Plant regeneration from encapsulated somatic embryos of pedunculate oak (Quercus robur L.)

Summary Cotyledonary Quercus robur L. somatic embryos from two cell lines were encapsulated in 4% (w/v) sodium alginate. An artificial endosperm was provided by the addition of P₂₄ medium plus 3% (w/v) sucrose. Oak somatic embryos and oak synthetic seeds were germinated on P₂₄ medium plus 0.1 μM indole-3-butyric acid and 0.9 μM 6-benzylaminopurine or were dehydrated prior to germination. The highest conversion rates (26%) were obtained with encapsulated somatic embryos as well as artificial endosperm-coated somatic embryos. Encapsulation improved the regeneration into oak plantlets in one of the two cell lines tested. The artificial endosperm had no additional beneficial effect on conversion frequency, but increased germination rate in one cell line tested. Significant higher conversion could be attributed to slow desiccation compared to the non-encapsulated control. Cold storage as a post-maturation treatment had no influence on the germination ability of oak synthetic seeds. Differences in the response of the cell lines with respect to conversion frequencies and timing of germination were observed. Fifty-six well-developed plantlets regenerated 12 wk after germination, and 29 plants were transferred to the greenhouse, where they have been successfully established in substrate.     

Occurrence of Temperature-Sensitive Phenotypic Plasticity in Chlorophyll-Deficient Mutants of Arabidopsis thaliana

A collection of 75 putative mutants with alterations in leaf pigmentation was visually selected from Arabidopsis thaliana plants (M₂ generation) grown at 26°C from seeds treated with the mutagen ethylmethanesulfonate. Fifty-eight of the plants were found to have chlorophyll contents decreased by at least 10% from the parental Columbia ecotype. These plants were screened for chlorophyll content and the ratio of chlorophyll b/a after growth at 20 or 26°C. Relative to the parental type, a significant number of individuals in which the chlorophyll-deficient phenotype was exacerbated at one of the growth temperatures were identified. We conclude that temperature-sensitive phenotypic plasticity for chlorophyll content is relatively common in mutant populations of higher plants.     

Effect of sodium fluoride on radiation sensitivity of barley seeds

Barley seeds soaked in 0.01 M sodium fluoride (NaF) in phosphate buffer (pH7) or in buffer alone for 18 h were dried and equilibrated to 10% moisture, either in air or in nitrogen. Pre-treated and re-dried seeds were irradiated in air or in nitrogen with 0, 13, 20, 26 and 32 kR of γ rays, and were immediately hydrated in oxygen- or nitrogen-bubbled water. Parameters of radiation effect considered were seedling injury, mitotic and meiotic cells with bridge aberrations at anaphase and pollen fertility in M₁, and the frequency of chlorophyll mutations in M₂. NaF at 0.01 M was not mutagenic by itself. Pre-treatment with NaF significantly enhanced the radiation effect, when the irradiation was done in air, in comparison with the buffer soaked seeds. The increased effect due to NaF was additional to the oxygen effect. In nitrogen, NaF pre-treatment increased the mutagenic effect but it was not always significant. Post-soaking of irradiated seeds in 0.01 M NaF for 5 h increased seedling injury in comparison with the irradiated seeds soaked in buffer alone or in 0.01 M NaCl. At least a part of the sensitizing effect of NaF may be due to the inhibition of repair.     

Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) — a recalcitrant grain legume

Agrobacterium-mediated transformation of Vigna radiata L. Wilczek has been achieved. Hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B₅ basal medium supplemented with 5×10⁻⁶ M BAP, 2.5×10⁻⁶ M each of 2,4-D and NAA and 50 mg l⁻¹ kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing β-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) marker genes. Transformed calli were found resistant to kanamycin up to 1000 mg^.l⁻¹. Gene expression of kanamycin resistance (nptII) and gusA in transformed calli was demonstrated by nptII assay and GUS histochemical analysis, respectively. Stable integration of T-DNA into the genome of transformed calli of mungbean was confirmed by Southern blot analysis. Transgenic calli could not regenerate shoots on B₅ or B₅ containing different cytokinins or auxins alone or in combination. However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B₅ medium containing 6-benzylaminopurine (5×10⁻⁷ M) and 75 mg l⁻¹ kanamycin. The putative transformed shoots were rooted on B₅+indole-3-butyric acid (5×10⁻⁶ M) within 10–14 days and resulted plantlets subsequently developed flowers and pods with viable seeds in vitro after 20 days of root induction. The stamens, pollen grains and T₀ seeds showed GUS activity. Molecular analysis of putative transformed plants revealed the integration and expression of transgenes in T₀ plants and their seeds.     

Factors influencing <Emphasis Type="Italic">in vitro</Emphasis> regeneration from protoplasts of wild cotton (<Emphasis Type="Italic">G. klotzschianum</Emphasis> A) and RAPD analysis of regenerated plantlets

Plants of a diploid wild cotton species (G. klotzschianum A.) were efficiently regenerated from protoplasts isolated from immature somatic embryos and suspension cultures by studying various factors affecting regeneration. Purified protoplasts were cultured with the density of 2–10×10⁵ ml⁻¹, and the medium was k₃ inorganic salts with modified KM₈P organic compositions, supplemented with several combinations of PGRs. Calluses were formed from protoplasts of suspension cultures and immature somatic embryos. The influences of carbon sources and GA₃ on callus differentiation and somatic embryo germination were analyzed. Somatic embryos germinated normally and formed regenerated plantlets. Regenerated plantlets were transferred to the soil and seeds were obtained. Random amplified polymorphic DNA (RAPD) analysis using 80 arbitrary oligonucleotide 10-mers showed 23 primers that gave 74 clear reproducible bands, with amplification products being monomorphic for 14 tested plantlets. A total of 1036 bands obtained exhibited no aberration in RAPD banding patterns in the 14 plants. Plants regenerated via somatic embryogenesis from the diploid cotton protoplasts have genetic homogeneity.     

Induction of somatic embryogenesis in Brassica juncea L. and analysis of regenerants using ISSR-PCR and flow cytometer

A new and simple protocol has been developed and standardized for direct somatic embryogenesis and plant regeneration from aseptic seedlings derived from immature Brassica juncea seeds. Depending on the age of immature seeds and nutrient media, in vitro occurrence of embryogenesis and the number of embryos from each seedling have varied greatly. The largest number of somatic embryos, producing 12.7 embryos per seedlings, have been developed by seedlings obtained from immature seeds collected after 21 days of pollination (DAP). Effect of different nutrient media [Gamborg (B5), Murashige and Skoog (MS) and Linsmaier and Skoog (SH)] and carbon sources (fructose, glucose, maltose and sucrose) were assessed to induce somatic embryos and the maximum response were achieved on Nitsch culture medium fortified with sucrose (3% w/v) followed by fructose and maltose. The somatic embryo converted into complete plantlets within 04-weeks of culture on Nitsch medium containing half-strength of micro and macro salts. The regenerated plantlets were successfully established in soil with 90% survival rate. The acclimated plants were subsequently transferred to field condition where they grew normally without any phenotypic differences. Genetic stability of B. juncea plants regenerated from somatic embryos were confirmed by inter-simple sequence repeat (ISSR)-PCR analysis and flow cytometry. No significant difference in ploidy level and ISSR banding pattern were documented between somatic embryo’s plants and control plants grown ex vitro.     

Anticholinesterase and mutagenic evaluation of in vitro-regenerated Agapanthus praecox grown ex vitro

Beyond establishing micropropagation protocols for medicinal plants, it is important that the efficacy and safety of propagated plants be ascertained for these plants to be accepted for use in traditional medicine. The use of propagated plants could alleviate/reduce over-exploitation of wild populations. The present study evaluated the anticholinesterase and mutagenic properties of 1-yr-old tissue culture-derived Agapanthus praecox grown ex vitro and naturally grown mother plants. The tissue culture-derived plants were regenerated using different plant growth regulators. A dose-dependent inhibition of acetylcholinesterase (AChE) enzyme was observed in all the tissue culture-derived and naturally grown mother plants. The leaf extract of tissue culture-derived plants regenerated with a combination of benzyladenine (BA) and thidiazuron (TDZ) demonstrated a significantly low AChE-inhibitory activity. Conversely, the root extract of plants regenerated with BA alone demonstrated the highest AChE-inhibition activity (IC₅₀ = 0.20 mg/mL) when compared to extracts from other treatments and the naturally grown mother plants. None of the samples were found to be mutagenic in the absence of metabolic activation. The present study indicated that regenerated plants could be used as potent substitutes for naturally grown plants in traditional medicine. However, the choice of treatment used during micropropagation operation may significantly influence the therapeutic potential of regenerated plants, even after 1 yr of growth.     

Induced Androgenesis in vitro in Mutated Populations of Barley,Hordeum vulgare

Graduated concentrations of chemomutagens ethylmethanesulphonate (EMS), N-nitroso-N-methylurea (MNU) and N-nitroso-N-ethylurea (ENU) and one concentration of sodium azide (NaN₃) were used to treat seeds of spring barley cultivars Heris, Tolar, Granát and Novum. Androgenesis in vitro was induced in mutagenised plant populations. The significant stimulation effect of mutagenic treatment on the mean number of androgenic anthers from all 36 treated variants was registered in 17 variants, on the mean number of regenerated plants in 15 variants and on the mean number of regenerated plants per androgenic anthers in seven variants only. Genotypes with a lower androgenic response were relatively more stimulated. Evaluation was made of the frequency of chlorophyll mutations within androgenic regenerants and in seed progeny of androgenic donors. Androgenesis was also induced in vital chlorophyll mutants and in the variants where the mutagen treatment resulted in less than 50% survival of the donor plants. Ratios between frequency of haploids and spontaneous dihaploids were similar in green and in albino androgenic plants. The results confirm that in barley it is possible to enhance the frequency of in vitro pollen embryogenesis by mutagenic treatment of seeds.     

Differential expression of β-conglycinin genes in nodulated and non-nodulated isolines of soybean

Nodulated (T202) and non-nodulated (T201) isolines of soybean (Glycine max [L.] Merr.) were cultivated in a rotated paddy field in Niigata, Japan. The pods, and seeds were harvested at 7-day intervals until maturity, and the subunit compositions of seed storage proteins were analyzed by SDS-PAGE. The β-subunit of β-conglycinin could scarcely be detected in the non-nodulated isoline, T201, at any period throughout seed development, although it was a major component in T202. The accumulation of α′- and α-subunits of β-conglycinin, together with the acidic and basic subunits of glycinin, appeared about one week later in seeds of T201 than in those of T202, perhaps due to a shortage of nitrogen and growth retardation. Northern hybridization could not detect the β-subunit mRNA in immature T201 seeds, while it was pronounced in T202. These results indicate that the suppression of the β-subunit in the non-nodulating isoline T201 is regulated at the level of mRNA accumulation. The α′(α)-subunit mRNAs were actively expressed in both isolines. Total nitrogen concentration was consistently lower in T201 than T202. No significant difference was observed in either the free amino acid or ureide concentrations in seeds, although the concentration of sucrose was considerably lower in T201 seeds and pods compared with T202. This result indicates the possibility that β-subunit accunmlation was regulated not only directly by total nitrogen concentration but also by carbohydrate concentrations. Nitrogen regulation of storage protein subunit levels of soybean seed were evaluated using T201 and T202. Greenhouse-grown plants were subjected to different levels and timing of nitrate treatments. The culture solution (2, 5 or 10 mM NO₃–was supplied from flowering, 42 days after planting (DAP), until maturation (137 DAP), or switched from 2 to 10 mM, or from 10 to 2 mM at 61 DAP. With a continuous 2 mM NO₃–treatment, seed dry weight and N concentration of the T201 plants were significantly lower than those in the T202 plants due to the lack of N₂ fixation by the non nodulated T201 plants. However, when adequate NO₃ was supplied, N concentration and dry weight were similar in T201 and T202 seeds. When 5 mM NO₃ was supplied, the subunit proportion of the seed storage protein was similar in non-nodulating and nodulating isolines. On the other hand, when plants received a low level of NO₃ (2 mM), the β-conglycinin proportion was lower in T201 than in T2O2. Furthermore, in the nodulating T202 plants treated with 10 mM NO₃–the proportion of β-conglycinin increased markedly. The results indicate that non-nodulated T201 has a normal, non-defective, β-subunit gene and that limited N availability decreases accumulation of β-conglycinin, whereas high N availability increases the proportion of β-conglycinin in soybean seeds, irrespective of whether N was derived from N₂ fixation or from NO₃ absorption.     

Assessment of Genetic Stability in Three Generations of In Vitro Propagated <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. Plantlets Using ISSR Markers

Tissue culture has been widely employed in Jatropha curcas L. for the clonal multiplication of superior genotypes. However, the evaluation of genetic stability is necessary to detect somaclonal variants. In this context, the present aim was to evaluate the genetic stability of J. curcas plantlets, obtained via indirect organogenesis, by means of ISSR markers. To supply the explant sources for in vitro propagation, the first generation of plants was produced from in vitro germination of J. curcas seeds. Fragments of cotyledonary leaves were inoculated into medium supplemented with 1.5 mg L^?1 BAP and 0.05 mg L^?1 of IBA for induction of callogenesis. The resulting calli were transferred to bud induction medium. Subsequently, the buds were cultured in medium for elongation, giving rise to the second generation of plants. These plants provided new buds, which were excised and subcultured in elongation medium, yielding a third generation of plants. To evaluate genetic stability in three plant generations, twelve ISSR primers were used, resulting in 124 bands showing 41.93 % of polymorphism. Increase was observed in the level of somaclonal variation (SV) over the generations. The present study reports, for the first time, the analysis of genetic stability in J. curcas plantlets regenerated via indirect organogenesis by means of ISSR markers. The results suggest that the indirect route is associated to higher levels of genetic instability, which also increased with successive subcultures. The ISSR markers were efficient in detecting SV, and the generated genetic variability may be useful for breeding programs.     

Inter Simple Sequence Repeat Analysis to Confirm Genetic Stability of Micropropagated Plantlets in Three Grape (Vitis spp) Rootstock Genotypes

Three grape rootstock genotypes — Dogridge (Vitis champini), SO4 (V. beriandieri × V. rupestris) and H-144 (V. vinifera × V. labrusca), and their 30 in vitro regenerated plantlets were subjected to Inter Simple Sequence Repeat (ISSR) analysis in order to ascertain the genetic stability of micropropagated plantlets. Out of 35 primers screened initially with three mother plants, 10 were finally selected based on sufficient polymorphism and appearance of clear and scorable banding patterns. Each primer generated a unique set of amplification products ranging in size from 100 to 1800 bp. These ten ISSR primers produced 81 distinct and scorable band classes with an average of 8.1 bands per primer. Based on similarity matrix and cluster analysis the rootstock genotypes and their tissue culture derivatives formed three distinct genetic groups indicating their genetic relationships. Furthermore, no variation was detected among in vitro regenerated grape plantlets and their field-grown mother plants corroborating the high level of clonal fidelity of the in vitro regenerated plantlets and supporting the multiplication protocol utilizing nodal segments as in vitro culture initiation material.     

β-N-Oxalyl-L-α, β-diaminopropionic Acid in Somaclones Derived from Internode Explants of Lathyrus sativus

Protocols have been developed for in vitro regeneration from internode explants from Lathyrus sativus. Callus raised on B₅ medium supplemented with 10.7 μM NAA + 2.2 μM BA permitted shoot regeneration upon transfer to modified MS medium containing 10.7 μM NAA + 2.2 μM BA. Rooting was obtained only on 1/2 MS media containing 0.5 μM IBA. The in vitro regenerated plants, after primary and secondary hardening, were taken to the field. Analysis of ODAP in leaves and seeds was carried out. The low toxin containing progeny of the somaclones were further grown in the field. The toxin contents varied from 0.015% to 0.460% in leaf and 0.030% to 0.539% in seed in R, generation, as compared to 0.258% in leaf and 0.406% in seed for the parent P-24. Statistical analysis showed a positive significant correlation between leaf and seed ODAP contents. Mean seed toxin in R₁ generation of some of the somaclones varied from 0.039–0.057% and single plant seed yield varied from 25.8 to 45.0 g. Some plants showed seed toxin content of less than 0.01% from 1–22 progeny. Thus, following in vitro culture of internode explant, toxin content in seeds in R₂ generation has been found to be substantially reduced with single plant seed yield either equal to or higher than that of parent cv. P 24.     

Male sterile mutant from somatic cell culture of rice

Summary Using MS medium supplemented with 6% sucrose and hormones, plantlets were regenerated from the expiants of mature seeds and young panicles of IR_s and IR₅₄. Out of 157 regenerated plants (R₁), three were found to be male sterile (ms): one from IR_s and two from IR₅₄, including a fertile and sterile chimaera. In the second generations (R₂) of IR₂₄ and IR₅₄, one line from each segregated into male sterile and fertile plants. These ms plants could be divided into two types with pollen failure: pollen free (without pollen) and pollen abortive. IR₂₄ was a semi-restorer for ms-plants of the pollen free type derived from the second generation of IR₅₄ somaclones. The segregation ratio of fertile: sterile in both R₂ of line 91 and the F₂ of ms-plant/IR₂₄ fitted the formula 15/161/16 quite well, showing that the male-sterile was controlled by two independent nuclear genes. Until now, as we know, male-sterile could be produced by hybridization or mutagenesis: sometimes it could be found in nature by spontaneous mutation. Recently the cytoplasmic male-sterile of tobacco was produced by protoplast fusion. This is the first paper to report male-steriles in regenerated plants and their offspring obtained from somatic cell culture.Some of the tissue culture and plant regeneration work in this study was conducted at IRRI, Manila     

Efficient plantlet regeneration from protoplasts isolated from suspension cultures of poplar (Populus alba L.)

We developed an efficient plant regeneration system from protoplasts for poplar (Populus alba L.). Protoplasts were isolated from 4-day-old suspension cultures derived from seed-induced calli with a yield of 6.96× 10⁶ cells/g fresh weight cells and then cultured at a concentration of 2.5×10⁵ cells/ml in NH₄NO₃-free Murashige and Skoog (MS) medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 μM thidiazuron (TDZ) and 0.5 M glucose as a osmoticum. The plating efficiency of the cultured protoplasts was calculated at 26.5% at day 7 and 31.7% at day 14. Cell colonies were observed after culturing for 4 weeks. Regenerated colonies were propagated through subculture in liquid MS medium supplemented with 5 μM 2,4-D. Buds were induced from regenerated calli on MS medium containing 10 μM kinetin or 1 μM TDZ. Regenerated shoots were rooted on half-strength MS medium, and the plantlets were transplanted in soil. Randomly amplified polymorphic DNA analysis did not detect any DNA polymorphism among the regenerated plants. Received: 7 March 1997 / Revision received: 16 June 1997 / Accepted: 5 July 1997     

Fertile plant regeneration from barley (Hordeum vulgare L.) protoplasts isolated from primary calluses

Barley (Hordeum vulgare L.) protoplasts were isolated from the immature embryo-derived primary calluses. These protoplasts were cultured with nurse cells, and they then divided to form colonies. After transfer of the colonies to regeneration medium, either green or albino shoots were regenerated from these colonies. A high agarose concentration (2.4% w/v) in the protoplast culture medium promoted protoplast division. The plantlets that developed strong root systems were transferred to the soil. These plants flowered and have set seeds.     

Segregation in the progeny of transformed rapeseed (Brassica napus)

The primary transformant of spring rapeseed cv. HM-81 contained TL- and TR-DNA of agropine plasmid pRi ofAgrobacterium rhizogenes 15834. The presence of TL-DNA corresponds to visible transformed phenotype in its progeny; the leaves are wrinkled and the plants are shorter than normal plants. R₁ R₂ and R₃ generations have mostly transformed phenotype. The normal phenotype appears in a low frequency in F₁ generation. Autogamised F₁ plants segregate in F₂ transformed and normal phenotype in 3:1 ratio. It is possible to suppose that TL-DNA is present in two differentloci of one pair of homologic chromosomes. The recombination frequency is 12 % (microsporogenesis) or 6 % (microsporogenesis and macrosporogenesis). In some crosses the transformed phenotype has a maternal type of inheritance. Maternal inheritance influences also several growth characteristics,e.g. length of plants and number of seeds/pods.     

Rapid regeneration of <Emphasis Type="Italic">Phaseolus angustissimus</Emphasis> and <Emphasis Type="Italic">P. vulgaris</Emphasis> from very young zygotic embryos

A rapid regeneration protocol for proembryos of Phaseolus angustissimus as young as 1 day after pollination (DAP) involving pod culture for 1 week followed by embryo culture for 2 weeks and embryo germination for 1 or 2 weeks is provided. Optimization of the media was conducted with pods collected 3 DAP. The best pod culture medium was composed of basal medium [(Phillips and Collins 1979) salts with (Geerts et al. 2001) vitamins], 1000 mg l⁻¹ glutamine, 1000 mg l⁻¹ casein hydrolysate, 3% sucrose and 0.5% agar. Embryo culture medium consisted of basal medium with 500 mg l⁻¹ glutamine, 250 mg l⁻¹ casein hydrolysate, 1.9 μM ABA, 3% sucrose and 0.5% bacto-agar. Embryos developed into plantlets on germination medium containing basal medium with 0.25 μM BA, 3% sucrose and 0.7% bacto-agar. Fertile, normal plants were recovered from direct embryogenesis and from micrografted embryo-derived shoots. Embryos obtained from pods collected 3 DAP regenerated plantlets at a rate of 29.3%, while embryos from pods collected 2 DAP and 1 DAP regenerated at rates of 20.2 and 4%, respectively. A second accession of P. angustissimusregenerated at a rate of 26.2%. Using this 5-week protocol for P. vulgaris resulted in a plantlet regeneration rate of 12.5%.     

Enhanced Alkaloid Production in Callus Cultures of Heliotropium indicum L and Regeneration of Plantlets

Heliotropium indicum L (Boraginaceae) contains the anticancer pyrrolizidine alkaloid, indicine-N-oxide (INO). To study the yield of INO as a function of plant development, plantlets were regenerated in vitro from nodal and hypocotyl explants and also from hypocotyl callus, on Murashige and Skoog’s (MS) medium supplemented with 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), asparagine (Asp) and glutamine (Glu). The regenerated plantlets were rooted on MS supplemented with Glu or gibberellic acid (GA₃). While 5-week-old seedlings showed a high amount of INO (0.12% dry wt) that depleted gradually as the plants attained maturity and flowered, fast growing callus produced a much higher yield of INO (0.32% dry wt). As the callus cultures differentiated into shoots and subsequently into plantlets, the INO content decreased to about 0.2% dry wt. It appears that INO is the primary product of pyrrolizidine alkaloid biosynthetic pathway in rapidly growing meristematic tissue of Heliotropium indicum, later reduced to its alkaloidal base and reallocated to other tissues. The article includes an efficient micropropagation method for Heliotropium indicum.     

Encapsulation of microcuttings for propagation and short-term preservation in Ruta graveolens L.: a plant with high medicinal value

Synthetic seeds technology is a potential tool for an efficient and cost-effective clonal propagation system. In the present study, synthetic seeds were produced by encapsulating nodal segments (synthetic or synseeds) of Ruta graveolens in calcium alginate gel. The best gel complex was achieved using 3 % sodium alginate and 100 mM CaCl₂.2H₂O. Maximum conversion response of synthetic seeds into plantlets was obtained on MS medium supplemented with 10 μM 6-benzyladenine (BA) and 2.5 μM α-naphthalene acetic acid (NAA). Encapsulated nodal segments could be stored at low temperature (4 °C) up to 4 weeks with a survival frequency of 86.7 %. The regenerated shoots rooted on MS medium containing 0.5 μM indole-3-butyric acid (IBA). Well-developed plantlets with proper root and shoot system from encapsulated nodal segments were hardened off successfully with 90 % survival rate. The high frequency of plant re-growth (conversion) from alginate-coated nodal segments coupled with high viability percentage after 4 weeks of storage is highly encouraging for the exchange of R. graveolens genetic resources.