Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.)

We report regeneration of fertile plants from barley (Hordeum vulgare L. cv. Igri) protoplasts isolated from regenerable suspension cultures initiated from anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine - PP Protoplasts     

Application of the salt stress to the protoplast cultures of the carrot (Daucus carota L.) and evaluation of the response of regenerants to soil salinity

This is the first study to generate carrot plants for enhanced salinity tolerance using a single-cell in vitro system. Protoplasts of three carrot accessions were exposed to treatment by seven different concentrations of NaCl (10–400 mM). Salt concentrations higher than 50 mM decreased plating efficiency and those of 200–400 mM of NaCl completely arrested mitotic divisions of cultured cells. The protoplast-derived plants from the control and 50–100 mM NaCl treatment were subjected to an 8-week salt stress in greenhouse conditions induced by salinized soil (EC 3 and 6 mS cm^?1). 50 mM NaCl stress applied in vitro induced polyploidy among regenerated plants. The regenerants obtained from the 50 and 100 mM NaCl-treated protoplast cultures grown in saline soil had a higher survival rate compared to the regenerants from the control cultures. The salt-stressed plants accumulated anthocyanins in petioles and produced denser hairs on leaves and petioles in comparison to the control plants. Salt stress influenced pollen viability and seed setting of obtained regenerants. The results suggest that salt stress applied in vitro in protoplast cultures creates variation which allows alleviating the negative effects of salt stress on the development and reproduction of the carrot.

     

Fertile plant regeneration from cell suspension and protoplast cultures of barley (t Hordeum vulgare cv. Schooner)

Fertile plants were regenerated from both cell suspension and protoplast-derived cultures of the two-row barley, Hordeum vulgare L. cv. Schooner. Embryogenic calluses, derived from immature embryos, were used to establish suspension cultures. More than 100 plants, with variable seed set, have been regenerated from six embryogenic cell suspension cultures. Protoplasts isolated from three suspension cultures divided and when the resultant embryogenic proto-calluses were transferred to regeneration medium both green and albino shoots were produced. The green shoots were transferred to growth regulator-free medium and plantlets that developed strong root systems were potted in soil and grown to maturity in the glasshouse. Root tip analysis of plants regenerated from cell suspension cultures revealed the expected 2N = 14 complement of chromosomes. However, chromosomal analysis of protoplast-derived plants showed numerical variation among a proportion of the regenerants. This revised version was published online in June 2006 with corrections to the Cover Date.     

In Vitro Selection Against Ascochyta Blight of Chickpea (Cicer arietinum L)

Callus cultures established on MS medium containing 2.0 mg l^-1 2, 4-D were inoculated on the regeneration medium supplemented with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3%, v/v) of culture filtrate (CF) of Ascochyta rabiei infesting chickpea. Out of 486 callus pieces and 270 regenerants obtained from immature embryo derived callus screened, 50 callus lines and 74 regenerants were found resistant. Further, these resistant callus lines and regenerants were subjected to stability test by growing them on a medium containing 3% CF. Seventeen callus lines and 28 regenerants of the selected lines showed normal growth on the selection medium. The regenerated plants were tested in pots under artificial epiphytotic conditions where they showed normal growth behaviour and high degree of resistance.     

In vitro clonal propagation and genetic fidelity of the regenerants of Spilanthes calva DC. using RAPD and ISSR marker

An efficient in vitro protocol has been established for clonal propagation of elite plant of Spilanthes calva DC., an important source of spilanthol, an antimalarial larvicidal compound. Nodal explants excised from field grown plant of S. calva DC. when reared on Murashige and Skoog’s medium augmented with different cytokinins, viz. N⁶-Benzyladenine (BA), N⁶-(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn), differentiated multiple shoots from the axils. BA at 10 μM proved optimum for elicitation of multiple shoots in 91.6 % cultures with an average of 7.12 shoots per explant within 6 weeks. The excised shoots rooted on half strength Murashige and Skoog’s medium supplemented with 0.1 μM IBA. Micropropagated plants were hardened and transferred to field for acclimatization, where 95 % plants survived and were phenotypically similar to the donor plant. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to evaluate the genetic fidelity amongst the regenerants. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 71 scorable bands, ranging in size from 100 bp to 1,100 bp were generated by a combination of the two markers in the aforesaid plants. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant. The similarity values amongst the aforesaid plants varied from 0.967 to 1.000. The dendrogram generated through UPGMA (Unweighted Pair Group Method with arithmetic mean) analysis revealed 98 % similarity amongst them, thus confirming the genetic fidelity of the in vitro clones.     

Plant growth regulator induced phytochemical and antioxidant variations in micropropagated and acclimatized Eucomis autumnalis subspecies autumnalis (Asparagaceae)

Eucomis species is a valuable plant with both medicinal and horticultural potential. The current study evaluated the role of plant growth regulator (PGR) on growth, phytochemicals, and antioxidant activity in Eucomis autumnalis subspecies autumnalis. Five cytokinins including topolins and benzyladenine (BA) at 2 µM in combination with varying (0–15 µM) concentrations of naphthalene acetic acid (NAA) were tested. In vitro regenerants were acclimatized in the greenhouse for 4 months. Highest number of shoots (9 shoots/explant) was observed with 15 µM NAA alone or when combined with BA. Acclimatized plants derived from the 15 µM NAA treatment had the highest number of roots, largest leaf area and biggest bulbs. While applied PGRs increased the iridoids and condensed tannins in the in vitro regenerants, total phenolics and flavonoids were higher in the PGR-free treatment. Among the in vitro regenerants, 5 µM NAA and 2 µM BA treatments produced the best antioxidant activity in the DPPH (55 %) and beta-carotene (88 %) test systems, respectively. A remarkable carry-over effect of the PGR was conspicuous in the phytochemical levels and antioxidant activity of the 4-month-old plants. In addition to the optimized micropropagation protocols, the current findings present a promising potential for manipulating the type and concentration of applied PGRs to improve phytochemical production and hence medicinal value in E. autumnalis subspecies autumnalis.     

Detection of somaclonal variation in garlic (Allium sativum L.) using RAPD and cytological analysis

Plants were regenerated by somatic embryogenesis from long-term callus cultures derived from five garlic (Allium sativum L.) cultivars. Thirty-five of these plants were subjected to RAPD analysis. The frequency of variation was found to be cultivar dependent: approximately 1% in the two clones Solent White and California Late and around 0.35% in another three clones, Chinese, Long Keeper and Madena. Certain band changes were found in regenerants of different cultivars, suggesting the existence of a mutation-sensitive part of the garlic genome. The karyotypes of another 75 regenerants derived from the same callus cultures of three parental garlic clones were examined. Of these plants, 9.3% were found to be tetraploids, 4% aneuploid and 2.6% showed a change in the position of the secondary constriction. No association could be shown between the rate of variation for molecular and cytological characters either by comparing cultivars or examining individual regenerants. Received: 30 July 1996 / Revision received: 28 October 1996 / Accepted: 12 November 1996     

Plant regeneration from leaf-derived protoplasts within the Daucus genus: effect of different conditions in alginate embedding and phytosulfokine application

Protoplasts of four Daucus carota subspecies and three wild Daucus species were isolated from 2-week-old shoot cultures during overnight incubation in an enzyme mixture composed of 1 % (w/v) cellulase Onozuka R-10 and 0.1 % (w/v) pectolyase Y-23. Before the culture, they were embedded in autoclave- or filter-sterilized alginate solution. Modified thin alginate layer (TAL) and extra thin alginate film (ETAF) techniques were applied for protoplast immobilization. A rich mineral-organic medium based on the formulation of Kao and Michayluk supplemented with 0.1 mg l^?1 2,4-dichlorophenoxyacetic acid, 0.2 mg l^?1 zeatin, and optionally 100 nM phytosulfokine (PSK), a peptidyl plant growth factor, was used for protoplast culture. Plating efficiency was genotype-dependent and in 40-day-old cultures, it varied from 10 % for Daucus pusillus to 73 % for D. carota subsp. sativus. A considerably higher ability in colony formation was observed in the modified TAL culture system using filter-sterilized alginate and in the presence of PSK in the protoplast culture medium. Plant regeneration through somatic embryogenesis stimulated by PSK application occurred for five out of the seven Daucus accessions used in the present study. We believe our data may facilitate the use of wild Daucus in somatic hybridization with cultivated carrot.     

In vitro selection of gamma irradiated shoots of ginger (Zingiber officinale Rosc.) against Fusarium oxysporum f.sp. zingiberi and molecular analysis of the resistant plants

In the present investigation, an attempt was made to develop Fusarium yellows resistant plants of ginger (Zingiber officinale Rosc.) var. Himgiri using in vitro mutagenesis and selection technique. Axenic in vitro cultures were subjected to gamma irradiation (10–100 Gy) for mutation induction. LD₅₀ was calculated after every 4 weeks, and was observed to be 15 Gy after 16 weeks of irradiation. Surviving 10 Gy irradiated shoots were cultured on selective medium containing different concentrations (0–20%) of fungal culture filtrate (FCF) obtained from Fusarium oxysporum f.sp. zingiberi for in vitro selection. FCF concentration of 17.5% in the selective medium was found to be the highest on which 5% shoots survived after first selection cycle, which further reduced to 1.1% after third continuous cycle of selection. It was followed by 4.3% shoot survival after third selection cycle on 15% FCF concentration. Surviving, selected shoots from 15 and 17.5% FCF were multiplied on multiplication medium and rooted plantlets were hardened with 100% survival. On in vivo evaluation 46.4 and 52% plants selected at 15 and 17.5% FCF were found to be highly resistant. Molecular analysis with disease specific SSR primers differentiated between FCF selected, tissue culture propagated and gamma irradiated plants. Two unique bands were obtained in 15 and 17.5% FCF selected plantlets with GIN 6 and GIN 9 primers, sequencing, of which showed 98 and 97% homology with disease resistance protein-like gene CC-NBS-LRR from clones ZwP627 and ZoP620 of Z. officinale. After in vivo bioassay, SSR analysis of selected highly resistant plants again confirmed the unique bands with both the primers. The DNA sequences obtained from these primers have been published in GenBank under accession number MN497252 and MN497253.

     

Production and molecular characterization of diploid and tetraploid somatic cybrid plants between male sterile Satsuma mandarin and seedy sweet orange cultivars

Seedlessness, an important economic trait for fresh fruit, is among the prior goal for all citrus breeding programs. Symmetric somatic hybridization provides a new strategy for citrus seedless breeding by creating cybrids transferring mitochondrial DNA (mtDNA) controlled cytoplasmic male sterility (CMS) from the callus parent Satsuma mandarin (C. unshiu Marc.) to seedy cultivars. In this study, protoplast fusion was adopted to transfer CMS from C. unshiu Marc. cv. Guoqing No. 1 (G1) to three seedy sweet oranges (C. sinensis L. Osb.), i.e. ‘Early gold’, ‘Taoye’ and ‘Hongjiang’. Flow cytometry analysis showed that 12 of 13 regenerated plants from G1 + ‘Early gold’, 9 of 12 from G1 + ‘Taoye’ and both two plants from G1 + ‘Hongjiang’ were diploids, while the remaining regenerated plants were tetraploids. Molecular analysis using 23 simple sequence repeat (SSR) markers previously proven to map to the citrus genome showed that the nuclear DNA from all recovered diploid and tetraploid plants derived from their corresponding leaf parent, while cleaved amplified polymorphic sequence analysis showed that the mtDNA of all regenerated plants derived from the callus parent, indicating that the regenerated 2X and 4X plants from all these three combinations are authentic cybrids. Furthermore, the Chloroplast SSR analysis revealed that somatic cybrid plants from the three combinations possessed either of their parental chloroplast type in most cases. These results demonstrated that mtDNA of G1 Satsuma mandarin was successfully introduced into the three seedy sweet orange cultivars for potential seedlessness via symmetric fusion.     

Micropropagation of Ajuga bracteosa, a medicinal herb

For conservation and genetic transformation, a successful in vitro micropropagation protocol for Ajuga bracteosa, a medicinal herb has been established for the first time. MS medium supplemented with IAA (2 mg/L) and BA (5 mg/L) induced 100 % shoot regeneration with an average of 41.4 shoots of 8.4 cm per culture. Excised in vitro shoots when transferred to MS + IBA (0.5 mg/L) produced 20 roots/shoot of 20.2 cm average length in 100 % cultures. Of the three explants, leaf, petiole and root, leaf displayed quickest response followed by petiole while root was the slowest. Hardening of plantlets was achieved with 82 % survival. The hardened plants were maintained in pots with garden soil under controlled (Temp. 25?±?2 °C) conditions. RAPD exhibited genetic fidelity with 100 % monomorphism in regenerants.     

In vitro induction of tetraploids in an interspecific hybrid of Calanthe (Calanthe discolor × Calanthe sieboldii) through colchicine and oryzalin treatments

Tetraploids were successfully produced from diploid seeds obtained through interspecific crossing between Calanthe discolor and Calanthe sieboldii by treating with colchicine or oryzalin. Colchicine was tested at concentrations of 0.05 and 0.1 % for 0, 3, or 7 days and oryzalin was tested at a concentration of 0.003 % for 1, 2, 4, and 7 days, and the ploidy of the seedlings was determined by flow cytometry. Tetraploids (4×) were obtained from the interspecific hybrid seeds treated with all colchicine and oryzalin concentrations. The most efficient condition for inducing tetraploids seemed to be treated with 0.003 % oryzalin for 1 or 2 days. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The stomatal density and sizes of the tetraploid plants were significantly higher and larger than those of the diploid plants. Differences in leaf shape were found between the tetraploid and diploid plants under the same growing conditions: the leaves of the diploids were elongated and those of the tetraploids were round.     

Morphological, physiological, cytological and phytochemical studies in diploid and colchicine-induced tetraploid plants of Echinacea purpurea (L.)

Echinacea purpurea (L.) is one of the important medicinal plant species. To obtain the tetraploid plants of Echinacea purpurea with improved medicinal qualities, the root tips of two true leaves seedlings were imbibed in 0.25 % (w/v) colchicine solution for 24, 48, 72, 96 and 168 h. The ploidy level of plants was determined by chromosome counting of root tip cells, and confirmed by flow cytometric analysis. Tetraploid induction occurred in seedlings treated for 24, 48 and 72 h at colchicine solution. The morphological, physiological, cytological, and phytochemical characteristics of diploid and colchicine-induced tetraploid plants were compared. Results indicated that tetraploid plants had considerable larger stomata, pollen grain, seed and flower. Moreover, chloroplast number in guard cells, amount of chlorophyll (a, b, and a + b), carotenoids as well as width and thickness of leaves were increased in tetraploids. However, stomata frequency, leaf index, plant height, and quantum efficiency of photosystem II in tetraploid were lower than diploid plants. High-performance liquid chromatography analysis showed that leaves of the tetraploid plants had more cichoric acid (45 %) and chlorogenic acid (71 %) than diploid plants. It was concluded that morphological and physiological characteristics can be used as useful parameters for preliminary screening of putative tetraploids in this species.     

In vitro regeneration and cytological characterization of shoots from leaf explants of three accessions of Solanum commersonii

In vitro culture of explants were used to apply genetic or cell engineering techniques to the sexually incompatible potato relative Solanum commersonii (2n=2x=24) Three accessions of S. commersonii were tested for regeneration from leaf explants using six different protocols. A two step-regeneration procedure gave the best results. Genetic variability for regeneration ability was found between accessions, and between clones within accessions. The accession PI 472834 regenerated at highest frequency. Clones with high regeneration ability were selected. Approximately 60% of regenerated plants were diploids and 40% were tetraploids. A very low frequency of chimeras was found. Leaf shape and chloroplast counts in guard cells were shown to be quick and reliable methods for estimating ploidy levels. Use of the diploid and tetraploid regenerants obtained for potato breeding is discussed.Abbreviations BAP 6-benzylaminopurine - EBN Endosperm Balance Number - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA 1-naphthalene-acetic acid - ZEA zeatin     

Chromosome variations in regenerants of Arabidopsis thaliana derived from 2- and 6-week-old callus detected using flow cytometry and FISH analyses

Shoot organogenesis was induced from 2- and 6-week-old callus derived from the leaves of Arabidopsis thaliana ecotype Columbia (2n = 10). Regenerated plants were evaluated for chromosomal variations by means of flow cytometry and fluorescent in situ hybridization (FISH). Flow cytometric measurements revealed the occurrence of diploid, tetraploid, and octoploid plants among the regenerants of 2-week-old calli, whereas only diploid and tetraploid plants were regenerated from the 6-week-old calli. Chromosome counting showed that plants developed from the 2-week-old calli exhibited mixoploidy and a high frequency of aneuploid cells. These plants were infertile and displayed altered morphology. FISH with 5S and 25S rDNA probes allowed to detect some structural chromosomal rearrangements in regenerated plants. Along with cells which exhibited correct localisation of rDNA loci, also cells bearing chromosomal translocations, deletions or duplications were found. The type of structural aberrations varied between diploid and tetraploid regenerants.     

Direct shoot regeneration from basal leaf segments of Lilium and assessment of genetic stability in regenerants by ISSR and AFLP markers

Here, we report a widely applicable procedure for direct shoot regeneration via basal leaf segments of Lilium. Leaf segments (0.8–1.0 cm long and 0.4 cm wide) were excised from leaves on shoot nodes 3 to 6 of 4-wk-old in vitro stock shoot cultures. The segments were wounded by three transverse cuts across the midvein on the abaxial side, with 1 mm between cuts, and cultured with the abaxial side in contact with a shoot regeneration medium composed of half-strength Murashige and Skoog medium supplemented with 1 mg/l naphthaleneacetic acid, 0.5 mg/l thidiazuron, 30 g/l sucrose, and 7 g/l agar (pH?5.8). The cultures were incubated for 4 wk under a 16-h photoperiod at 23?±?2°C for adventitious shoot regeneration. With this procedure, a mean shoot regeneration frequency of 92–100% and mean number of shoots of 4.7–7.0 per segment were obtained in five Lilium species and hybrids, which represent diverse genotypes of Lilium and are commercially popular lilies. Histological studies with Lilium Oriental hybrid “Siberia” revealed that meristemoids initiated from subepidermal cells on the adaxial side of the explant and eventually developed into adventitious buds, without callus formation. In an assessment of genetic stability in the regenerants of “Siberia”, no polymorphic bands were detected by intersimple sequence repeat and only 0.73% polymorphic bands were detected by amplified fragment length polymorphism. The morphologies of the regenerants were identical to those of the control. These results demonstrated that the regenerants were genetically and morphological stable. Thus, this procedure has great potential application for micropropagation, genetic transformation, and preparation of shoot tips for cryopreservation and cryotherapy for virus eradication of Lilium.     

A morphological study of species boundaries of the wild potato Solanum brevicaule complex: replicated field trials in Peru

The Solanum brevicaule complex contains about 20 species of diploids (2n = 2x = 24), tetraploids (2n = 4x = 48) and hexaploids (2n = 6x = 72), distributed from central Peru south to northwestern Argentina. The complex is defined entirely by morphological similarity of its constituent members, which are very similar to each other and to some landraces of the cultivated potato, Solanum tuberosum. Conflicting taxonomic treatments are common among authors. Species boundaries within the complex have been studied with morphological phenetics from germplasm accessions planted in a field plot in the north central US, and with molecular marker data from RAPDs, low-copy nuclear RFLPs, and AFLPs. The present study compares these results with an additional replicated morphological study of the same germplasm accessions in a greenhouse environment in the high Andes of central Peru. The results support extensive reduction of species in the complex.     

Production of plants resistant to <Emphasis Type="Italic">Alternaria carthami</Emphasis> via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamus tinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 10⁵ spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min⁻¹ mg⁻¹ protein) from organogenesis and 47% (0.23 μmol min⁻¹ mg⁻¹ protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg⁻¹ protein) and 19.5% (145 U mg⁻¹ protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.     

柑桔细胞电融合再生两个种间体细胞杂种

朋娜脐橙(Citrussinensis Osbeck)胚性细胞悬浮系原生质体分别与粗柠檬(C.jambhiri Lush)、枸头橙(C.aurantium)叶肉原生质体经电场诱导而融合。经培养,两组合均获得再生植株。对朋娜脐橙+粗柠檬的再生胚状体进行染色体计数,随机取样的52个胚状体中,26个为四倍体,另外26个为二倍体;对74棵再生植株进行染色体计数,由此说明都为四倍体;表明体细胞杂种在植株再生过程中具有明显的竞争优势。朋娜脐橙+枸头橙再生的14棵植株都为四倍体。对朋娜脐橙+粗柠檬部分植株进行POX同工酶和RAPD分析,表明所有检测植株都为杂种。朋娜脐橙+枸头橙再生植株经RAPD分析,表明也为杂种。     

Reversion of Texas male-sterile cytoplasm maize in culture to give fertile,T-toxin resistant plants

Summary Plants carrying Texas male-sterile (Tms) cytoplasm are normally sensitive to Drechslera maydis T-toxin. Tissue cultures were initiated from immature embryos of maize carrying Tms-cytoplasm, and plants were regenerated after selection for resistance to T-toxin. Fertile, T-toxin resistant plants were obtained from the unselected control cultures as well as from the selected material. In addition, one regenerant from an unselected culture was fertile and T-toxin sensitive. The progeny of the regenerants showed the phenotype of the female parent with respect to pollen-fertility, and T-toxin resistance. The data are consistent with the heritable changes observed being the result of the expression of an altered mitochondrial genome.