Standardization of zygotic embryo culture from Nerium oleander L. and comparative analysis of biosynthesized cardiac glycosides within in vitro and acclimatized plants

The primary result of our experiment revealed that the germination percentage of N. oleander mature seeds is only 30%. From this observation, the concept of protocol standardization for zygotic embryo culture of this plant was originated. Zygotic embryo culture was proved an efficient in vitro multiplication system of N. oleander. The maximum germination percentage (96%) of zygotic embryos was observed on ¼ MS medium with 15 gm/L sucrose, whereas the best growth medium was optimized as ½ B5 with same sucrose concentration. The second part of this study was aimed to find out the cardiac glycoside accumulation pattern in both in vitro and acclimatized plants. For this purpose, one-month-old in vitro plantlets and acclimatized plants were subjected to LC-MS analysis and 09 cardiac glycosides were detected and quantified in both the systems. Most of the cardiac glycosides including odoroside A (32.71 mg/gm DW), odoroside H (4.69 mg/gm DW) and oleandrin (0.52 mg/gm DW) were found to be accumulated at maximum level within in vitro plantlets. CG 840b (1.89 mg/gm DW) is the only cardiac glycoside, which was maximally accumulated in acclimatized plants. From this study, it can be concluded that, zygotic embryo culture is a better choice for in vitro multiplication of N. oleander when compared to matured seeds and in vitro grown plantlets of this species favor cardiac glycosides biosynthesis in comparison to acclimatized plants. Therefore, all future research on the enrichment of cardiac glycosides from this plant may be conducted on zygotic embryos derived in vitro grown plantlets or cultures.

Development of a protocol for genetic transformation of Malus spp

A protocol to produce transgenic shoots of Malus X domestica cv Greensleaves was optimized using two gene constructs previously used to create parthenocarpic tomato, Ino-IaaM and DefH9-IaaM. The aim was to obtain sufficient nº of transgenic shoots for in vitro multiplication, transfer to soil, grafting and testing for parthenocarpy in the next years. We investigated the effects of two modifications of a previous published protocol: 1) co-transformation with an Agrobacterium containing “VIP” genes in the gene construct and 2) two different hormones or hormone combinations. More shoot regeneration was obtained with a combination of three hormones (BA:NAA:TDZ) during co-cultivation instead of IBA and no co-transformation was performed using the VIP gene. For the DefH9-IaaM transgene, 21.04% regeneration was achieved for this treatment instead of 8.95% achieved with “IBA treatment” and 4.42% with the Agrobacterium co-transformation treatment. More shoot regeneration occurred with the combination of three hormones (BA:NAA:TDZ) instead of with only IBA and no co-transformation was performed using VIP gene. Experiments using Ino-IaaM confirmed the results shown for the DefH9-IaaM transgene. The regenerated shoots were multiplied in selective media containing kanamycin and roots were obtained.

THE INFLUENCE OF LIGHT QUALITY IN THE in vitro CULTIVATION OF Cattleya crispata

ABSTRACT: Micropropagation technique is a valuable alternative for high quality genetic preservation of endemic species such as the orchid Cattleya crispata from “Campo Rupestre Ferruginoso”. This study aims to evaluate the influence of light quality on in vitro multiplication and elongation phases, offering new insights on the limiting factors of C. crispata. Seeds extracted from capsules were used for inoculation in the culture medium. Four light sources were evaluated for in vitro culture, namely: fluorescent lamp, white LEDs, red LEDs and red/blue LEDs. Data about the number of shoots, shoot length, shooting vigor and pigment content were assessed at 90 days of in vitro culture. Based on the recorded results, white LEDs are the most suitable ones for in vitro multiplication and elongation phases of C. crispata. It offers higher quality for seedling production and increases the chances of genetic conservation of the species. Keywords: ‘Campo Rupestre Ferruginoso’; in vitro propagation; wavelength; LEDs.

Role of Organic Growth Supplement In vitro Multiplication of Orchid Species- A Review

The main purpose of this article is to review role of several organic growth additives such Apple juice, coconut water (CW), maize extract, banana homogenate (BH), peptone and protocorms etc which stimulate the multiplication rate of various orchid species in in vitro multiplication. These organic growth supplements help to increase the number of shoots, root and leaf in culture medium. In many orchid tissue culture, organic growth supplements, which are the most essential medium aspect to stimulate tissue growth, production and facilitate the regeneration of shoot. The banana homogenate (BH) had the highest rate of regeneration and root developments. The use of organic growth supplements resulted in increased regeneration, the creation of more shoots and the development of fresh plantlets. Amino acids, proteins, vitamins, carbohydrates and various types of organic compounds are present in these growth supplements. These components have the potential to play a significant role in the development and creation of culture. Now more research is needed to figure out which factors are responsible for the organic additives’ promoter effect.

Multi-Walled Carbon Nanotubes Improved Development during In Vitro Multiplication of Sugarcane (Saccharum spp.) in a Semi-Automated Bioreactor

Carbon nanotubes play an important role in plant biotechnology due to their effects on the growth and differentiation of cells, tissues, organs, and whole plants. This study aimed to evaluate the effect of multi-walled carbon nanotubes (MWCNTs) during in vitro multiplication of sugarcane (Saccharum spp.) using a temporary immersion system. Morphological characterization of MWCNTs was carried out under a transmission electron microscope. Different concentrations (0, 50, 100, 200 mg L−1) of MWCNTs were added to Murashige and Skoog liquid culture medium in the multiplication stage. At 30 d of culture, number of shoots per explant, shoot length, number of leaves per shoot, total chlorophyll, dry matter percentage, carbon percentage, and macro- and micronutrient content were evaluated. Results showed an increase in the development of sugarcane shoots at concentrations of 100 and 200 mg L−1 MWCNT. Total chlorophyll content increased at concentrations of 50 and 100 mg L−1 MWCNT, whereas macro- and micronutrient content was variable at the different MWCNT concentrations. Results suggest a hormetic effect, characterized by stimulation at low concentrations. In conclusion, the use of low concentrations of MWCNTs had positive effects on development, total chlorophyll, carbon percentage, and macro- and micronutrient (N, Ca, S, Fe, Cu, Zn and Na) contents during in vitro multiplication of sugarcane and may have a potential use in other species of agricultural interest.

In vitro multiplication protocol for Curcuma mangga : Studies on carbon, cytokinin source and explant size

Mango ginger (Curcuma mangga Valeton & Zijp.) is an underutilized rhizomatous species that has been valued in tropical Asian countries as a source of vegetable, spice, salad, medicine, and essential oil. This species is hardy and requires less care for obtaining good yields. Rhizomes are the commonly used propagules for the species, which are also the economic part of the crop. Huge quantity of seed rhizomes is required to promote this crop in larger areas. An efficient in vitro multiplication protocol is one of the options to meet the planting material requirement. Effects of carbon source (glucose, fructose and sucrose) and concentration (1 and 3%, w/v), cytokinins (BAP and meta topolin) and concentration (1 mg/L and 2 mg/L), size of explants (one/ two/ three bud) and IBA treatment (0, 250, 500 and 1,000 mg/L) for concurrent ex vitro rooting cum hardening were studied. Results revealed that for facilitating efficient multiplication, the medium should be supplemented with glucose (3%) as a carbon source and meta topolin (1 mg/L) as cytokinin. Two-bud explant should be used for subculture as it promoted superior shoot proliferation. Concurrent ex vitro rooting cum hardening was possible even without auxin treatment. The present protocol could be useful for large-scale production of quality planting material of this underexploited tropical species.

Butășirea prin metoda tradițională a murului roșu (Rubus loganobaccus L. H. Bailey) versus micropropagarea in vitro

The rooting rate of lignified and non-lignified hybridberry cuttings was tested. The experiment was conducted both with treated (with growth regulator) and untreated cuttings, at different times of the year, to prove the much higher efficiency of in vitro multiplication of the culture as compared with traditional methods of multiplication by cuttings.