Micropropagation and field evaluation of micropropagated plants of turmeric

A protocol was developed for in vitro propagation of turmeric cv `elite' using young vegetative buds from sprouting rhizomes. The shoot buds produced multiple shoots when cultured on MS solid medium supplemented with benzyladenine and 1-naphthalene acetic acid. The effect of various cytokinins on shoot multiplication was studied by culturing the shoot tips on MS liquid medium supplemented with benzyladenine, benzyladenine riboside, kinetin, kinetin riboside, zeatin, 6-,-dimethylallylaminopurine, adenine, adenine sulfate or metatopolin each at 10 M in combination with 1-naphthalene acetic acid (1 M). Significant differences were observed between the treatments. Liquid medium was more favourable than agar medium for shoot multiplication. Among the various concentrations of agar tested, 0.4% and 0.6% were the best and produced the highest number of shoots per explant. Among the different carbohydrates tested, sucrose, fructose, glucose, sugar cubes, maltose, levulose and market sugar were found to be equally effective for shoot multiplication and xylose, rhamnose, lactose and soluble starch were inhibitory. Ninety five percent of the micropropagated plants survived in sterilized soil in paper cups and all of them survived in the field. Among 48 plants, two plants showed variegated leaves on the tillers. The micropropagated plants showed a significant increase in shoot length, number of tillers, number and length of leaves, number of fingers and total fresh rhizome weight per plant when compared with conventionally propagated plants. RAPD analysis of 11 regenerated plants using sixteen 10-mer primers did not show any polymorphism.     

Miscanthus×giganteus plant regeneration: effect of callus types,ages and culture methods on regeneration competence

The perennial rhizomatous grass, Miscanthus×giganteus is an ideal biomass crop due to its rapid vegetative growth and high biomass yield potential. As a naturally occurring sterile hybrid, M. ×giganteus must be propagated vegetatively by mechanically divided rhizomes or from micropropagated plantlets. Plant regeneration through somatic embryogenesis is a viable approach to achieve large‐scale production of plantlets in tissue culture. Effect of the callus types, ages and culture methods on the regeneration competence was studied to improve regeneration efficiency and shorten the period of tissue culture in M. ×giganteus. Shoot‐forming calli having a yellow or white compact callus with light‐green shoot‐like structures showed the highest regeneration frequency. Percentage of shoot‐forming callus induction from immature inflorescence explants was 41% on callus induction medium containing 13.6 μM 2,4‐d and 0.44 μM benzyladenine (BA). The use of a regeneration medium containing 1.3 μM NAA and 22 μM BA was effective at shortening the incubation period required for plantlet regeneration, with 69% of total regenerated plantlets obtained within 1 month of incubation on regeneration medium. Embryogenic‐like callus morphotype could maintain regeneration competency for up to 1 year as suspension cultures. Field grown regenerated plants showed normal phenotypic development with DNA content and plant heights comparable to rhizome propagated plants. Winter survival rates of the regenerated plants planted in 2006 and 2007 at the University of Illinois South Farm, Urbana‐Champaign, Illinois, were 78% and 56%, respectively.     

Domestication of micropropagated plants of the spice damiana (Turnera diffusa)

Tissue culture propagation was performed on the spice shrub damiana (Turnera diffusa. Willd.) using MS medium (Murashige and Skoog 1962) supplemented with different combinations of the plant growth regulators, 6-benzyl adenine (BA) and indole-3-butyric acid (IBA). Organogenesis of leaf explants from wild plants and explants from propagated cuttings was compared; only the former regenerated complete plants. The highest shooting rate (92%) occurred at a concentration of 10^–7 M BA plus 10^–6 M IBA. Regenerated shoots were rooted in MS medium without any plant growth regulators. Foliage productivity of the micropropagated plants under field cultivation was determined yearly over 3 years. The yield increased annually for the first two years. The quantity of essential oils in propagated plants was similar to that of wild plants growing nearby. We propose tissue culture propagation of damiana as a viable means of domestication of this wild plant for semi-arid agriculture in Mexico. Commercial propagation would help to conserve wild populations of damiana that are currently threatened by overharvesting.Abbreviations BA 6-benzyl adenine - IBA indole-3-butyric acid     

In vitro and ex vitro evaluation of long-term micropropagated turmeric as analyzed through cytophotometry,phytoconstituents, biochemical and molecular markers

Turmeric (Curcuma longa L.), a high valued medicinal plant, was micropropagated through induction of multiple shoots using latent axillary buds of rhizome. Cytophotometric and random amplified polymorphic DNA (RAPD) as well as inter simple sequence repeats (ISSR) analysis were used to periodically monitor the genetic stability of micropropagated clones of Curcuma longa conserved in vitro up to 7 years at every 6 months interval. A total of eighteen RAPD and eight ISSR primers gave 45,537 distinct and reproducible bands, monomorphic across all 353 plants analyzed. Micropropagated turmeric after being conserved for 7 years in vitro was transplanted into soil in field. Drug yielding potential of tissue culture derived plants was evaluated in field through estimation of phytoconstituents like curcumin and essential oil contents. The result of 2 years of field trial showed that micropropagated turmeric retained stability in all the characteristics examined when compared with the field performance of conventionally propagated plants. Thus long term conservation of an elite genotype of turmeric with epigenetic and genetic stability is significant for stable supply of drug i.e., curcumin and essential oil to the market.     

Study on the chemical composition and antioxidant activity of extracts from shoot culture and regenerated plants of <Emphasis Type="Italic">Scutellaria altissima</Emphasis> L.

The flavonoid (baicalin, wogonoside, luteolin, luteolin-7-glucoside) and verbascoside contents of Scutellaria altissima in both shoot cultures, and the shoots and roots of micropropagated plants grown in the greenhouse for 12 weeks or in the field for 2 years were determined. The level of secondary metabolites was found to be strongly affected by the age and type of plant organ. A comparative analysis of S. altissima plants propagated in vitro and from seeds revealed no differences in the level of secondary metabolites when plants of the same age were studied. The antioxidant potential of methanolic extracts from shoot cultures, and the shoots and roots of S. altissima plants propagated in vitro, were evaluated using ABTS radical scavenging, FRAP metal reduction power and the lipid peroxidation test, in relation to the content of baicalin, wogonoside, verbascoside, total phenolic and total flavonoid compounds. Extracts from the roots of field-grown regenerated plants at the flowering stage were found to possess the strongest antioxidant activity. Correlation analysis revealed that the antioxidant activity of extracts correlated most closely with their total phenolic content estimated by the Folin-Ciocalteu method.     

Regeneration and assessment of genetic fidelity of the endangered tree Moringa peregrina (Forsk.) Fiori using Inter Simple Sequence Repeat (ISSR)

Moringa peregrinais an endangered species of Moringaceae.M. peregrinais a multipurpose tree with a wide variety of potential uses including its medicinal activity. In our study, a rapid and efficient micropropagation protocol for M. peregrina has been established. In vitro germinated seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different levels of either 6-benzyladenine (BA) or kinetin (Kin). The maximum shoot proliferation of 6.5 shoots per explant with 100 % shoot proliferation rate was observed on MS medium supplemented with 1.0 mg/l BA. On the other hand, MS medium supplemented with 1 mg/l indole-3-butyric acid (IBA) resulted in the maximum number of roots. Micropropagated plants were successfully acclimatized. Genetic stability of micropropagated plants was assessed using Inter-Simple Sequence Repeat (ISSR). The amplification products were monomorphic in all in vitro grown plants. No polymorphism was detected indicating the genetic integrity of in vitro propagated plants. This micropropagation protocol could be useful for raising genetically uniform plants for plant propagation and commercial cultivation.     

Rapid in vitro adventitious shoot propagation of <Emphasis Type="Italic">Scopolia parviflora</Emphasis> through rhizome cultures for enhanced production of tropane alkaloids

A rapid micropropagation system for Scopolia parviflora Nakai (Solanaceae), a rare medicinal plant native to Korea, was established using rhizome cultures. Shoots that originated from adventitious shoots of the rhizome were multiplied when the rhizomes were cultured on half-strength B5 liquid medium supplemented with various growth regulators. Optimum shoot multiplication was observed in half-strength B5 medium containing 3% (w/v) sucrose and 5.77 M gibberellic acid (GA₃). Each rhizome gave rise to an average of 12 shoots. Shoot elongation and root induction from multiple shoots occurred on growth regulator-free half-strength B5 solid medium. Healthy plantlets were transferred to a peat moss:vermiculite mixture for acclimatization, which was successful. The concentrations of tropane alkaloids, hyoscyamine and scopolamine were determined in different tissues of native growing plants, in vitro-propagated plants and acclimatized plants by high-performance liquid chromatography. The analysis revealed that the levels of hyoscyamine and scopolamine were higher in in vitro-propagated plants than in the native growing plants. When the rhizome was cut into segments and transferred to optimal culture conditions for multiple shoot propagation, only 12 weeks were required to produce a mature plant. We conclude that in vitro propagation techniques through rhizome cultures provide an efficient and rapid method for shoot propagation of S. parviflora.Abbreviations BA Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - HPLC High-performance liquid chromatography - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid     

CCC and Prohexadione-Ca Enhance Rhizome Growth and Lateral Bud Production in Rhubarb (<Emphasis Type="Italic">Rheum rhabarbarum</Emphasis> L.)

Accelerating rhizome growth is crucial to enhancing propagule production in rhubarb (Rheum rhabarbarum L.) because the crop is propagated through rhizome divisions. This can be achieved through manipulating source-sink activity. This study tested the hypothesis that synthetic plant growth retardants Prohexadione-Ca and CCC enhance rhizome growth in rhubarb. Two different concentrations of these plant growth retardants and GA₃ (positive control) were foliarly applied on the cultivar German Wine at three stages of shoot growth under greenhouse conditions. Both Prohexadione-Ca and CCC favorably enhanced rhizome growth through suppressing shoot growth. CCC at 3000 mg L⁻¹ produced the best results and the effect was apparent when applied at 12 weeks after shoot emergence. The rhizome diameter, fresh weight, and the number of viable buds were enhanced significantly in plants sprayed with CCC 3000 mg L⁻¹. Both Prohexadione-Ca and CCC were equally effective in enhancing dry mass and starch allocation preferentially toward the rhizome. Prohexadione-Ca- and CCC-induced rhizome growth enhancement could possibly be due to their known role as GA biosynthesis inhibitors or through increasing photosynthetic efficiency and preferentially reallocating carbohydrates to the rhizome.     

Genetic stability, <Emphasis Type="Italic">ex vitro</Emphasis> rooting and gene expression studies in <Emphasis Type="Italic">Hagenia abyssinica</Emphasis>

Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic stability of 80 micropropagated Hagenia abyssinica plants, 40 of axillary origin and 40 of adventitious origin. The shoots were isolated from the same mother tree and micropropagated for over two years. Among the 83 RAPD primers screened, 16 gave reproducible band patterns. These 16 primers produced 115 bands for each plant. One plant from axillary origin showed two unique bands with primer OPC-11. All other plants showed identical band patterns. Generally, there was no significant difference in the shoot multiplication rate between shoots of axillary and adventitious origin. Indole-3-acetic acid (IAA) resulted in better ex vitro rooting compared to indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA). Non-micropropagated plants that were grown in the greenhouse for about one year were better in ex vitro rooting compared to those of juvenile material and mature tree derived micropropagated plants of the same treatment. Adventitious rooting related oxygenase gene (ARRO-1) isolated from apple (Malus domestica) was not expressed in H. abyssinica using a complementary DNA representational difference analysis fragment (cDNA RDA14) as a probe.     

Plant regeneration through shoot formation from callus of Areca catechu L.

In order to establish and optimize an in vitro micropropagation protocol of Venus fly trap (Dionaea muscipula Ellis), a carnivorous plant, the effects of medium type, MS medium concentration, pH, and cytokinin and auxin types on shoot proliferation and root formation were investigated using 3-month-old shoots. The shoot proliferation was most effective in 2.3 M kinetin-supplemented 1/3MS medium at pH 5.5. The best conditions for rooting were 1/3MS medium supplemented with 0.5 M IBA. All subcultured shoots produced extensive root systems after 5–6 weeks culture. When plantlets after rooting were planted in plastic pots filled with 1:1 peat moss and sand, the survival rate of plantlets was almost 100%, exhibiting normal development. With subculture every 8 weeks, hundreds of the plants were propagated from a single plant within a year.     

High-frequency clonal propagation of <Emphasis Type="Italic">Curcuma angustifolia</Emphasis> ensuring genetic fidelity of micropropagated plants

A high-frequency clonal propagation protocol was developed for Curcuma angustifolia Roxb., a high valued traditional medicinal plant. Axillary bud explants of C. angustifolia were explanted on Murashige and Skoog (MS) medium fortified with 4.4–22.2 µM 6-benzyladenine (BA), 2.9–5.7 µM indole-3-acetic acid (IAA), 2.3–23.2 µM kinetin (Kin), 2.7–5.4 µM naphthalene acetic acid (NAA) and 67.8-271.5 µM adenine sulphate (Ads) in different combinations. The maximum number of shoots per explants (14.1?±?0.55) and roots per shoot (7.6?±?0.47) was achieved on media containing 13.3 µM BA, 5.7 µM IAA and 135.7 µM Ads. Stability in phytomedicinal yield potential of micropropagated plants was assessed through GC–MS and HPTLC. Gas chromatogram of essential oil of conventional and micropropagated plants of C. angustifolia had similar essential oil profile. HPTLC analysis of rhizome extracts of in vitro and field grown plants revealed no significant differences in the fingerprint pattern and in curcumin content. Genetic integrity of in vitro and field grown derived plants were evaluated with inter-simple sequence repeat (ISSR) primers and flow cytometry using Glycine max as an internal standard. A total of 1260 well resolved bands were generated by 12 ISSR primers showing monomorphic banding patterns across all plants analyzed. The mean 2C DNA content of conventionally and micropropagated plant was estimated to be 2.26 pg and 2.31 pg, respectively. As no somaclonal variations were detected in tissue culture plantlets, the present micropropagation protocol could be applied for in vitro conservation and large-scale production of C. angustifolia.     

Comparative study of morphological parameters of Grand Nain banana (<Emphasis Type="Italic">Musa</Emphasis> AAA) after <Emphasis Type="Italic">in vitro</Emphasis> multiplication with growth retardants

Variation in morphological traits and increased disease susceptibility were observed in micropropagated plants of rhubarb (Rheum rhaponticum L.), PC49. Our investigations have demonstrated that micropropagated plants can vary substantially in morphological traits and the variation of quantitative traits was substantially greater than conventionally propagated plants. Micropropagated plants produced significantly more leaves than conventional plants under the same growth period, with a more bushy growth habit and/or recumbence. A higher incidence of disease (petiole spotting) was also observed in micropropagated plants. It is concluded that micropropagated PC49 had substantially higher incidence of somaclonal variation and regenerants were not suitable to establish an economic crop comparable to the conventional plants.     

Plant regeneration from mesophyll protoplasts of Centaurea cyanus,Senecio x hybridus and Callistephus chinensis

Protoplasts were isolated from leaves of glasshouse-grown plants of Centaurea cyanus and axenic shoot cultures of Senecio x hybridus. Upon culture, using modified MS-based media, protoplasts of both systems entered division to produce callus, followed by plant regeneration. Leaf protoplasts of Callistephus chinensis entered sustained division only following the preconditioning for 24h of peeled leaf tissues on agar-solidified MS-based medium. Protoplasts were also isolated from cell suspensions of C. chinensis and divided in MS-based or KM media. However, only leaf mesophyll protoplasts of Callistephus produced callus, which developed shoots.The establishment of protoplast-to-plant protocols for these ornamental species has provided a basis for broadening their gene pools through somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - KM Kao and Michayluk (1975) - g.f.wt. gram fresh weight     

Screening and selecting arbuscular mycorrhizal fungi for inoculating micropropagated apple rootstocks in acid soils

Santa Catarina state is the largest producer of apples in Brazil. Soils in this region have low pH and high levels of aluminum and manganese, requiring high inputs of fertilizers and amendments increasing costs of apple production. Inoculation of arbuscular mycorrhizal fungi can improve the establishment of micropropagated apple plants in such adverse soil conditions. Soil samples were collected from apple orchards in the Caçador, Fraiburgo and São Joaquim regions to develop a corn bioassay to identify mycorrhizal communities with high infectivity. Eleven fungal species were identified from one Caçador soil with the highest infectivity. Glomus etunicatum SCT110, Scutellospora pellucida SCT111, Acaulospora scrobiculata SCT112 and Scutellospora heterogama SCT113 were brought into single-species culture and used in a plant growth and nutrient uptake experiment using micropropagated apple (Malus prunifolia), cultivated at three soil pH. Colonization by fungal isolates significantly affected plant height, shoot and root dry weights, and root:shoot ratio. Soil pH also significantly affected all growth parameters except shoot dry weight. Mycorrhizal inoculation also significantly altered tissue concentrations of P, Zn, Cu, Ca, S, Na, N, K, Fe and Al. Association with mycorrhizal fungi increased P concentration and also decreased Al concentrations in the shoots. Overall, G. etunicatum and S. pellucida were the most effective isolates to promote plant growth and nutrient uptake. Inoculation of apple rootstock with selected fungal isolates during the acclimatization stage represents a useful strategy for producing micropropagated apples that can withstand acidic soil conditions.     

Micropropagation of Hemidesmus indicus for cultivation and production of 2-hydroxy 4-methoxy benzaldehyde

Caulogenic responses of various explant types from 12-month-old plants of Hemidesmus indicus were tested. Second and third visible nodes (0.5 cm) from the apex and root segments (0.5 cm) were the most and least regenerative respectively, with the formation of 9.37 and 2.6 shoots in 4 weeks on half strength MS medium supplemented with 2.22 μM BA and 1.07 μM NAA and 4.44 μM BA and 2.69 μM NAA respectively. Caulogenic ability of the nodes decreased with increasing maturity. Shoot buds initiated upon the young nodes on day 10 developed into 7.2 cm long shoots within 4 weeks thereby making a shoot elongation phase unnecessary. Nodal explants of the in vitro raised shoots subcultured in the same medium produced 9.32 shoots of 7.1 cm length in 3–4 weeks, similar to those of the mature plant-derived nodes. Multiplication through subculture of the nodes up to 25 passages of 4 weeks each was achieved without decline. Shoot cultures were rooted in quarter salt strength MS medium containing 9.8 μM IBA and the rooted plants were hardened for establishment in pots at 96% rate. Four months after establishment, the micropropagated plants were stable and showed uniform morphological and growth characteristic. After 12 months of cultivation in the field, on an average micropropagated plant consisted of 4–5 shoots, 5–8 branches per shoot and increased root biomass (13.5 g) compared to the poor growth (single shoot and 2–3 branches) and root production (4.6 g) values obtained with plants raised from conventional rooted stem cuttings. The concentration of the root specific compound, 2-hydroxy 4-methoxy benzaldehyde per plant was 2–3 fold higher in micropropagated plants though on unit dry root biomass (0.12% per g dry wt) basis it remained the same between two sources of plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Conservation and Propagation of High Altitude Medicinal and Aromatic Plant: Hedychium spicatum

The micropropagation of H.spicatum, a medicinal and aromatic plant was investigated as an option for conservation and propagation, as wild populations are fast depleting. The source of raw material is rhizomes of plants that are collected from the wild. There is no planned cultivation of the plant. Multiple shoot cultures were established on MS medium supplemented with BAP and IAA from the pre-existing buds on the rhizome. Prolonged cultivation on the same medium or transfer to hormone free medium induced roots/rhizome formation; liquid medium proved more suitable. Greenhouse hardened plants were transferred to field. A successful protocol with 99% root formation and 80–85.5% field survival has been formulated.     

Factors influencing in vitro multiplication and rooting of peach cultivars

Success at propagating peach (Prunus persica (L.) Batsch) scion cultivars in vitro has been limited. This study describes factors influencing in vitro multiplication and rooting of 8 peach scion cultivars and one rootstock, as well as acclimatization and genetic stability of these cultivars. Shoot multiplication was best when 8.8 M 6-benzylamino purine (BA) was added to the shoot proliferation medium. Maximum rooting occurred when shoots were placed on 1/2-strength Murashige and Skoog (MS) medium, stored in the dark at 4°C for 35 to 40 days and then incubated on rooting medium in the dark at 26°C for 14 days. All cultivars exposed to 1/2-strength MS medium supplemented with 28.5M of either indoleacetic acid (IAA), indolebutyric acid (IBA) or -napthaleneacetic acid (NAA) rooted best on NAA medium. A 5-fold reduction in NAA concentration to 5.8 M, the use of IAA plus phenolic rooting cofactors, and length of time shoots were in vitro prior to rooting each increased the percentage of rooting for most cultivars. No plant loss occurred during acclimatization. Cytogenetic analysis of micropropagated plants indicated that all plants were diploid, 2n=2x=16. Examination of the performance of in vitro propagated plants under field conditions is now in progress.     

Effect of in vitro shoot multiplication and somatic embryogenesis on 5-methylcytosine content in DNA of Myrtus communis L.

Reversed-phase HPLC analysis of 2-deoxynucleosides was performedto study the amount of 5-methylcytosine in genornic DNA of Myrtuscommunis L. About 11% cytosines were found to be methylated inDNA of field growing shoots. This amount showed no variation after theestablishment of shoots in vitro or their subsequentrooting and acclimatisation to ex vitro conditions.Therefore, adult elite plants can be micropropagated and transferred to thefield without global DNA methylation changes. Likewise, no trend in5-methylcytosine content was introduced by increasing the number of subculturesin either adult- or seedling-originated shoot stocks. On the other hand, nodifference was found in DNA methylation after plant regeneration fromembryogenic calli. The significance of a tissue culture model system with noglobal hypo- or hypermethylation is discussed.     

Micropropagation of Ranunculus Asiaticus: A Review and Perspectives

Ranunculus asiaticus is an important ornamental species mainly cultivated in the countries surrounding the Mediterranean sea. So far the multiplication of this plant has been mainly carried out by seed and rhizome division; however, these systems present many drawbacks. Tissue culture is an attractive alternative for accelerated propagation of selected and indexed genotypes. In this paper we review the work carried out on in vitro culture of R. asiaticus and present a flow chart for its commercial production, using axillary budding and embryogenesis. Although the price of micropropagated plants is higher compared to traditional material (seedlings and rhizomes from seed populations), it should be considered that tissue cultured plants have a better rhizome yield per plant; moreover, the tissue culture approach allows to offer clonal material of selected lines.