Membrane-associated phosphoinositides-specific phospholipase C forms from <Emphasis Type="Italic">Catharanthus roseus</Emphasis> transformed roots

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membrane-associated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.     

Somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm⁻³ of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA₃, 1.0 mg dm^{− 3}) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm⁻³ BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm⁻³ BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

Growth regulators affect primary and secondary somatic embryogenesis in Madagaskar periwinkle (<Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don) at morphological and biochemical levels

An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don in which primary and secondary embryogenic calluses were developed from hypocotyls and primary cotyledonary somatic embryos (PCSEs), respectively. Two types of calluses were different in morphology and growth behaviour. Hypocotyl-derived embryogenic callus (HEC) was friable and fast-growing, while secondary callus derived from PCSE was compact and slow-growing. HEC differentiated into somatic embryos which proliferated quickly on medium supplemented with NAA (1.0 mg l⁻¹) and BA (1.5 mg l⁻¹). Although differentiation and proliferation of somatic embryos were faster in primary HEC, maturation and germination efficiency were better in somatic embryos developed from primary cotyledonary somatic embryo-derived secondary embryogenic callus (PCSEC). At the biochemical level, two somatic embryogenesis systems were different. Both primary and secondary/adventive somatic embryogenesis and the role of plant growth regulators in two modes of somatic embryo formation have been discussed.     

Somatic embryogenesis and two embryo specific proteins (38 and 33 kD) in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

In the present study, the regeneration pathway, especially the different events of somatic embryogenesis (SE) have been studied morphologically and biochemically in Catharanthus roseus. Firstly, the calluses were induced from different explant sources (hypocotyl, epicotyl and root) by using various auxins. Embryogenic and non-embryogenic calluses were identified based on their morphology, colour and dry weight. Embryogenic callus was later cultivated on MS added with 0.45 μM 2,4-D, 6.62 μM BAP and 1.44 μM GA3 for obtaining various developmental stages of embryos. Different stages of embryos have been assayed for the establishment of marker based embryogenesis, particularly on embryo specific proteins whose presence or absence will ensure a rapid and efficient production of embryos that has a special application to clonal biotechnology. Two embryo specific proteins (38 and 33 kD) have been identified for the first time in C. roseus during torpedo stage of embryogenesis. Besides, multiple shoot formation from in vitro raised emblings was also attempted to examine the role of BAP and kinetin for shoot proliferation. The shoots were rooted with 5.37 μM NAA and 5.71 μM IAA before transplantation.     

Effect of <Emphasis Type="Italic">Glomus</Emphasis> species on physiology and biochemistry of <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

The present study on efficacy of different Glomus species, an arbuscular mycorrhizal (AM) fungus (G. aggregatum, G. fasciculatum, G. mosseae, G. intraradices) on various growth parameters such as biomass, macro and micronutrients, chlorophyll, protein, cytokinin and alkaloid content and phosphatase activity of pink flowered Catharanthus roseus plants showed that all Glomus species except G. intraradices enhanced the chlorophyll, protein, crude alkaloid, phosphorus, sulphur, manganese and copper contents of C. roseus plants along with phosphatase activity significantly over uninoculated plants. However only G. mosseae and G. fasciculatum exhibited superior symbiotic relationship with the plant. G. mosseae was found to be the best for increasing the crude alkaloid content (8.19%) in leaf and also in increasing the quantity of important alkaloids vincristine and vinblastine.     

Impact of hypoxia on the growth and alkaloid accumulation in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell suspension

The Madagascar periwinkle (Catharanthus roseus) produces numerous indole alkaloids, several of which have an important pharmaceutical uses such as ajmalicine, vinblastine and vincristine. The relationship between hypoxia and ajmalicine production in a cell suspension culture of C. roseus were investigated during the cycle of cell culture, in correlation with the effects on growth. The results show that the lack of oxygenation in C20D cells provokes a very strong inhibition in accumulation of the alkaloids and of other possible substances. Moreover, the addition of loganin, a metabolic intermediate of the biosynthetic pathway, in the culture medium of cells subjected to hypoxia restored the alkaloid production. Also, the results showed that the addition of benzyladenine (BA) to the culture medium increased the ajmalicine production and that the inhibitory effect of hypoxia was almost absent in these conditions. Therefore, it could be suggested that BA can without doubt decrease the effects of the hypoxia and increase the ajmalicine production in periwinkle cell suspensions.     

Construction of genetic linkage map of the medicinal and ornamental plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

An integrated genetic linkage map of the medicinal and ornamental plant Catharanthus roseus, based on different types of molecular and morphological markers was constructed, using a F₂ population of 144 plants. The map defines 14 linkage groups (LGs) and consists of 131 marker loci, including 125 molecular DNA markers (76 RAPD, 3 RAPD combinations; 7 ISSR; 2 EST-SSR from Medicago truncatula and 37 other PCR based DNA markers), selected from a total of 472 primers or primer pairs, and six morphological markers (stem pigmentation, leaf lamina pigmentation and shape, leaf petiole and pod size, and petal colour). The total map length is 1131.9 cM (centiMorgans), giving an average map length and distance between two markers equal to 80.9 cM and 8.6 cM, respectively. The morphological markers/genes were found linked with nearest molecular or morphological markers at distances varying from 0.7 to 11.4 cM. Linkage was observed between the morphological markers concerned with lamina shape and petiole size of leaf on LG1 and leaf, stem and petiole pigmentation and pod size on LG8. This is the first genetic linkage map of C. roseus.     

Plant regeneration from protoplasts of <Emphasis Type="Italic">Musa acuminata</Emphasis> cv. Mas (AA) via somatic embryogenesis

A protocol for plant regeneration from protoplasts of Musa acuminata cv. Mas (AA) via somatic embryogenesis was developed. Viable protoplasts were isolated from embryogenic cell suspensions at a yield of 1.2 × 10⁷ protoplasts/ml packed cell volume (PCV). Liquid and feeder layer culture systems with medium-A and medium-B were used for protoplast culture. In liquid culture system, medium-B was more efficient for inducing cell division (17.5% at 14 days) and colony formation (6.7% at 28 days) than medium-A. However, all protoplast-derived cell colonies (PDCC) obtained from liquid culture system could not develop further. In feeder layer culture system, there was no significant difference between medium-A and medium-B on cell division and colony formation of the cultured protoplasts, and the cell division frequency at 14 days and colony formation frequency at 28 days were 24.5% and 11.2%, respectively, in medium-B. Comparative study on the effects of BAP (2.2 μM, 4.4 μM, 8.8 μM), zeatin (0.4 μM, 0.8 μM, 1.2 μM) and TDZ (0.2 μM, 0.4 μM, 0.6 μM) on embryo formation of PDCC from feeder-layer culture indicated that TDZ was best. TDZ at 0.4 μM induced 7906 mature embryos per ml PCV PDCC, which was 4-fold the frequency as with BAP at 4.4 μM, 7.5-fold as with zeatin at 0.8 μM and 150-fold as control medium (no mentioned cytokinins) after 45 days on M3 medium. About 44% of the mature embryos were converted into plantlets with poor root system after subculture on M4 medium. Root further development of regenerated plantlets was promoted by addition of activated charcoal (AC) to MS basal medium.     

The role of BAP in somatic embryogenesis induction from seed explants of <Emphasis Type="Italic">Arachis</Emphasis> species from Sections <Emphasis Type="Italic">Erectoides</Emphasis> and <Emphasis Type="Italic">Procumbentes</Emphasis>

Somatic embryogenesis was induced from seed explants of Arachis archeri, A. porphyrocalix (Section Erectoides) and A. appressipila (Section Procumbentes) in response to 6-benzylaminopurine (BAP). Embryo axes first developed into single shoots in response to 4.4 μM BAP. Friable embryogenic calluses were produced from the hypocotyl region of these explants in response to different BAP concentrations. Embryonic leaflets also gave rise to friable calluses, but somatic embryos were only observed in explants of A. archeri and A. appressipila. Histological analyses revealed the presence of heart-shaped, torpedo and cotyledonary stages embryos, both as isolated and fused structures. A low frequency of embryo-to-plant conversion was achieved by inducing shoot development on medium solidified with 0.5% phytagel and supplemented with 1.5% or 3% sucrose. Rooting was induced on MS supplemented with indole-3-acetic acid (IAA).     

Highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.) through somatic embryogenesis

A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166 and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin (Km) were then recovered on CIM + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM α-naphthaleneacetic acid (NAA) + 25 mg l⁻¹ Km + 250 mg l⁻¹ timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and 18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production of transgenic celery plants.     

Improved somatic embryogenesis of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) with focus on induction parameters and efficient plant regeneration

A study of four parameters (induction medium, floral explant, developmental stage and year) was carried out to determine the best combination for the embryogenesis induction of eight grapevine (Vitis vinifera L.) cultivars. Anthers and ovaries were extracted from flower buds at three developmental phases and incubated in two induction media over two consecutive years. As average, the percentage of embryogenesis on Nitsch and Nitsch-derived medium (9.1%) was higher than in Murashige and Skoog-derived medium (5.9%) and embryogenesis from ovaries (10.1%) was 2-fold higher than from anthers (4.9%). Earlier flower developmental stages (II–III) favored embryogenic induction from anthers, while later stages (III–V) did it from ovaries. Induction of embryogenic cultures was genotype dependent. Two years after the establishment of the embryogenic lines, an average of 48.0% of the pro-embryogenic masses were viable and suitable to initiate cell suspensions. Embryogenic cultures of four genotypes showed a high percentage of conversion from embryos to plants: Albariño (61.8%), Garnacha (48.8%), Tempanillo (71.0%) and Sultanina (69.0%). Moreover, cell suspensions were competent for transient transformation based on β-glucuronidase assay, as up to 6,387 blue spots per Petri plate after Biolistic bombardment were obtained. Here, we present the advantage of ovaries over anthers for the embryogenesis induction of several grapevine cultivars. This is the first report of embryogenesis from the cultivars Albariño, Verdejo and Muscat Hamburg as well as transient transformation of Albariño and Tempranillo.     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan     

Protoplast isolation and culture for somatic hybridization of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> and <Emphasis Type="Italic">L</Emphasis>. <Emphasis Type="Italic">subcarnosus</Emphasis>

A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 10⁵ protoplasts g⁻¹ fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks) and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids. However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and micropropagation of <Emphasis Type="Italic">Givotia rottleriformis</Emphasis> Griff.

The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA₃). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts) supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively.     

Assessing competence for adventitious shoot formation in hypocotyl explant cultures from<Emphasis Type="Italic">Catharanthus roseus</Emphasis> cultivars

Hypocotyl expiants from 22 cultivars ofCatharanthus roseus were cultured on various shoot-inducing media to assess their competence for adventitious shoot formation. The Murashige and Skoog (MS) media had been supplemented with 14 μM zeatin and 2.5 μM indole-3-butyric acid (IBA), 4.5 μM BA and 0.5 μM α-naphthaleneacetic acid (NAA), or 14 μM thidiazuron and 2.5 μM IBA. After eight weeks, the expiants from ‘Cooler Raspberry Red’ showed the greatest frequency of adventitious shoot formation, followed by ‘Cooler Orchid’ and ‘Cooler Treated’. The highest frequency (86.7%) for ‘Cooler Raspberry Red’ was attained on the medium enhanced with 14 μM zeatin and 2.5 μM NAA. Excised adventitious shoots were then readily rooted on a half-strength MS basal medium. Afterward, the regenerated plantlets were transferred to potting soil and grown to maturity in a greenhouse.     

Somatic embryogenesis and plant regeneration from seeds of wild<Emphasis Type="Italic"> Dicentra spectabilis</Emphasis> (L.) LEM

Regeneration via somatic embryogenesis from callus was studied in Dicentra spectabilis. To obtain somatic embryogenic callus, we cultured D. spectabilis seeds on MS basal media supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The highest percentage of embryogenic callus formation was observed on media containing 1.0 mg/l 2,4-D under dark conditions. Somatic embryogenesis was studied by transferring the callus onto MS basal medium containing different concentrations (0.0, 0.1, 0.5, 1.0, 2.0 mg/l) of KIN (kinetin) and/or BAP. Somatic embryogenesis on MS basal media with 1.0 mg/l of KIN was excellent under light conditions. Somatic embryos were rooted by transferring them to half-strength MS basal media containing 2 g/l Phytagel. About 64.2% of the somatic embryos converted to rooted plantlets, 4% showed secondary embryogenesis and 31.8% did not develop and died. Rooted plantlets showed a 46% survival rate when acclimatized ex vitro.Abbreviations BAP 6-Benzylaminopurine - 2.4-D 2,4-Dichlorophenoxyacetic acid - KIN Kinetin - SEM Scanning electron microscopyCommunicated by H. Lörz     

Transient gene expression of recombinant terpenoid indole alkaloid enzymes in<Emphasis Type="Italic">Catharanthus roseus</Emphasis> leaves

We developed a transient expression assay for Madagascar periwinkle (Catharanthus roseus [L.] G. Don.) that is based on vacuum infiltration of intact leaves with recombinantAgrobacterium tumefaciens. This simple and rapid technique was used to overexpresstryptophan decarboxylase (tdc) andstrictosidine synthase (str1) genes, which encode 2 key enzymes of the terpenoid indole alkaloid (TIA) biosynthesis pathway. Immunoblot analysis of crude leaf extracts demonstrated that recombinant TDC and STR1 accumulated to detectable levels when targeted to their native subcellular compartments (i.e., the cytosol and vacuole, respectively) or to the chloroplast. In this article, we discuss possible applications of the transient assay in studies on the overexpression of enzymes of the TIA pathway in intactC. roseus leaves.     

Proteome analysis of the medicinal plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.