Stability of salt tolerance at the cell level after regeneration of plants from a salt tolerant tobacco cell line

Plants were regenerated from both the wild type and a stable NaCI-tolerant line of tobacco cells ( Nicotiana tabacum/gossii ). The regeneration process was much more difficult in the case of the NaCI-tolerant line and was only successful in the absence of NaCI. These plants differed morphologically from those regenerated from the wild type cell line, exhibiting abnormally short internodes, small leaves and reduced growth. Cell suspension cultures derived from plants regenerated from the stable NaCI-tolerant line retained a high level of tolerance to salt. The NaCI-concentration required to reduce fresh and dry weight gain by 50% was about twice that observed in the case of the cells obtained from wild type plants.
The results presented here, together with those of Watad et al. (1985), indicate that resistance to salt is operating and stable at the cellular level before and after plant regeneration. When the regenerated plants were grown in increasing levels of salt their growth response was not clearly different from that of the plants regenerated from the wild type cell line. However, the survival of plants on high concentrations of NaCI tended to be higher in the case of plants regenerated from the NaCI-tolerant cell line.     

Vegetative regeneration on sexual organs inPhycomyces blakesleeanus

Phycomyces blakesleeanus produced an abundance of sexual organs when two mating types met on solid medium, but only about 14.7% of the sexual organs developed to the final stages. On the sexual organs showing arrested development, vegetative hyphae or dwarf sporangiophores (microphores) often regenerated. This vegetative regeneration was accelerated when the paired and looped progametangia were isolated from mycelia, when the counterparts of the progametangial cells constructing the loop were surgically incised, and whenPhycomyces was mated at high temperature (25–27°C). A leaky-carotenogenic mutant, whose sexual reaction was imperfect and arrested at an intermediate stage even when mated with the wild type, also regenerated hyphae with a high frequency on these arrested intermediate organs. The vegetative regeneration seems to result from interruption of a cell-to-cell recognition system between cells of different mating type, which is believed to be essential for the mating process of this fungus in addition to the pheromonal actions.     

In vitro propagation of four saponin producing <Emphasis Type="Italic">Maesa</Emphasis> species

A successful micropropagation system was developed for four different medicinal Maesa species. Multiple shoots were induced through both axillary bud formation and adventitious shoot regeneration from leaf explants. The explants were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), thidiazuron (TDZ) and/or α-naphthalene acetic acid (NAA). The success of regeneration varied for different species and depended on the type and concentration of plant growth regulators. Regenerated shoots spontaneously developed roots within 6 weeks on MS hormone-free medium. The rooted shoots were transferred to the greenhouse with a 100% success rate. Furthermore, flow cytometry analysis indicated that there were no changes in ploidy level of those regenerated shoots as compared with wild type adult plants. Thin layer chromatography (TLC) analysis revealed that common and distinguishing spot of saponins were similarly observed in regenerated shoots compared to the control plants. Therefore, the protocol also provides an effective means for the in vitro conservation of Maesa spp. that produce pharmaceutically interesting saponins.     

Plant regeneration from protoplasts of cytoplasmic male sterile lines of rice (Oryza sativa L.)

This study compared plant regeneration from protoplasts isolated from suspension cultures of threeJaponica rice (Oryza sativa L.) lines with different male sterile cytoplasms. More than 180 green plants were regenerated from protoplasts from 5–8 month old suspensions of IR58024A, a line with the WA type of cytoplasmic male sterility (CMS). About 40% of the calli recovered from protoplasts produced green plants. ShuangbaiA (BT type of CMS) and Tai2A (Dian I type of CMS), both from Zhejiang province of China, responded less well in culture. ShuangbaiA produced green plants from 6.6% of calli, although initial protoplast yield per gram fresh weight was higher than for IR58024A. Tai2A showed lower protoplast yield, and only 1.1% of the calli produced green plants. Flow cytometric analyses of nuclear DNA content indicated that many of the regenerated plants were tetraploid. The percentage of tetraploids varied in the different lines. The male sterile characteristics of the original lines were maintained in the regenerated plants. Pollen abortion occured earliest in IR58024A and latest in Tai2A. IR58024A is a promising rice genotype for use as a recipient in direct gene transfer experiments.Abbreviations BAP 6-benzylaminopurine - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxyacetic acid - IRRI International Rice Research Institute - LS Linsmaier and Skoog's (1965) medium - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid - WA wild abortive     

High capacity of plant regeneration from callus of interspecific hybrids with cultivated barley (Hordeum vulgare L.)

Callus was induced from hybrids between cultivated barley (Hordeum vulgare L. ssp. vulgare) and ten species of wild barley (Hordeum L.) as well as from one backcross line ((H. lechleri x H. vulgare) x H. vulgare). Successful callus induction and regeneration of plants were achieved from explants of young spikes on the barley medium J 25–8. The capacity for plant regeneration was dependent on the wild parental species. In particular, combinations with four related wild species, viz. H. jubatum, H. roshevitzii, H. lechleri, and H. procerum, regenerated high numbers of plants from calli.     

Transformation by Agrobacterium rhizogenes and regeneration of transgenic shoots of the wild soybean Glycine argyrea

Glycine argyrea accession G1420 was evaluated for its response to inoculation with Agrobacterium rhizogenes strains LBA9402 and A4T, carrying wild type Ri plasmids, and by strains R1601 and A4TIII with engineered plasmids. Hypocotyls from young seedlings were the most responsive in producing roots at inoculation sites. Root production was also dependent on bacterial concentration. Excised, cultured roots produced green nodular callus which regenerated shoots on SC2 medium containing 1.1 mg l^–1 6-benzylaminopurine and 0.005 mg l^–1 indole-3-butyric acid. The transformed nature of the roots and of callus regenerating shoots was confirmed by the presence of opines and by dot blot analysis for Ri TL-DNA. Tissues regenerated from roots transformed by A. rhizogenes strains R1601 and A4TIII exhibited NPTII enzyme activity, confirming the stable integration and expression of the chimaeric kanamycin resistance gene in transgenic tissues.Abbreviations BAP 6-benzylaminopurine - IBA indole-3-butyric acid - NPTII neomycin phosphotransferase II - SDS sodium dodecyl sulphate     

Isolation and characterization of a sporeless mutant in <Emphasis Type="Italic">Pleurotus eryngii</Emphasis>

 A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains. Received: September 5, 2002 / Accepted: October 16, 2002 Acknowledgments We are grateful to Mrs. Motoe Masuda for her skillful technical assistance. Contribution no. 358 from the Tottori Mycological Institute Correspondence to:Y. Obatake     

The neurotrophin receptors,trkB and p75, differentially regulate motor axonal regeneration

Neurotrophic factors that support neuronal survival are implicated in axonal regeneration after injury. Specifically, a strong role for BDNF in motor axonal regeneration has been suggested based on its pattern of expression after injury, as well as the expression of its receptors, trkB and p75. Despite considerable in vitro evidence, which demonstrate specific and distinct physiological responses elicited following trkB and p75 activation, relatively little is known about the function of these receptors in vivo. To investigate the roles of the trkB and p75 receptors in motor axonal regeneration, we have used a tibial (TIB)‐ common peroneal (CP) cross suture paradigm in p75 homozygous (?/?) knockout mice, trkB heterozygous (+/?) knockout mice, as well as in their wild‐type controls. Contralateral intact TIB motoneurons, and axotomized TIB motoneurons that regenerated their axons 10 mm into the CP distal nerve stump were identified by fluorescent retrograde tracers and counted in the T11‐L1 spinal segments. Regeneration was evaluated 2, 3, 4, 6, and 8 weeks after nerve repair. Compared to wild‐type animals, there are significantly fewer intact TIB motoneurons in p75 (?/?), but not trkB (+/?) mice. The number of motoneurons that regenerated their axons was significantly increased in the p75 (?/?) knockout mice, but significantly attenuated in the trkB (+/?) mice compared to wild‐type controls. These results suggest that p75 is important for motoneuronal survival during development, but p75 expression after injury serves to inhibit motor axonal regeneration. In addition, full expression of trkB is critical for complete axonal regeneration to proceed. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 314–325, 2001     

Production of haploids and doubled haploids from unfertilized ovule culture of various wild species of gentians (Gentiana spp.)

The production of haploids and doubled haploids (DHs) on unfertilized ovule culture was examined in 19 wild species of gentians (Gentiana ssp.) classified into eight sections. Of the 19 species including 35 strains, embryo-like structures (ELSs) were obtained from unfertilized ovule culture in 15 species, and regenerated plants were produced in 11 species. ELS production has varied greatly among the15 species, i.e., 0.5–79.2% frequency of responding flower buds and 0.01–1.99 ELSs per flower bud. Of the ELS-producing species, almost all were classified into the sects. Pneumonanthe or Cruciata. Species in sect. Pneumonanthe showed higher responses than those in sect. Cruciata. In examining the effect of flower bud stage on ELS production, more than twice as many ELSs were observed at the anther-dehiscent stage than that at the anther-indehiscent stage. Ploidy level was determined in 117 randomly selected regenerated plantlets, which suggests that most were diploid (46.2%) and haploid (32.5%). When 12 diploid plants were examined using simple sequence repeat (SSR) markers, 8 (66.7%) were DH. This study revealed that unfertilized ovule culture can be applied not only on cultivated gentian species but also on a number of wild species. The production of haploids and DHs in wild gentians provides novel prospects for ornamental and/or medicinal gentian breeding.

     

Characteristics of carrot plant regenerated from two cell line selected as either ionic-Al tolerant cell or Al-phosphate utilizing cells

Plantlets of carrot (Daucus carota L.) were regenerated from two types of cell lines. One type was selected as ionic-Al tolerant (IAT) cells, while the second type featured Al-phosphate utilizing cells (IPG). Their tolerance characteristics were investigated. The plantlets from IAT were directly regenerated, whereas those from IPG were regenerated after somatic hybridization with wild-type cells previously inactivated with iodacetamide, because the IPG cells had completely lost the ability to regenerate naturally.The sexual progeny of IAT showed Al-tolerant properties, established by testing their root elongation in the presence of 500 µM Al ions. Most of the calli obtained from the somatic hybrids grew more rapidly than the wild-type cells when Al-phosphate was used as a sole source of phosphorus. Thus, we obtained two types of carrot plantlets, regenerated from IAT and IPG. Both possessed the tolerant characteristics as observed with the stress-selected cells.     

Ultraviolet mutagenesis and genetic analysis of resistance to methylglyoxal-bis (guanylhydrazone) in tobacco

Summary We have isolated cell lines of Nicotiana tabacum resistant to methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of S-adenosylmethionine decarboxylase. We obtained 31 resistant lines from ultraviolet light mutagenized cultures, representing at least 13 independent events. No resistant lines were obtained from non-mutagenized control cultures. The increase in mutation rate due to the ultraviolet light treatment was 22 to 62 fold increased over an estimate of the maximum possible spontaneous rate. In reconstruction experiments we reselected resistant cell lines from varying dilutions into a background of a constant concentration of wild type cells; at the minimum ratio of resistant cells to wild type cells, 1:125,000, we recovered resistant colonies at an estimated plating efficiency of 12.5%. A number of resistant lines have been regenerated into plants. All of the ones that flowered are male sterile, sometimes associated with morphological transformations. Some are female sterile as well. Meiotic genetic analysis of one resistant line, Mgr12, suggests that the MGBG resistance is segregating as a nuclear dominant trait. The male sterility and abnormal floral development of Mgr12 cosegregate with the MGBG resistance, suggesting the two phenotypes are coincident.     

Selection, characterization and regeneration of hydroxyproline-resistant cell lines of Solanum tuberosum: Tolerance to NaCl and freezing stress

Sixty-seven hydroxyproline-resistant (hyp^r) cell lines were selected from cell suspensions of a diploid potato ( Solanum tuberosum L., clone H²578) after plating on 5 and 10 m M hydroxyproline (hyp). Resistant colonies were obtained with a spontaneous frequency of 2.9×10⁻⁶. No clear influence could be shown from treatment with N-ethyl-N-nitrosourea (10 or 50 μ M ). Ninety % of the variant lines contained more proline than the wild type when cells were grown away from hyp for 1 month. Total free amino acid content was increased 2.2 to 6.8 times. When the lines were grown for another 2–5 months on non-selective medium, the content of proline and other amino acids and hyp resistance decreased. After this period the values were, however, still substantially higher than in the wild type. When tested for growth on media with other amino acid analogues (azetidine-2-carboxylic acid and dehydroproline, analogues of proline; aminoethyl-cysteine, analogue of lysine and 3-fluorotyrosine, analogue of tyrosine) and on media with inhibitory concentrations of lysine + threonine. lines H4a and H4b4 were cross resistant to these compounds. When tested on media with inhibitory NaCl concentrations, variant lines H2a, H4a, and H6 showed better tolerance than the wild type. One variant cell line (H4a) was successfully regenerated into plants. Preliminary results showed an increased frost tolerance in the leaves of these plants (−4.5°C compared to −3°C for the wild type), accompanied by a higher leaf proline content. Callus initiated from leaves of the regenerated clones was more resistant to hyp than wild type callus, indicating that the variant trait might be due to a mutation.     

Investigation of chloroplast DNA heteroplasmy in Medicago sativa L. using cultured tissue

Summary Medicago sativa L. cv Regen S is heteroplasmic for chloroplast DNA (cpDNA). Previous analyses of regenerated plants have shown a predominance of one of the cpDNAs which we have designated type A (the other we have designated type B). Studies of the replication of the two cpDNAs in tissue culture were carried out using leaflet expiants with defined cpDNA types and a distinguishing probe. The explants obtained showed a bias toward type A cpDNA during tissue culture. The data suggest that chloroplasts with different DNAs in a common nuclear background can multiply at different rates.     

Improved isolation of dihaploidsolanum tubersoum protoplasts and the production of somatic hybrids between dihaploidS. tuberosum andS. brevidens

Summary A modified protoplast isolation technique, applicable to a range of dihaploidSolanum tuberosum genotypes, has been developed. A combination of high calcium and high pH was used to fuse mesophyl protoplasts of dihaploidS. tuberosum (PDH40) and the diploid wild speciesS. brevidens. Large numbers of colonies were obtained after fusion and putative hybrids selected on the basis of phenotype from regenerated shoots. From these, 11 somatic hybrid plants have been identified by their isoenzyme patterns and morphologic characteristics. Four of these hybrids had the expected chromosome number of 48. The approach of mass culture after fusion followed by selection of hybrids from regenerated shoots and the application of somatic hybridization to potato breeding are discussed.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

Loss of AtPIN1 does not influence the <Emphasis Type="Italic">in vitro</Emphasis> morphogenic potential of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> suspension cultures

Suspension cultures from Arabidopsis thaliana wild type and AtPIN1-deficient lines were initiated and maintained for more than 3 years. A protocol for efficient regeneration from long-term suspension cultures was established. Arabidopsis wild-type and respectively AtPIN1 mutant plants have been regenerated from these cultures and characterized. Additionally, transgenic suspension cultures expressing the uidA ( -glucuronidase) reporter gene under the control of AtPIN1 promoter have been used for morphogenic studies. Our studies suggest that a lack of AtPIN1 function affects shoot differentiation and development, but does not influence in vitro regeneration of plants.     

苜蓿抗甲硫氨酸变异体的筛选

紫花苜蓿(Medicago sativa L.)下胚轴愈伤组织用NaN_3溶液诱变处理后,在含有全致死浓度甲硫氨酸的MS培养基上进行了6个月的连续筛选培养,获得了能抗100mmol/L甲硫氨酸的变异细胞系,并分化成再生植株。所获变异细胞系在脱离选择压力6个月后,对甲硫氨酸的抗性仍比对照高7.2倍,并表现出对乙硫氨酸的交叉抗性(为对照抗性的3.3倍)。抗性细胞系及其再生植株的甲硫氨酸、赖氨酸、苏氨酸和异亮氨酸含量均比对照有大幅度增加。抗性系的SDS-PAGE电泳图谱及过氧化物酶同工酶谱带均与对照有显著不同,并出现了新带,表明变异系已经产生变化了的基因产物。     

Nuclear DNA content of <Emphasis Type="Italic">Hydrastis canadensis</Emphasis> L. and genome size stability of in vitro regenerated plantlets

Knowing the genome size is an important step towards deciding and planning for genome sequencing of a given species. Using flow cytometry, nuclear DNA content of Hydrastis canadensis was estimated, and genome size stability of its in vitro regenerated plantlets were assessed. The nuclear DNA content of H. canadensis was estimated to be 2.62 ± 0.020 pg/2C. This is the first report to estimate nuclear DNA content of H. canadensis; therefore this study provides valuable information that will facilitate genome sequencing and subsequent molecular studies of this economically important medicinal plant. Comparison of genome size between in vitro regenerated (explant source was from wild plants) and wild plants of H. canadensis did not reveal any significant difference (P ≤ 0.05) in nuclear DNA content. This suggests that micropropagation of H. canadensis, even after numerous sub-culturing and long-term culture periods produced in vitro plantlets with a stable genome size. These results provide further evidence that micropropagation techniques have the potential to be used as a source of planting stock, along with seeds, for restoring locally threatened H. canadensis wild populations and for commercial cultivation to supply the growing herbal market.