Protoplasts from <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-transformed cell line of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. regenerated to hairy roots

Summary Protoplasts were isolated from Agrobacterium rhizogenes A4-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×10⁶ per g fresh weight) was obtained from 12-d-old calluses after being subeultured on fresh medium. The viability of protoplasts reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented with 2 mgl⁻¹ (9.05 μM) 2,4-dichlorophenoxyacetic acid, 0,2mgl⁻¹ (0.93 μM) kinetin, 0.3 M mannitol, 2% (w/v) sucrose, and 500 mgl⁻¹ casein hydrolyzate at a plating density of 1.0×10⁵ per ml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency was about 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without growth regulators. Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast culture system would be valuable for further somatic hybridization in forage legumes.     

Plant regeneration by somatic embryogenesis and organogenesis in commercial pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> L.)

Summary Axillary and terminal buds from suckers of Ananas comosus cv. Phuket were established on Murashige and Tucker-based (MT) medium with 2.0 mgl⁻¹ (9.8 μM) indolebutyric acid, 2.0 mgl⁻¹ (10.74 μM) naphthaleneacetic acid, and 2.0 mgl⁻¹ (9.29 μM) kinetin, followed by multiplication on Murashige and Skoog-based (MS) medium containing 2.0 mgl⁻¹ (8.87 μM) benzyladenine (BA) to provide a continuous supply of axenic shoots. Leaves, excised from such cultured shoots, produced adventitious shoots from their bases when these explants were cultured on MS medium containing 0.5 mgl⁻¹ (2.26 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mgl⁻¹ (8.87 μM) BA. Embryogenic callus was produced when leaf explants were cultured on MS medium with 3.0 mgl⁻¹ (12.42 μM) 4-amino-3,5,6-trichloropicolinic acid (picloram). Somatic embryos developed into shoots following transfer of embryogenic tissues to MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. Cell suspensions, initiated by transfer of embryogenic callus to liquid MS medium with 1.0 mgl⁻¹ (4.14 μM) picloram or 1.0 mgl⁻¹ (4.52 μM) 2,4-D, also regenerated shoots by somatic embryogenesis, on transfer of cells to semisolid MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. All regenerated shoots rooted on growth regulator-free MS medium, prior to ex vitro acclimation and transfer to the glasshouse. These studies provide a baseline for propagation, conservation, and genetic manipulation of elite pineapple germplasms.     

Improvement of artemisinin production by chitosan in hairy root cultures of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Artemisinin production by hairy roots of Artemisia annua L. was increased 6-fold to 1.8 μg mg⁻¹ dry wt over 6 days by adding 150 mg chitosan l⁻¹. The increase was dose-dependent. Similar treatment of hairy roots with methyl jasmonate (0.2 mM) or yeast extract (2 mg ml⁻¹) increased artemisinin production to 1.5 and 0.9 μg mg⁻¹ dry wt, respectively.     

Abscisic acid and gibberellin-like substances in roots and root nodules ofGlycine max

Summary The content of endogenous gibberellin (GA)-like substances of roots and root nodules of SOya, and GA production byRhizobium japonicum cultures, were investigated by a combined thin layer chromatographic (TLC)-dwarf pea epicotyl bioassay technique. GAs were more concentrated in root nodules than in the roots, totalling 1.34 and 0.16 nM GA₃ equivalents g⁻¹ dry wt. respectively. GA production byR. japonicum cultures was demonstrated (1.00 nM GA₃ equivalentsl ⁻¹) and comparison of the GA components of plant and bacterial culture medium extracts, suggested that rhizobial GA production may contribute to the nodule GA content. Cis-trans abscisic acid (ABA) was identified in root and nodule extracts by TLC-gas liquid chromatography (GLC), and amounted to 0.18 and 2.21 nM g⁻¹ dry wt. respectively, whereas 0.30 and 4.63 nM ABA equivalents g⁻¹ dry wt. were detected by a TLC-wheat embryo bioassay technique. ABA was not detected in extracts of bacterial cultures.     

Thidiazuron-induced <Emphasis Type="Italic">in vitro</Emphasis> shoot organogenesis of the medicinal plant <Emphasis Type="Italic">Arnebia euchroma</Emphasis> (Royle) Johnst

Summary An efficient in vitro propagation system was developed for Arnebia euchroma, an important Chinese traditional medicinal plant. The present study utilized thidiazuron (TDZ) for the induction of shoot organogenesis on cotyledon and hypocotyl explants. The maximal number of shoots was obtained on the modified Linsmaier and Skoog (LS) medium supplemented with 1.0 mgl⁻¹ (4.5 μM) TDZ for 12d on cotyledon explants (8.6 shoots per cotyledon explant). Other cytokinins (kinetin and 6-benzyladenine) and auxin (α-naphthaleneacetic acid) were not efficient in inducing regeneration on cotyledon explants. Browning of the basal portion of the subcultured shoots could be significantly reduced when they were cultured on the modified LS medium supplemented with 100 mgl⁻¹ (33.3 μM) polyvinylpyrrolidone. Well-developed shoots formed roots on the same medium containing 1.0 mgl⁻¹ (4.9 μM) indole-3-butyric acid. The efficient regeneration protocol reported here provides an important means of micropropagation of this plant. Furthermore, this protocol is essential to future genetic improvement of plants via transformation protocols.     

Direct shoot regeneration from leaf segments of mature plants of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> (L.) moench

Summary This is the first communication of direct shoot regeneration from fully developed leaves of potted mature Echinacea purpurea plants. Shoot buds were induced directly on the adaxial surface of mature leaf tissues of E. purpurea 30 d after culture initiation on Woody Plant Medium (WPM) supplemented with various levels of 6-benzyladenine (BA). Maximum shoot organogenesis, with 12–20 shoots per leaf segment, was obtained with 5% coconut milk and 2.5 mg l⁻¹ (6μM) BA in 30 d. Callus was induced using 0.5 mgl⁻¹ (1μM) α-naphthaleneacetic acid and 2.5 mgl⁻¹ (6μM) BA. The regenerated shoots were rooted on WPM supplemented with 1.5 mgl⁻¹ (3μM) of indole-3-butyric acid, 3% sucrose, and 0.85% agarose. Rooted plants were successfully transferred to soil in pots and appeared morphologically normal and flowered in a growth chamber.     

Isolation and production of artemisinin and stigmasterol in hairy root cultures of Artemisia annua

Scaled-up hairy root culture of Artemisia annua L. was established in three-liter Erlenmeyer flask. Both artemisinin and stigmasterol that derive from the common precursors of isopentenyl diphosphate and farnesyl pyrophosphate were isolated from hairy roots. The production rate of artemisinin isolated by column chromatography from hairy root cultures was 0.54% (mg.gDW⁻¹). Stigmasterol was identified by mass spectrometry and nuclear magnetic resonance analysis. The production of stigmasterol isolated by column chromatography from hairy root cultures was 108.3% (mg.gDW⁻¹). In hairy root cultures, the production rate of stigmasterol was estimated to be 201 times greater than that of artemisinin. Our results suggest that investigation of secondary metabolites may provide a new insight to study artemisinin production in hairy root cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

The development of an efficient cultivar-independent plant regeneration system from callus derived from both apical and non-apical root segments of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.)

Summary Callus induction and later plant regeneration were studied in four widely grown garlic (Allium sativum L.) cultivars from Europe. Root segments from in vitro plantlets were used as starting material. In addition to cultivar effects, the effects of auxin and cytokinin levels and the position of the segments on the root were studied. There were no statistically significant differences among cultivars for the number of root segments that induced callus in the two series of experiments. The average induction frequency was 34.7% in the first series of experiments. Callus induction on apical root segments was significantly higher compared to callus induction on non-apical root segments in the second series of experiments. Two months after callus induction, callus lines were transferred to a regeneration medium consisting of Murashige and Skoog basal medium supplemented with 30gl⁻¹ sucrose and 1 mgl⁻¹ (4.6μM) kinetin. Calluses derived from different experiments were quite uniform with respect to their regeneration potential. Also it was found that our regeneration system was cultivar-independent. The average shoot regeneration frequency was 17.9% in the first series of experiments. Highly significant differences were found in the frequency of shoot regeneration among different callus induction treatments. When the cytokinin 6-(γ,γ-dimethylallylamino)purine (0.1mgl⁻¹∶0.5 μM) was present during callus induction, shoot regeneration ranged from 30.10 to 47.60%. Shoot regeneration from callus induced on non-apical segments was higher, although not significant, compared to callus induction from apical root segments in the second series of experiments. All in all, an efficient callus induction and plant regeneration system was developed from both apical and non-apical segments taken along the entire length of the roots. This system has potential to be used for garlic transformation.     

Highly efficient embryogenesis and plant regeneration of tall fescue (Festuca arundinacea Schreb.) from mature seed-derived calli

Summary We report a protocol for efficient plant regeneration of four tall fescue (Festuca arundinacea Schreb.) cultivars (‘Surpro’, ‘Coronado’, ‘Summer Lawn’, and ‘Fawn’) via somatic embryogenesis. Calli were initiated from mature seeds grown on modified Murashige and Skoog (MMS) medium supplemented with 7.0mgl⁻¹ (31.7μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mgl⁻¹ (0.23 μM) kinetin (Kin). Calli were maintained and proliferated by subculture at monthly intervals on MMS medium containing 4.5 mgl⁻¹ (20.4 μM) 2,4-D and 0.2mgl⁻¹ (0.9 μM) Kin. Somatic embryos (SE) were induced from seed-derived calli on SE-induction medium (MMS supplemented with 2.0 mgl⁻¹ 2,4-D and 0.2mgl⁻¹ Kin). Plantlets were regenerated from somatic embryogenic calli grown on modified SH medium supplemented with 2 mgl⁻¹ Kin. Using this optimized protocol, 78.6–82.3% of mature seeds of all four cultivars produced SE clusters, of which 93.5–95.3% regenerated into plants within 10 wk. The regenerants showed no phenotypic abnormalities.     

Factors influencing shoot regeneration from cotyledons of tetraploid <Emphasis Type="Italic">Isatis indigotica</Emphasis> fort

Summary An efficient in vitro plant regeneration system from cotyledons was established in tetraploid Isatis indigotica Fort. Factors influencing shoot regeneration from cotyledons, including culture medium type, combinations of plant growth regulators, and sucrose concentrations in the medium, as well as illumination were investigated. Murashige and Skoog's (MS) medium was found to be best for promoting shoot regeneration, followed by Gamborg's B₅ and White's medium. The highest shoot regeneration frequency was achieved from cotyledons cultured on MS medium supplemented with 2.0 mgl⁻¹ (8.9 μM) 6-benzyladenine and 1.0 mgl⁻¹ (5.4 μM) α-naphthaleneacetic acid (NAA), with 97.9% regeneration, associated with a high number of multiple shoots developed per explant (8.6 shoots per explant). A sucrose concentration of 3% present in the medium and light conditions were beneficial for shoot regeneration. The shoots developed were rooted in a half-strength MS medium supplemented with 1.0 mgl⁻¹ (5.4 μM) NAA and successfully transplanted in soil in pots with over 85% survival. The establishment of an efficient plant regeneration procedure from cotyledons provides a basis for the rapid in vitro multiplication of tetraploid Isatis indigotica Fort., one of the most extensively used medicinal plants in China currently under great shortage.     

Aluminum inhibition of shoot lateral branches ofGlycine max and reversal by exogenous cytokinin

Aluminum effects on the morphological development of soybean (Glycine max (L.) Merr.) were characterized in greenhouse and growth chamber experiments. An Al-sensitive cultivar, ‘Ransom’, was grown in an acid soil (Aeric Paleudult) adjusted to 3 levels of exchangeable Al. Lateral shoot development at the nodes of the main stem was extensive in the limed soil containing 0.06 cmol(+) Alkg⁻¹. However, lateral shoot length and weight were severely inhibited in the unlimed soil containing 2.19 cmol(+) Alkg⁻¹, and in the unlimed soil amended to 2.63 cmol(+) Alkg⁻¹ with AlCl₃. This inhibition by the high Al/low pH condition was reversed by the exogenous application of a synthetic cytokinin 6-benzylaminopurine (BA). The daily application of 20 μg mL⁻¹ BA applied locally to the lateral meristems of plants grown in the unlimed soil stimulated lateral shoot growth substantially, such that it was either comparable to or greater than that observed in the limed treatment without BA. Accumulation of K, Ca, and Mg in lateral shoot branches was also stimulated by the local application of BA. The inhibitory effects of Al on lateral shoot development were confirmed in solution culture. In addition, differential sensitivity to Al was evident among the primary root, first order lateral roots, and second order lateral roots. The length of the primary root was only slightly decreased by increasing concentrations of Al up to 30 μM. In contrast, the length of basipetally located first order lateral roots was restricted to greater extent; up to 50% by 30 μM Al. Second order lateral lengths were inhibited even more severely; up to 86% by 30 μM Al. Substantial evidence in the literature indicates that the root apex is a major site for the biosynthesis of cytokinin that is supplied to shoots, and cellular function and development in this region of the root are impaired during Al toxic conditions. This suggests that one mode of action by which Al may affect shoot growth is by inhibiting the synthesis and subsequent translocation of cytokinin to the meristematic regions of the shoot. The present observation of a reversal of Al-inhibited lateral shoot development by exogenously applied cytokinin supports this hypothesis. However, the inability of applied cytokinin to counter the restriction imposed by Al on total shoot dry matter production implies the impairment by Al toxicity of other root functions, such as ion and water transport, also played an important role in altering shoot morphology.     

Improvement of ginsenoside production by jasmonic acid and some other elicitors in hairy root culture of ginseng (Panax ginseng C. A. Meyer)

Summary Hairy root cultures of Panax ginseng, established after the infection of root sections with Agrobacterium rhizogenes KCTC 2703, were cultured in phytohormone-free Murashige and Skoog (MS) liquid medium containing different concentrations of jasmonic acid and some other elicitors, in order to promote ginsenoside accumulation. Jasmonic acid in the range 1.0−5.0 mg l⁻¹ (4.8–23.8 μM) strongly improved total ginsenoside production in ginseng hairy roots. Peptone (300 mg l⁻¹) also showed some effect on ginsenoside improvement; however its effect was much weaker than that of jasmonic acid. Ginsenoside content and productivity were 58.65 and 504.39 mg g⁻¹, respectively. The Rb group of ginsenoside content was increased remarkably by jasmonic acid, while Rg group ginsenoside content changed only slightly compared to controls. However, jasmonic acid also strongly inhibited ginseng hairy root growth.     

High-frequency plant regeneration by <Emphasis Type="Italic">in vitro</Emphasis> shoot proliferation in cotyledonary node explants of grasspea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.)

Summary Multiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N⁶-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium augmented with 2.0 mgl⁻¹ (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8% of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mgl⁻¹ (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil.     

Improved method of regeneration of black gram (Vigna mungo L.) through liquid culture

Summary A very rapid and efficient regeneration method of Vigna mungo L. has been established using liquid culture. A highly regenerable explant, viz., young multiple shoots obtained by germinating the seeds in 2 mgl⁻¹ (8.9μM) N⁶-benzyladenine-supplemented Murashige and Skoog (MS) medium, was used as a source of tissue to initiate the liquid culture. The liquid medium consisted of half-strength B5 or MS salts supplemented with MS organics, α-naphthaleneacetic acid (0.1 mgl⁻¹, 0.54μM) and N⁶-benzyladenine (0.5mgl⁻¹, 2.2μM). Transferring the growing tissues to fresh medium every third day resulted in ca. 142% increase in the number of shoot buds produced after 24d. Shoot buds elongated on one-third-strength MS (MS_1/3) semisolid medium and plantlets were obtained by transferring the shoots onto MS_1/3 semisolid medium supplemented with indolebutyric acid (1 mgl⁻¹, 4.9 μM).     

Nickel tolerance and hyperaccumulation in shoot cultures regenerated from hairy root cultures of <Emphasis Type="Italic">Alyssum murale</Emphasis> Waldst et Kit

Shoot cultures of nickel hyperaccumulating Alyssum murale were established from epicotyl explants of seedlings aseptically germinated on hormone-free MS medium. They were further maintained on media with 0–0.92 μM kinetin. Optimal shoot multiplication was at 0.46 μM kinetin. Inoculation by shoot wounding was performed with overnight suspension of A. rhizogenes A4M70GUS which contains GUS gene cointegrated in pRiA4. After 30 days hairy roots were produced at the wounding site in 31 explant (25% out of 124). Hairy roots were excised and further propagated on hormone-free medium as separate clones. In the first passage clones 3 and 6 could be distinguished by fast growth and spontaneous shoot regeneration. In other clones (12, 23 and 25) shoot regeneration required presence of cytokinins. The five shoot culture clones regenerated from hairy roots were further cultured on media with 0.46 μM kinetin. These shoots were characterized by good elongation and lateral shoot branching, short internodes, minute slightly curled leaves and well developed plagiotropic root system spreading over the surface of media. Thus all plants regenerated from hairy root cultures manifested the characteristic Ri syndrome phenotype. They all had a strong positive GUS reaction. PCR analysis confirmed presence of uidA sequence from the gus construct. They were also tolerant to nickel accumulating up to 24,700 μg g⁻¹ dry weight.     

Hairy roots cultures from different Solanaceous species have varying capacities to produce E. coli B-subunit heat-labile toxin antigen

The gene encoding enterotoxigenic Escherichia coli B-subunit heat-labile toxin (LTB) antigen was co-transformed into hairy root cultures of Nicotiana tabacum (tobacco), Solanum lycopersicum (tomato) and Petunia parodii (petunia) under the CaMV35S promoter. Tobacco and petunia roots contained ~65–70 μg LTB g⁻¹ tissue whilst hairy roots of tomato contained ~10 μg LTB g⁻¹. Antigen at ~600 ng ml⁻¹ was detected in growth medium of tobacco and petunia. Tobacco roots with higher LTB levels showed growth retardation of ~80% whereas petunia hairy roots with similar levels of LTB showed only ~35% growth retardation, relative to vector controls. Regeneration of plants from LTB-containing tobacco hairy roots was readily achieved and re-initiated hairy roots from greenhouse-grown plants showed similar growth and LTB production characteristics as the original hairy root cultures.     

Plant regeneration from mesophyll-and cell suspension-derived protoplasts of <Emphasis Type="Italic">Dianthus acicularis</Emphasis> and characterization of regenerated plants

Summary Plant regeneration systems from mesophyll- and cell suspension-derived protoplasts were established in Dianthus acicularis (2n=90), a species with resistance to Burkholderia caryophylli (Psedomonas caryophylli). Protoplasts were isolated from both leaves of in vitro-grown plants and cell suspension cultures established from the calluses originated from leaves of in vitro-grown plants. Protoplasts isolated from both sources showed about the same response to the type and concentration of cytokinins, and gave the highest frequencies of cell division and colony formation in 0.1% (w/v) Gelrite^?-solidified Murashige and Skoog (MS) medium supplemented with 0.5M glucose, 1.0mgl⁻¹ (4.53 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5 mg l⁻¹ (2.28 μM) zeatin. Numerous plantlets were regenerated after transfer of the colonies to 0.8% (w/v) agar-solidified half-strength MS medium supplemented with 0.5 mgl⁻¹ (2.28 μM) zeatin. Most plantlets exhibited normal phenotypes, but some showed variations, such as abnormal morphology with reduced chromosome number, precocious flowering, and vigorous growth with a tetraploid chromosome number. Possible mechanisms responsible for the observed somaclonal variation are discussed.     

High-frequency multiple shoot regeneration from cotyledonary nodes of guar (Cyamopsis tetragonoloba L. Taub)

Summary Guar (Cyamopsis tetragonoloba L. Taub) is a drought-tolerant multipurpose cash crop. A rapid regeneration system has been developed for four Indian guar genotypes. Investigations were carried out to assess the effect of different growth regulators and their combinations on a variety of explants such as the embryo, cotyledons, and cotyledonary nodes for shoot morphogenesis. It was established that Murashige and Skoog's culture medium containing 6-benzylaminopurine (13.3 μM or 3 mgl⁻¹) in combination with indole-3-acetic acid (11.4 μM or 2mgl⁻¹) with cotyledonary node explants gave the highest frequency of multiple shoot induction. In vitro rooting from cultured shoots was maximal on a half-salt concentration of Murashige and Skoog's culture medium fortified with indole-3-butyric acid (4.9 μM or 1 mgl⁻¹). In vitro-regenerated plants were grown to pod setting and subsequent maturity in greenhouse conditions.     

Long-lasting <Emphasis Type="Italic">Capsicum baccatum</Emphasis> ‘Organogenic callus’ formation

Summary Capsicum regeneration is often obtained through direct (adventious) regeneration and only a few reports claim indirect regeneration (through callus) as an option to obtain complete plants. A possible reason for this may be because Capsicum cultures offer a narrow window in time for the regeneration process to occur, and after that, the ability to regenerate plants rapidly diminishes. In this study, the C. baccatum radicle-side half-seed was the explant of choice to induce the formation of callus on semisolid Murashige and Skoog (1962) medium supplemented with 5 mgl⁻¹ (22.2μM) 6-benzylaminopurine, 1 mgl⁻¹ (5.7μM) indole-3-acetic acid, and 2 mgl⁻¹ (6.1μM) gibberellic acid. Organogenic calluses developed within 4–5 mo. and were subcultured every 2 mo. thereafter. Eventual bud elongation occurred and these shoots developed into complete plants after transfer to medium without plant growth regulators. The organogenic callus type retained its organogenic ability for more than 3 yr.     

Direct plantlet regeneration from male inflorescences of medicinal yam (Dioscorea floribunda Mart. & Gal.)

Summary Segments of male inflorescences of medicinal yam (Dioscorea floribunda) cultured on Murashige and Skoog (MS) medium supplemented with 13.94 μM kinetin (Kn) resulted in the conversion of floral buds into vegetative buds and these later developed into plantlets. Growth and multiplication of shoots could be obtained by culturing individual shoots in MS modified basal medium, replacing the MS standard three vitamins with 10.0 mgl⁻¹ thiamine in addition to 13.94 μM Kn. Root induction was also obtained simultaneously from the base of the shoots in the same medium. Such plantlets have been successfully transferred to potted soil, where they grew normally. Plantlets were also made to develop tubers on MS medium with 18.91 μM abscisic acid (ABA) and also with 2.68 μM α-naphthaleneacetic acid (NAA) and 40–50 gl⁻¹ sucrose.