Artemisinin production by shoot regeneration of Artemisia annua L. using thidiazuron

An efficient in vitro method for multiple shoot bud induction and regeneration has been developed in Artemisia annua L. using leaf and stem explants in various concentrations and combinations of plant growth regulators to evaluate the frequency of regeneration. The sources of explants as well as plant growth regulators in the medium were found to influence the multiple shoot induction. The result shows that the stem segment cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l thidiazuron (TDZ) gave a perfect shoot formation (100%) and good shoot multiplication (57 shoots/explant) after 2 weeks of culture. Healthy regenerated shoots were elongated and rooted in MS medium without hormones. The artemisinin content in plants regenerated from stem explants using 0.1 mg/l TDZ was (3.36 +/- 0.36) microg/mg dry weight and two-fold higher than that of in vitro grown plants of the same age [(1.73 -/+ 0.23) microg/mg DW]. This system exhibited a potential for a rapid propagation of shoots from the stem explant and makes it possible to develop a clonal propagation of A. annua.     

Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Helianthus annuus L.

We have developed a highly efficient three-stage protocol for plant regeneration in sunflower (Helianthus annuus L.) from embryonal cotyledons. This protocol uses phenylacetic acid (PAA) for both shoot-bud induction and the elongation of smaller buds. The medium used for inducing bud formation from the cotyledons was modified MS medium supplemented with 3 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l PAA. Buds were elongated on MS medium supplemented either with only 0.2 mg/l gibberellic acid (GA₃) or with 0.2 mg/l GA₃ + 0.1 mg/l PAA + 0.3 mg/l BAP. The elongated shoots were then transferred onto rooting medium containing 1 mg/l PAA. The complete plantlets with well-developed roots were transferred to field conditions where they survived and set normal seeds. The induction of shoot buds from embryonal cotyledons was also observed on modified MS medium supplemented with 0.5-5 mg/l BAP in combination with 0.5-5 mg/l !-naphthaleneacetic acid (NAA). In this case, the formation of callus took place along with shoot-bud formation, which hindered further development of the latter. The presence of PAA with BAP in the primary bud induction medium promoted normal development and elongation of shoot buds.     

Improved plant regeneration in Capsicum annuum L. from nodal segments

Multiple shoots were induced by culturing nodal explants excised from 1-month-old aseptic seedlings of red pepper (Capsicum annuum L. cv. Pusa Jwala) on Murashige and Skoog (MS) medium supplemented with (0.1–10 μM) thidiazuron (TDZ). The rate of multiple shoot induction per explant was maximum (14.4 ± 0.06) on MS medium supplemented with 1.0 μM TDZ. Regenerated shoots were elongated well on growth regulator free MS medium. Adventitious roots were induced two weeks after transfer of elongated shoots to MS medium supplemented with auxins (IAA, IBA or NAA) in different concentrations. Optimum root formation frequency was obtained in medium containing 1.0 μM IBA. Ex-vitro rooting was also achieved by pulse treatment with 300 μM IBA for 10 min. Rooted shoots were transplanted in plastic pots containing garden soil (with 90 % survival rate), where they grew well and attained maturity. Regenerated plants were phenotypically and cytologically normal.     

Factors influencing the regeneration capacity of oilseed rape and cauliflower in transformation experiments

The efficiency ofAgrobacterium-based transformation technique in oilseed rape and cauliflower was influenced by cultivar specificity, donor plant age and explant type. Marked differences in demands for plant hormone contents in the regeneration medium were recorded already among different types of nontransformed explants. The highest regeneration capacity was recorded with stem and leaf segments isolated from one-month-old aseptically grown plants. The regeneration was markedly species-dependent. Regeneration of transformed plants from stem segments and thin layers isolated from field-grown oilseed rape plants (at the most 2% of regenerating explants) and from oilseed rape hypocotyls (0.8% of regenerating explants) and cauliflower (1.2% of explant regenerated transformed shoots) was achieved after disarmedAgrobacterium treatment. Hypersensitive reaction of explants could be prevented by using prolongedin vitro precultivation and delayed application of the selective agent.     

Micropropagation of Acacia mearnsii from ex vitro material

Multiple shoots were produced from nodal explants, of 30-day-old in vitro grown seedlings and from pretreated 3- and 9-month-old greenhouse grown Acacia mearnsii plants, respectively. Explants were sterilized using 0.1% and 0.2% HgCl2 for 15 min for 3- and 9-month-old explants, respectively. Nodal explants were induced to form multiple shoots when placed on MS medium supplemented with 2.0 mg l –1 benzyladenine. Rooting of these shoots was achieved on MS medium supplemented with 1.0 mg l –1 indole-3-butyric acid. Plantlets were acclimatized in transparent plastic containers under greenhouse conditions with a 90% success rate.     

Efficient regeneration and antioxidant potential in regenerated tissues of <Emphasis Type="Italic">Piper nigrum</Emphasis> L.

The organogenic potential and antioxidant potential (1, 1-diphenyl-2-picrylhydrazyl-scavenging activity) of the medicinal plant Piper nigrum L. (black pepper) were investigated. Callus induction and shoot regeneration were induced from leaf explants of potted plants cultured on MS medium supplemented with different plant growth regulators. The best callogenic response was observed on explants cultured for 30 days on MS medium supplemented with either 0.5 or 1.5 mg l⁻¹ 6-benzyladenine (BA) + 1.0 mg l⁻¹ α-naphthaleneacetic acid. Subsequent transfer of the callogenic explants onto MS medium supplemented with 1.5 mg l⁻¹ BA + 1.0 mg l⁻¹ gibberellic acid (GA₃) achieved 85% shoot organogenesis after 30 days of culture. The maximum number (7.2) of shoots/explant was recorded for explants cultured in MS medium supplemented with 1.0 mg l⁻¹ BA. Following the transfer of shoots to an elongation medium, the longest shoots (5.4 cm) were observed on MS medium supplemented with 1.0 mg l⁻¹ BA + 1.0 mg l⁻¹ GA₃. The elongated shoots were rooted on MS medium supplemented with different concentrations of indole butyric acid. An assay of the antioxidant potential of the in vitro-grown tissues revealed that the antioxidant activity of the regenerated shoots was significantly higher than that of callus and the regenerated plantlets.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

In vitro plant regeneration from cotyledon explants of Swainsona salsula Taubert

An effective protocol has been developed for plant regeneration from cotyledon explants of Swainsona salsula Taubert (Saline swainsona), a medicinal and agronomic shrub. Adventitious shoots were obtained from 83.2% of cotyledon explants from 3-day seedlings cultured on Murashige and Skoog (MS) medium containing 2.0 mg l⁻¹ thidiazuron (TDZ), with an average of 9.3 shoots per explant. Individual elongated shoots were rooted on half strength MS medium supplemented with 2.0 mg l⁻¹ indole-3-butyric acid (IBA), with 59.3% success. Regenerated plants with well developed shoots and roots were successfully transferred to soil, without detectable variants. Histological observation revealed that shoots developed from cotyledon explants via organogenesis, with little callus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic Embryogenesis and Plant Regeneration in Kinnow Mandarin

Somatic embryogenesis and plant regeneration from stem explants of kinnow mandarin, a hybrid of king and willow mandarins, are reported. It is an economically important cash crop of India with great deal of production and export. This is for the first time that callus induction and internodal regeneration has been successfully achieved in this citrus cultivar. Callus was induced from nodal segment (1-2 cm) of kinnow mandarin on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) and benzyl amino purine (BAP). Cotyledonary stage somatic embryos with regenerated shoots were induced on BAPenriched medium. Roots developed when regenerated shoots were excised and cultured on half strength MS medium supplemented with NAA only.     

Efficient In vitro Plant Regeneration Protocol from Leaf Explant of Jatropha curcas L — A Promising Biofuel Plant

An efficient in vitro plant regeneration from leaf-disc culture of Jatropha curcas L has been established. Adventitious shoot buds along with callus were induced from leaves of 2-year-old J. curcas plants cultured on Murashige and Skoog’s (MS) medium supplemented with TDZ (2 μM) BAP (2 μM) and IBA (1 μM), wherein 63.3% leaf explants responded. The multiplication of shoots was achieved from the adventitious shoot buds after transferring them to shoot induction medium. The highest number of shoots (9.7/explant) was achieved after 6 weeks of culture on MS medium containing 3 μM of BAR The welldeveloped shoots were rooted on MS medium supplemented with IBA (1.5 μM) with the rooting frequency of 53.3%. Addition of phloroglucinol (200 μM) to the medium enhanced the frequency of rooting to 76.7%. Regenerated plantlets were successfully transferred to field after initial acclimatization.     

Callus induction and plant regeneration from different explant types of Miscanthus x ogiformis Honda ‘Giganteus’

Different explants of Miscanthus x ogiformis Honda Giganteus were tested in order to develop an efficient tissue culture system. Shoot apices, leaf and root sections from in vitro-propagated plants, and leaf and immature inflorescence sections from 6-month-old greenhouse-grown plants were used. The explants were cultured on Murashige and Skoog medium supplemented with 4.5, 13.6, 22.6 or 31.7 M 2,4-dichlorophenoxyacetic acid. Three types of callus were formed but only one was embryogenic and regenerated plants. Callus induction and formation of embryogenic callus depended on the type and developmental stage of the explants. Shoot apices formed the highest percentage of embryogenic callus. There was a difference in the formation of embryogenic callus between leaf explants from in vitro-propagated shoots and greenhouse-grown plants. The best results were obtained from newly formed leaves of in vitro-propagated shoots and older leaves of greenhouse-grown plants. Immature inflorescences smaller than 2.5 cm produced a higher percentage of embryogenic callus than larger more mature inflorescences. Embryogenic callus derived from immature inflorescences had the highest regeneration capacity. Differences in 2,4-dichlorophenoxyacetic acid concentrations had no significant effect on callus induction, embryogenic callus formation and plant regeneration.Abbreviations MS Murashige & Skoog - 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzyladenine - NAA 1-naphthaleneacetic acid - PPFD photosynthetic photon flux density     

In vitro regeneration in Primula ssp. via organogenesis

Culturing pedicle segments of primroses on a medium supplemented with 2,4-dichlorophenoxyacetic acid (2, 4-D) and thidiazuron (TDZ) resulted in callus induction rates of about 80%. The highest shoot regeneration rate (1.8 shoots per explant; mean of ten genotypes) was achieved with the combination of 2.0 mg/l 2, 4-D and 2.0 mg/l TDZ. Culture on a medium containing a high concentration of nitrate (for example, B5 medium) negatively affected the survival of regenerated shoots of one genotype, Gelb IV 48, probably due to an increase in the pH value of the medium. Consequently, the highest efficiency was obtained using a basal medium containing half-strength Murashige and Skoog macroelements. A protocol to regenerate shoots of Primula vulgaris and P. elatior is described.     

Genetic tranformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp) using Agrobacterium tumefaciens

Mature de-embryonated cotyledons with intact proximal end of Vigna unguiculata were cultured on B5 basal medium containing varying concentrations of BAP. Thirty-six percent of the explants produced shoots on B5 medium supplemented with 8× 10^–6 M BAP. Cotyledon explants were pre-incubated for 24 h, inoculated with A. tumefaciens pUCD2614 carrying pUCD2340, co-cultivated for 48 h and transferred to hygromycin-B (25 mg/l) containing shoot induction medium. Approximately 15–19% of the explants produced shoots on the selection medium. The elongated shoots were subsequently rooted on B5 basal medium containing hygromycin. The transgenic plants were later established in pots. The presence of hpt gene in the transgenic plants was confirmed by Southern blot hybridization.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - hpt hygromycin phosphotransferase - IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

Plant Regeneration from Decapitated Mature Embryo Axis and Agrobacterium Mediated Genetic Transformation of Pigeonpea

A reliable method of plant regeneration has been achieved from decapitated mature embryo axes (DCMEA) explants. Shoots appear directly from explants of genotype T-15-15 when cultured on Maheswaran and Williams (EC₆) basal medium supplemented with N⁶-benzylaminopurine (BAP) and indole-3-acetic acid (IAA) at various combinations. The shoots elongated on half strength Murashige and Skoog (MS) medium fortified with 3 μM gibberellic acid. Elongated shoots were rooted with 80 – 85 % efficiency on half strength MS medium with 0.5 μM indole-3-butyric acid. Survival of plants in the pots was 75 – 80 %. This protocol was used in Agrobacterium mediated transformation. The DCMEA explants were treated independently with two A. tumefaciens (LBA 4404) strains harbouring a binary vector carrying the green fluorescent protein (GFP) and β-glucuronidase (GUS) reporter genes, respectively. Both the strains contained neomycin phosphotransferase selectable marker gene. After co-cultivation, the explants were cultured on EC₆ basal medium supplemented with 5 μM BAP and 1 μM IAA. The selection of putative transformants was on a medium containing 50 mg dm⁻³ kanamycin. Expression of GUS and GFP gene was confirmed by histochemical assay and fluorescence microscopy, respectively. The elongated shoots expressing GFP reporter gene were rooted and transferred to pots for hardening. The integration of GFP gene into the genome of putative transformants was confirmed by Southern blotting. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of 2,4-dichlorophenoxyacetic acid and donor plant environment on plant regeneration from immature inflorescence-derived callus of Lolium perenne L. and Lolium multiflorum L.

Callus cultures were obtained from immature inflorescences of perennial ryegrass (Lolium perenne) and Italian ryegrass (Lolium multiflorum). Inflorescence segments were cultured on Murashige and Skoog medium (MS) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The response in culture with regard to compact callus induction, embryogenesis and plant regeneration was determined for different varieties. The in vitro response was compared for explants from field-grown plants and explants from greenhouse-grown plants. The effect of different 2,4-D concentrations on the in vitro response was also investigated in one L. perenne variety and one L. multiflorum variety. The percentage of explants that formed compact callus and embryogenic callus differed strongly with the cultivar. There was no consistent effect of the growth conditions of the donor plants or the 2,4-D concentration of the medium on this response. Green plants were regenerated from all the cultivars tested. Explants from field-grown plants showed a higher tendency to form albino shoots than explants from greenhouse-grown plants. In the L. perenne variety tested higher 2,4-D concentrations (up to 15 mg/l) resulted in a lower regeneration frequency of green shoots and a higher regeneration frequency of albino shoots (up to 12.5 mg/l). In the L. multiflorum variety tested the effect of 2,4-D on regeneration was less pronounced.     

Production of glycyrrhizin in callus cultures of licorice

Licorice plants, Glycyrrhiza glabra, G. uralensis, and G. inflata, were investigated for callus induction using Murashige and Skoog (MS) medium combined with auxins and cytokinins. After 4 weeks of culture, 33-100% of leaf or stem explants formed calli. Maximum of shoot induction from callus cultures was achieved by G. inflata stem explants cultured on MS medium supplemented with 1 mg/l alpha-naphthaleneacetic acid (NAA) and 0.5 mg/l 6-benzyladenine (BA) (67%) which also gave maximum shoot formation per explant (two shoots per explant). These results indicated that all three Glycyrrhiza species regenerated shoots from callus cultures on MS medium combined with NAA and BA or only thidiazuron (TDZ; 0.1 and 0.5 mg/l). Glycyrrhizin contents of G. uralensis calli induced using MS medium in combination with NAA and BA [(27.60 +/- 8.47) microg/g DW] or TDZ alone [(36.52 +/- 2.45) microg/ g DW] were higher than those found in other combinations.     

Agrobacterium-mediated transformation of Campanula glomerata

A transformation system for Campanula glomerata 'Acaulis' based on the co-cultivation of leaf explants with Agrobacterium tumefaciens LBA4404 or EHA105 was developed. A. tumefaciens was eliminated when the explants were cultured on medium containing 400 mg/l vancomycin and 100 mg/l cefotaxime. Transgenic plants containing the uidA gene that codes for #-glucuronidase (gus) were obtained following co-cultivation with either strain of A. tumefaciens, LBA4404 or EHA105, both of which harbored the binary vector pGUSINT, coding for the uidA and neomycin phosphotransferase II (nptII) genes. While the transformation frequency (2-3%) was similar for both strains, A. tumefaciens LBA4404 was effectively eliminated from Campanula at a lower concentration of antibiotic as compared to EHA105. The concentration of individual antibiotics required to eliminate EHA105 resulted in a decreased rate (55-67%) of regeneration. The highest percentage of explants that regenerated plants (79%) and the highest regeneration rate was achieved with 100 mg/l cefotaxime combined with 400 mg/l vancomycin. Plants were also transformed with the isopentenyl transferase (ipt) gene using LBA4404 containing the 35S-ipt vector construct (pBC34).     

Indirect regeneration of the Cancer bush (<Emphasis Type="Italic">Sutherlandia frutescens</Emphasis> L.) and detection of <Emphasis Type="SmallCaps">l</Emphasis>-canavanine in <Emphasis Type="Italic">in vitro</Emphasis> plantlets using NMR

The present study reports a simple protocol for indirect shoot organogenesis and plant regeneration of Sutherlandia using rachis and stem segments. Different concentrations (0.0–68.08 μmol l⁻¹) of thidiazuron (TDZ) were used for callus induction and shoot organogenesis. The highest percentage of callus formation (97.5%) and the highest percentage of explants forming shoots (88.8%) were obtained from rachis explants cultured onto Murashige and Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15:473–495, 1962) supplemented with 45.41 μmol l⁻¹ TDZ. Scanning electron microscopy demonstrated the early development of adventitious shoots derived from callus cultures. Shoot clusters were further developed and grown in MS hormone-free medium. The presence of l-canavanine was determined by thin-layer chromatography and confirmed after column fractionation using silica gel and nuclear magnetic resonance spectroscopy. Individual shoots were rooted on different concentrations and combinations of MS salt strength and IBA. Half-strength MS salt medium supplemented with 24.6 μmol l⁻¹ IBA was optimal for root induction in which 78% of shoots were rooted. The in vitro plants were successfully acclimatized in a growth chamber with a 90% survival rate.     

Rapid propagation of Holostemma ada-kodien Schult., a rare medicinal plant, through axillary bud multiplication and indirect organogenesis

Efficient protocols of axillary bud multiplication and indirect organogenesis were established for Holostemma ada-kodien Schult. (Asclepiadaceae). Murashige and Skoog (MS) medium supplemented with 2.0 mg l^-1 N⁶-benzylaminopurine (BAP) and 0.5 mg l^-1 indole-3-butyric acid (IBA) induced an average of eight shoots per node and was the best for axillary bud proliferation. Subsequent cultures enhanced the number of shoots. The explant source of callus and the growth regulator inducing the callus exhibited significant influence on organogenesis. Callus developed from the basal cut end of the node explants differentiated more than 15 shoots on MS medium fortified with 1.5 mg l^-1BAP. Callus from internode explants developed fewer shoots than callus from the basal cut ends of node explants. Leaf-derived callus did not undergo organogenesis. The abscission of leaves and shoot tips of the developed shoots was prevented by the addition of AgNO₃ or CoCl₂, but with a concomitant significant reduction in the number of shoots. Half-strength solid MS or liquid medium with 0.05 mg l^-1 IBA exhibited the best in vitro rooting. Ninety percent of the rooted shoots survived in the field.