Different Methods for Overcoming Integumental Dormancy during in vitro Germination of Red Araza Seeds

Red Araza, or Red Strawberry Guava (Psidium cattleianum Sabine) is a native Brazilian Atlantic Forest species of the Myrtaceae family, whose seeds exhibit integumental dormancy. Due to its importance to different industries worldwide, recent research efforts are seeking to expand this species’ micropropagation processes using in vitro seedling germination, especially since in vitro micropropagation of adult plant material has, so far, been limited. This research effort evaluated different methods of overcoming integumental dormancy during in vitro germination of the Red Araza, so as to allow future micropropagation of the species. The seeds’ emergence and vigor were evaluated based on mechanical and acid scarification, using different substrates and immersions in solutions with different levels of gibberellic acid (GA3), and on the influence of the pre-immersion of seeds in water and sulfuric acid. The mechanical and acid scarification of the seeds, combined or separate, resulted in higher in vitro germination percentages and a higher germination rate index (GRI). Pre-immersion in distilled water (20 hours) also proved to be efficient for the germination of the Red Araza seed, with 76.2% of the seeds germinating and a higher speed of emergence (GRI = 0.18). When compared to a Murashige and Skoog (MS-zero) medium, sowing in a hydrophilic cotton substrate showed greater emergence and vigor, with approximately 70% of the seeds germinating. Treating the seeds by pre-immersing them in GA3 turned out to be unnecessary. The methods used for overcoming integumental dormancy during in vitro germination of Red Araza seeds proved to be efficient, and could be used to develop micropropagation protocols of seminal origin for this species.

In vitro Propagation of Critically Endangered Endemic Rhaponticoides mykalea by Axillary Shoot Proliferation

Turkey is one of the richest countries in variability of flora. It has nearly 9000 plant species about 3000 of which are endemic [1]. Asteraceae, is represented by 50 species in Turkey with an endemism of nearly 54% [2]. Rhaponticoides mykalea (Hub.-Mor.) M.V. Agab. & Greuter which belongs to the Asteraceae family, falls within the CR (Critically Endangered) category in the Red Data Book of Turkey [1]. While R. mykalea (Hub.-Mor.) was classified under the section Centaurea as Centaurea mykalea (Hub.-Mor.) before now. Today it has been separated from the section Centaurea [3]. It spreads very scarce in Kuşadası (Aydın), Muğla and Isparta, and faces with the danger of extinction. R. mykalea that has very limited number of individuals is under strong anthropogenic pressure such as the gradually increase in ongoing urbanization due to rapid developments of tourism sector, the conversion of natural habitats into human dominated lands, the over-grazing and collecting capitula of R. mykalea by local people for food. The species has already been under the threat of extinction and the situation above will increase the risk of extinction of this species even more [4]. For this reason, local protection measures and global conservation strategies are necessary [5].

Nowadays, the conservation of wild plant genetic resources is very important for preventing a decrease in genetic variability. Conservation of the endemic or threatened plants is carried out using different strategies. In vitro culture is an

In vitro Regeneration, Acclimatization and Antimicrobial Studies of Selected Ornamental Plants

Tissue culture has been applied to diverse research techniques such as viral elimination, clonal propagation, gene conservation, in vitro fertilization, mutation, induction for genetic diversity, genetic transformation, protoplast isolation and somatic hybridization, secondary metabolite production and other related techniques. The commercial production of ornamental plants is growing worldwide. Its monetary value has significantly increased over the last two decades and there is a great potential for continued further growth in both domestic and international markets. About 156 ornamental genera are propagated through tissue culture in different commercial laboratories worldwide. About 212.5 million plants including 157 million ornamental plants amounting to 78% of the total production were reported [1]. These plants are over exploited due to their high medicinal value and hence, propagation of the plants by tissue culture may be mandatory, which offers a greater potential to deliver large quantities of disease-free, true-to-type healthy stock within a short span of time. Biotechnological interventions for in vitro regeneration, mass micropropagation and gene transfer methods in forest tree species have been practiced with success, especially in the last decade. Against the background of the limitations of long juvenile phases and lifespan, developments of plant regeneration protocols of ornamental species are gaining importance. Ornamental industry has applied immensely in vitro propagation approach for large-scale plant multiplication of elite superior varieties. During in vitro condition, plantlets are grown under fixed and controlled environment in sterile formulated medium which contained macronutrients, micronutrients, vitamins and plant growth regulators. After the plantlets reached optimum growth in the culture containers after

Micropropagation of Anthurium spp.

Micropropagation as an alternative method to conventional propagation, the culture of somatic cells, tissues and organs of plants under controlled conditions is a suitable way to produce a large number of progeny plants which are genetically identical to the stock plant in a short time. The important property of the plant cells is totipotency which is a capacity to produce the whole plant from different plant parts. Micropropagation has some features to be chosen in commercial production such as multiplicative capacity in a relatively short time, healthy and disease-free production capacity and ability to generate population during a year [1-5].

The genetic pattern of the plant is key element to select the propagation method. Using micropropagation techniques in plant biotechnology applications are costlier than conventional propagation methods. Propagation by using in vitro techniques instead of conventional methods offer some advantages like utilizing small pieces of plants called as explants to maintain the whole plant and increase their number. The main point is to evolve new strategies to

Effect of Additives on Micropropagation of an Endangered Medicinal Tree Oroxylum indicum L. Vent

Sonpatha (Oroxylum indicum (L.) Vent.) is a threatened medicinal tree species [1,2] belonging to family Bignoniaceae. It is valued for its antimicrobial, antiarthritic, anticancerous and antihepatitic properties possessed by its various parts. Root extract of this tree has been used for long in ayurvedic preparations like Dashmularisht and Chyawanprash [3,4].This tree possesses a flavonoid viz. Baicalein used to check proliferation of human breast cancer cell line MDA - MB - 435 [5]. Sonpatha grows in India, Sri Lanka, South China, Celebes, Philippines and Malaysia[6,7]. In India, it is distributed throughout the country up to an altitude of 1200 m and found mainly in ravine and moist places in the forests [8].Owing to indiscriminate collection, over exploitation and uprooting of whole plants with roots, this valuable tree has become vulnerable in different states of India like Karnataka, Andhra Pradesh, Kerala, Maharastra, M.P. and Chhatisgarh [9,10]. Hence research towards mass multiplication, conservation and higher production of the active compound under in vitro culture conditions is essential [11]. Few reports are available on the in vitro regeneration of the species [12,13]. Optimum factors influencing growth and morphogenesis vary with the genotype and types of explants used for micropropagation. [14]. Murashige and Skoog (MS) medium with a high content of nitrate, ammonium and potassium is of widespread use in the successful culture of a wide variety of plants. Sometimes it requires supplementation of additional substances in the medium.

Application of additives is adapted to the cultural needs[15] i.e. objectives of the experimental studies like micropropagation, regeneration, cytodifferentiation, androgenesis, biosynthesis of secondary metabolites and biotransformation of cells as well as

Tissue Culture Techniques for Native Amazonian Fruit Trees

The fruits of the Amazon have attracted great interest in recent years, both nationally and internationally, according to its exotic flavors and pleasant and varied ways to use its pulp by agribusiness [1], pharmaceutical industry [2], high vitamin and antioxidant content[3].

In recent decades, the production of native fruits of the Amazon showed significant growth, mainly due to expansion of area for fruit production. It is noteworthy that this activity has had little impact on native vegetation, since most of the orchards were planted in areas previously occupied by other crops for market problems or environmental issues and pressure for sustainable agriculture, ceased to be interesting for farmers [4].

The Amazon forest has large number of non-domesticated fruit species and a minority being exploited through crop in place of natural occurrence [5]. According to the

Tissue Cultured Versus Traditionally Grown Pineapples

Ananas comosus, commonly known as pineapple, is an herbaceous perennial belonging to the order Bromeliales, family Bromelaceace and sub-family Bromelioideae [1-3]. According to Carlier et al. [4] there are 56 genera of the pineapple which include 2921 species. According to Gene Technology Regulation [5] and Carr [6], pineapples originated in South America and through travels and migration of different peoples, its cultivation spread to other parts of the world. The Jamaican pineapple is said to have been discovered by Christopher Columbus on his voyage to the New World in the 15th century.

Bartholomew et al. [1], in describing the pineapple, states that the plant grows to 1-2 m high and wide with a club shaped stem having dimensions of 25-50 cm in length, width of 2-5 cm at the base and 5-8 cm at the top. The leaves are sessile and enclose the stem on two thirds of its circumference. They tend to be sword like with sharply dentate edges often variegated or

Characterization of the Embryogenic Tissue of the Norway Spruce Including a Transition Layer between the Tissue and the Culture Medium by Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) is a non-invasive method widely applied in the study of molecules. The MRI approach is frequently employed not only in medicine but also in biological, biochemical, and chemical research. Most of the papers describing the use of MRI to investigate plant physiology have emphasized certain aspects of anatomy or functional morphology [1]. In plant biology, MRI supports several major activities, namely, the research of the water and mineral compounds transported within a plant [2], [3], the determination of plant metabolites [4], [5], the investigation of cellular processes [6], and the examination of the growth and development of plants [7]. MRI is also instrumental towards monitoring water changes in early somatic embryos (ESEs). Šupálková et al. [8] examine the influence of the ESEs of spruce treated with cadmium and/or lead ions for twelve days using multi-instrumental analytical procedures; these authors employ image analysis to estimate the growth, a fluorimetric sensor to detect the viability of enzyme-treated ESEs, and the MRI technique to facilitate non-destructive measurement of the volume of the ESEs.

 The interaction between the cells plays a fundamental role in the growth and development of multicellular organisms. In this context, let us note that

Somaclonal Variation in Tissue Culture: A Case Study with Olive

Micro propagation of woody plants and fruit crops constitutes a major success in the commercial application of in vitro cultures. An important aspect to be considered when deriving perennial plants from micro propagation is the maintenance of genetic integrity with regard to the mother plant. In this regard, soma clonal variation has been reported at different levels (morphological, cytological, cytochemical, biochemical, and molecular) in micro propagated plants [1]. The economic consequence of soma clonal variation among regenerated plants is enormous in fruit crops and woody plants, because they have long life cycles. In consequence, the behavior of micro propagated plants should be assessed after their long juvenile stage in field conditions. The occurrence of soma clonal variation is a matter of great concern for any micro propagation system. In order to evaluate its presence several strategies were used to detect soma clonal variants, based on one or more determinants from among morphological traits, cytogenetic analysis (numerical and structural variation in the chromosomes), and molecular and biochemical markers [2]. In addition, studies on soma clonal variation are important for its control and possible suppression with the aim of producing genetically identical plants, and for its use as a tool to produce genetic variability, which will enable breeders the genetic improvement. Soma clonal variation has been studied extensively in herbaceous plants, whereas few studies have focused on

Regeneration of in Vitro Shoot and Root Structure through Hormone Manipulation of Coconut Zygotic Embryos

Zygotic embryos tissues have found to be more responsive explants for clonal propagation of coconut. In the present study, the feasibility of using zygotic embryos explants for clonal propagation of a local coconut variety MATAG F2 was assessed. Callus was induced by incorporating of cytokinin and auxin into the medium. The sliced embryos explants were immersed in 1 M maltose for 60 mins, then with 0.05 M maltose for 1 min and followed by 0.01 M maltose for 5 mins was the best for prevention of phenolic compounds excretion. The callus formation depended on the concentration of 2,4-D in the media and the best effect was observed with the high level (2,4-D and BAP) tested. Attempts at inducing multiple shoot from the zygotic embryos callus were unsuccessful. No multiple shoots was present; most of the callus became root structure.

In vitro propagation of a medicinal important plant Bacopa monnieri from nodal explants

Bacopa monnieri, a traditional Indian medicinal plant with high commercial
importance, is used as a neurotonic, immuno modulator, adaptogen transquilizing, memory
and learning enhancing, cerebral activator, anti-ulcer, antispasmodic, anti-asthmatic
ayurvedic herb. The study was undertaken in order to elaborate the tissue culture technique
for Brahmi (Bacopa monnieri). In the present study nodal explants were taken for in vitro
propagation. For sterilization, 0.01% HgCl2 was used as the sterilant. The explants gave best
results when treated with the sterilant for 5 minutes. To induce bud-break, nodal segments
were inoculated onto MS media supplemented with 1mg/l BAP. Basal MS medium without
any plant growth hormone was used as control. Further multiplication was obtained on MS
medium + BAP (1.0 mg/l) +IAA (0.5 mg/l). The shoots were rooted and best rooting was
observed by IBA when incorporated in MS at different concentrations (0.1 - 0.3 mg/l) and
0.2 mg/l gave good results. After 30 days about 2.2 cm length was observed. Almost 70%
of the rooted shoots survived during hardening.

In vitro callus induction and isolation of volatile compounds in callus culture of Lallemantia iberica

A modern biotechnological technique to obtain useful natural products from plants is to isolate them from their callus cultures. Lallemantia iberica is an annual herb of the Lamiaceae family known for its stimulant, diuretic, and expectorant effects in Iranian folk medicine. The present study investigated the induction of plant callus tissue and identification of its volatile compounds. For this purpose, plant seeds of Lallemantia iberica were sterilized and cultured in petri dishes lined with an MS medium. After the emergence of seedlings, cotyledon segments were transferred to another MS medium supplemented with different combinations of the plant hormones BAP and 2,4-D. The petri dishes were incubated in a growth chamber at 25 °C for a given photoperiod. The fresh weights of the calli thus produced in the hormonal treatments were measured. In a second stage of the study, the essential oil of the fresh calli was obtained using a Clevenger type apparatus and subjected to analysis by gas chromatography-mass spectrometry (GC-MS). Results showed that callus induction from the otyledon
segments of the seedlings was better accomplished in the MS medium containing phytohormones and in a dose-dependent manner. Maximum callus production was induced in the MS medium supplemented with 2, 4-D (4 mg/L) and BAP (1.5 mg/L) as 3.5g. GC/MS analysis showed that the dominant compounds in the essential oil were Thymol (53.03%), Octane (19.90%), Decane (5.73% ), Carvacrol (5.63%), and Octadecane (3.73%).

Tissue Culture of Gerbera

Gerbera (Gerbera jamesonii) somatic tissues and seeds were tried for raising tissue cultures. The explants of shoot tips, immature inflorescences, leaf sections, capitulum explants, axillary buds and receptacles explants from field grown plants had contamination problems. Another trouble was slow growth of explant cultures as they were treated with sterilizing chemicals which damage their growing regions. However, callusing was in all explants and shoot regenerations were obtained from shoot tip and axillary buds. The calluses were obtained from clean seeds which were pre-soaked (20mg/liter for six hours) in colchicine for induction of polyploidy. The germinating seedlings were crushed and tissue mass was put on callusing medium containing MS with BA+2,4-D (each 3 mg/liter). After six weeks, the calli were transferred to MS+BA (4mg/liter) +IBA (1mg/liter). The developed plantlets were separated and cultured on MS containing BA and IBA (each @ 0.5mg/liter). The plantlets were transferred to pots under 100% humidity during initial weaning period, acclimatized and grown as normal plants.

Morphology of callus, shoots, roots and leaves of Withania somnifera (Cultivated and Wild) in vitro tissue culture conditions with different hormones concentration

Modern plant tissue culture is practiced under aseptic or favourable conditions under filtered air. Living plant materials which was taken from the environment or free space are being naturally contaminated or will show contamination on their external surfaces (and sometimes interiors) with pathogenic microorganisms, so surface sterilization of initial materials (explants) in chemical solutions (usually Sodium or calcium hypochlorite or mercuric chloride) is done. Mercuric chloride is mostly used as a plant sterilant now a days, unless other sterilizing agents which require for removal of contamination are dispose found to be least effective, as it is very dangerous and harmful to use, and is very difficult to dispose of (Aniel, et al., 2011) [1]. 

Explants are then generally placed or maintained on the upper surface of a solidified culture medium (normally MS) (Pawar & Maheshwari, 2004) [11], but during some cases it will be inoculate directly into a liquid nutrient medium, normally when cell suspension cultures are required. Solid nutrient and liquid nutrient media usually consists of hormones. Solidified media are prepared or produced from the liquid media with the use of a gelling or solidified agent, generally a purified agar. The constituents of the