Advancing Coconut Micropropagation through Cell Suspension Cultures

Morus alba L. has been used in Asian traditional medicine as an anti-inflammatory, anti-asthmatic, anthelmintic and as a whitening agent in cosmetic products. Mulberroside A is the major active compound from M. alba root bark. In this study, cell suspension and root cultures of M. alba were established, and the effect of the elicitors on the enhancement of mulberroside A production in M. alba was investigated. The cell suspension and root cultures of M. alba were exposed to elicitors and then mulberroside A contents were determined by an indirect competitive ELISA method. High levels of mulberroside A were obtained by addition of 100 and 200 μM salicylic acid with 24 h exposure time in cell suspension cultures (37.9 ± 1.5 and 34.0 ± 4.7 mg/g dry wt., respectively). Furthermore, addition of yeast extract at 2 mg/mL with 24 h exposure time can significantly increase mulberroside A contents from both cell suspension (3.2-fold) and root cultures (6.6-fold). Mulberroside A contents from both cell suspension and root cultures after treatment with elicitors are similar or higher than those found in the intact root and root bark of several years old M. alba. These results indicate that mulberry tissue cultures using the elicitation method are interesting alternative sources for mulberroside A production.

Withania somnifera, is an important medicinal plant in India, also known as Ashwagandha, that contains the bioactive compounds- withanolides and withaferin A. The endophytic fungus Piriformospora indica has been shown to be an elicitor stimulating plant growth and metabolism. Different concentrations of cell homogenate, culture filtrate and individual culture discs of P. indica were added to cell suspension and callus cultures of W. somnifera at different time intervals (10, 15, 20, 25 and 5, 10, 15, 20 days respectively) to observe the effect on cell biomass and withaferin A production. Of all the concentrations of P. indica used to study the effect on withaferin A production in cell suspension cultures, the maximum enhancement was achieved with 3 % cell homogenate (2.04 times), followed by 3 % culture filtrate (1.78 times) and culture disc (1.46 times). Quantitative PCR analysis showed an effect of P. indica elicitation on the regulatory genes of MVA, MEP and withanolides biosynthetic pathways, viz. hmgr, fpps, ss, se, cas, dxs and dxr in callus and cell suspension cultures. The highest gene expression of 11.2, 8.7 and 6.9 times was observed with hmgr among all the expressed genes in cell suspension and callus cultures with 3 % cell homogenate, 3 % culture filtrate and disc respectively, in comparison with the controls.

Fast-growing callus, cell suspension and root cultures of Vernonia cinerea, a medicinal plant, were analyzed for the presence of alkaloids. Callus and root cultures were established from young leaf explants in Murashige and Skoog (MS) basal media supplemented with combinations of auxins and cytokinins, whereas cell suspension cultures were established from callus cultures. Maximum biomass of callus, cell suspension and root cultures were obtained in the medium supplemented with 1 mg/L alpha-naphthaleneacetic acid (NAA) and 5 mg/L benzylaminopurine (BA), 1.0 mg/L NAA and 0.1 mg/L BA and 1.5 mg/L NAA, respectively. The 5-week-old callus cultures resulted in maximum biomass and alkaloid contents (750 microg/g). Cell suspension growth and alkaloid contents were maximal in 20-day-old cultures and alkaloid contents were 1.15 mg/g. A 0.2-g sample of root tissue regenerated in semi-solid medium upon transfer to liquid MS medium containing 1.5 mg/L NAA regenerated a maximum increase in biomass of 6.3-fold over a period of 5 weeks. The highest root growth and alkaloid contents of 2 mg/g dry weight were obtained in 5-week-old cultures. Maximum alkaloid contents were obtained in root cultures in vitro compared to all others including the alkaloid content of in vivo obtained with aerial parts and roots (800 microg/g and 1.2 mg/g dry weight, respectively) of V. cinerea.

Establishment of hairy root cultures of Ammi majus

Fertile plants were regenerated from both cell suspension and protoplast-derived cultures of the two-row barley, Hordeum vulgare L. cv. Schooner. Embryogenic calluses, derived from immature embryos, were used to establish suspension cultures. More than 100 plants, with variable seed set, have been regenerated from six embryogenic cell suspension cultures. Protoplasts isolated from three suspension cultures divided and when the resultant embryogenic proto-calluses were transferred to regeneration medium both green and albino shoots were produced. The green shoots were transferred to growth regulator-free medium and plantlets that developed strong root systems were potted in soil and grown to maturity in the glasshouse. Root tip analysis of plants regenerated from cell suspension cultures revealed the expected 2N = 14 complement of chromosomes. However, chromosomal analysis of protoplast-derived plants showed numerical variation among a proportion of the regenerants.

Peroxidase production by cell suspension and hairy root cultures of horseradish ( Armoracia rusticana)

Callus cultures, cell suspension cultures and shoot cultures of Leonurus cardiaca L. (Motherwort) were established and growth conditions optimized. Shoot cultures showed constant growth whether in the dark or under continuous light, accumulating varying amounts of the furanic labdane diterpenes leosibiricin, preleosibirin, leosibirin and isoballotenol acetate, which are also present in the soil-grown plants. Only traces of leosibiricin were detected in callus cultures, while cell suspension cultures did not produce any furanic diterpenes. A small amount of furanic labdane diterpenes was found in the medium of shoot cultures. Callus and shoot culture induction of several other Lamiaceae species is also described.

For 18 sugarcane cultivars, four distinct callus types developed on leaf explant tissue cultured on modified MS medium, but only Type 3 (embryogenic) and Type 4 (organogenic) were capable of plant regeneration. Cell suspension cultures were initiated from embryogenic callus incubated in a liquid medium. In stage one the callus adapted to the liquid medium. In stage two a heterogeneous cell suspension culture formed in 14 cultivars after five to eight weeks of culture. In stage three a homogeneous cell suspension culture was developed in six cultivars after 10 to 14 weeks by selective subculturing to increase the proportion of actively dividing cells from the heterogeneous cell suspension culture. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures for up to 148 days of culture but plants could not be regenerated from homogeneous cell suspension cultures. High yields of protoplasts were obtained from homogeneous cell suspension cultures (3.4 to 5.2 × 106 protoplasts per gram fresh weight of cells [gfwt-1]) compared to heterogeneous cell suspension cultures (0.1 × 106 protoplasts gfwt-1). Higher yields of protoplasts were obtained from homogeneous cell suspension cultures for cultivars Q63 and Q96 after regenerating callus from the cell suspension cultures, then recycling this callus to liquid medium (S-cell suspension cultures). This process increased protoplast yield to 9.4 × 106 protoplasts gfwt-1. Protoplasts isolated from S-cell suspension cultures were regenerated to callus and recycled to produce SP-cell suspension cultures yielding 6.4 to 13.2 × 106 protoplasts gfwt-1. This recycling of callus to produce S-cell suspension cultures allowed protoplasts to be isolated for the first time from cell lines of cultivars Q110 and Q138.

A culture of callus cells has been developed from a transgenic line of tobacco which contains an introduced phyA-cDNA encoding phytochrome A. Suspension cultures of the cells were shown to accumulate a significant immunodetectable level of the heterologous phytochrome, but not of the native phyA-gene product. The red-irradiated form (Pfr) of the heterologous phytochrome was specifically degraded in vivo, and the red-irradiated (Pfr) and far-red-irradiated (Pr) forms demonstrated different patterns of in vitro proteolytic cleavage. These results strongly suggested that the phytochrome apoprotein was associated with a chromophore moiety which mediated red/far-red sensitive conformational changes of the molecule. Exogenous application of 4-amino-5-hexynoic acid (AHA) to the transgenic suspension cultures resulted in the accumulation of a population of phytochrome which was stable under red light and gave identical patterns of in vitro digestion in the red and far-red irradiated forms, i.e. the spectral activity of phytochrome was inhibited. Application of exogenous 5-aminolevulinic acid (ALA) or biliverdin overcame the inhibitory effects of AHA to restore spectral sensitivity of the phytochrome pool. These results are consistent with the proposed pathway of phytochrome chromophore biosynthesis in intact plant systems. Thus, the transgenic suspension cultures provided a single-cell system in which spectrally-active phytochrome, apparently indistinguishable from the native phytochrome synthesized in etiolated seedlings, was accumulated. Photoregulation of expression of the genes encoding the small subunit of ribulosc-1,5-tephosphate carboxylase and chlorophyll aib binding proteins demonstrated that the heterologous phytochrome population mediated rapid changes in gene expression in the de-differentiated cells. It is therefore proposed that such a suspension culture of transgenic cells offers a model system for the study of phytochrome function.

In this study, we compared two in vitro culture systems, dedifferentiated cell suspension culture and differentiated adventitious root culture, using inoculum from long-term (20-year culture period) and short-term (1-year culture period) cultures, for the production of ginsenosides in wild ginseng (Panax ginseng). Increases in biomass and ginsenoside content were monitored in a 3-L bioreactor. The biomass in short-term cell suspension and adventitious root cultures increased at a faster rate than that in the long-term cultures, reaching a peak after 4 and 8 weeks of culture, respectively. In long term cultured materials, the biomass of cell suspension and adventitious root cultures decreased by 1.67- and 1.52-fold, respectively. Although the amount of ginsenosides in cell suspension and adventitious root cultures varied during the culture periods, the total ginsenoside content remained constant. These data suggest that both culture systems are advantageous for the production of different ginsenosides for pharmaceutical purposes.

Alkannin, a red-purple dye and bioactive compound found in the roots of Arnebia hispidissima has antibiotic and anti-inflammatory properties and is also used in cosmetic and textile industries at a large-scale. In the present communication, we demonstrate the establishment of callus and cell suspension culture of A. hispidissima with the aim of optimizing the production of alkannin. Highest alkannin content was recorded in cell suspension and callus culture established on M-9 medium. Production of alkannin was influenced by the different culture medium. Evaluation of alkannin content of roots of field-grown plants and in vitro grown cell, tissue and organ showed that alkannin production was higher in all in vitro grown culture systems (cell suspension, callus and roots) than the roots of field-grown plants. The present investigation may be applicable in designing systems for the large-scale cultivation of A. hispidissima cell suspensions for the production of alkannin.

Elicited Teucrium chamaedrys cell cultures produce high amounts of teucrioside, but not the hepatotoxic neo-clerodane diterpenoids

Plants have been used for more than 20 years to produce recombinant proteins but only recently has the focus shifted away from proof-of-principle studies (i.e. is my protein expressed and is it functional?) to a serious consideration of the requirements for sustainable productivity and the regulatory approval of pharmaceutical products (i.e. is my protein safe, is it efficacious, and does the product and process comply with regulatory guidelines?). In this context, plant tissue and cell suspension cultures are ideal production platforms whose potential has been demonstrated using diverse pharmaceutical proteins. Typically, cell/tissue cultures are grown in containment under defined conditions, allowing process controls to regulate growth and product formation, thus ensuring regulatory compliance. Recombinant proteins can also be secreted to the culture medium, facilitating recovery and subsequent purification because cells contain most of the contaminating proteins and can be removed from the culture broth. Downstream processing costs are therefore lower compared to whole plant systems, balancing the higher costs of the fermentation equipment. In this article, we compare different approaches for the production of valuable proteins in plant cell suspension and tissue cultures, describing the advantages and disadvantages as well as challenges that must be overcome to make this platform commercially viable. We also present novel strategies for system and process optimization, helping to increase yields and scalability.

Properties of cell cultures containing the citrus exocortis viroid

In order to develop a sustainable source of metabolism-enhancing phytoecdysteroids, cell suspension and hairy root cultures were established from shoot cultures of wild-harvested Ajuga turkestanica, a medicinal plant indigenous to Uzbekistan. Precursors of phytoecdysteroids (acetate, mevalonic acid cholesterol) or methyl jasmonate (an elicitor) were added to subculture media to increase phytoecdysteroid accumulation. In cell suspension cultures, 20-hydroxyecdysone (20E) content increased 3- or 2-fold with the addition of 125 or 250 μM methyl jasmonate, respectively, compared to unelicited cultures. Precursor addition, however, did not provoke phytoecdysteroid accumulation. In hairy root cultures, addition of sodium acetate, mevalonic acid, and methyl jasmonate, but not cholesterol, increased phytoecdysteroid content compared to unelicited cultures. Hairy root cultures treated with 150 mg l−1 sodium acetate, or 15 or 150 mg l−1 mevalonic acid, increased 20E content approximately 2-fold to 19.9, 20.4 or 21.7 μg mg−1, respectively, compared to control (10.5 μg mg−1). Older hairy root cultures, extracted after the seventh subculture cycle, also showed increases in 20E content (24.8 μg mg−1), turkesterone (0.9 μg mg−1) and cyasterone (8.1 μg mg−1) compared to control cultures maintained for a shorter duration of four subculture cycles. Doses of 10 or 20 μg ml−1 hairy root extract increased protein synthesis by 25.7% or 31.1%, respectively, in a C2C12 mouse skeletal cell line. These results suggest that sustainable production of metabolically active phytoecdysteroid can be achieved through hairy root culture systems.