A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica

Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25±2°C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained.     

Gradual Depletion of 2,4-D in the Culture Medium for Indirect Shoot Regeneration from Leaf Explants of Camellia sinensis (L.) O. Kuntze

Adventitious shoot regeneration via callus phase from in vitro leaf explants is reported for the first time in tea. Callus was obtained on Murashige and Skoog medium supplemented with varied concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5, 5.0, 7.5 and 10.0 mg/l). Rhizogenesis was observed at all concentrations of 2,4-D. Adventitious shoot buds developed indirectly on leaf explants after prolonged culture for 16 weeks on medium supplemented with 10.0 mg/l 2,4-D. GC analysis of the medium and the tissues at different stages of development showed that specific levels of 2,4-D in the tissue were responsible for morphogenesis. Shoot buds developed on rhizogenic calli, only when 2,4-D declined to undetectable or negligible concentrations in the tissue probably due to detoxification and metabolism. Alternatively, shoot buds could also be evoked when rhizogenic calli were transferred to medium supplemented with low concentration of 2,4-D (1.5 mg/l). The adventitious nature of the shoots was confirmed through histological studies.     

Direct somatic embryogenesis and plantlet regeneration from seedling leaves of winged bean,Psophocarpus tetragonolobus (L.) DC

Excised seedling leaf segments of winged bean [Psophocarpus tetragonolobus (L.) DC.] underwent direct somatic embryogenesis under appropriate incubation conditions. Initiation and development of the somatic embryos occurred using a two-step culture method. The culture procedure involved incubation for 28 days on MS basal medium supplemented with 0.1–0.5 mg/l NAA and 1.0–2.0 mg/l BA (induction medium) before transfer to MS medium supplemented with 0.1 mg/l IAA and 2.0 mg/l BA (embryo development medium). The initial exposure to low levels of NAA coincident with high levels of BA in the induction medium was essential for embryogenic induction. Maximum embryogenesis (43.3%) was obtained with 0.2 mg/l NAA and 2.0 mg/l BA, and at least 14 days on induction medium were required prior to transfer to the embryo development medium. The conversion frequency of cotyledonary embryos was 53.3% upon culture on MS medium containing 0.1 mg/l ABA for 7 days followed by transfer to MS medium supplemented with 0.1 mg/l IBA and 0.2 mg/l BA. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid     

Genetic transformation and regeneration of transgenic plants from precultured cotyledon and hypocotyl explants of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis> Sm. using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

An efficient transformation protocol was developed for Eucalyptus tereticornis Sm. using cotyledon and hypocotyl explants. Precultured cotyledon and hypocotyl explants were cocultured with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pBI121 containing the uidA and neomycin phosphotransferase II genes for 2 d and transferred to selective regeneration medium containing 0.5 mg/l 6-benzylaminopurine (BAP), 0.1 mg/l naphthalene acetic acid, 40 mg/l kanamycin, and 300 mg/l cefotaxime. After two passages in the selective regeneration medium, the putatively transformed regenerants were transferred to Murashige and Skoog (MS) liquid medium containing 0.5 mg/l BAP and 40 mg/l kanamycin on paper bridges for further development and elongation. The elongated kanamycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1.0 mg/l indole-3-butyric acid and 40 mg/l kanamycin. A strong β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. This protocol allows effective transformation and direct regeneration of E. tereticornis Sm.     

Isolation and characterization of a new carbendazim-degrading <Emphasis Type="Italic">Ralstonia</Emphasis> sp. strain

A bacterial strain 1-1 capable of utilizing carbendazim was isolated from carbendazim-treated Qiyang red soils Hunan Province, China. It is gram-negative, rod-shaped, motile with peritrichous flagella, which formed round, smooth, convex and transparent colonies of about 1.1 mm diameter after 3 days of incubation on the isolation and purification medium using carbendazim as the sole carbon and energy sources. The degradation ratios of carbendazim by strain 1-1 were 19.16 and 95.96 in the carbendazim (500 mg/l)-degrading medium and the carbendazim (500 mg/l)-degrading medium supplemented with yeast extract (150 mg/l) within 24 days, respectively. Strain 1-1 was identified as Ralstonia sp. (β-Proteobacteria) based on the results of phenotypic features, G+C mol and phylogenetic analysis of 16S rDNA. Strain 1-1 could become a new bacterial resource for biodegrading carbendazim and might play a bioremediation role for soils contaminated by carbendazim.     

Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons

A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO₃ supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l α-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.     

Genetic transformation of NERICA,interspecific hybrid rice between <Emphasis Type="Italic">Oryza glaberrima</Emphasis> and <Emphasis Type="Italic">O. sativa</Emphasis>, mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T₀ and T₁ generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.     

Somatic embryogenesis and plantlet regeneration in the genus Secale

Summary A tissue culture of five wild species of the Secale genus, i.e., S. africanum (Stapf.), S. ancestrale (Zhuk.), S. kuprianovii (Grossh), S. segetale (Rosher.), and S. vavilovii (Grossh), from immature embryos of sizes (stages) varying between 1.0 mm to 3.0mm, cultured on MS (1962) mineral nutrient medium supplemented with 0.62 mg/1–5.0 mg/1 of 2,4-D, was established. Initially various types of callus were observed and a correlation between genotype, size of explant and 2,4-D concentration was found. The best embryogenic response was observed when explants were smaller than 1.0 mm. Induction of somatic embryogenesis of 2.0 mm–3.0 mm explants required a higher concentration of 2,4-D. Most embryoids were formed in the presence of 5.0 mg/l of 2,4-D. Secale africanum and S. kuprianovii appeared to have the highest embryogenic capacity among the five investigated species. For embryoids germination to plantlets the MS medium supplemented with GA₃ and cytokinins was used. Ultimately, out of the 932 regenerants obtained 364 originated from somatic embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA3 deGibberellic acid - BAP Benzylaminopurine     

Copper resistance in Desulfovibrio strain R2

A sulfate-reducing bacterium, designated as strain R2, was isolated from wastewater of a ball-bearing manufacturing facility in Tomsk, Western Siberia. This isolate was resistant up to 800 mg Cu/l in the growth medium. By comparison, Cu-resistance of reference cultures of sulfate-reducing bacteria ranged from 50 to 75 mg Cu/l. Growth experiments with strain R2 showed that Cu was an essential trace element and, on one hand, enhanced growth at concentrations up to 10 mg/l but, on the other hand, the growth rate decreased and lag-period extended at copper concentrations of >50 mg/l. Phenotypic characteristics and a 1078 bp nucleotide sequence of the 16S rDNA placed strain R2 within the genus Desulfovibrio. Desulfovibrio R2 carried at least one plasmid of approximately of 23.1 kbp. A 636 bp fragment ot the pcoR gene of the pco operon that encodes Cu resistance was amplified by PCR from plasmid DNA of strain R2. The pco genes are involved in Cu-resistance in some enteric and aerobic soil bacteria. Desulfovibrio R2 is a prospective strain for bioremediation purposes and for developing a homologous system for transformation of Cu-resistance in sulfate-reducing bacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium × formolongi

An efficient system for Agrobacterium-mediated transformation of Lilium × formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 μM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.     

Micropropagation of <Emphasis Type="Italic">Swertia chirata</Emphasis> Buch.-Hams. ex Wall.: a critically endangered medicinal herb

An efficient in vitro plant regeneration protocol for Swertia chirata Buch.-Ham. ex Wall (Gentianaceae), a critically endangered Himalayan medicinal herb, was developed using shoot tip explants derived from in vitro grown seedlings. Media with 2% sucrose and various types of hormones markedly influenced in vitro propagation of S. chirata. An in vitro shootlet production system using Murashige and Skoog (MS) medium with various hormones such as BAP, KN and TDZ was established. BAP at 1.0 mg/l and KN, 0.1 mg/l induced highest number of multiple shoots (42.16 ± 1.05) per explant. Micro-proliferated shoots were transferred to elongation medium amended with GA₃ (0.1 mg/l) and hormone free basal medium, after which they were transferred to rooting medium. The highest frequency of rooting (22.48 ± 1.08) was obtained in half-strength MS medium supplemented with NAA, 0.1 mg/l after testing with different auxins at various concentrations within 4 weeks of transfer to the rooting medium. Hardening was successfully attained under controlled conditions inside the plant tissue culture room. This method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands.     

Direct somatic embryogenesis and plant regeneration from leaf, petiole, and stem explants of Golden Pothos

Somatic embryos directly formed at cut edges or on the surface of leaf explants, around cut ends or along side surfaces of petiole and stem explants of Golden Pothos [Epipremnum aureum (Linden & Andre) Bunt.] on Murashige and Skoog (MS) medium supplemented with N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) or N-phenyl-N-1, 2, 3-thiadiazol-5-ylurea (TDZ) with -naphthalene acetic acid (NAA) and a medium called MK containing MS salts with Kaos vitamins, supplemented with 2.0 mg/l TDZ and 0.2 mg/l NAA. Somatic embryos were also produced on MS medium containing 2.0 mg/l kinetin (KN) and 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) from leaf and petiole explants, MS medium supplemented with 2.0 mg/l CPPU and 0.5 mg/l 2,4-D from petiole and stem explants, and 2.0 mg/l TDZ and 0.2 mg/l or 0.5 mg/l 2,4-D from stem explants. In addition, somatic embryos occurred from stem explants on Chus N₆ medium containing 2.0 mg/l CPPU and 0.2 mg/l NAA. Somatic embryos matured and grew into multiple buds, shoots, or even plantlets after 2–3 months on the initial culture medium. Germination was optimal on MS medium containing either 2 mg/l 6-benzylaminopurine (BA) and 0.2 mg/l NAA or 2 mg/l zeatin and 0.2 mg/l NAA. Shoots elongated better and roots developed well on MS medium with no growth regulators. Approximately 30–100 plantlets were regenerated from each explant. The regenerated plants grew vigorously after transplanting to a soil-less container substrate in a shaded greenhouse.     

In vitro culture of Cuminum cyminum regeneration of flowering shoots from calli of hypocotyl and leaf explants

Calli from hypocotyl explant of Cuminum cyminum L. (Cumin) grew rapidly on Gamborg's B₅ basal medium with the following supplements, (i) 0.5 mg/l — 2,4-D (ii) 4 mg/l — NAA plus 2 mg/l — Kinetin and (iii) 0.2 mg/l — NAA plus 0.2 mg/l — BAP, whereas calli from leaf explant in these media grew slowly. Hypocotyl and leaf calli produced roots when transferred to basal medium only and shoots in basal medium with 0.5 mg/l NAA and 0.1 mg/l BAP. Ninety percent of the shoots produced roots when they were transferred to half strength MS inorganic salts supplemented with 0.5 mg/l each of IBA and NAA.Fifty to sixty percent of rootless as well as rooted shoots produced terminal umbellate flowers on this medium.     

Induction of root and shoot formation from root meristems ofPetunia hybrida

Summary A procedure has been developed for the induction of root or shoot formation from root meristems of germinated seeds ofPetunia hybrida. Root formation was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 6-benzylaminopurine (BA) (0–0.5 mg/l) and naphtaleneacetic acid (NAA) (0.05–2.0 mg/l). Induction of predominantly shoot formation was obtained on MS medium containing the following combinations of hormones (in mg/l): 0.05–0.5 NAA and 0.25–2.0 BA. Complete plant formation was obtained after rooting of the shoots on MS medium supplemented with IAA (0–2.0 mg/l) or NAA (0-0.5 mg/l).     

Somatic embryogenesis and plant regeneration in Quercus acutissima

Immature embryos of Quercus acutissima were collected weekly beginning 5 weeks post-fertilization and cultured on modified MS(Murashige and Skoog) medium containing 1,000 mg/l glutamine and 5 mM proline with different combinations of IBA(0.5–10.0 mg/l) and BA(0 or 1.0 mg/l) in light. The highest percentage of embryogenic cultures occurred on the medium containing 0.5 mg/l IBA or 1.0 mg/l BA and 0.5 mg/l IBA. Four weeks after initiation, the embryogenic cultures were transferred to MS medium without plant growth regulators and cultured for 4 weeks. The somatic embryos were then transferred to germination medium. The best germination results were achieved from WPM(Woody Plant Medium) containing 0.1 mg/l BA. Plantlets from somatic embryos were incubated on WPM supplemented with 0.2 mg/l BA for 4 weeks and plantlets with well developed shoots and roots were transplanted to perlite and peat moss(11, v/v) mixtures and placed in a culture room. After being hardened off for 8 weeks, they were transferred outdoors where they grew.Abbreviation BA N⁶-benzyladenine - IBA indole-3-butyric acid - GA₃ gibberellic acid - ABA abscisic acid - MS Murashige & Skoog Medium - WPM Woody Plant medium     

Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.)

Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding.     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

Genetic polymorphism analysis of somatic embryo-derived plantlets of Cymbopogon flexuosus through RAPD assay

The genetic status of somatic embryo-derived plantlets of Cymbopogon flexuosus was examined by randomly amplified polymorphic DNA (RAPD) analysis. Auxins such as 2, 4-dichlorophenoxyacetic acid (2, 4-D) (1–4 mg/l) were used in Murashige and Skoog (MS) medium for induction of calli from rhizomatous explants of Cymbopogon flexuosus. Optimum calli were induced on MS medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D) (3.5 mg/l) alone or in combination with N ⁶-benzyladenine (2 mg/l). Somatic embryogenesis was achieved from long term calli when cultured on MS medium containing 2, 4-dichlorophenoxyacetic acid (2, 4-D) (2 mg/l) along with N ⁶-benzyladenine (BA) (1–2 mg/l). Regeneration was achieved when freshly induced embryogenic calli were sub-cultured on MS medium supplemented with N ⁶-benzyladenine (3 mg/l) alone. Long-term cultured embryos showed profuse minute rooting on regeneration medium supplemented with N ⁶-benzyladenine (3 mg/l). Microshoots were rooted in the presence of indole-butyric acid (IBA) (2 mg/l). DNA samples from the mother plant and 18 randomly selected regenerated plants from a single callus were subjected to RAPD analysis with 6 arbitrary decamer primers for the selection of putative somaclones. A total of 64 band positions were scored, out of which 19 RAPD bands were polymorphic. From genetic similarity coefficient based on RAPD band data sharing, it was found that the majority of the clones were almost identical or more than 92% similar to the mother plant, except CL2 and CL9 (66%) which showed highest degree of genetic change with CL2 and CL9 showing presence of two non-parental bands each.     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace Gaza—The “African Potato”

We present efficient protocols for the regeneration of fertile plants from corm explants of Hypoxis hemerocallidea Fisch. & C. A. Mey. landrace Gaza, either by direct multiple shoot formation or via shoot organogenesis from corm-derived calluses. The regeneration efficiency depended on plant growth regulator concentrations and combinations. Multiple direct shoot formation with high frequency (100% with 5–8 shoots/explant) was obtained on a basal medium (BM) supplemented with 3 mg/l kinetin (BM1). However, efficient indirect regeneration occurred when corm explants were first plated on callus induction medium (BM2) with high kinetin (3 mg/l) and naphthalene acetic acid (NAA 1 mg/l), and then transferred to shoot inducing medium (BM3) containing BA (1.5 mg/l) and NAA (0.5 mg/l). Shoot regeneration frequency was 100% and 30–35 shoots per explant were obtained. The regenerated shoots were rooted on a root inducing medium (BM4) containing NAA (0.1 mg/l). Rooted plantlets were transferred to the greenhouse. The regenerants were morphologically normal and fertile. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. Efficient cloning protocols described here, have the potential not only to substantially reduce the pressure on natural populations but also for wider biotechnological applications of Hypoxis hemerocallidea—an endangered medicinal plant.