explant and the mean shoot length (Sivanesan et al., 2016).
2. LITERATURE SEARCH
Aronia is known as the black chokeberry. Aronia berry is among the other small fruit as well as ornamental shrub which its original area is North America. Now days however it is well known in Eastern Europe. The fruits are richest source of antioxidant phytonutrients among the fruits as well as vegetables. Aronia could be propagating by seeds but this process is not recommended. Although Aronia berry can be propagated by cutting methods, it is recommended to use the in vitro methods of propagation due to the short duration in vitro propagation (Brand and Cullina, 1992). In Asian and European countries the cultivation of berries is for both fruits production as well as for ornamental plants industry. Aronia can be easily propagated with using seeds, but while using this way of vegetation the fruits berries production is late and therefore not recommended to propagate them in this method (Litwińczuk, 2004). There are genus Crataegus species close Aronia berry which belong to the Rosacea family that known as hawthorn. This species has many important functions including small fruits trees, medicinal plants as well as horticulture shrubs (Phipps et al., 2003). Breaking of seed dormancy, germination and seedling emergence of the common hawthorn have also been studied (Bujarska-Borkowska, 2002). In Turkey, wild-grown hawthorn plants are often top worked with pears and less often with apples (Nas et al., 2012). In regard with growth regulators, a study showed the effect of abscisic acid acclimation Aronia outside the laboratory. However, the results showed that the treatment with abscisic acid did not provide any physiological advantage on the Aronia plant, which would facilitate the adaptation outside the laboratory (Colón-Guasp et al., 1996). Using of 6-benzyladenine alone or in combination with auxin affected the regeneration of shoot (Huetteman and Preece, 1993). BA (benzyladenine) can be used as a cytokinin, because cytokinin is more effective than thidiazuron and metatopolin while using hawthorn micro propagation (Nas et al., 2012). The use of tissue culture for production of commercial plants especially for plants that has economically importance is due to modern and sophisticated developments of this field in biotechnology. Consider new methods to produce uniform planting material of high quality and diseases–free plants. Biotechnology tools such as micro propagation and in vitro culture provide a valuable alternative in the fight against viruses and management of genetic resources. The micro propagation method is the most recent and widely used in the spread of commercial plants for horticulture and forests (Isac et al., 2010). While conducted the 2017 study on
the Aronia plant in vitro, by using micropropagation technology, two varieties of Aronia plant ‘Nero’ and ‘Melrom’ have been used. Increased concentrations of auxin and cytokinin led to high rate of shoot multiplication in ‘Melrom’ cultivar, and in a significantly higher rate in cultivar ‘Nero’ (Şuţan et al., 2017). However, the great difference in the shoot proliferation ability of the two studied Aronia cultivars can be attributed to the influence of genotype (Litwińczuk, 2013). Active buds produced mostly callus instead of shoots (Pruski, 1987). Nowadays in the food industry is popular industrial dyes are the most widely used. As these industrial dyes can cause the development of several diseases that including the heart or cancer disease, one of the most valuable natural pigments are anthocyanins which are found in Aronia in abundant quantities. The investigations have been presented by Nano filtration on the concentration of anthocyanins. Have been used Na2SO3 – one of the most efficient extractants of anthocyanins that are found in Aronia solutions. Where it proved that sodium sulfate (IV) is not only a good extracting and preserve but also its presence highly increases the effectiveness of the filtration process (Gilewicz-Łukasik et al., 2007). Aronia has unique properties promoting health that is due to the high content of polyphenols and vitamins in its fruit. Chokeberry fruits may get contaminated with mycotoxin-producing fungi, in spite of their high resistance to spoilage; experiments have shown that Penicillium sp. is one of the most common fungi that infect fresh chokeberry (Genchev et al., 2014).
2.1. What is Aronia medical benefits
Prevent urinary tract infection, to contain Aronia on high levels of quinic acid. Antioxidant capacity increase in blood by Aronia helps to protect the body against oxidative stress. Furthermore, red wine and green tea they are among the benefits of Aronia, because both of type containing anthocyanins, catechin and polyphenols, this is due to content of antioxidant sources significantly. Urinary tract health: millions of people suffer from UTIs (urinary tract infections) from various around the world, and Aronia can effective against urinary tract infections because of, Aronia content on high levels quinic acid. Weight control: Aronia controls weight through preventing the body from storing fat around the abdomen zone. That chokeberries offer protection against neurological diseases, cancer, inflammation, bacterial infections, diabetes, and aging. All these evidence by scientific studies have been shown (Anonymous, 2018d-b). There
are others benefits relate in Aronia fruit such as treat inflammations, hypertension as well as can be very beneficial in cases of arthritis, cardiovascular conditions and other diseases. Aronia also contributes to strengthening immunity, blood vessels, lower blood pressure levels, and Aronia also delays the natural aging process (Anonymous, 2018d-c). Vitamins, alkaloids, minerals, essential oils, saponins, and polyphenolic compounds, inter into within active compounds, where medicinal plants are rich sources of active compounds and characterized by numerous beneficial actions in the human body (Vidavalur et al., 2006). Aronia an additive to fruit juices and energy drinks, where aronia uses in the U.S. in primarily used by large food processors. Moreover, aronia's extract increases antioxidant content and uses as a coloring agent. In addition, Aronia using as a food coloring and is added to fruit syrups, jellies, jams, and teas in Europe. In all the U.S. a number of wineries trying to develop dessert wines from Aronia fruit. To add color to the wine, often wine makers mix Aronia with other fruit. Aronia fruit is easy-to-grow and therefore the future of Aronia production largely depends on what uses people will find for this fruit (Jansky and Bell, 1994). After know the health benefits which Aronia fruit characterized where it is impressive, in order to planted Aronia for purpose fruit production, Americans first done that purpose. In the scientific literature some examples about Aronia health benefits is found after consuming high anthocyanin Aronia extracts, patients suffered reduced blood pressure as well as heart attacks (Kulling and Rawel, 2008). Blood pressure infect polish men , after they drank over six weeks 250 milliliters of Aronia juice daily the result was a decrease in systolic and diastolic blood pressure readings of 13 and 7 respectively in addition a significant decrease in HDL cholesterol and tricglycerides along . In animal studies similar results was achieved (Skoczyñska et al., 2007).
Within Aronia using medical there are more important factor inter in this major that is:
2.1.1. Anthocyanin
Anthocyanin effects: Anthocyanins are colored water-soluble found in plants. Anthocyanin's pigments belonging to the phenolic group as well as the pigments are in glycosylated shapes. Anthocyanins responsible for many colors, such as purple, red, and blue, these colors can found in vegetables and fruits (Khoo et al., 2017). Therapeutically they show positive effect in the anti-inflammatory and anti-oxidative activity and, also in the treatment several of neoplasms (Kowalczyk et al., 2003). There are studies about
immunomodulation activity for polyphenols in Aronia fruits suggested that polyphenolic compounds inside of aronia fruit may have beneficial effects as anti-inflammatory and immunomodulators agents (Ho et al., 2014). Aronia juice works to killing teratocarcinomal cancer stem-like cells. AMJ inhibited cell proliferation, publically the results strongly indicate that AMJ is functionally able to counteract the carcinogenesis process through targeting CSCs (cancer stem cells) (Sharif et al., 2013). From main subgroups for flavonoid are anthocyanins in fruits and berries. Phenolic acids present in fruits and berries are considered hydroxylated derivate of cinnamic and benzoic acid (Macheix et al., 1990). Studies suggested that the flavonoids and other phenolics have been played to preventive role in the development of heart and cancer disease (Cao et al., 1998). Black chokeberry considered of species which contains higher anthocyanin significantly, blueberry, cranberry and lingonberry crops contain on phenolic and, also antioxidant activity in that crops (Zheng et al., 2003). And in a study conducted on Aronia melanocarpa phenolics and their antioxidant activity it was shown significantly that the phenolics content in the pomace higher than that which in fruits and juice, whereas in aronia juice the concentration of phenolic acids content was higher than in pomace which indicated it has good solubility in water. Result showed that Aronia melanocarpa products, such as pomace and juice as well as aronia fruit contained antioxidant activity with high level of polyphenols. Aronia melanocarpa fruit might be a potential source of potent natural antioxidants (Oszmiański and Wojdylo, 2005). There are study conducted on red pigment of black chokeberry, where it was shown that red pigment fraction of black chokeberry cultivar in both in vivo and in vitro systems has a potent oxidative activity as well as played a significant role in anti-ulcerative activity on acute gastric hemorrhagic lesions in rats (Matsumoto et al., 2004). The proanthocyanidines and their potent anti-oxidant capacity and possible protective effects on human health make them very important in the field of medicine and nutrition (Santos‐Buelga and Scalbert, 2000). Procyanidins has free radical scavenging properties where that may reduce the risk of cardiovascular diseases (Reed, 2002; Steinberg et al., 2003), cancer disease (Bagchi et al., 2000), and blood clotting (Murphy et al., 2003), as well as appointed types of trimetric PAs may protect against urinary tract infections (Foo et al., 2000).
2.2. Chemical compound in different cultivars of Aronia
The date of harvest for Aronia melanocarpa berries is influence factor on the total content for polyphenols (including anthocyanins, proanthocyanidins, and hydroxycinnamic acids) as well as pigmentation of chokeberry juice and sugars in aronia berries, in addition the antioxidative activity (Bolling et al., 2015). “Aron” cultivar is considered the poorest in polyphenols where per g f.w. containing 15.865 ± 1.237 mg of gallic acid equivalents. The content of total polyphenolic compounds and anthocyanins is an effected at the time and temperature of storage exert and also on the antioxidative properties of freeze‐dried chokeberries. when kept compounds at 23 °C or 40 °C for 6 months The amount of these compounds decreases by 90% and also by as much as about 80% when kept at 3 °C for the same period. Therefore the temperature of storage has a particularly great influence on the content of anthocyanins (Misiak and Irzyniec, 2009). Usually the berries of Aronia stored in the frozen form. As that sterols, tocopherol fraction, and phospholipids it is found in Aronia seed oil in large quantities (Zlatanov, 1999). In young leaves of Aronia polyphenolic compounds also found but in smaller amounts than in the berries such as neochlorogenic acid, caffeic acid, and including chlorogenic acid mainly (Skupień et al., 2008; Do Thi and Hwang, 2014). Chromatographic analyses (HPLC) and GAE showed the ‘Hugin’ cultivar is a rich in polyphenols for chokeberry fruits. Among all polyphenols there was the largest amount of cyanidin3-galactoside, which it constituted 50% of phenolic compounds after was determined. The ‘Hugin’ cultivar juice became darker and has a more intense blue color after it was light-colored as a result of Maceration of fruit pulp although the ‘Hugin’ cultivar was characterized by light-colored fruits. The fruits of the two varieties ‘Nero’ and ‘Viking’ Characterized as darkest and the juice that obtained from them are dark. As well as the ‘Nero’ and ‘Viking’ cultivars have the largest amount of substances coloring fruits, red and blue juices, and pulp. It can be concluded that the chemical composition and morphological features of the fruits and leaves are very similar for both ‘Viking’ and ‘Nero’ cultivars, while The ‘Hugin’ cultivar among the other three cultivars is fundamentally different (Ochmian et al., 2012). Generally chokeberry fruits contain on low level of organic acid from 1 to 1.5% approximately (Lehmann, 1990; Tanaka and Tanaka, 2001; Skupień et al., 2008). The mainly acids that was identified are L-malic and citric acid. Under study the fruit were characterized in low level of organic acid where it was at the level of 0.75 g in 100 grams of in the ‘Galicjanka’
cultivar (Ochmian et al., 2012). Normally Aronia berries, edible as a fresh fruit, and it is more tastier than that processed, where the level of sugar in the fruit is high (12 to 20%soluble solids), have a pH of 3.3 to 3.7, and 0.7 to 1.2% titratable acidity, and anthocyanins (560 to 1050 mg/100 g fresh weight).The fruit is also very suitable in the industry (Oszmianski and Sapis, 1988; Jeppsson and Johansson, 2000). In many rose family fruit species sorbitol is found where considered a natural sweetener for Aronia berry and rose family fruits especially sweet cherries. Often the sorbitol contents of fresh weight are between 2 and 4% in sweet cherries. Absorbed sorbitol is easily as well as a small amount from it is digested, whereas large amount of sorbitol gives people an upset stomach or become as a laxative. Humans metabolize sorbitol not fastly but slowly. Sometimes sorbitol added to sweeten wine. Sorbitol does not ferment with regular sugars, because most yeast strains used in wine making cannot metabolize sorbitol (Kulling and Rawel, 2008). The only Aronia varieties grown on an Industrial scale are Viking or Aronia mitschurinii types. These types have made up nearly in all the materials used in the studies and associated with the health benefits of aronia. In addition there are types of Viking has lowest ORAC (Oxygen Radical Absorbance Capacity) value, and it has the same amount of anthocyanin or less (Brand and Connolly, 2018). All that relates to the contents of total phenol, monomeric anthocyanins, procyanidins, and flavonoids, in addition to with phytochemical composition, the antioxidant activity study was evaluated for two Aronia melanocarpa cultivars "Viking" and "Aron" and of Aronia prunifolia hybrid. Aronia prunifolia hybrid richest in anthocyanins, and procyanidins than the Aronia melanocarpa cultivars (AmA or AmV), as well as have the highest antioxidant activity. Whereas in viking cultivar other flavonoids (quercetin equivalents) has found. In relation to Aronia prunifolia berries antioxidant potentials, their phytochemical content, richest in procyanidins and anthocyanins compared to Aronia melanocarpa cultivars (Rugină et al., 2012). The higher amounts of polyphenols with high percentage proanthocyanidin and anthocyanin can found in Aronia prunifolia berries cultivar. Also the highest amount of total anthocyanins as well as total phenolics have found within chokeberry capsules and powders compared to other products (Tolić et al., 2015). But the phenolic content in chokeberry juices had lower. The high antioxidant capacity, characterized in it chokeberry products and her juices (Shin et al., 2008). In addition chokeberries considered richest source of hydroxycinnamic acids, anthocyanins, and proanthocyanidins. For chokeberry juices and dried chokeberries, total flavonoids
content was higher in both of two cases. Quercetin is the main contributor for total flavonoid content, according to the literature. In many quercetin glycosides (quercetin-3-galactoside, quercetin-3-glucoside and quercetin-3-rutinoside) and in chokeberries, quercetin had been detected (Denev et al., 2012).
2.3. Tissue culture
The key element in plant biotechnology is plant culture, in addition a part from mass multiplication of elites. And also multiply and regenerate novel plants can provides from genetically engineered cell. Tissue culture is widely used in rapid propagation of plants which are difficult to propagate in order to obtaining plants free of bacterial and fungal infection. Through the enhanced multiplication of axillary bud, micro propagation used as main methods in tissue culture generally. By using optimum concentrations of cytokinin and auxin or mix both of them axillary buds can be broken. The axillary buds development into shoots in case dormancy is broken. By using media containing optimum concentrations of plant growth regulators by that, they can be made to multiply very rapidly. The morphology of the tissues that grow from the initial explant is deeply affected in both the particularly composition of the plant growth regulators and the source of the nitrogen (amino acids or nitrates versus ammonium salts). For example, the surplus of auxin will produce roots, while cytokinin is producing yield shoots. Unorganized growth of cells or callus will produce when auxin and cytokinin be balance, whereas the fruit morphology of the outgrowth depends off the composition of the medium and plant species (Sharma et al., 2015). Plant tissue culture helps to understand the requirements of cell, tissue, culture, organ, their development and growth, that requirements are such as chemical and physical. there are new avenues were discovered by the creation of tissue culture of cell, tissue and organ culture and regeneration of plantlets under in vitro conditions, in the area of plant biotechnology (Dagla, 2012). And also organ and protoplast culture as well as aseptic cell and tissue a general term for tissue culture (Libby and Ahuja, 1993). In addition to that, tissue culture term is the growth and maintenance of cell outside the tissue which they are normally a part from higher organisms in vitro. In addition the tissues excised from a plant and the culture of pollen or seeds cultivate in a sterile, synthetic medium (Prüller; Maynard, 1996b). With the discovery and characterization of plant hormones, the development of plant tissue culture was closely linked with this discover as a fundamental science, as well as has facilitated our understanding to growth and
development of plant. Many practical applications in agriculture and horticulture industrial chemistry basically formed through control of growth each of cells and tissues in culture and to ability culturing their and this is considered a prerequisite for plant genetic engineering. In plant tissue culture widely used grow of masses of unorganized cells (callus) on agar or in liquid suspension in biochemical and growth studies (Puhan and Martin, 1971; Street, 1973). To study differentiation, morphogenesis and plant regeneration, must be provide systems by the culture of segments of stems, roots, callus or of leaves (Puhan and Martin, 1971; Vasil and Vasil, 1972). In order to propagate plant and production of virus free stock, shoot apex culture methods leading to plant regeneration have been adopted (Vasil and Vasil, 1972). To haploid plant formation, the culture of anthers and pollen provides new approaches for this purpose (Kasha, 1974). The technique is not without problems, but a vast amount has been recorded of knowledge and experience in numerous publications included a variety of observations on cell nutrition, differentiation, biochemistry, growth, differentiation and physiology differentiation (Steward, 1969; Puhan and Martin, 1971; Dougall, 1972; Koblitz, 1972; Vasil and Vasil, 1972; Murashige, 1974; Hildebrandt, 1981). Successful plant tissue culture depends on the selection of nutrient medium. May be the grown on completely defined media the cells of most plant species. The Murashige Skoog (MS) most used widely; generally MS media consist of a carbon source (generally sucrose), vitamins, and mineral salts as well as plant hormones. (MS) very extensively designed for tobacco cells in addition obvious success for culturing callus on agar through it (Classic Murashige and Skoog, 1962). To improve cell growth and development, (AC) is often used in tissue culture, where activated charcoal works to micropropagation, somatic embryogenesis, orchid seed germination, synthetic seed production, anther culture, protoplast culture, rooting, bulb formation, elongation of stem, thus, AC (activated charcoal) has played important role in this regard (Thomas, 2008). Activated charcoal (AC) they are small, compact particles made up of carbon by removal of all noncarbon impurities and the oxidation of carbon surface thus being distinguished from elementary carbon, as well as it is a porous and tasteless material (Mattson, 1971; Budavari et al., 1996). AC used in to both of liquid and semi-solid media, as well as AC works to adsorption of inhibitory substances in the culture medium and this is due to its influence on development and growth (Horner et al., 1977; Fridborg et al., 1978; Weatherhead et al., 1978; 1979; Theander and Nelson, 1988). There are other effects for AC represented in drastic decrease both of the phenolic oxidation and brown exudate accumulation
(Carlberg et al., 1983; Liu, 1993; Teixeira et al., 1994), change of medium pH to an optimum level for morphogenesis (Owen et al., 1991), in addition hence simulate soil conditions and also establishment of a darkened environment in media culture (Dumas and Monteuuis, 1995). Drastic dip in concentration of PGRs and other organic supplements result of adsorption of these chemicals by AC (Ebert and Taylor, 1990). Growth regulators, especially oxytocin and cytokinin, increase the level of phenolic substances. When removed from the medium or reduced the concentration, plants improve. Activated charcoal in this case reduces the amount of phenolic substances that are secreted into the medium by adsorbing them and also adsorbing some media compounds, especially hormones (Anonymous, 2018e).
2.4. Micropropagation
Micropropagation is an art and science of plant propagation under in vitro conditions and also is a precise and well adapted technique. It has a great profit-making potential, the ability to produce disease-free plants and the high plant quality. considerable progress has been made in the culture of plant tissues and cells in vitro and in the experimental management of higher plant parts and cells as microorganisms although, the micro propagation industry being only 15 to 20 years old (Afreen-Zobayed et al., 2000). And also micropropagation is adventitious means or refers to propagation in culture by axillary. In vitro propagation, micropropagation mean a general term for vegetative (asexual) (Klopfenstein et al., 1997) . In tissue culture, micropropagation considered one of the most popular techniques particularly one hand of vegetative growth (Sharma et al., 2015). Micropropagation means part of plant such as tissue, cell, or small plant parts has been cultivated in a sterile manner in test tube or other container, where can be controlled the environment and nutrition rigidly. Often micropropagation term used as synonymous with tissue culture (Schmidt, 1997). Generally “Micro propagation through tissue culture technique is called clonal propagation”, that is means“through tissue culture, regeneration of whole plant is popularly called micro propagation”. In a short time and space micropropagation can be achieved. Thus, from a single individual it is possible to produce plants in large numbers. Use of tissue culture for microprogation was initiated by (Morel, 1960; Anonymous, 2018f) in orchid. “By cloning micro propagation has been a propagated plant in vitro. Typically, to encourage axillary bud development meristematic tissue must excise on a special medium to achieve develop purpose, after that new shoot will
separated and cultured, to obtain on mean to promote root growth, the cycle is will repeat until finally by moving shoots to culture medium, in order to produce plants. In agriculture, horticulture, and forestry, micropropagation is used, as special genotypes can be maintained and bred and also this process is rapid as well as plants can be remain free of diseases (Anonymous, 2018g-b). Usually employs grafting method for mature trees, because micropropagation for these trees is difficult. For rejuvenation of the scion and subsequent propagation, grafting older scions onto younger rootstocks (Ahuja, 1993; Mátyás, 1997). Usually by grafting propagate a plant asexually, during tissue culture, rooting cutting, or apomictic seed (Maynard, 1996a). Micropropagation is a produce a large number of progeny plants using modern plant tissue culture methods through rapidly multiplying of stock plant material. To multiply novel plants, such as those that have been genetically modified or bred through conventional plant breeding methods, micropropagation is used for this purpose. In addition from a stock plant which does not produce seeds provide a sufficient number of plantlets for planting, or doesn't respond well to vegetative reproduction. In the late 1950s and early 1960s Cornell University botanist Frederick Campion Steward discovered and pioneered micropropagation and plant tissue culture (Anonymous, 2018h). In the end it is a fast method to propagation disease-free plants especially plant that has a great potential to develop high quality. Several techniques are development in this field and these techniques for improvement of a wide range of horticultural crops and their production systems and rapid multiplication. There are three types of vegetative propagation within micropropagation: 1. adventitious shoot production, 2.somatic embryogenesis, and 3. Axillary shoot production, where provide excellent opportunities for successful vegetable crops production (Butt et al., 2015). And finally, there is study in Basrah university by Ahmed Al mayahi called photoautotrophic tissue culture “micropropagation without sugar in the (MS), where during photosynthesis and inorganic nutrient uptake, the growth or accumulation of carbohydrates of cultures are occurring". To using this form of propagation there are multiple advantages, result to this system actively encourages plant growth. Especially, due to the lack of sugar in the growing medium there are lower contaminations, as well as more catering to plants that can’t to be successfully multiplied by conventional means. When make root formation from cuttings be difficult, many plants have trouble being propagated due to physiological or morphological inhibitions which are reason in difficulty root formation from cuttings. Then the ex-planting process can often be very stressful and result in
plant death, and that isn't be a problem for pre-acclimated plantlets grown via photoautotrophic tissue culture (In this context, pre-acclimated indicate the plantlets that have developed a cuticle and have been cultivated in an open air system instead of the closed system in typical operations)(Anonymous, 2018i).
2.5. The most important plant hormones and her importance for growth and develops plants
2.5.1. Plant growth regulators (PGRs)
Plant growth regulators are formed from group of naturally organic substances, and the low concentrations of PGRs influence physiological processes which are development, differentiation, and growth, while other processes like stomata movement they also may be affected. Usually the transport of cytokinins from roots to leaves important to prevents senescence as well as to maintain metabolic activity (Davies, 2010). And also PGRs have great influences on both shoot elongation and shoot proliferation. BA hormone used most in micro-propagation for wood species, whereas TZD has been used less and has been used rarely compared to the first one (Nas et al., 2010; Valero-Aracama et al., 2010; Amoo et al., 2011). TDZ caused large amounts of callus as well as had detrimental effects, caused formation of short shoots. On the other hand, the majority of PGRs are used to retard the growth of plant shoots. The situation is difficult particularly for PGRs in fruit production. Fruit trees are more complex to deal with, and the market size of a particular application is often relatively small. In addition, public opinion may be sensitive particularly in this area. For these reasons, most new plant growth regulators will only be available for fruit production if this is an additional indicator (Rademacher, 1992). In modern horticulture the use of plant growth regulators is well established. In fact certain fields of horticulture are highly dependent on the use of synthetic plant growth regulators especially in asexual propagation (Cutting and Wolstenholme, 1992). In this study the most used PGRs are:
2.5.1.1. Auxin
Is the most important growth regulator among other growth regulators. This hormone used in two cases first, in tissue culture, and second in the natural. In tissue culture auxin have been used generally for rooting differentiation as well as for cell division, while in the natural using involved with abscission rooting, tropism, apical dominance, and also with elongation of steam and inter nodes. There are several types of auxin that often used in tissue culture are: NAA, IBA, IAA, NOA, 2,4,5-D,P-CPA, and 2,4-D. Of these there are two hormones used widely for rooting that are IBA and NAA, whereas for shoot proliferation in interaction with a cytokinin. Other hormones such as 2, 4, 5-T and 2,4-D used for induction growth of callus and their very effective for this purpose, in addition 2,4-D very important for induction of somatic embryogenesis. However, auxins are dissolved in either ethanol or dilute NaOH. IAA considered the least stable among various auxins. Therefore, IBA and NAA are synthetic auxins, have been preferred for use in tissue culture media. Because of its strong tendency to induce callusing, their concentrations must be from 0.1 to 1 mg l-1, while this ranges of concentration helping to shoot multiplication. 2,4-D is the most effective auxin but, during shoot multiplication must be avoid this type of auxin because, it is attempted through adventitious bud development or axillary branching for somatic embryogenesis (Bhojwani and Razdan, 1986). In order to achieve rooting, auxin is suitable for this purpose where IBA, IAA, and NAA have been used at (0.1-1 mg l-1) concentration most often, but IAA was more effective for rooting (George and Sherrington, 1984). Rooting of micro cutting in some woody species may require high concentration of auxin, but the result may be undesirable, such as leaf cholorosis, inhabitation of root elongation, and callusing, and also quiescence or dormancy in the shoot tip which is difficult to overcome during acclimatization stage (Maynard et al., 1991). ). Without an auxin best rooting micro cuttings occurred on agar-water medium of Leucopogon obtectus plant, while with increasing MS salt level the response was worsened (Bunn et al., 1989). The promoting effect diluted mineral salt solution on rooting is probably due to reduce reduced nitrogen level, as for shoot multiplication (Driver and Suttle, 1987). with lower nitrogen and high sucrose content was beneficial for invitro rooting, where observed in case that pre conditioning of walnut micro cuttings on medium. The best rooting, in respons to 9.8 µm IBA, when reduction of NH4NO3 level to ½ or ¼ strength in MS medium also when NH4NO3 was deleted
from MS where that showed for all the three apple scion cultivars by (Gala, Royal Gala, Jonagold), studied by (Sriskandarajah et al., 1990). As well as significantly increased the rooting respons in cvs Gala and Royal Gala but not in Jonagold. (Riffaud et al., 1981) reported a quick dip in 3 mµ IAB, because Micro cuttings of Prunus serotina were rooted on 1/5th strength MS salt medium. (Litwińczuk, 2004) reported optimal root formation (90%) has been found at low concention of IBA (0.05 mg l−1) in Aronia
melanocarpa. However for root induction IBA was found to be the best hormon for that
purpose, in addition in 1/2 MS medium containing 1.0 mg L−1 concentration of IBA, where 83% of regenerated shoots of Aronia melanocarpa were rooted, reported by (Brand and Cullina, 1992). The composition of shoot induction medium happens through it the variation in the optimal level of IBA for root formation. The better response on rooting had been at 0.5 mg l−1NAA various concentrations of NAA that for the various concentrations of NAA studied. The frequency of rooted shoots, number and length of roots, and induced callus on the shoot base significantly decreased at high concentrations of NAA (1.0–2.0 mg l−1) reported by (Sivanesan et al., 2016). There are interactive effects between IBA and D vitamin on adventitious root formation of
Populus tremula (Gorst and De Fossard, 1980; Pythoud et al., 1986). Raising the
temperature from 25 to 30 C0 during the begining (3-7 days) of the rooting stage by dark treatment and the purpose of this treatment is to improve the rooting response of several apple scion cultivars. This treatment helped to achieve up to 100% rooting in some cultivars which hard to rooting for hours (Zimmerman, 1984; Zimmerman and Fordham, 1985). Under higher light intensity (66µmol m-2 s-1 ), shorter photoperiod (17h) and lower temperature (19 0C) the shoots were placed, during shoot multiplication to promote lignification, during treatment in auxin (Lakso et al., 1986). There are other studies relate in auxin. By the application of weak electric current (1µA), tobacco calluse growth 70% increase in such a way that the callus was made negative and the medium positive. This stimulation achieve only on IAA containing medium (Rathore and Goldsworthy, 1985c; 1985b). By exposure to electrical treatments, the callus which only formed roots is the callus derived from mature embryo of wheat, where it was induce to form several shoots (Rathore and Goldsworthy, 1985a).
Some physiological phenotypes of auxins
1. Efficiency of photovoltaic construction: It was found that the auxins especially IAA, NAA lead to activate of efficient of photosynthesis, as well as the activation of enzymes and the synthesis of dyes competent with the interaction of light
2. Apical dominance: It is noted that the buds tip affects the growth of lateral buds. It is observed that when the bud is absent, the lateral buds are active in the growth because the buds produce high concentrations of auxins and when these high concentrations move, they inhibit the growth of lateral buds, which leads to the occurrence apical dominance of the terminal bud
3. Seedless fruits: Seedless fruits can obtain on by adding lanoline paste added IAA to the flower stigma. It is noted that the ovaries of the plants capable of producing seedless fruits because auxins content be more than those of species ovaries which need to be fertilized but produce fruits (Anonymous, 2018g-a).
2.5.1.2. Cytokinin
In tissue culture cytokinin function mainly are differentiation of adventitious shoots from callus and organ and cell division. In addition these hormones are modification of apical dominance, shoot differentiation and also concered with cell division, as well as these hormones may have other uses such as, by the release of axillary buds from apical dominance shoot proliferation was used. There are several types of sytokinin widly used are: TDZ, zeatin, BAP, and 2-ip. Of these thidiazuron used at low concentration (0.1 µg l-1) compared with others. Usually cytokinin have been dissolved in NaOH or in dilute HCL as well as Dmso (Dimethylsulfoxide) may be used as solvent for thidiazuron (Bhojwani and Razdan, 1986). In many cases the use of young and meristematic tissue has enabled rising of regenerative cultures, but mature and differentiated explants failed to show that response (Wernicke and Brettell, 1980). Three days after germination which coincides with the complete depletion of lipid in the cells of the cotyledons, the cotyledons lose the potential to form adventitious shoot buds in Pinus radiata, In contrast, in Pinus gerardiana show higher potential to form shoot buds in addition, Pinus gerardiana the cotyledons derived from ungerminated seeds (Banerji, 2009) To achieve regeneration the explant must be ideal as well as peto length
must be short (1mm) almost with a lamina together, regeneration istiole is achieve. This is also consisting with Brassica oleracea (Lazzeri and Dunwell, 1986; Horeau et al., 1988). Culture failed to form roots or shoots within 3-5 days after the petiole lost contact with the medium during that period (Klimaszewska and Keller, 1985). In
Cunninghamia lanceolato the explants if planted vertically produce of shoots will be
less, reverse horizontally way on the medium produce three times more shoots than those planted vertically (Bigot and Engelmann, 1987). BAP induces shoot bud differentiation at the cut end of petiole in cotyledon cultures of Brassica juncea, but only roots are formed at the same site during the absence of BAP (Sharma et al., 1990). After 11 days of incubation on BAP-containing medium the cotyledon transfer to basal medium and form only shoots not roots, if cotyledon is pre-cultured on BAP-free medium for longer than 7 days they lose the potentiality to form shoots on BAP-medium. For leaf explants of convolvulus arvensis each of part in this plant such as shoots, roots, and callus formation, have different media culture to form on. form only shoots on MS+7mg 1-1 2ip+0.05mg 1-1 IAA (SIM), for roots form only on MS +12 mg 1-1 IBA (RAM) , and in the end callus form only on MS 0.3 mg 1-1 kinetin +3 mg1-1 IAA (CIM) (Christianson and Warnick, 1983; Christianson and Warnick, 1984). In order to find best culture medium, cytokinin type and concentration for micropropagation, that by using single-axillary bud explants (from cultures regularly subcultured over 2 years), where two independent experiments were conducted about this. Large amount of callus with a short shoots were they obtained at the lowest level, 1.25 lM TDZ. While explants produced only large amounts of callus or/ and rosette shoots, at higher TDZ levels. BA considered the only cytokinin that provided a reasonable shoot length as well as shoot number. To obtain the highest number of shoots long enough to be rooted that 5.0 lM would be the best BA concentration, that according to the results which indicated. In hawthorn micropropagation BA could be used as the choice cytokinin more effective than mT and TDZ although the effects of species and genotypes could not be excluded. Generally among plant hormones, benzyl adenine was the only cytokinin that promoted shoot elongation and shoot proliferation together (Nas et al., 2012).
2.5.1.3. Gibberellins
Generally there are over 20 types known from gibberellins, of these types GA3 the most used type compared with auxins in addition, among all plant hormones gibberellins are used very rarely. Gibberellins stimulate normal development of plantlets from in vitro formed adventive embryos, and GA3 is easily soluble in cold water up to 1000 mg l-1, according to they reported (Bhojwani and Razdan, 1986). In some woody species to improve shoot elongation GA3 has been used in the shoot multiplication medium (Wochok and Sluis, 1980; Brand and Lineberger, 1992). (Murashige, 1961; 1964) reported, in tobacco GA3 inhibits shoot-bud differentiation. The differentiating callus in tobacco during exposure of the dark to period as short 30-60 min reduce shoot bud (Goldsworthy and Rathore, 1985) differentiation (Thorpe and Meier, 1973; 1975). At the stage of mereistemoid formation GA3 was most effective. GA3 did not inhibit their further development once shoot buds were formed. Only in the dark the complete inhabitation by gibberellin were accrued (Thorpe and Meier, 1973). Also giberellic acid considered tetracyclic di-terpenoid compound where, this hormon stimulating development and growth of plant, as well as stimulate seed germination by amino acids. In addition the trigger transitions from meristem to shoot growth, adult leaf stage, vegetative and also to flowering. Other GA functions are grain development, and sex expression along with an interaction of different environmental factors such as temperature, water, and light. Stamens are the major site of bioactive GA, and the stamens are influencing on pedicel growth and male flower production, in addition Bioactive GAs are endogenous hormones it's functions regulate the natural developmental processes such as stem growth in plants. During stem growth an increase in both cell elongation and cell division occurs. To understand the appropriate mechanism of GA movement in sex expression, seed germination, plant’s growth, floral and grain development that presently, it is a great challenge for scientific community. for the survival of plant species and successful crop production, the appropriate elucidation of GA transport mechanism is essential in this area (Sun, 2010). By releasing DELLA mediated inhibition of BZR1 (Brassinosteroid signaling positive regulator) transcription factor, GA promotes cell elongation. For pollen development the tapetum considered essential for this purpose, and also provid nutrients and contains pollen coat and allowing dehiscence (Goldberg et al., 1993; Izhaki et al., 2002). The site that seems to be major of GA biosynthesis is tapetum in developing anthers in
Arabidopsis and rice (Itoh et al., 2001; Kaneko et al., 2003; Hu et al., 2008). The
embryo responsible about produce the gibberellin, where gibberellin stimulates cells of the aleurone layer to synthesise and secrete -amylase and other hydrolases which degrade starch and other polymeric reserves in the endosperm, and also providing nutrients for the developing seedling. The tissue that surrounds the starchy endosperm and the embryo is the cereal aleurone, where cereal aleurone considered a layer secretory. To secrete a range of hydrolytic enzymes, GA3 works on stimulate aleurone cells for that purpose. There are study of the response of cereal aleurone to gibberellin and abscisic acid respectively (Woodger et al., 2010). The aleurone layer is unable to synthesize GA during seed germination but perceive the GA signals (Fincher, 1989; Gubler et al., 1995). As a model for hormone action in plants the effect of incubating isolated aleurone layers in media containing specified concentrations of GA3 and/or
ABA has been widly studied. If GA3 present in excess, ABA prevents the action of GA3,
and ABA considered the key regulator of aleurone. Because the aleurone is a uniform tissue with well-defined target responses to its two key regulators, ABA and GA, through that tissue can study GA-response pathways. There are rapid increases in the concentration of cytosolic Ca2, [Ca2] i, and that one of the earliest observed in GA-stimulated aleurone, whereas [Ca2] i increases in barley aleurone protoplasts approximately 3-fold within 5 h of GA treatment. ABA prevents the GA-induced increases in [Ca2] i and the expression of CaM , during treatment of barley aleurone protoplasts with GA3. Through Ca2 and/or CaM levels (2, ABA antagonism of GA-action might work largely, however block the ABA inhibition of both -amylase gene expression and -amylase secretion (Woodger et al., 2010). During the endogenous growth regulating hormones (GA and ABA), and by some physical factors (light, temperature and moisture) the breaking of seed dormancy controlled to germination (Debeaujon and Koornneef, 2000). In the end, the level concentration of GA3 has
influence on female flower, where the low concentration of GA3 promoted induction of
these flowers, in addition the castor bean, corn and hyoscyamus stimulates the pistillate flower development (Katsumi et al., 1983).
