Modified MS Medium Research Articles

Notes on In-vitro Propagation of Tecomella undulata (Sm.) Seem

Different explants of Tecomella undulata (Sm.) Seem. cultured on modified MS media gave significant differences for shoot regeneration and root differentiation. Plantlets were successfully developed using axillary bud and cotyledon as explants. There was no shoot formation from leaf explants. Maximum multiple shoots from axillary buds and cotyledons were obtained on medium containing MS major salts with BAP 4.0 mg/l + IAA 0.1 mg/l and BAP 5.0 mg/l + NAA 0.5 mg/l, respectively. Healthy roots developed from two step procedure. Isolated shoots were rooted by culturing on half strength MS liquid medium having IBA 2.5 mg/l for 48 hrs and then transferring to hormone free half strength solid MS medium. An initial dark period for 5-7 days favoured root induction. About 55 percent of in-vitro produced shoots were rooted.

A Novel Medium for Enhancing Callus Growth of Hazel (Corylus avellana L.)

Paclitaxel is a powerful antimitotic agent with excellent activity against a range of cancers. Hazel has been described as a paclitaxel-producing species among angiosperms. Fast-growing callus is a prerequisite for the success of callus production and then paclitaxel production. Therefore, optimizing the medium culture for enhancing callus growth is a crucial step for paclitaxel production. In this research, Murashige and Skoog (1962) (MS) medium was optimized for improving callus growth of hazel (Corylus avellana L.). The M10 medium (MS medium with pH 6.0 and supplemented with 1000 mg l−1 spirulina powder, 1000 mg l−1 casein hydrolysate and 3 g l−1 gelrite) significantly improved hazel callus growth. This modified MS medium increased callus fresh weight (55.8%) as compared to the control. M10 medium increased fatty acids yield of callus (66.7%) as compared to the control. Liquid M10 medium maintained growth over a longer period of time and also increased slightly, the paclitaxel production as compared to the control. This novel medium is promising for facilitating the mass production of hazel callus as a source of valuable metabolites including paclitaxel, linoleic and oleic acids.

Water stress effects on biochemical traits and antioxidant activities of wheat (Triticum aestivum L.) under In vitro conditions

ABSTRACTWater stress is one of the major environmental stresses that affect agricultural production worldwide, especially in arid and semi-arid regions. This research investigated the effect of water deficit, induced by PEG-6000 on wheat genotypes (GA-2002, Chakwal-97, Uqab-2000, Chakwal-50 and Wafaq-2001) grown in modified MS medium solution. Osmotic stress caused a more pronounced inhibition in leaf relative water content and leaf membrane stability more sensitive (index in Wafaq-2001 and Uqab-2000) genotypes compared with the tolerant (Chakwal-50, GA-2002 and Chakwal-97) genotypes. Upon dehydration, an incline in proline, total soluble sugar, total soluble protein, superoxide dismutase, peroxidase, catalase and malondialdehyde activity content were evident in all genotypes, especially at osmotic stress of −8 bars. The observed data showed that status of biochemical attributes and antioxidant enzymes could provide a meaningful tool for depicting drought tolerance of wheat genotypes. The present study shows that genotypic differences in drought tolerance could be likely attributed to the ability of wheat plants to induce antioxidant defense under drought conditions. In order to develop genotypes with stable, higher yields in dry farming conditions, it is necessary to characterise genetic resources based on drought adaptation, determine suitable genotypes, and then use them in breeding programmes.

Micropropagation of some Sewa Oasis Date Palm (Phoenix dactylifera L.) Cultivars Grown in Egypt

Shoot tip and leaf primordial explants of Phoenix dactylifera L. cvs. (Sewi and Oshkingbeel) were cultured in modified MS medium supplemented with various combinations between different growth regulators cytokinins (2iP or BA) and Auxins (2, 4-D or NAA). Friable callus was produced from shoot tip and leaf primordial explants. The highest percentage of callus initiation was achieved on modified MS medium supplemented with 100 mg/L 2,4-D and 3 mg/L 2iP for shoot tip and leaf primordial explants. The highest percentage of embryogenic callus was achieved on modified MS medium supplemented with 10 mg/L 2,4-D, 5 mg/L NAA, 3 mg/L 2iP and 3.0 g/L activated charcoal which gave 87.5% and 75% for Sewi and Oshkingbeel cvs., respectively. Somatic embryos formation were obvious on modified MS medium plus 0.1 mg/L NAA which gave the highest average number (5.16, 4.94) for Sewi and Oshkingbeel, respectively after three subcultures. Greatest embryonic elongation resulted at 0.5 mg/L 2iP + 0.5 mg/L kinetin + 40 g/L sucrose in solid full strength MS medium. Also, the highest value of leaves number was shown on the same medium. The complete plantlets developed from individual embryos (5-10 cm) were transferred to rooting MS medium supplemented with 1 mg/L NAA with good growth in solid and liquid media. Rooted date palm plantlets from both cultivars were transferred to plastic pots (5cm diameter) containing peat-moss, sand and vermiculite at equal volume. Acclimatization percentage decreased gradually during four months in the first acclimatization stage. Date palm plantlets produced from the first acclimatization stage were transferred to plastic pots (20 cm diameter) containing peatmoss, washed sand and vermiculite at equal volume for more growth and development to be ready to cultivate in the field. Although survival percentage was low during the first acclimatization stage, it was very high in the second acclimatization stage and date palm survival percentage became constant after another six months with good growth.

Effects of environmental factors on seed germination and seedling establishment in bilberry ( Vaccinium myrtillus L.)

Abstract The bilberry (Vaccinium myrtillus L.) is a spontaneous dwarf shrub with potential for national and international markets due to food and medicinal uses of its fruits, being considered an important source of sugars, antioxidants, vitamins and minerals. Request for bilberry fruits are on the rise, but the land area covered in bilberry bushes and locally collected berries have declined in Central Italian Apennine regions in recent last decades. The objective of this study was to assess the most suitable conditions for in vivo and in vitro seed germination in order to improve the reproductive performance of this species under artificial and controlled environments. Freshly matured bilberry seeds were tested for germination before and after cold stratification and air-room storage under different light conditions and temperatures. Seeds were then in vivo and in vitro germinated on different soil types and culture media. Fresh seeds showed a better germination capacity (76%) than stratified ones, being 16-h photoperiod and air temperatures of 22.5 °C the optimal conditions for germination. At 25 °C the germination rate decreased to 66% and very few seeds (8%) germinated at 15 °C. Darkness always had a negative influence on the germination capacity. However, after cold stratification germination increased significantly at 15 °C, both in light (51%) and darkness (24%); thus, seeds of this species are conditionally dormant at maturity. Although the effect was different depending on temperature and light condition, cold seed stratification had an overall negative effect on all the considered germination parameters, and seed viability was strongly reduced after storage at room temperature for 90 days. Soil type and culture media did not significantly affect the final germination percentage (≈62 and 78%) in the in vivo and in vitro trials performed on fresh non stratified seeds, respectively. Nevertheless V. myrtillus turf (in vivo experiment) and modified MS medium (in vitro experiment) gave rise to the most germination in the shortest time. Temperature proved to be the most influential physical factor on bilberry seed germination. Seedlings obtained from in vivo experiments showed good development.

ОСОБЕННОСТИ ПОЛУЧЕНИЯ И СОХРАНЕНИЯ CANNA × HYBRIDA HORT. EX BACKER В УСЛОВИЯХ IN VITRO

The aim of the study was to investigate means of morphogenesis and explant regeneration characteristics in in vitro culture and to develop biotechnological methods for micropropagation and conservation of promising garden varieties of Canna. The experiments were conducted in the plant biotechnology and virology laboratory of the Nikitsky Botanical Garden, National Centre of Science. For the induction of adventitious shoot development of explants, a Murashige and Skoog (MS) nutrient medium was used having 30 g / l of sucrose, 10 g / l of agar-agar with BAP and TDZ growth regulators. For the in vitro deposition, explants (vegetative buds and meristemoids) were placed on a modified MS medium having 60 g / l of sucrose and 0.2-0.4 g / l of CCС. The culture vessels with explants were transferred to cooling chambers with a low positive temperature (2-6 ° C) and an illumination of 0.1-0.5 klx. It was found that for active adventitious shoot formation during cultivation of Dar Vostoka and Suevia explant varieties, it is necessary to add 1.27 mg / l of TDZ to the MS nutrient medium, and 1.91 mg / l of TDZ for the Livadia variety. It is shown that for the deposition of meristemoids of Dar Vostoka, Livadia and Suevia varieties, the nutrient medium of ½ MS with 0.15-0.25 g / l of CCC is optimal; for adventitious shoots of the Suevia variety, the optimal nutrient was ½ MS with 0.2 g / l of CCC.

In vitro organogenesis from Tinospora cordifolia (Willd.) Miers — A highly valuable medicinal plant

Efficient in vitro regeneration protocols were established for Tinospora cordifolia through direct and indirect organogenesis, using cotyledon (C), young leaf (YL) and mature leaf (ML) explants. Highest response of 97.9–100.0% organogenic callus was induced on Murashige and Skoog (MS) medium containing indole-3-acetic acid (IAA) at 2.0mg/L. Morphology of the callus varied from yellow friable to compact on auxin treatments. Shoot bud induction from callus was rapid on modified MS medium (mMS) containing IAA or 1-naphthalene acetic acid (NAA) with 6-benzyladenine (BA), kinetin (KN) and ascorbic acid (AA). Among the explants, cotyledons produced the highest shoot number (24.1 shoots) followed by YL (19.0) and ML (16.1) explants. Direct organogenesis was better on C than YL explant. Highest of 14.5 and 11.0 shoots were achieved on BA, KN, AA and IAA (0.5mg/L) from C and YL explants, respectively. Best shoot length of 8.3cm was achieved on MS medium containing gibberellic acid (GA3) at 0.5mg/L. All the shoots were rooted on MS medium at half strength macro salts (½ MS) with indole-3-butyric acid (IBA) 0.5mg/L and NAA 0.5mg/L. Rooted plantlets were successfully hardened under in vitro conditions and transferred to the glasshouse with 75% survival rate. This is the first report on successful organogenesis via callus and from different explants in T. cordifolia. The same protocols using different medium and plant growth regulators (PGRs) at different stages of organogenesis can be utilized for mass production to aid commercial needs and eco-restoration of this plant and to the related genera.

Microbial volatile compounds alter the soil microbial community

Volatile organic compounds (VOCs) from soil bacteria are likely to have an important role in the interactions among soil microorganisms. However, their effects on the soil microbial community have not been extensively studied. In this study, the effect of bacterial VOCs generated by growing Bacillus amyloliquefaciens NJN-6 on modified MS medium on soil microbial community was evaluated. B. amyloliquefaciens NJN-6 was able to produce 48 volatile compounds as determined by solid-phase microextraction-GC/MS. MiSeq sequencing data showed that bacterial VOCs could alter the composition of both soil bacterial and soil fungal communities and could decrease the alpha-diversity of the soil microbial community. Taxonomic analysis revealed that bacterial VOCs significantly increased the relative abundance of Proteobacteria, Bacteroidetes, and Firmicutes. Moreover, bacterial VOCs significantly increased the relative abundance of Ascomycota. The qPCR data showed that bacterial VOCs of strain NJN-6 decreased the soil fungal biomass and increased the soil bacterial biomass. Further evaluation of the effect of bacterial VOCs on functional genes revealed that VOCs could reduce the copies of nifH, nirS, and a gene encoding nonribosomal peptide synthase, while increasing the copy number of the ammonium-oxidizing bacteria gene. The effect on gene encoding polyketide synthase was insignificant. Results from this study indicated that bacterial VOCs could influence the soil microbial community as well as functional gene abundance.

The exploit of cereal embryo structure for productive reasons by in vitro techniques

There are two main sides of our works exploiting embryo structure in durum wheat and some other cereals. First is haploid (or doubled haploid) embryo production using anther or microspore culture or intergeneric crosses, to ameliorate desirable characters genetically homozygote. Secondly, to develope convenient embryo culture technique in order to be stored and cultivated longer time of genotypes without being alien pollination etc. in field conditions. For that reason, two different auxin and also their combination with kinetin were used for mature embryos of wheat genotypes (hexaploid and tetraploid), to understand efficient dose for calli production and plant regeneration in plant tissue culture. Modified MS media were used adding a single dose of arabinogalactan protein (AGP) and without adding for regeneration. In further step of this study, most efficient auxin+kinetin combination which is determined previous research, it was used in the same modified MS medium to produce calli production and plant regeneration in three different genotypes (hexaploid and tetraploid wheat and diploid barley). Data were calculated in five different developmental stages of treatments. All statistical analysis of data were performed and means were compared with Duncan's test. Genetics and morphological effects of AGP on genotypes were discussed with the results of variance analysis. Simple correlation coefficient (r) was calculated base on the main values of replications.

In vitro microrhizome induction in Hedychium stenopetalum Lodd.

Hedychium stenopetalum Lodd. is a potential species as a source for raw materials for pharmaceutical and cosmetic products. In this study, the possibility of in vitro microrhizome induction in H. stenopetalum was studied. Sterile young shoot tips were cultured on modified Murashige and Skoog medium (MS) (1962) supplemented with various concentrations of 6-benzylaminopurine (BAP) at 0, 1, 2, 3 and 4 mg L-1. Cultures were incubated under 16 h illumination day-1 at 25±2°C for 8 weeks. The 1 mg L-1 BAP supplemented MS medium increased both number of shoots explant-1 and shoot height with higher levels showing no further increase in number of shoots and decreased shoot height. Six-week-old in vitro plantlets were selected as materials for an experiment on microrhizome induction. Plantlets were cultured on modified MS medium supplemented with sucrose concentrations of 3, 6, 9 and 12% in combination with ancymidol at 0, 1, 2, 3 and 4 μM. The heaviest microrhizome production was obtained from medium supplemented with 6 and 9% sucrose. Ancymidol had no significant effect on microrhizome induction. These results will be used to develop protocols for large-scale production of uniform and pathogen-free H. stenopetalum microrhizomes for field plantations, germplasm conservation and large-scale production of rhizomes for the herbal cosmetics and pharmaceutical industries.

Effect of Light Quality, Sucrose and Trehalose on In Vitro Organogenesis of Cymbidium devonianum (Lindl.)

The present study investigated the effect of sucrose, trehalose and combination of sucrose and trehalose with modified MS medium on in vitro regulation of protocorm-like bodies (PLBs) of Cymbidium devonianum under different quality of lights (white fluorescent tube, green, red and blue LED). As a result of this study, new PLB and shoots were successfully regenerated on modified MS medium under different quality of lights. The highest PLB formation rate (100%) and the highest shoot formation rate (85%) were observed amongst explants cultured on medium supplemented with 10 g/l sucrose + 10 g/l trehalose under green LED. The maximum fresh weight of PLBs, the highest average number of PLBs and shoots were recorded on medium containing 10 g/l sucrose + 10 g/l trehalose under green LED. For plant tissue culture, sucrose is considered an indisputably important carbon and energy source and biosynthesis of trehalose is similar to that of sucrose. The hereby study concluded that the contribution of LED lights, sucrose and trehalose (combined) can induce PLB and shoot formation of Cymbidium devonianum tissue culture without the use of any other plant growth regulator, whereas the green light showed the best formation rate compare with the other studied qualities of lights.

The Issues of Apricot (Prunus Armeniaca L.) Micropropagation

The effect of four modified mediums for apricot multiplication was observed in this study. A total number of 1864 single nodes of 20 Prunus armeniaca L.varieties were established. Explants surface was disinfected with 0.2 % mercuric chloride for 5 minutes. MS (1962) medium with 0.5 mg.l−1 BA, 0.01 mg.l−1 NAA and 0.5 mg.l−1 GA3 was used as a medium for primary culture. ‘Velkopavlovická’, ‘Bergeron’, genotype 1128 and genotype LE 2927 Š9 were successfully transferred to aseptic conditions and multiplied. Modified MS medium (1962), DKW/Juglans medium, Quoirin, Lepoivre (1977) medium and Marino et al. (1991) medium were used for multiplication. Modified MS medium and modified DKW/Juglans medium were not suitable for apricot multiplication at all and explants did not grow. The best results were observed in the case of Quoirin, Lepoivre (1977) medium with 0.4 mg.l−1 BA and 0.01 mg.l−1 NAA. Young plants multiplied well, were fresh and vital and no damage was observed. The highest number of new shoots was observed in the case of Marino et al. (1991) medium. The average growth of new shoots after the last passaging was 600 %, rate 7.33 (Velkopavlovická); 566 %, rate 7.0 (Bergeron); 475 %, rate 6.25 (1128) and 483 %, rate 6.33 (LE 2927 Š9)). However, new shoots in clusters were too dense and stunted and this medium is not recommended for apricot multiplication.

Production of Δ7-Sterols from In Vitro Root Cultures of Endangered Gypsophila trichotoma

Species from the genus Gypsophila are known for their medicinal, industrial and decorative applications. G. trichotoma Wend. is an endangered plant species for the Bulgarian flora according to the Red Data Book. Δ⁷-Sterols, which are unusual and rare in the plant kingdom, are present in the roots of this species. In previous studies different in vitro cultures were established from aerial parts of the species. The objective of this study was to explore the possibility for production of Δ⁷-sterols from in vitro cultured roots of G. trichotoma. The root cultures were grown on six modified MS media and the quantity of sterols was analyzed. These findings will serve to solve the important matter of the role of nutrients on sterols biosynthesis.

Rescue and in vitro culture of herbaceous peony immature embryos by organogenesis

Herbaceous peony (Paeonia lactiflora Pall.) is a landscaping plant that has considerable development prospects worldwide. However, its embryo dysplasia severely limits the breeding of high-quality P. lactiflora cultivars by the cross-breeding method. In this study, the immature natural hybrid embryos of P. lactiflora ‘Zi Fengyu’ with double flower were used to study its rescue and in vitro culture by organogenesis. The results showed that the best medium for adventitious bud induction was modified Murashige and Skoog (MS) medium supplemented with 1.0mg/L thidiazuron (TDZ), 600mg/L acidic hydrolysis casein (ACH), 10% coconut water and 100g/L sucrose. The best medium for adventitious bud multiplication was modified MS medium supplemented with 4.0mg/L TDZ, 15% coconut water, 60g/L sucrose and 0.1% activated charcoal (AC). Modified MS medium containing 0.5mg/L gibberellic acid (GA3), 100g/L sucrose and 0.1% AC was the more suitable medium for adventitious bud growth. For root regeneration, indole-3-acetic acid (IAA) was a more effective plant growth regulator (PGR), and 1/2 MS medium supplemented with 0.5mg/L IAA, 30g/L sucrose and 0.1% AC was the more suitable medium, resulting in a rooting rate of up to 92.12%, and the survival rate was 70.8%. Additionally, histological observation confirmed that the rescue and in vitro culture of P. lactiflora immature embryos by organogenesis was successful. These results provide useful technical support for breeding high-quality P. lactiflora cultivars by the cross-breeding method.

In Vitro Mid-Term Conservation of Acorus calamus L. via Cold Storage of Encapsulated Microrhizome

ABSTRACT In vitro rhizome production, encapsulation and cold storage of Acorus calamus were attempted for its propagation and ‘true-to-type’ conservation. Shoot cultures were initiated using underground rhizome buds, on 6-benzyladenine (BA) containing Murashige and Skoog (MS) medium. Maximum microrhizome production was observed in presence of 33.3 µM BA, on modified MS medium containing 6% sucrose, 100 mg/L citric acid and 1 g/L polyvinyl pyrrolidone-40. Synthetic seeds were produced from regenerated microtubers by encapsulation in calcium alginate beads. These synthetic seeds were stored in complete darkness at 100C temperature for different durations for mid-term conservation. After cold storage, synthetic seeds were re-cultured in vitro, 100% survival was recorded after the storage of 1, 3 or 6 months; and 80% survival was observed after the storage of 12 months. The microrhizomes were produced roots in 4.9 µM indole-3-butyric acid containing half strength MS medium. All the regenerated plantlets were successfully transferred to field after acclimatization. It is the first report on successful one year in vitro cold storage of A. calamus.

Indirect Somatic Embryogenesis from Mature Inflorescence Explants of Date Palm.

Due to the limitations associated with shoot tip explants in the micropropagation of date palm, inflorescence explants are an ideal alternative. This chapter focuses on the protocol for the induction of callus from inflorescence tissue, establishment for proliferation of somatic embryos, germination, elongation, rooting, and acclimatization. Female inflorescences, 30-40cm in length, cv. Shaishee, were used for culture initiation. After disinfection, the outer inflorescence cover (spathe) is cut open, and the spikelet explants, 1cm long, are cultured on modified Murashige and Skoog (MS) medium containing 100mg/L 2,4-D, 3mg/L kinetin, and 3mg/L 2ip and incubated at 25±2°C in the dark. Callus obtained after 6-8months of culturing is transferred to the culture medium to induce somatic embryogenesis and plant regeneration. Well-developed regenerated shoots are cultured on MS medium containing 0.2mg/L NAA for root induction and plantlets acclimatized in the greenhouse before transfer to the field.

In-vitro Mutagenesis Induction to Improve Abiotic Stress in Tissue Cultured Plantlet of Picrohiza kurroa Royle ex. Benth: An Endangered Plant of Western Himalayas, India

The study is focused to establish an improved salt and drought tolerant Picrohiza kurroa Royle ex Benth, an endangered medicinal plant of Western Himalayas through mutagenesis system in conjunction with in- vitro regeneration technique. Regeneration using leaf as explants from mature plant of characterized germplasm is beneficial because the source plant is not damaged. Efficient callus formation i.e., 85% was achieved in modified MS medium at 2,4-D at 3.5 mg L-1. Ethyl methane sulfonate (EMS) a chemical mutagen was used to induce mutation in the callus biomass under in-vitro condition. Relatively decreases in callus biomass were observed as the dose of mutagenic chemical increased from 0.0 to 0.8 mM. Selection of mutants callus lines were also investigated against salt (NaCl) and drought (mannitol) tolerance level by using its various concentrations i.e., 50 to 250 mM L-1. In mutagenized callus (MC) under both stress responses there was increase in callus biomass with the successive increase in concentration of NaCl and Mannitol till 100 mM L-1, after that it started decline. Stress tolerated mutant callus line were also characterized by the accumulation of proline and glycine betaine (GB) content. At 100 mM L-1 concentrations of NaCl and mannitol, higher proline and GB content were accumulated in the mutagenized callus i.e., 5.23 and 5.18 µmol g-1 FW and 11.23 and 11.98 µg g-1 FW respectively which is significantly higher in comparison to non-mutagenized callus (UM). For shoot proliferation in mutant callus line, various concentrations with combination of plant growth regulators (PGR’s) were used in treatments (T1, T2 and T3). Invariably, in treatment T3 the concentration of 1.0+0.5 mg L-1 resulted in highest shoot regeneration i.e., 85% while minimum 20% was obtained in T1 at concentration 0.5+0.1 mg L-1. NAA fortified MS medium was found superior to IAA and IBA with respect to the induction of roots. The mutant and stress selected grown through tissue engineering were evaluated for their ex situ agronomic performance in saline as well as drought condition for 30 days. Under both stress condition the mutant plant revealed a remarkable increase in all the parameters studied i.e., shoot and root length, fresh and dry root, shoot biomass and number of leaves with respect to normal plant (control).

Indirect Somatic Embryogenesis of Date Palm Using Juvenile Leaf Explants and Low 2,4-D Concentration.

This chapter describes an efficient protocol for large-scale micropropagation of date palm. Somatic embryo-derived plants are regenerated from highly proliferating suspension cultures. Friable embryogenic callus is initiated from juvenile leaves using slightly modified Murashige and Skoog (MS) medium supplemented with 0.1mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures consisting of proembryonic masses are established from highly competent callus for somatic embryogenesis using half-strength MS medium enriched with 0.1mg/L 2,4-D and 300mg/L activated charcoal. The productivity of cultures increased 20-fold when embryogenic cell suspensions were used instead of standard protocols on solidified media. The overall production of somatic embryos mostly exceeds 10,000units per liter per month. Partial desiccation of mature somatic embryos, corresponding to a decrease in water content from 90 down to 75%, significantly improved germination rates.

Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition.

Cell growth medium composition has profound impacts on the O -glycosylation of a "designer" arabinogalactan protein-based module; full glycosylation is essential in directing efficient extracellular secretion of the tagged recombinant protein. Expression of recombinant proteins in plant cells as fusion with a de novo designed hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) tag, termed HypGP engineering technology, resulted in dramatically increased secreted protein yields. This is due to the function of the HypGP tag as a molecular carrier in promoting efficient transport of conjoined proteins into culture media. To optimize the cell culture to achieve the best secreted protein yields, the medium effects on the cell growth and protein secretion were investigated using as a model system the tobacco BY-2 cell expressing enhanced green fluorescence protein (EGFP) fused with a (SP)32 tag (32 tandem repeats of "Ser-Pro" motif). The (SP)32 tag was found to undergo two-stage Hyp-O-glycosylation in plant cells with the dramatic secretion of the conjoined EGFP correlating with the triggering of the second-stage glycosylation. The BY-2 cell culture in SH medium generated a high secreted protein yield (125mg/L) with a low cell biomass accumulation (~7.5gDW/L). In contrast, very low secreted protein yields (~1.5mg/L) with a high cell biomass accumulation (13.5gDW/L) were obtained in MS medium. The macronutrients, specifically, the nitrogen supply greatly impacted the glycosylation of the (SP)32 tag and subsequent protein secretion. Modified MS medium with reduced nitrogen levels boosted the secreted EGFP yields to 168mg/L. This study demonstrates the profound impacts of medium composition on the secreted yields of a HypGP-tagged protein, and provides a basis for medium design to achieve the highest productivity of the HypGP engineering technology.

Indirect Organogenesis and Somatic Embryogenesis of Pineapple Induced by Dichlorophenoxy Acetic Acid

<p>This research aimed to study the effect of 2,4-D,<br />AdS, and basal media to the regeneration of pineapple<br />through indirect organogenesis and somatic embryogenesis,<br />and to study the complete event of somatic embryogenesis.<br />Callus formation was induced by 21, 41, and 62 μM 2,4-D<br />with addition of 9 μM TDZ. The non embryogenic calli were<br />transferred onto 4.65 μM Kn containing medium.<br />Embryogenic callus formation was induced on MS or Bac<br />basal media consisted of N-organic compounds with<br />addition of AdS (0, 0.05 and 0.1 μM). The embryogenic calli<br />were regenerated on modified MS medium with addition of<br />0.9 μM IBA, 1.1 μM BA, 0.09 μM GA3 or MS medium<br />supplemented with 0.018 mM BA. The result proved that the<br />single auxin of 2,4-D was not enough to induce embryogenic<br />cells. Therefore the non embryogenic calli were regenerated<br />through organogenesis. The 21 μM 2,4-D yielded high level of<br />callus formation (80%), higher fresh weight (0.2 g/explant)<br />and higher number of shoot (25 shoots/explant in two<br />months). Embryogenic calli were produced on N-organic<br />compounds enriched media. The regeneration medium<br />significantly affected the level of browning, where the MS<br />medium with addition of 0.018 mM BA yielded lower level of<br />browning. There was an interaction of embryogenic callus<br />induction medium and regeneration medium to the number<br />of mature somatic embryos. The embryogenic callus<br />induction on MS medium enriched with N-organic<br />compounds and 0.05 μM AdS followed by the regeneration<br />of somatic embryos on MS medium with addition of 0.018<br />mM BA was the best treatment which yielded 17 mature<br />somatic embryos/explant.</p>