Somatic cell cryopreservation and protoplast regeneration of important disease-resistant wild riceOryza meyeriana Baill

Oryza meyeriana Baill is one of the three wild rice species found in Chiia.O. mcyeriana possesses valuable characteristics but is reluctant in cell culturein vitro. In a series of experiments, callus with no regeneration ability was induced from young panicle ofO. meyeriana. The callus was subcultured and propagated. Embryogenic cell clones were obtained after cryopreswation. Suspension cultures were established and protoplasts were isolated and regenerated into plants. Results of artificial inoculation ofXanthomonas campestris pv.Oryzae showed that the strong resistance did not change in the regenerated plants. The development of protoplast-to-plant system is an important progress towards utilization ofO. meyeriana via cellular engineering. The experiments demonstrated that cryopreservation of plant calli was a new way to obtain embryogenic cell line.     

Somatic cell cryopreservation and protoplast regeneration of important disease-resistant wild rice Oryza meyeriana Baill

Oryza meyeriana Baill is one of the three wild rice species found in Chiia.O. mcyeriana possesses valuable characteristics but is reluctant in cell culturein vitro. In a series of experiments, callus with no regeneration ability was induced from young panicle ofO. meyeriana. The callus was subcultured and propagated. Embryogenic cell clones were obtained after cryopreswation. Suspension cultures were established and protoplasts were isolated and regenerated into plants. Results of artificial inoculation ofXanthomonas campestris pv.Oryzae showed that the strong resistance did not change in the regenerated plants. The development of protoplast-to-plant system is an important progress towards utilization ofO. meyeriana via cellular engineering. The experiments demonstrated that cryopreservation of plant calli was a new way to obtain embryogenic cell line. Project partially supported by the National Natural Science Foundation of China (Grant No. 392704361, Foundation for Outstanding Young Teachers of China, State Key Program of China and Natural Science Foundation of Hubei Province, China.     

云南野生稻不同染色体组型和外植体材料的离体培养研究

云南野生稻不同外植体愈伤组织诱导能力差别较大。花粉培养中愈伤组织诱导率差异在0％～11．8％之间,用成熟胚诱导愈伤组织,其诱导率在18．0％～35．2％之间,茎叶培养则在12．0％～25．0％之间。云南野生稻不同外植体诱导的愈伤组织再分化为绿苗的分化率在8．3％～100．0％之间。疣粒稻组培特性最好,东乡普通野生稻和景洪普通野生稻次之,药用稻最难组培。本文建立了疣粒、东乡、景洪普野3种野生稻的离体无性系,为长期保存云南野生稻资源奠定了基础。     

Isolation of paraquat-tolerant mutants from tomato cell cultures

Summary Tomato callus clones selected for the ability to grow at paraquat concentrations lethal to wild-type cells were found at an approximate frequency of 5×10^–8 per viable cell. Diploid plants were regenerated from nine of the nineteen paraquat-tolerant callus clones isolated. Although some of these plants appeared normal, others had altered morphology and reduced vigor and fertility. New callus cultures initiated from these regenerated plants typically had at least a 30-fold increase over the wild type in tolerance to paraquat. Tests on callus from sexual progeny showed that the paraquat-tolerant phenotypes of clones PQT⁴, PQT⁶, and probably also PQT¹³ resulted from dominant nuclear mutations, but the number of loci involved is not yet known. Paraquat spray experiments indicated that slight paraquat-tolerance was expressed at the plant level in PQT¹³, but not in any of the other clones tested.     

Retention of the capacity to produce plants from protoplasts in cryopreserved cell lines of rice (Oryza sativa L.)

A method is described for cryopreservation of cell suspension lines of rice (Oryza sativa L.) for use in protoplast research and as a way of retaining desirable characteristics of cell lines. The procedure involves pre-culture with mannitol, addition of a cryoprotectant solution of sucrose, dimethyl sulfoxide, glycerol and L-proline, two step freezing and storage in liquid nitrogen. Cells have been preserved for up to 14 months (the longest period tried in these experiments). Cryopreserved cells proliferated after plating on solid medium and new cell suspensions could be initiated within 15 days. Viable protoplasts, capable of divisions and callus formation, could be obtained 15–21 days after thawing. Variation between cell lines in terms of recovery rate after cryopreservation occurred. Differences between cell lines in plating efficiencies on solidified medium, however, contributed to this variation. Protoplasts from cryopreserved regenerable cell lines gave rise to embryogenic callus from which plants could be regenerated. These plants developed to maturity. A transformed cell line was also cryopreserved and it had retained the hygromycin resistance and regenerative capacity of the original cell line.Abbreviations DMSO dimethyl sulfoxide - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphtylacetic acid - FDA fluorescein diacetate     

Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture

Summary Previous experiments have revealed that the maize transposable element Activator (Ac) may become active during tissue culture. The objective of the present study was to determine whether a second transposable element, Suppressor-mutator (Spm), could also be activated in tissue culture and detected in regenerated maize plants. Approximately 500 R₁ progeny of 143 regenerated plants (derived from 49 embryo cell lines) were crossed as males onto an Spm-responsive tester stock. Spm activity was observed in two R₁ progeny of a single regenerated plant. This plant had been regenerated from Type II (friable embryogenic) callus of an A188 × B73 genetic background after 8 months in culture; the absence of Spm activity in four other plants regenerated from this same callus demonstrates that Spm activity was not present before culturing. Approximately 20 Spm-homologous DNA sequences were detected in each of the inbreds used to initiate the tissue cultures; it is presumed that one of these became active to give rise to Spm activity.     

Genetic and biochemical stability assessment of plants regenerated from cryopreserved shoot tips of a commercially valuable medicinal herb <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) Wettst

Shoot tips from four accessions (IC249250, IC 426442, IC 375976, and IC468878) of Bacopa monnieri (L.) Wettst., a commercially valuable memory revitalizing medicinal plant, were cryopreserved using a vitrification technique. Depending on the genotype, 0 to 20% plant regeneration without intermediary callus was achieved from cryopreserved shoot tips. Genetic stability of plants derived from cryopreserved shoot tips was assessed using biochemical and molecular markers. The regenerated plants from non-frozen controls and cryopreserved shoot tips exhibited morphological similarity to respective parental material when transferred to soil. On the basis of ten random amplified polymorphic DNA (RAPD) and bacoside A content using HPLC analysis, no significant reproducible variation was observed between the controls and in vitro-cryopreserved plants. Thus, after cryopreservation treatment, the regenerated plants exhibited molecular and biochemical genetic stability.     

Selection of ethionine-resistant variants with increased accumulation of methionine from embryogenic protoplasts of the forage legume <Emphasis Type="Italic">Astragalus adsurgens</Emphasis>

In vitro selection was carried out to obtain ethionine-resistant plants with increased contents of free methionine in the vegetative tissues of the forage legume Astragalus adsurgens Pall. Three-week-old cell colonies were derived from protoplasts mutagenized with N-methyl-N-nitrosoguanidine from embryogenic callus and were selected with 0.6mM ethionine. Four colony lines were isolated and their resistance to ethionine was 7–8 times that of the wild-type callus. No plant regeneration occurred on these colony lines in the differentiation medium containing ethionine. Only one colony line (R-1) regenerated plants through somatic embryogenesis in the absence of ethionine. Stem and leaf segments from the regenerated plants showed the same potential to produce callus in the presence of ethionine as in the absence of ethionine. The formed callus kept continuously growing in ethionine-containing medium. Free amino acid analysis revealed that colony line R-1, its regenerated plants and callus from the regenerated plants accumulated methionine at levels at 5–9 times higher than in wild-type. These results suggested that ethionine resistance and methionine over-accumulation were also expressed at plant level. Thus, the obtained resistant colony line that could regenerate plants with over-accumulation of methionine might provide an alternative approach to improve the nutritional quality of this forage.     

Ploidy levels of plants regenerated from mixed ploidy maize callus cultures

Summary Haploid and diploid anther-derivedZea mays callus lines were treated with the antimicrotubule herbicide pronamide to produce mixed ploidy callus as determined by flow cytometry. The ploidy levels of the plants regenerated from the callus were determined by counting the leaf epidermal guard cell chloroplast numbers. The proportion of diploid regenerated plants was somewhat lower than the proportion of diploid cells of the callus. The diploid plants regenerated somewhat faster than the haploids. The proportion of tetraploids regenerated from the pronamide treated diploid callus, which originated by spontaneous chromosome doubling, was much lower than the proportion of cells indicating that tetraploid cells survive or regenerate plants at a lower frequency than diploid cells.     

Secondary metabolite content in rhizomes, callus cultures and in vitro regenerated plantlets of Solidago chilensis

An in vitro culture system leading to the formation of callus and plant regeneration, starting from nodal sections and shoot tips, was developed for Solidago chilensis (Asteraceae). The content of the gastroprotective diterpene solidagenone as well as the phenolics chlorogenic acid (CA) and rutin was determined either in rhizomes from wild growing plants and in callus and in in vitro regenerated plantlets by analytical HPLC. Additionally, total phenolic and flavonoid content was assessed in plant samples, callus and cell suspensions. In terms of dry starting material, the percentual solidagenone content in nine S. chilensis samples ranged from 0.5-3.5% for rhizomes from wild growing plants, 0.1-0.3% for callus and 0.3% for an in vitro regenerated plantlet, respectively. The highest solidagenone contents were found in the wild plant during the late summer in the months of March and April (3.5-2.2%) while highest values for chlorogenic acid (0.5%) and rutin (0.4%) were detected in May, before senescence. The callus tissue and cell suspensions contained some 1.8-2.0 and 1.2% of total phenolics, respectively. CA was the main phenolic in the cell suspension while only traces were found in the callus. Rutin was not detected in the callus nor cell culture.     

Plant regeneration from tissue cultures of Pokkali rice is promoted by optimizing callus to medium volume ratio and by a medium-conditioning factor produced by embryogenic callus

The frequency of plant regeneration from seed-derived Pokkali rice callus has been substantially increased. Four conclusions were drawn from the study: (1) Non-embryogenic callus consisting of elongated, highly-vacuolated cells did not produce regenerated plants. Embryogenic callus consisting of small, non-vacuolated cells produced somatic embryos and regenerated plants. (2) The numbers of plants could be markedly increased by optimizing a medium for embryogenic callus production and a second medium for plant regeneration from embryogenic callus. (3) The optimization of callus to medium volume ratio of 6.5 mg embryogenic callus per 1.0 ml of medium significantly increase plant production on regeneration medium. (4) A further significant increase was obtained by using regeneration medium previously conditioned for one or two weeks by optimal amounts of embryogenic callus. At present, the callus derived from a single seed in six months could theoretically be used in the seventh month to produce 127500 plants.This research was supported by the Agency for International Development under Contract No. AID/DSAN-C-0273     

中国野生稻遗传资源的保护及其在育种中的利用

我国有三种野生稻,即普通野生稻(Oryza rufipogon)、药用野生稻(O.officinalis)和瘤粒野生稻(O.meyeriana)。这三种野生稻均被列为国家二级保护植物(渐危种)。调查结果表明,野生稻由于其自然群落大量丧失而濒危,濒危程度为普通野生稻>药用野生稻>瘤粒野生稻。造成濒危的主要原因是人为的破坏活动。人类的经济活动导致了野生稻生境丧失、生境质量不断恶化、栖息地越来越少;人类的活动也导致了外来种的入侵。目前,对野生稻的保护措施主要有就地保护(原地保护或原位保护)和迁地保护(易地保护或异位保护)。易地保护包括以种子保存的种质厍、以种茎保存的种质圃和以器官培养物作为材料的超低温保存。野生稻具有许多优良特性,如特强的耐寒性、高的抗病虫性、优质蛋白质含量高、功能叶片耐衰老的特异性、特强的再生性、良好的繁茂性及生长优势等等,这些优良特性已被广泛用于水稻常规育种和杂交育种中,并取得了巨大的社会效益和经济效益。有关野生稻生物技术方面的研究,如花药培养、原生质培养、体细胞杂交和基因工程等方面已取得了较大的进展。野生稻将在水稻育种中发挥越来越重要的作用。     

A low-temperature method for maintaining plant regeneration activity in embryogenic callus of rice (Oryza sativa L.)

 A method was developed to maintain plant regeneration activity of rice cells (Oryza sativa L.) using embryogenic callus. Calluses were cultured in suspension, then on solid medium, to form compact globular callus resistant to low-temperature stress and with high plant regeneration activity. Callus preserved at 5  °C for 5 months regenerated plants from protoplasts at a frequency higher than from non-preserved callus from cv. Nipponbare, and cv. Koshihikari, but at lower rates from cv. Akitakomachi. Similar results were obtained from protoplasts of the three cultivars. Callus preserved at 5  °C for 8 months incurred cell damage, yet some surviving cells divided in suspension culture and eventually regenerated whole plants. Preserved and non-preserved regenerated plants showed similar levels of somaclonal variation. Received: 7 January 1999 / Revision received: 28 April 1999 / Accepted: 26 May 1999     

Steroidal alkaloids in tissue cultures and regenerated plants of Solanum dulcamara

Callus and cell suspension cultures were established with shoots of the soladulcidine variety of the bittersweet Solanum dulcamara L. Plantlets were regenerated from undifferentiated callus. From mixotrophic callus as well as mixotrophic suspension cultures soladulicidine, solasodine and the corresponding neutral spirostanes tigogenin and diosgenin were isolated and identified by thin layer chromatography and mass spectrometry. Total alkaloid concentrations were about 0.2 mg/g dry weight (callus) and 0.1 mg/g dry weight (green suspension cultures). In the heterotrophic cell line only the neutral sapogenins could be detected. Alkaloid accumulation in callus of Solanum dulcamara could be enhanced by the induction of organogenesis. The shoots of the regenerated plants from the mixotrophic callus contained soladulcidine (1.6 mg/g dry weight) and tigogenin. Thus, in concentration and composition the regenerated plants equalled the source plant.Abbreviations 2.4-D 2.4-dichlorophenoxyacetic acid - NAA naphthylacetic acid - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Regeneration of flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) plants from anther culture and somatic tissue with increased resistance to<Emphasis Type="Italic"> Fusarium oxysporum</Emphasis>

The aim of this study was to establish a protocol for the efficient production of flax plants of microspore origin. The results were compared to those obtained for plants regenerated from somatic explants from hypocotyls, cotyledons, leaves, stems and roots. All the plants obtained during the experiments were regenerated from callus that was grown for periods from a few weeks to a few months before the regeneration was achieved. Anther cultures were less effective in plant regeneration than somatic cell cultures. However, regenerants derived from anther cells showed valuable breeding features, including increased resistance to fungal wilt. The age of the donor plants and the season they grew in had a noticeable effect on their anther callusing and subsequent plant regeneration. Low temperature had a negative effect and dark pre-treatment a positive effect on callusing and plant regeneration. Different media were most effective for callus induction, shoot induction and rooting. For callus induction two carbon sources (2.5% sucrose and 2.5% glucose) were most effective; for shoots, only sucrose at lower concentration (2%) was effective. Rooting was most efficient in 1% sucrose and reduced (50%) mineral concentration in the medium. It was found that the length of in vitro cultivation significantly increases the ploidy and affects such features as regenerant morphological characteristics, petal colour, and resistance to Fusarium oxysporum-induced fungal wilt. The established plant regeneration system provides a basis for the creation of transgenic flax.Abbreviations BAP 6-Benzyl-aminopurine - IAA Indole-3-acetic acid - MS Murashige and Skoog medium - NAA -Naphthalene-acetic acidCommunicated by H. Lörz     

Occurrence of chromosomal variations and plant regeneration from long-term-cultured citrus callus

Summary Embryogenic callus of Anliucheng sweet orange (Citrus sinensis Osbeck) is theoretically diploid. However, significant chromosomal variations occurred when the calluses were subcultured and preserved for a long time. Cytological observation revealed a variety of mitotic irregularities underlying the occurrence of chromosomal variations. Despite the ubiquitous existence of chromosomal variations, long-term-cultured calluses were still capable of producing somatic embryos and plants. Interestingly, chromosomal variants were selected against when somatic embryos and plants regenerated from the embryogenic callus. Randomly amplified polymorphic DNA (RAPD) analysis was also carried out to detect DNA sequence variation in regenerated plants derived from the embryogenic callus. No difference in banding patterns was detected. It was clear that the plant regeneration from long-term-cultured callus was inclined to select against somaclonal variations.     

玉米未成熟胚乳愈伤组织和器官的发生及其倍性变化的初步研究

本试验在附加和不附加外源激素的MS培养基上,均得到了玉米未成熟胚乳愈伤组织。愈伤组织在附加外源激素的MS培养基上达到器官分化,获得了发育正常的和许多畸形的胚乳植株。所得到的愈伤组织细胞和植株根尖细胞染色体数目和倍性是不稳定的,二者有相同的趋向,其中有整倍体的细胞(2n=10,20,30,40,50),也有各种非整倍体的细胞(2n=5—49)。     

Response to NaCl of alfalfa plants regenerated from non-saline callus cultures

Salinity restricts crop productivity in many arid environments. Inadvertent selection for tolerance to osmotic stress may occur under cell or tissue culture conditions and could affect the performance of regenerated plants. The effect of NaCl on forage produced by alfalfa (Medicago sativa L.) plants regenerated from non-saline callus cultures was examined in this study. Plants of Regen-S, which was selected for improved callus growth and regeneration in non-saline cultures, had higher forage weight when grown on SHII medium at NaCl levels up to 100 mM compared to its parental cultivars, Saranac and DuPuits. Five additional original-regenerant plant pairs, each derived from non-saline callus cultures of different alfalfa plants, were evaluated in a solid (soil-like) substrate under saline and non-saline conditions. Weight of forage produced by rooted stem cuttings of regenerated plants was 33% higher at 50 mM NaCl compared to cuttings of explant donor plants. Self progenies from four of five regenerants had higher relative forage weight at 100 mM NaCl (percent of 0 NaCl treatment) than the original plants indicating increased NaCl tolerance.     

Plant cells express telomerase activity upon transfer to callus culture, without extensively changing telomere lengths

Changes in telomere lengths and telomerase activity in tobacco cells were studied during dedifferentiation and differentiation; leaf tissues were used to initiate callus cultures, which were then induced to regenerate plants. While no significant changes in the range of telomere lengths were observed in response to dedifferentiation and differentiation, there was a conspicuous increase in telomerase activity in calli compared to the source leaves, where the activity was hardly detectable. In leaves of regenerated plants, the telomerase activity fell to almost the same level as in the original plant, showing on the average 0.04% of the level in callus. The process was then repeated using the regenerants as the source material. In the second round of dedifferentiation and differentiation, telomerase activity showed a similar increase in calli derived from regenerated plants and a drop in plants regenerated from these calli. Telomere lengths remained unchanged both in calli and in leaves of regenerants. The conservation of telomere lengths over repeated rounds of dedifferentiation and differentiation, which are associated with dramatic changes in cell division rate and corresponding variation in telomerase activity may reflect the function of a regulatory mechanism in plant cells which controls telomerase action to compensate for replicative loss of telomeric DNA. Received: 31 July 1998 / Accepted: 21 September 1998     

Variation in nuclear DNA content of isonicotinic acid hydrazide-resistant cell lines and mutant plants of Nicotiana tabacum

Summary Isonicotinic acid hydrazide (INH)-resistant lines of Nicotiana tabacum have been maintained in callus culture for six years and mutant plants have been regenerated from a number of these lines. This study examines variations in DNA content in nuclei of several of these callus cultures, regenerated plants, and secondary callus from the regenerated plants. The lines selected for study include three easily regenerated lines (I 21, I 24, and I 9) and two lines of poor regenerating capacity (I 1 and I 18). Two of the regenerating lines eventually led to fertile plants and the third produced only sterile plants. In general, the range of total nuclear variability was not as high as anticipated from other studies of long-term tobacco callus cultures. The majority of nuclei in all the distributions were between 3 and 20 pg, and the most frequently encountered distributions concentrated in the 7–18 pg region corresponding to 2–5C by our estimate of the C value for tobacco. Distributions were not identical for plants regenerated from the same culture simultaneously, and the nuclear DNA content of secondary callus cultures from one of the plants examined did not reflect the quantitative DNA pattern of the plant from which it was derived. The greatest degree of variability and highest DNA content for individual nuclei were observed in the primary callus of the poorly- and non-regenerating lines. The variability in DNA content was not associated with the INH-resistant trait.