Effect of 2,4-Dichlorophenoxy Acetic Acid and Activated Charcoal on Callus Induction of Cocos Nucifera L. Hybrid MATAG Inflorescence

Cocos Nucifera Linn. Var. MATAG is a Dwarf coconut variety that had high demand in Malaysia but low supply. Vegetative propagation of high-yielding MATAG coconut by using in vitro cloning must be considered in contributing to increase coconut productivity. Thus, attempts were made to develop a protocol that would enhance callogenesis as a first preliminary step towards a protocol for mass propagation of C. nucifera L. var. MATAG. The anther isolated from immature inflorescence was used as explants and cultured on modified Eeuwens Y3 media in different concentrations of 2,4-dicholorophenoxy acetic acid (2,4-D) and activated charcoal. The highest callus induction percentage (31.25 ± 12.18) was observed in 20 mg/L 2,4-D. However, 2,4-D at any level tested were not statistically significant. Callus induction media supplemented with 0.5 mg/L activated charcoal gave the highest callusing percentage (25.89 ± 13.59 %) indicating a positive effect of activated charcoal on callusing even though the result obtained not significant compared to control (15.95 ± 6.76 %). But, activated charcoal supplemented in media produced a significant effect compared to control in reducing the percentage of browning. In conclusion, media supplemented with activated charcoal produced a higher rate on callus induction and preventing tissue browning in explant. Besides that, the anther and ovule explant may serve as an efficient explant to study the callus induction of C. nucifera L. var. MATAG and as a basis to screen the potential useful plant growth regulators for somatic embryogenesis.

Cryopreservation of Arecanut (Areca catechu L.) Embryogenic Calli by V Cryo-plate Method

Aims: Arecanut, a perennial palm species of Arecaceae family, has huge commercial value, and is grown mainly for its masticatory nuts. The ever-increasing demand for uniform quality plantlets from growers necessitates putting in place In vitro mass multiplication and other crop improvement programmes. The present study was carried out to standardize the procedure for cryopreservation of embryogenic calli of arecanut, derived from immature inflorescence cultures, by vitrification based cryo-plate technique. Study Design: Completely randomized design (CRD) with three replications. Place and Duration of Study: ICAR-Central Plantation Crops Research Institute, Kerala, India during 2019. Methodology: The embryogenic calli were precultured in Eeuwen's Y3 basal medium supplemented with sucrose (0.2, 0.3 and 0.4 M) for three days. Explants were affixed on cryo-plates and later dehydrated using plant vitrification solution 3 (PVS3) for 30 min. Cryoplates were inserted in cryovials and cryopreserved. Explants with no cryostorage served as control. Explants were rewarmed quickly in a water bath (40ºC) for 2 min and treated with unloading solution and cultured on recovery medium. Results: The results showed 8-10 % recovery of embryogenic calli that resulted in normal plantlet production. The clonal fidelity studies, using Start Codon Targeted (SCoT) marker, showed no variation of cryopreserved calli in comparison to the original calli. Conclusion: This preliminary study demonstrated the successful use of vitrification (V) cryo-plate technique in cryopreservation of embryogenic calli of arecanut. With better recovery percentage, the optimal concentration of sucrose in the preculture medium was found to be 0.3 M. Desiccation in PVS3 solution for 30 min had no adverse effect.

Thidiazuron-induced direct organogenesis from immature inflorescence of three date palm cultivars

Background Inflorescence explants of date palm proved to be a promising tool for micropropagation of elite cultivars or rare males and females as organogenesis and somatic embryogenesis could be achieved. These plant materials are abundantly available every year and can be used as cheap and potent explants. Nevertheless, many difficulties could be faced in this protocol according to selection of the spathe size and age, media components, growth regulators, etc. The aim of this study was to determine the influence of various cytokinins on direct organs induction of three date palm cultivars (Selmi, Barhee, and Medjool) from immature inflorescence. An additional objective of this study was to investigate the effect of cytokinins and auxins on growth and development of Medjool cultivar. Results Various combinations of cytokinins were investigated on three date palm inflorescences as N6-(2-isopentenyl) adenine (2iP), kinetin, benzyleadenine (BA), and thidiazuron (N-phenyl-N′-1,2,3-thidiazol-5-yl urea) (TDZ). TDZ alone or in combination with BA proved to be superior for direct organogenesis in all three cultivars so that another combination of TDZ with BA was conducted. Results showed that moderate concentration of BA, with TDZ, gave superior response. Medjool cultivar response surpassed other two cultivars that made the possibility to conduct some growth regulators treatments on its multiplication and regeneration. TDZ at 0.5 + BA at 1.0 mg/l without activated charcoal seemed to enhance multiplication rate. Medium containing 0.5 mg/l of both naphthaleneacetic acid and indole butyric acid in addition to 1.0 mg/l indole acetic acid appeared to be more suitable for rooting stage of Medjool shootlets. Conclusion In this study, we created an innovation sequence of growth regulators included in nutrient media for date palm direct organogenesis from inflorescence. Organogenesis has been accelerated from immature inflorescence explants and developed to healthy plantlets which acclimatized in greenhouse.

Siliplant1 protein precipitates silica in sorghum silica cells

Silicon is absorbed by plant roots as silicic acid. The acid moves with the transpiration stream to the shoot, and mineralizes as silica. In grasses, leaf epidermal cells called silica cells deposit silica in most of their volume using an unknown biological factor. Using bioinformatics tools, we identified a previously uncharacterized protein in Sorghum bicolor, which we named Siliplant1 (Slp1). Slp1 is a basic protein with seven repeat units rich in proline, lysine, and glutamic acid. We found Slp1 RNA in sorghum immature leaf and immature inflorescence. In leaves, transcription was highest just before the active silicification zone (ASZ). There, Slp1 was localized specifically to developing silica cells, packed inside vesicles and scattered throughout the cytoplasm or near the cell boundary. These vesicles fused with the membrane, releasing their content in the apoplastic space. A short peptide that is repeated five times in Slp1 precipitated silica in vitro at a biologically relevant silicic acid concentration. Transient overexpression of Slp1 in sorghum resulted in ectopic silica deposition in all leaf epidermal cell types. Our results show that Slp1 precipitates silica in sorghum silica cells.

Acute and Subacute Toxicity Studies of the Ethyl Acetate Soluble Proanthocyanidins of the Immature Inflorescence of Cocos nucifera L. in Female Wistar Rats

Ayurvedic and traditional medical practitioners of Sri Lanka use the decoction of the immature inflorescence of Cocos nucifera L. (IC) variety aurantiaca for the treatment of menorrhagia. The progestogenic effect of the ethyl acetate soluble proanthocyanidins (EASPA) of the IC in female rats at a dose of 3.5 mg/kg body weight has been reported. Acute and subacute toxicity studies of EASPA of the IC carried out using female Wistar rats according to Organization for Economic Co-operation and Development (OECD) guidelines 423 and 407, respectively, are reported herein. In the acute toxicity study, a single dose of EASPA (2000 mg/kg body weight) was orally administered to rats, which were monitored for 14 days. In the subacute toxicity study, rats were orally administered with EASPA daily for 28 days at doses of 1.75, 3.5, 7, and 14 mg/kg body weight. No rat in either the acute or subacute toxicity study exhibited mortality or clinical signs of toxicity. Further, these rats did not show any significant change in their mean body weight, food, and water intake, haematological and biochemical parameters as well as in the results of their histopathological examinations compared to those of control group rats. According to results of the acute toxicity, the LD50 of EASPA is estimated to be greater than 2000 mg/kg body weight. Considering the results of the subacute toxicity study, the oral administration of EASPA daily for 28 days was well tolerated up to the dose, 14 mg/kg by rats. These results will be useful in the development of a novel therapeutic agent from EASPA of the IC for the treatment of menorrhagia, which incapacitates a considerable proportion of women worldwide.

Development of In vitro Tetraploid Plants of Hevea brasiliensis

Increase in global consumption of natural rubber necessitates crop improvement of Hevea aimed at increased productivity. As conventional breeding of Hevea is very elaborate and time consuming. Hence in the present study development of tetraploids through chromosome doubling of diploid callus obtained from cultured immature inflorescence of Hevea using colchicines were attempted. Chromosome doubling of the diploid callus occurred when treated with 1.25 µM colchicine for 3 days. In higher concentrations as well as at longer exposure periods, the callus texture and viability were affected. 48 % embryo induction and a maturation frequency of 45 % were obtained. Embryo germination and plant regeneration with a germination frequency (30 %) and a regeneration frequency (20 %) were obtained. Cytological and flow cytometric analyses confirmed the tetraploid nature of the colchicines treated callus. In vitro tetraploid plant developed through these in vitro techniques can be further used in Hevea brasiliensis breeding.

Coconut Tissue Culture: The Indian Initiatives, Experiences and Achievements

Coconut is one of the principal crops of India cultivated in over 35 districts mainly in the southern states. The productivity of the crop is declining in many of the traditionally cultivated regions owing to ageing plantations as well as biotic and abiotic stresses. These plantations are to be replanted with high yielding varieties/hybrids for which adequate quantity of quality planting material is not available. Even though tissue culture research was initiated in many laboratories in the country, the work was eventually phased out in most of the laboratories for want of a repeatable protocol. At ICAR-CPCRI, coconut tissue culture programs have been continuing for the past three decades. The attempts made include experimentation with different explants viz., immature inflorescence, plumular tissues, mature palm shoot meristem, ovary and anthers and different culture media supplemented with varying levels and types of hormones. Some of the successful protocols developed at the Institute include coconut zygotic embryo culture for collection and exchange of germplasm, cryopreservation and retrieval of zygotic embryos and pollen and plantlet regeneration from plumular tissues. Even though ICAR-CPCRI has succeeded in obtaining plantlets via direct organogenesis from inflorescence explants, the absence of friable calli formation from explants, the low rate of somatic embryo formation, large number of cultures turning to abnormal shoot development, non conversion of somatic embryos into plantlets, and formation of abnormal somatic embryos remain the major bottlenecks. Gene expression studies are being currently undertaken to decipher the molecular basis of in vitro recalcitrance.