Effect of Cryopreservation Method Supported with Biochemical Analyses in the Axillary Bud of Jewel Orchid, Ludisia discolor.

This study investigated conserving an endangered terrestrial jewel orchid Ludisia discolor, using in vitro grown axillary buds. Excised segments of axillary buds (4-5 mm in length) were precultured on a modified Murashige and Skoog (MS) medium supplemented with 0.2 M sucrose for 24 h and osmoprotected in a loading solution for 20 min. Then, axillary buds were dehydrated in plant vitrification solution 2 (PVS2) for 10 min at 0 °C and incubated in liquid nitrogen for 1 h. Subsequently, axillary buds were rewarmed rapidly by dilution solution and transferred to a growth recovery medium supplemented with 0.05 µM melatonin, which led to an improved survival chance (16.67%) for cryopreserved L. discolor. The osmotic stress and the overproduction of reactive oxygen species (ROS) during cryopreservation stages may result in cryoinjuries and poor survival as increased levels of proline (5.51 µmol/g), catalase (85.64 U/g), peroxidase (565.37 U/g), and ascorbate peroxidase activities (12.19 U/g) were detected after dehydration, preculture, rewarming, and loading stage, respectively. Results obtained from this study indicate that further experimental designs which apply different PVS and exogenous antioxidants are needed for improved survival and regrowth of L. discolor.

Impact of culture vessels on micro-morphological features of in vitro Dendrobium Sabin Blue orchid.

Different designs of the plant tissue culture vessel, such as size, material, and shape, may alter its microenvironment atmosphere. The present study was conducted on protocorm-like bodies (PLBs) of Dendrobium Sabin Blue orchid to determine the development of PLBs on plastic and glass culture vessels of different sizes. PLBs were cultured in half-strength Murashige and Skoog (MS) medium with the same initial weight of 0.5 g in 10 replicates. The growth index of the PLBs was calculated after 11 weeks to study their growth in every vessel; additionally, biochemical analysis was performed to determine carbohydrate content, proline concentration, and photosynthesis pigments in the PLBs. Scanning electron microscopy (SEM) was performed to study stomata development on PLBs in each vessel, and histological analyses were conducted to study the cell structure. Overall, the PLBs cultured in a large 470 ml plastic vessel showed successful growth with a high growth index, high carbohydrate content, low-stress condition, and high chlorophyll content. SEM confirmed that the presence of trichome and rhizoid in PLBs cultured in the 470 ml plastic vessel. Histological analysis showed the formation of the shoot on the PLBs and the presence of starch granules. Thus, the use of plastic as a culture vessel provides a good impact for culturing PLBs and has low cost.

Morphogenetic and physiological effects of LED spectra on the apical buds of Ficus carica var. Black Jack.

The use of artificial light sources such as light-emitting diodes (LEDs) has become a prerequisite in tissue culture studies to obtain morphogenetic enhancements on in vitro plants. This technology is essential for developmental enhancements in the growing plant cultures due to its light quality and intensity greatly influencing the in vitro growing explants at a cellular level. The current study investigates the effects of different light-emitting diode (LED) spectra on the growth of apical buds of Ficus carica var. Black Jack. Ficus carica, commonly known as figs is rich in vitamins, minerals, and phytochemicals capable of treating microbial infections and gastric, inflammatory, and cardiac disorders. Apical buds of Ficus carica var. Black Jack, presented morphogenetic changes when grown under six different LED spectra. The highest multiple shoots (1.80 per growing explant) and healthy growing cultures were observed under the blue + red LED spectrum. Wound-induced callus formation was observed on apical buds grown under green LED spectrum and discolouration of the growing shoots were observed on the cultures grown under far-red LED spectrum. Multiple shoots obtained from the blue + red LED treatment were rooted using 8 µM indole-3-acetic acid (IAA), and the rooted plantlets were successfully acclimatised. Compared with the other monochromatic LEDs, blue + red proved to be significantly better for producing excellent plant morphogeny. It is apparent that blue and red LED is the most suitable spectra for the healthy development of plants. The findings have confirmed that the combination of blue + red LED can potentially be used for enhancing growth yields of medicinally and commercially important plants.

Elevation of secondary metabolites production through light-emitting diodes (LEDs) illumination in protocorm-like bodies (PLBs) of Dendrobium hybrid orchid rich in phytochemicals with therapeutic effects.

Gas-chromatography-mass-spectrometry revealed the presence of various bioactive compounds with anticancer properties in protocorm-like-body (PLB) cultures of a Dendrobium hybrid orchid (Dendrobium Enopi x Dendrobium Pink Lady). Pre-illumination of red fluorescent light lessened the stimulating effects of light-emitting diodes (LEDs) on secondary metabolites production among in vitro PLB cultures, possibly due to habituation. The highest flavonoid content of 16.79 µmol/ g of fresh weight (FW) was achieved under blue-red (1:1) LED for PLBs pre-treated with white LED for more than 3 subculture cycles. Phenolics content significantly reduced as PLBs pre-cultured under red fluorescent light for 2 subculture cycles were exposed to LED illuminations, where far red LED resulted in the lowest total phenolic content (18.85 µmol/ g FW). High intensity green LED (16.9 µmol/s) enhanced the accumulation of phenolics while amino acids such as L-leucine, glycine and proline exhibited no significant stimulating effect for secondary metabolites production.

Droplet-vitrification of Aranda Broga Blue orchid: Role of ascorbic acid on the antioxidant system and genetic fidelity assessments via RAPD and SCoT markers.

A droplet-vitrification cryopreservation protocol has been successfully developed for Aranda Broga Blue orchid hybrid using protocorm-like bodies (PLBs). However, maximum growth regeneration percentage was recorded at 5% only based on previous report. Thus, to improve growth recovery of cryopreserved PLBs, cryopreservation stages were supplemented with ascorbic acid, tested at 50, 100 and 150 mg/L. However, results demonstrated that exogenous ascorbic acid was not favorable in regeneration of cryopreserved explants (maximum value of 1.67 % with 50 mg/L ascorbic acid supplementation). Total soluble protein and various antioxidant enzyme activities such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) were evaluated after each cryopreservation stages in conjunction with the application of exogenous ascorbic acid. Addition of antioxidant must be carefully evaluated and its application may not guarantee successful growth recovery. RAPD and SCoT molecular analysis confirmed the genetic stability of regenerated cryopreserved PLBs as no polymorphism was detected compared to control PLBs culture.

Shoot Induction in White Eggplant (Solanum melongena L. Cv. Bulat Putih) using 6-Benzylaminopurine and Kinetin.

Solanum melongena L. commonly known as the eggplant or brinjal comes from the family of Solanaceae, sharing the same ancestor with the tomato and potato. It is an economically important crop worldwide, being well studied for its medicinal properties, nutritional values and its role as an alternative model plant. The eggplant fruit has been previously used for treatments of various diseases such as bronchitis, asthma, arthritis and diabetes as well as its nutritive properties that are beneficial to the human diet. Plant transformation studies on the eggplant have been widely done for the production of transgenic eggplants harbouring genes that are beneficial for optimal plant growth and fruit production. Shoot induction is an essential step required for the successful regeneration of transformed plant tissues and therefore is an essential pre-requisite in Agrobacterium-mediated transformation. The local eggplant cv. Bulat Putih is a local cultivar of eggplant in Malaysia with white and round fruits making it a potential model plant colour pigment accumulation studies in fruit crops. The current work aims to investigate the shoot induction potential of 6-benzylaminopurine (BAP) and Kinetin from cotyledon explants of eggplant cv. Bulat Putih. Results indicated that both BAP and Kinetin were able to induce the regeneration of callus from cotyledon explants. On the other hand, Kinetin at the concentration of 2.0 mg/L successfully induced shoots at the value of 1.50 ± 0.22 shoots per explant, whereas BAP alone did not trigger any formation of shoots. This study indicated that kinetin alone is sufficient to induce shoots in eggplant cv. Bulat Putih without the presence of BAP.

Shoot Induction from Axillary Shoot Tip Explants of Fig (Ficus carica) cv. Japanese BTM 6.

Fig, or Ficus carica, is a fruit tree from the Moraceae family and is widely grown in tropical and subtropical regions of the world. Fig plants are mainly propagated through grafting, air layering, and hardwood cutting whereby these methods were found to be less efficient. Plant tissue culture is efficient method to propagate plants, particularly to produce true-to-type platelets for mass multiplication. The aim of this study is to induce multiple shoot formation on Ficus carica cv. Japanese BTM 6 through identifying and optimising the concentrations of 6-Benzylaminopurine (BAP) and Zeatin suited for shoot formation. The axillary shoot tip explants were cultured in MS media supplemented with different concentrations of BAP and Zeatin (0, 0.5, 1.0, 1.5 and 2.0 mg/L) to determine the optimal concentration for the formation of multiple shoots. Number of shoots per explants and the differences in shoot height of explants were calculated after 8 and 12 weeks of culture respectively. Of all the treatments of BAP, MS media containing with 2 mg/L BAP marked the highest number of shoots per explant with the average value of 1.67 ± 0.33 while 1.5 and 2 mg/L of BAP produced the highest differences in shoot height with 0.51 ± 0.08 cm and 0.51 ± 0.07 cm after 12 weeks respectively. Murashige and Skoog (MS) media supplemented with 2 mg/L Zeatin showed the highest production of multiple shoots and differences in shoot height with the average of 0.83 ± 0.219 and 0.32 ± 0.04 cm respectively among all the different treatments of Zeatin. In this study, BAP performed better in shoot induction and elongation as compared to Zeatin for the cultivar Japanese BTM 6.

The effect of adenosine monophosphate deaminase overexpression on the accumulation of umami-related metabolites in tomatoes.

KEY MESSAGE: This study highlights the changes in umami-related nucleotide and glutamate levels when the AMP deaminase gene was elevated in transgenic tomato. Taste is perceived as one of a combination of five sensations, sweet, sour, bitter, salty, and umami. The umami taste is best known as a savoury sensation and plays a central role in food flavour, palatability, and eating satisfaction. Umami flavour can be imparted by the presence of glutamate and is greatly enhanced by the addition of ribonucleotides, such as inosine monophosphate (IMP) and guanosine monophosphate (GMP). The production of IMP is regulated by the enzyme adenosine monophosphate (AMP) deaminase which functions to convert AMP into IMP. We have generated transgenic tomato (Solanum lycopersicum) lines over expressing AMP deaminase under the control of a fruit-specific promoter. The transgenic lines showed substantially enhanced levels of AMP deaminase expression in comparison to the wild-type control. Elevated AMP deaminase levels resulted in the reduced accumulation of glutamate and increased levels of the umami nucleotide GMP. AMP concentrations were unchanged. The effects on the levels of glutamate and GMP were unexpected and are discussed in relation to the metabolite flux within this pathway.